WO2022187241A1 - Compositions and methods for targeting card8 - Google Patents

Compositions and methods for targeting card8 Download PDF

Info

Publication number
WO2022187241A1
WO2022187241A1 PCT/US2022/018333 US2022018333W WO2022187241A1 WO 2022187241 A1 WO2022187241 A1 WO 2022187241A1 US 2022018333 W US2022018333 W US 2022018333W WO 2022187241 A1 WO2022187241 A1 WO 2022187241A1
Authority
WO
WIPO (PCT)
Prior art keywords
hiv
dpp9
cells
card8
inhibitor
Prior art date
Application number
PCT/US2022/018333
Other languages
French (fr)
Inventor
Liang Shan
Qiankun Wang
Original Assignee
Washington University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Washington University filed Critical Washington University
Publication of WO2022187241A1 publication Critical patent/WO2022187241A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/536—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/69—Boron compounds

Definitions

  • the present disclosure encompasses compositions and methods for the treatment of subjects infected with retroviruses. Specifically, aspects of the present disclosure are directed to compositions and methods for inducing pyroptosis of HIV-infected cells.
  • HIV-1 the causative agent of AIDS
  • ART antiretroviral therapy
  • ART antiretroviral therapy
  • eradication of infection is not achieved with conventional treatment strategies, necessitating lifelong ART.
  • accumulating data suggest that “non-AIDS” cardiovascular, renal and hepatic diseases are amplified by HIV infection, and the immune system may exhibit premature senescence even among patients with viral suppression.
  • LRAs latency reversing agents
  • vorinostat and disulfiram have failed to demonstrate significant reduction in reservoir size, although transient elevation in viral RNA has been observed. Accordingly, in vitro experiments have revealed that the majority of existing LRAs exert weak effects on HIV-1 transcription and reactivation. The future success of shock and kill will depend on our capacity to design highly efficacious new LRAs and/or adjuvant therapies to boost the reactivation potential of existing LRAs.
  • the present disclosure provides methods to reduce the number of HIV-infected cells in a subject or cell population by administering to the subject a therapeutically effective amount or contacting the cell population with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • DPP9 dipeptidyl peptidase 9
  • the present disclosure provides methods to induce pyroptosis in a HIV-infected cell by contacting the HIV-infected cell with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • the present disclosure provides methods to sensitize the CARD8 inflammasome to non-nucleoside reverse transcriptase inhibitor-induced activation in a HIV-infected cell by contacting the HIV infected cell or administering to a subject having an HIV-infected cell an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • DPP9 dipeptidyl peptidase 9
  • the present disclosure provides method of treating a subject with a HIV-infection by administering to the subject a therapeutically effective amount of a corn-position comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • a corn-position comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • the present disclosure provides methods of treating a subject with a NNRTI resistant HIV-infection by administering to the subject a therapeutically effective amount of a composition comprising a non nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • a composition comprising a non nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • the present disclosure provides methods to reduce HIV reservoirs in a subject with an HIV-infection by administering to the subject a therapeutically effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • the present disclosure provides methods of activating HIV-1 protease activity and inhibiting DPP9 activity in an HIV- infected cell by contacting the cell with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • the NNRTI can be selected from nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), etravirine (ETR), or rilpivirine (RPV), or a combination thereof.
  • NTP nevirapine
  • DLV delavirdine
  • EMR efavirenz
  • RPV rilpivirine
  • the NNRTI is selected from rilpivirine (RPV) or efavirenz (EFV) or a combination thereof.
  • the DPP9 inhibitor can be selected from talabostat, talabostat mesylate, or 1G244.
  • the NNRTI is administered in an amount effective to induce premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of H I V-1 -infected cells.
  • the DPP9 inhibitor is administered in an amount effective to positively regulate CARD8 function, which triggers CARD8 inflammasome activation and rapid killing of virus-infected cells.
  • the combination of the NNRTI and DPP9 inhibitor is administered in an amount effective to induce the clearance of latent HIV-1 in a HIV-infected CD4+ T cell after viral reactivation.
  • the combination of the NNRTI and DPP9 inhibitor is administered in an amount effective to induce clearance of persistent HIV-1 infection.
  • the combination of the NNRTI and DPP9 inhibitor is administered to blood, lymphoid tissues, or hematopoietic-derived cells, simultaneously or separately.
  • the present disclosure provides methods of screening for CARD8 activating agents by contacting a test agent or compound with primary CD4+ T cells infected with clinical viral isolates; and detecting CARD8- dependent caspase-1 activation and/or pyroptosis by intracellular p24 staining.
  • the present disclosure provides methods for measuring the clearance of HIV-1 , by obtaining blood CD4+ T cells from a subject under suppressive ART to measure the size of viral reservoirs; and detecting viral replication after test agent or test compound treatment.
  • FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E and FIG. 1F depicts The CARD8 inflammasome senses HIV-1 protease activity.
  • FIG. 1A shows domain architecture of the CARD8 protein. CARD8 undergoes autoproteolytic processing in the FUND domain at position 296, generating the N-terminal ZU5 and C-terminal UPA- CARD fragments that remain associated non-covalently. Two HIV-1 protease cleavage sites are in the non-structured N-terminus and ZU5 domain, respectively.
  • FIG. 1B shows HIV-1 protease cleaves the N-terminus of CARD8.
  • HEK293T cells were co transfected with HA-CARD8-expressing plasmid together with pNL4-3-GFP or PR- D25H. Cells were collected 24 hours after transfection. Anti-HA, anti-CARD8-N, anti- CARD8-C, and anti-p24 antibodies were used sequentially on the same blot. Cleaved fragments are denoted with red asterisks.
  • FIG. 1C shows HIV-1 protease triggers CARD8 inflammasome activation. Control or CARD8- KO HEK293T cells were co transfected with plasmids encoding CASP1, pro-IL-1 b, and pNL4-3-GFP.
  • FIG. 1D shows autoproteolytic processing is required for HIV-1 protease triggered CARD8 inflammasome activation.
  • CARD8- KO HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and CARD8 or CARD8 S297A , together with pNL4-3-GFP.
  • LPV was added immediately after transfection.
  • FIG. 1E shows HIV-1 protease triggered CARD8 inflammasome activation is proteasome-dependent.
  • HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and pNL4-3-GFP.
  • FIG. 1F shows HIV-1 protease cleaves CARD8 in vitro. HA-tagged CARD8 was immunoprecipitated and incubated with lysed HIV-1 particles with the presence of indicated drugs. The eluate was collected for immunoblotting.
  • FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D and FIG. 2E depicts HIV-1 protease cleaves the N-terminus of CARD8.
  • FIG. 2A shows CARD8 A21 70 is cleaved at the second site.
  • HEK293T cells were transfected with plasmids encoding CARD8 or CARD8 A21 70 , and pNL4-3-GFP or PR-D25H.
  • FIG. 2B shows cleavage in the ZU5 domain does not activate the CARD8 inflammasome.
  • CARD8- KO HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and WT or mutant CARD8, together with pNL4-3-GFP.
  • FIG. 2C and FIG. 2D show mapping of the first cleavage site.
  • HEK293T cells were transfected with plasmids encoding truncated or mutated CARD8, and pNL4-3-GFP.
  • FIG. 2E shows cleavage in the N-terminus is required to activate the CARD8 inflammasome.
  • CARD8- KO HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and WT or mutant CARD8, together with pNL4-3-GFP. Cell lysates were evaluated by immunoblotting. Data are representative of three or more independent experiments. Empty vector (EV).
  • FIG. 3H and FIG. 3I depict HIV-1 protease triggers pyroptosis of infected macrophages upon NNRTI treatment.
  • FIG. 3A to FIG. 3E show HIV-1 protease activation by NNRTIs induced rapid pyroptosis of infected monocyte-derived macrophages (MDMs). MDMs were infected with replication-competent HIVNL4-3/BaL. On day 4, RAL and T-20 were added to block new infection. Cells were then treated with RPV, EFV, LPV, or combinations for 24 hours or as indicated. GFP + cells were detected by flow cytometry. DMSO controls were used to determine cell killing by NNRTIs (FIG. 3A to FIG. 3C).
  • FIG. 3D Representative images of infected MDMs were taken at 0 and 3 hours post RPV treatment. Scale bars represent 100 pm (FIG. 3D). Culture supernatant was collected after NNRTI treatment for IL-1 b ELISA (FIG. 3E).
  • FIG. 3F and FIG. 3G show pyroptosis of H I V-1 -infected MDMs is CASP1 -dependent. MDMs were infected and treated as described above. Cleavage of pro-CASP1 and cleaved CASP1 (p10 and p20) in infected MDMs after RPV treatment for one hour (FIG. 3F).
  • FIG. 3G shows HIV-1 protease mediated inflammasome activation is proteasome-dependent.
  • MDMs were infected and treated as described above.
  • Infected MDMs were pretreated with proteasome inhibitors MG132, bortezomib, or Me-Bs for 30 min and then treated with RPV for 4 hours.
  • Cell killing was determined by flow cytometry (FIG. 3H).
  • Culture supernatant from (FIG. 3H) was collected for IL-1 b measurement by ELISA (FIG. 3I).
  • FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E depict CARD8 inflammasome is required for pyroptosis of HIV-1 -infected THP-1 cells.
  • FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E depict CARD8 inflammasome is required for pyroptosis of HIV-1 -infected THP-1 cells.
  • FIG. 4A and FIG. 4B show NNRTIs induce death of HIV-1 -infected THP-1 cells.
  • THP-1 cells were infected with VSV-G pseudotyped HIV-1 reporter virus NL4-3-GFP. On day 4 post infection, cells were treated with NNRTIs for another 2 days before flow cytometry analysis.
  • FIG. 4C to FIG. 4E show NNRTI-triggered cell death is CARD8 inflammasome-dependent. Bulk populations of knockout THP-1 cells were used. Knockouts of CARD8, ASC, CASP1 or NLRP3 in THP-1 cells were confirmed by immunoblotting (C).
  • Infected THP-1 cells were pre-treated with LPS (100 ng/ml) for 3 hours before RPV treatment. GFP expression was analyzed by flow cytometry 24 hours post RPV treatment. Data were normalized to the control group (FIG. 4D). Culture supernatant was collected 48 hours post RPV treatment for IL-1 b detection (FIG. 4E
  • FIG. 5H, FIG. 5I and FIG. 5J depict HIV-1 Protease induces CARD8 inflammasome activation and subsequent cell death in CD4+ T cells.
  • FIG. 5A shows the analysis of CARD8, caspase 1, and caspase 3 expression levels in unstimulated (U) and activated (FIG. 5A) primary CD4+ T cells by immunoblotting.
  • FIG. 5B and FIG. 5C show HIV-1 protease activation by NNRTIs leads to killing of infected primary CD4+ T cells.
  • Activated primary CD4+ T cells were infected with HIV-1 reporter virus NL4-3-Pol (FIG. 5B) or different HIV-1 reporter viruses (FIG. 5C) for 3 days before treatment with RPV or indicated NNRTIs.
  • FIG. 5D and FIG. 5E show HIV-1 protease activation by NNRTIs induced CASP1 activation in infected primary CD4+ T cells.
  • Activated primary CD4+ T cells were infected with the NL4-3-Pol.
  • cells were treated with EFV, RPV, LPV or combinations for 3 hours before staining for active CASP1.
  • FIG. 5F shows chemical inhibition of caspase-1 blocks NNTRI-induced cell death.
  • Activated primary CD4+ T cells were infected with the NL4-3-Pol.
  • FIG. 5G shows knockout of CARD8 in primary CD4+ T cells was confirmed by immunoblotting.
  • FIG. 5H to FIG. 5G show the CARD8 inflammasome is required for pyroptosis of HIV-1 -infected primary CD4+ T cells.
  • CARD8-, ASC- or CASP1 -knockout primary CD4+ T cells were co-stimulated and then infected with NL4-3-Pol.
  • cells were treated with RPV.
  • FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D and FIG. 6E depict the Induction of CARD8 inflammasome activation clears infection of clinical HIV-1 isolates.
  • FIG. 6A shows killing of primary CD4 + T cells infected with different subtypes of clinical HIV-1 isolates. Activated CD4 + T cells were infected with a panel of international HIV-1 isolates. RAL and T-20 were added with or without EFV, RPV or NVP on day 6 post infection. Viral infection was measured by intracellular p24 staining on day 8 and normalized to the DMSO controls.
  • FIG. 6B shows killing of infected primary CD4 + T cells is proteasome-dependent.
  • Activated primary CD4 + T cells were infected with different subtypes of clinical HIV-1 isolates. RAL, T-20, and RPV were added with or without bortezomib on day 6 post infection. Viral infection was measured by intracellular p24 staining 6 hours after drug treatment.
  • FIG. 6C shows the CARD8 inflammasome is required for the killing of infected primary CD4 + T cells.
  • Activated CARD8- KO or control primary CD4 + T cells were infected with different subtypes of clinical HIV-1 isolates.
  • FIG. 6E shows the clearance of latent HIV-1 by RPV treatment. Frequency of latent HIV-1 was determined by QVOA. Red: ARV for 2 days and RPV at 2.5 mM. Blue: ARV for 3 days and RPV at 5 pM. Open circle: no detectable HIV-1 infection by p24 ELISA.
  • FIG. 6B and FIG. 6C P-values were calculated using multiple t test.
  • FIG. 6E P- value was calculated by ratio paired t test. *P ⁇ 0.01 , **P ⁇ 0.01 , ***P ⁇ 0.001 , and ****PO .001.
  • FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G and FIG. 7H show NNRTIs induce death of HIV-1 -infected cells in a dose-dependent manner.
  • FIG. 7A is a depiction of how CD4 + T Cells were treated with NNRTI’s and assayed for killing.
  • FIG. 7B is a dose response curves for various NNRTI’s in successive three-fold dilutions in three healthy donor CD4 + T cells isolated from PBMC.
  • EC50 Values for EFV, RPV, and ETR, are as follows: 266.1 nM, 87.8nM, and 786.6nM respectively. ETR and DOR did not provide sufficient killing for EC50 calculation.
  • FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G and FIG. 7H show NNRTIs induce death of
  • FIG. 7B is a dose response curves for THP-1 infected with NL4-3-GFP and treated with EFV, RPV, or DOR as in panel A. ECso for EFV is 2128nM and 438 for RPV. FIG. 7D is a dose response curves for killing of NL4-3-Avif-vpr infected THP-1 CARD8KO or Cas9 transduced control cells treated with EFV, RPV, or NVP. The ability of EFV and RPV to kill infected cells is CARD8 dependent regardless of concentration.
  • FIG. 7G show treatment of CD4 + T cells with EFV and RPV with or without the presence of 50% human serum in the culture media (NL4-3-pol).
  • FIG. 7G shows the log fold change increase in EC50 due to the presence of human serum.
  • FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D and FIG. 8E show DPP9 inhibition sensitizes the CARD8 Inflammasome to NNRTI-induced pyroptosis.
  • FIG. 8A is a graphical depiction of CARD8 inflammasome activation and sensitization through VBP.
  • CARD8 first undergoes autoprocessing of its FUND domain leaving two non- covalently bound fragments (N-terminal and C-terminal). HIV-1 protease, after premature activation due to Gag-Pol dimerization through NNRTIs, can cleave the N- terminal fragment leading to proteasomal degradation.
  • FIG. 8C shows log fold changes in EC50 due to varying VBP concentrations are plotted for EFV (left) and RPV (right).
  • FIG. 8D shows VBP and EFV combination treatment denotes a synergistic relationship.
  • SynergyFinder2.0 Bosset, HSA, Loewe, and ZIP
  • FIG. 8E shows VBP toxicity in CD4 + T Cells as denoted by the heatmap of MTS assay results of three separate donors of primary CD4 + T Cells treated for two days with EFV and/or VBP.
  • FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F and FIG. 9G show characterization of VBP enhancement of NNRTI-induced cell killing.
  • FIG. 9A is a time course treatment of HIV-1 -infected primary CD4 + T cells treated with DMSO, EFV ( 5pM), VBP (.5mM), or combination. Killing of infected cells plateaus after 48 hours in all conditions.
  • FIG. 9B shows fold change enhancement of combination treatment in comparison to EFV alone treatment from panel A.
  • FIG. 9D show time course treatment and fold change enhancement of H I V-1 -infected THP-1 cells treated with DMSO, EFV (1 mM), VBP (.5mM), or combination.
  • FIG. 9E shows NNRTI-based killing and VBP enhancement is specific to the CARD8 inflammasome.
  • FIG. 9F shows CASP1 activation by NNRTIs and VBP. Cells were simultaneously treated with EFV and VBP and stained with CASP1 staining dye.
  • FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E and FIG. 10F show VBP sensitization of the CARD8 inflammasome can overcome NNRTI resistance.
  • FIG. 10A shows killing and enhancement of clinical isolates from Clades A, B, C, and D.
  • One Donor of primary CD4 + T Cells were treated with DMSO, EFV (.5mM), or EFV with VBP ( 5pM) in the presence of media containing T-20 (1 mM), Raltegravir (1 pM). Each strain showed significant enhancement upon addition of VBP (Two-way ANOVA with Tukey’s multiple comparison test).
  • FIG. 10B is a graphical depiction of the location of NNRTI RAMS.
  • FIG. 10C shows NNRTI RAMS show diminished capacity for killing HIV infected primary CD4 + T Cells from one donor upon treatment with 5pM of EFV with respective fold change from no RAM control shown in panel FIG. 10D (Two-way ANOVA with Tukey’s multiple comparison test).
  • FIG. 10E shows upon VBP treatment, these same mutants show significantly enhanced killing with increasing VBP concentrations (One-way ANOVA with Tukey’s multiple comparison test). Additionally, some RAM containing viruses have increased levels of killing efficiency as depicted by the fold change of killing by 1 mM VBP treatment from no RAM virus in panel FIG.
  • FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F and FIG. 11G show VbP enhances clearance of HIV-1 infected cells in humanized mice.
  • Primary CD4+ T cells were infected with NL4-3-Pol. 3 days post infection; these cells were infused into mice (5-10 million cells per mouse).
  • EFV and VbP were provided by IP and IV injection, respectively.
  • FIG. 11A shows killing of HIV-1 -infected human CD4+
  • FIG. 11 A, FIG. 11B, FIG. 11C and FIG. 11 D show killing of infected human CD4+ T Cells in blood (FIG. 11B) and lung tissues (FIG. 11C and FIG. 11D) at 24hr post treatment with 60 pg VbP, 0.5 mg EFV, or combo.
  • FIG. 11E shows cross cohort comparison of four independent cohorts of mice treated with EFV or combo as described above. CD4+ T cells from four healthy blood donors were used.
  • FIG. 11G show killing of infected human CD4+ T cells in blood (FIG. 11E) or lung (FIG. 11 F) with single dose or multidose combination treatment regimens.
  • the multi dose regimen group received 0.5 mg EFV injections on days 0, 1, and 2, and 60 pg VbP injections on days 0 and 2.
  • the single-dose regimen received both EFV and VbP only on day 0. Error bars show mean values with SEM.
  • FIG. 12 shows DPP8/9 inhibitor 1G244 enhances EFV-triggered killing of HIV-infected cells.
  • Primary CD4+ T cells were infected with a HIV reporter virus. 1G244 at indicated concentration (nM) with or without 3mM EFV was added to the culture on day 3 post infection. Cell killing was measured by FACS 2 days post drug treatment. DETAILED DESCRIPTION
  • compositions and methods comprising a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor to kill HIV infected cells.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • DPP9 dipeptidyl peptidase 9
  • NNRTI non-nucleoside reverse transcriptase inhibitor
  • DPP9 inhibitor-mediated sensitization of the CARD8 inflammasome in HIV-infected cells is shown to overcome NNRTI resistance. Additional aspects of the disclosure are described below.
  • the term “about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ⁇ 5%, but can also be ⁇ 4%, 3%,
  • a “biological sample” refers to a sample of tissue, cells, or fluid isolated from a subject, including but not limited to, for example, blood, buffy coat, plasma, serum, immune cells (e.g., macrophages), sputa, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, organs, biopsies and also samples of in vitro cell culture constituents, including, but not limited to, conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.
  • the term “blood sample” refers to a biological sample derived from blood, preferably peripheral (or circulating) blood.
  • the blood sample can be whole blood, plasma or serum, although plasma is typically preferred.
  • treat refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disease/disorder.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented.
  • the terms “effective amount” or “therapeutically effective amount” of a drug used to treat a disease is an amount that can reduce the severity of a disease, reduce the severity of one or more symptoms associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition.
  • An “effective amount” may be determined empirically and in a routine manner, in relation to the stated purpose.
  • Retroviruses are a family of enveloped viruses that replicate in a host cell through the process of reverse transcription.
  • a retrovirus is a single-stranded positive sense RNA virus with a DNA intermediate and, as an obligate parasite, targets a host cell.
  • the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern.
  • This new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus.
  • the host cell treats the viral DNA as part of its own genome, translating and transcribing the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus. It is difficult to detect the virus until it has infected the host. At that point, the infection will persist indefinitely.
  • HTLV 1 T-cell leukaemias/lymphomas, Tropical spastic paraparesis
  • HTLV 2 no known pathology
  • HIV 1 & 2 - AIDS HIV 1 & 2 - AIDS
  • HTLV-3 and HTLV-4 no known pathology
  • HTLV-1 is known to cause a type of cancer called adult T- cell leukemia/lymphoma and a demyelinating disease called HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP).
  • HTLV-2 is a virus closely related to HTLV-I and shares approximately 70% genomic homology with HTLV-I.
  • HTLV-3 and HTLV-4 are two new human retroviruses recently identified which are related to HTLV1 and 2.
  • HIV human immunodeficiency virus
  • AIDS acquired immunodeficiency syndrome
  • AIDS acquired immunodeficiency syndrome
  • average survival time after infection with HIV is estimated to be 9 to 11 years, depending on the HIV subtype.
  • HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells.
  • HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including apoptosis of uninfected bystander cells, direct viral killing of infected cells, and killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells.
  • CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections.
  • HIV establishes a persistent infection in its host and only causes death many years later. Most individuals experience a primary infection resulting in a febrile illness about 2-4 weeks after exposure. This illness coincides with seroconversion. The symptoms are similar to those of glandular fever, namely fever, sore throat, night sweats, lymphadenopathy, diarrhea. Following the primary infection, the patient enters a stage of clinical latency. During this time the patient feels fine, but they are infectious as they have ongoing viral replication and have HIV antibodies in their blood. As the CD4 counts drop, there is a gradual onset of a variety of prodromal disorders, such as weight loss, fever, persistent lymphadenopathy, oral candidiasis and diarrhea. These symptoms precede the progression to AIDS.
  • the human immunodeficiency virus causes the acquired immunodeficiency syndrome (AIDS), a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. Without treatment, average survival time after infection with HIV is estimated to be 9 to 11 years, depending on the HIV subtype.
  • HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including apoptosis of uninfected bystander cells, direct viral killing of infected cells, and killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells. When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections. [0052] HIV establishes a persistent infection in its host and only causes death many years later.
  • helper T cells specifically CD4+ T cells
  • macrophages specifically CD4+ T cells
  • dendritic cells dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including apoptosis of uninfected bystander cells, direct viral killing of infected cells, and
  • the present disclosure provides a composition comprising a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • DPP9 dipeptidyl peptidase 9
  • a composition of the disclosure comprises Efavirnez and a DPP9 inhibitor.
  • a composition of the disclosure comprises Rilpivirine and a DPP9 inhibitor.
  • a composition of the disclosure comprises a NNRTI and talbostat (Val-boroPro) or 1G244.
  • a composition of the disclosure may optionally comprise one or more additional drugs or therapeutically active agent in addition to the non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • DPP9 dipeptidyl peptidase 9
  • a composition of the disclosure may further comprise a pharmaceutically acceptable excipient, carrier or diluent.
  • a composition of the disclosure may contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (substances of the present disclosure may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants.
  • NRTI Non-Nucleoside reverse transcriptase inhibitors
  • Non-nucleoside reverse transcriptase inhibitor is an antiretroviral (ARV) HIV drug class.
  • NRTI Non-Nucleoside reverse transcriptase inhibitors
  • NNRTIs can inhibit HIV reverse transcriptase (an HIV enzyme) by binding to an allosteric site of the enzyme; NNRTIs can act as non-competitive inhibitors of reverse transcriptase.
  • NNRTIs affect the handling of substrate (nucleotides) by reverse transcriptase by binding near the active site. HIV uses reverse transcriptase to convert its RNA into DNA (reverse transcription). Blocking reverse transcriptase and reverse transcription prevents HIV from replicating.
  • NNRTIs can be further classified into 1st generation and second generation NNRTIs.
  • 1st generation NNRTIs include nevirapine and efavirenz.
  • Second generation NNRTIs are etravirine and rilpivirine. HIV-2 is naturally resistant to NNRTIs.
  • Non-limiting examples of suitable NNRTIs include efavirenz, nevirapine, etravirine, doravirine, nevirapine, delavirdine, and rilpivirine.
  • a NNRTI of the disclosure is selected from the group consisting of efavirenz, rilpivirine, and etravirine.
  • a NNRTI of the disclosure is not nevirapine.
  • of the disclosure is not doravirine.
  • a NNRTI according to the disclosure drive Gag-Pol dimerization and intracellular protease activation which cleaves CARD8 and leads to activation of the CARD8 inflammasome.
  • Dipeptidyl Peptidase 9 (HGNC: 18648 NCBI Entrez Gene: 91039 Ensembl: ENSG00000142002 OMIM®: 608258 UniProtKB/Swiss-Prot: Q86TI2) is encoded by the DPP9 gene (chr19:4,675,224-4,724,673). This gene encodes a protein that is a member of the S9B family in clan SC of the serine proteases. The protein has been shown to have post-proline dipeptidyl aminopeptidase activity, cleaving Xaa-Pro dipeptides from the N-termini of proteins.
  • DPP9 polynucleotide within the meaning of the disclosure, shall be understood as being a nucleic acid molecule selected from a group consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
  • a “DPP9 polypeptide”, within the meaning of the disclosure, shall be understood as being a polypeptide selected from a group consisting of (i) polypeptides having the sequence of SEQ ID NO: 1 , (ii) polypeptides comprising the sequence of SEQ ID NO: 1, (iii) polypeptides encoded by DPP9 polynucleotides; and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), or (iii); wherein said polypeptide has DPP9 activity.
  • a “DPP9 inhibitor” is any compound that is capable of reducing DPP9 activity and/or expression.
  • inhibitor is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists.
  • a compound with the ability to inhibit DPP9 may include, without limitation, a compound, a drug, a small molecule, a peptide, a nucleic acid molecule, a protein, an antibody, and combinations thereof.
  • a nucleic acid molecule may be an antisense oligonucleotide, a ribozyme, a small nuclear RNA (snRNA), a long noncoding RNA (LncRNA), or a nucleic acid molecule which forms triple helical structures.
  • DPP9 nucleic acid expression DPP9 protein expression
  • DPP9 activity may be measured as described in more detail below and in the Examples.
  • a DPP9 inhibitor may be Talabostat (Val- boroPro, PT100) or Talabostat mesylate.
  • a DPP9 inhibitor compound of the disclosure is a compound of formula (I):
  • a DPP9 inhibitor may be 1 G244.
  • a DPP9 inhibitor compound of the disclosure is a compound of formula (II):
  • a DPP9 inhibitor may be SLRFLYEG (SEQ ID NO: 3) described in Ross et al. (PNAS 115(7) 2018; Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer) incorporated herein by reference in its entirety.
  • Additional DPP9 inhibitors useful according to the present disclosure include those disclosed in WO 2005106487; WO 2014068023; WO2014127747; US 20050215784; WO 2011113895, US 20110218142; US 2008/0293618; WO 2005/106487 describes antibodies and siRNAfor modulating DPP9 activity; the disclosures of which are herein incorporated by reference in their entirety.
  • Dosages of a compound that inhibits DPP9 can vary between wide limits, depending upon the disease or disorder to be treated and/or the age and condition of the subject to be treated.
  • concentration of the compound that inhibits DPP9 may be from about 1 mM to about 40 pM.
  • concentration of the compound that inhibits DPP9 may be from about 5 pM to about 25 pM.
  • the concentration of the compound that inhibits DPP9 may be about 1 , about 2.5 about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 12, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 30, about 35, or about 40 pM. Additionally, the concentration of the compound that inhibits DPP9 may be greater than 40 pM. For example, the concentration of the compound that inhibits DPP9 may be about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 or about 100 pM.
  • the concentration of the compound that inhibits DPP9 may be from about 1 pM to about 10 pM, from about 10 pM to about 20 pM, from about 20 pM to about 30 pM, or from about 30 pM to about 40 pM. In a specific embodiment, the concentration of the compound that inhibits DPP9 may be from about 1 pM to about 10 pM.
  • the dose of the compound that modulates DPP9 may be from about 0.1 mg/kg to about 500 mg/kg.
  • the dose of the compound that inhibits DPP9 may be about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, or about 25 mg/kg.
  • the dose of the compound that inhibits DPP9 may be about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, or about 250 mg/kg. Additionally, the dose of the compound that inhibits DPP9 may be about 300 mg/kg, about 325 mg/kg, about 350 mg/kg, about 375 mg/kg, about 400 mg/kg, about 425 mg/kg, about 450 mg/kg, about 475 mg/kg or about 500 mg/kg.
  • DPP9 nucleic acid expression may be measured to identify a compound that inhibits DPP9.
  • DPP9 nucleic acid expression is decreased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
  • DPP9 mRNA may be measured to identify a compound that inhibits DPP9.
  • nucleic acid expression or “level of nucleic acid expression” as used herein refers to a measurable level of expression of the nucleic acids, such as, without limitation, the level of messenger RNA (mRNA) transcript expressed or a specific variant or other portion of the mRNA, the enzymatic or other activities of the nucleic acids, and the level of a specific metabolite.
  • mRNA messenger RNA
  • nucleic acid includes DNA and RNA and can be either double stranded or single stranded.
  • Non-limiting examples of suitable methods to assess an amount of nucleic acid expression may include arrays, such as microarrays, PCR, such as RT-PCR (including quantitative RT-PCR), nuclease protection assays and Northern blot analyses.
  • determining the amount of expression of a target nucleic acid comprises, in part, measuring the level of target nucleic acid mRNA expression.
  • the amount of nucleic acid expression may be determined by using an array, such as a microarray.
  • an array such as a microarray.
  • Methods of using a nucleic acid microarray are well and widely known in the art.
  • a nucleic acid probe that is complementary or hybridizable to an expression product of a target gene may be used in the array.
  • the term “hybridize” or “hybridizable” refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid.
  • the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
  • probe refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence.
  • the probe hybridizes to an RNA product of the nucleic acid or a nucleic acid sequence complementary thereof.
  • the length of probe depends on the hybridization conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length.
  • the amount of nucleic acid expression may be determined using PCR.
  • Methods of PCR are well and widely known in the art, and may include quantitative PCR, semi-quantitative PCR, multiplex PCR, or any combination thereof.
  • the amount of nucleic acid expression may be determined using quantitative RT-PCR. Methods of performing quantitative RT-PCR are common in the art.
  • the primers used for quantitative RT-PCR may comprise a forward and reverse primer for a target gene.
  • primer refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH).
  • the primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used.
  • a primer typically contains 15-25 or more nucleotides, although it can contain less or more.
  • the amount of nucleic acid expression may be measured by measuring an entire mRNA transcript for a nucleic acid sequence, or measuring a portion of the mRNA transcript for a nucleic acid sequence.
  • the array may comprise a probe for a portion of the mRNA of the nucleic acid sequence of interest, or the array may comprise a probe for the full mRNA of the nucleic acid sequence of interest.
  • the primers may be designed to amplify the entire cDNA sequence of the nucleic acid sequence of interest, or a portion of the cDNA sequence.
  • One of skill in the art will recognize that there is more than one set of primers that may be used to amplify either the entire cDNA or a portion of the cDNA for a nucleic acid sequence of interest.
  • Methods of designing primers are known in the art.
  • Methods of extracting RNA from a biological sample are known in the art.
  • the level of expression may or may not be normalized to the level of a control nucleic acid.
  • a control nucleic acid should not specifically hybridize with an mRNA nucleotide sequence of the disclosure. This allows comparisons between assays that are performed on different occasions.
  • DPP9 protein expression may be measured to identify a compound that inhibits DPP9. For example, when DPP9 protein expression is decreased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
  • DPP9 protein expression may be measured using immunoblot. In another specific embodiment, DPP9 protein expression may be measured using immunofluorescence staining.
  • Methods for assessing an amount of protein expression are well known in the art, and all suitable methods for assessing an amount of protein expression known to one of skill in the art are contemplated within the scope of the disclosure.
  • suitable methods to assess an amount of protein expression may include epitope binding agent-based methods and mass spectrometry based methods.
  • the method to assess an amount of protein expression is mass spectrometry.
  • MS mass spectrometry
  • the method to assess an amount of protein expression is an epitope binding agent-based method.
  • epitope binding agent refers to an antibody, an aptamer, a nucleic acid, an oligonucleic acid, an amino acid, a peptide, a polypeptide, a protein, a lipid, a metabolite, a small molecule, or a fragment thereof that recognizes and is capable of binding to a target gene protein.
  • Nucleic acids may include RNA, DNA, and naturally occurring or synthetically created derivative.
  • an antibody generally means a polypeptide or protein that recognizes and can bind to an epitope of an antigen.
  • An antibody as used herein, may be a complete antibody as understood in the art, i.e. , consisting of two heavy chains and two light chains, or may be any antibody-like molecule that has an antigen binding region, and includes, but is not limited to, antibody fragments such as Fab’, Fab, F(ab’)2, single domain antibodies, Fv, and single chain Fv.
  • the term antibody also refers to a polyclonal antibody, a monoclonal antibody, a chimeric antibody and a humanized antibody.
  • aptamer refers to a polynucleotide, generally a RNA or DNA that has a useful biological activity in terms of biochemical activity, molecular recognition or binding attributes. Usually, an aptamer has a molecular activity such as binging to a target molecule at a specific epitope (region).
  • an aptamer which is specific in it binding to a polypeptide, may be synthesized and/or identified by in vitro evolution methods.
  • Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well known in the art (See, e.g. US 7,939,313; herein incorporated by reference in its entirety).
  • an epitope binding agent-based method of assessing an amount of protein expression comprises contacting a sample comprising a polypeptide with an epitope binding agent specific for the polypeptide under conditions effective to allow for formation of a complex between the epitope binding agent and the polypeptide.
  • Epitope binding agent-based methods may occur in solution, or the epitope binding agent or sample may be immobilized on a solid surface.
  • suitable surfaces include microtitre plates, test tubes, beads, resins, and other polymers.
  • An epitope binding agent may be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art.
  • the epitope binding agent may either be synthesized first, with subsequent attachment to the substrate, or may be directly synthesized on the substrate.
  • the substrate and the epitope binding agent may be derivatized with chemical functional groups for subsequent attachment of the two.
  • the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the epitope binding agent may be attached directly using the functional groups or indirectly using linkers.
  • the epitope binding agent may also be attached to the substrate non-covalently.
  • a biotinylated epitope binding agent may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
  • an epitope binding agent may be synthesized on the surface using techniques such as photopolymerization and photolithography. Additional methods of attaching epitope binding agents to solid surfaces and methods of synthesizing biomolecules on substrates are well known in the art, i.e. VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No. 6,566,495, and Rockett and Dix, Xenobiotica 30(2): 155-177, both of which are hereby incorporated by reference in their entirety).
  • Contacting the sample with an epitope binding agent under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the epitope binding agent composition to the sample and incubating the mixture for a period of time long enough for the epitope binding agent to bind to any antigen present. After this time, the complex will be washed and the complex may be detected by any method well known in the art. Methods of detecting the epitope binding agent-polypeptide complex are generally based on the detection of a label or marker.
  • label refers to any substance attached to an epitope binding agent, or other substrate material, in which the substance is detectable by a detection method.
  • Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, stretpavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, and luciferase).
  • Methods of detecting an epitope binding agent-polypeptide complex based on the detection of a label or marker are well known in the art.
  • an epitope binding agent-based method is an immunoassay.
  • Immunoassays can be run in a number of different formats. Generally speaking, immunoassays can be divided into two categories: competitive immmunoassays and non-competitive immunoassays.
  • competitive immunoassay an unlabeled analyte in a sample competes with labeled analyte to bind an antibody. Unbound analyte is washed away and the bound analyte is measured.
  • the antibody is labeled, not the analyte.
  • Non-competitive immunoassays may use one antibody (e.g. the capture antibody is labeled) or more than one antibody (e.g. at least one capture antibody which is unlabeled and at least one “capping” or detection antibody which is labeled.) Suitable labels are described above.
  • the epitope binding agent-based method is an ELISA. In other embodiments, the epitope binding agent-based method is a radioimmunoassay. In still other embodiments, the epitope binding agent-based method is an immunoblot or Western blot. In alternative embodiments, the epitope binding agent-based method is an array. In another embodiment, the epitope binding agent-based method is flow cytometry. In different embodiments, the epitope binding agent-based method is immunohistochemistry (IHC) or immunofluorescence. IHC uses an antibody (or fluorescently labeled antibody in the case of immunofluorescence) to detect and quantify antigens in intact tissue samples.
  • IHC immunohistochemistry
  • immunofluorescence immunofluorescence
  • the tissue samples may be fresh- frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC or immunofluorescence. Methods of preparing tissue block for study by IHC or immunofluorescence, as well as methods of performing IHC or immunofluorescence are well known in the art. iii. DPP9 activity
  • DPP9 activity may be measured to identify a compound that inhibits DPP9.
  • DPP9 is a protease and therefore its ability to cleave peptides can be a measure of DPP9 activity.
  • Methods for measuring protease activity are standard in the art and also described below.
  • the ability of DPP9 to bind to CARD8 can be measured as a determination of DPP9 activity, where a compound which disrupts the ability of DPP9 to bind with CARD8 is an inhibitor of DPP9 activity.
  • HIV-infected cell viability may be measured as an indication of DPP9 activity.
  • Cell viability may be measured using methods standard in the art as described below in the Examples. For example, when cell viability of a HIV-infected cells is decreased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
  • caspases may be measured as an indication of DPP9 activity.
  • caspase 1 may be measured as an indication of DPP9 activity.
  • Inhibition of DPP9 leads to CASP1 activation in HIV-infected cells. For example, when CASP1 activation in HIV-infected cells is increased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
  • CARD8 inflammasome activation may be measured as an indication of DPP9 activity. Inhibition of DPP9, leads to CARD8 inflammasome activation in HIV-infected cells. For example, when CARD8 inflammasome activation in HIV-infected cells is increased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
  • the present disclosure also provides pharmaceutical compositions.
  • the pharmaceutical composition comprises a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor, as active ingredients, and at least one pharmaceutically acceptable excipient.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • DPP9 dipeptidyl peptidase 9
  • the pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant, or a coloring agent.
  • the amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science.
  • the excipient may be a diluent.
  • the diluent may be compressible (i.e. , plastically deformable) or abrasively brittle.
  • suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylit
  • Non-limiting examples of suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate.
  • the excipient may be a binder.
  • Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.
  • the excipient may be a filler.
  • suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone.
  • the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.
  • the excipient may be a buffering agent.
  • suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).
  • the excipient may be a pH modifier.
  • the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.
  • the excipient may be a disintegrant.
  • the disintegrant may be non-effervescent or effervescent.
  • Suitable examples of non- effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.
  • the excipient may be a dispersant or dispersing enhancing agent.
  • Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.
  • the excipient may be a preservative.
  • suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.
  • the excipient may be a lubricant.
  • suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.
  • the excipient may be a coloring agent.
  • Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).
  • the weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1 % or less of the total weight of the composition.
  • compositions can be formulated into various dosage forms and administered topically by a number of different means that will deliver a therapeutically effective amount of the active ingredients.
  • Such compositions administered topically in dosage unit formulations may contain conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired.
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. Formulation of drugs is discussed in, for example, Gennaro, A.
  • Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules.
  • the active ingredient is ordinarily combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above.
  • Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups.
  • the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.
  • the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
  • the preparation may be an aqueous or an oil-based solution.
  • Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as etheylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol.
  • the pH of the aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyo
  • compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • penetrants appropriate to the barrier to be permeated are generally included in the preparation.
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • the pharmaceutical composition is applied as a topical ointment or cream.
  • the active ingredient may be employed with either a paraffinic or a water-miscible ointment base.
  • the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base.
  • Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent.
  • compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes.
  • Transmucosal administration may be accomplished through the use of nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal administration may be via ointments, salves, gels, patches, or creams as generally known in the art.
  • a composition comprising a non nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells (e.g., HIV-infected cells), to increase the stability of the composition, or to minimize potential toxicity of the composition.
  • a suitable vehicle e.g., a variety of vehicles are suitable for delivering a composition of the present disclosure.
  • suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating compositions into delivery vehicles are known in the art.
  • a liposome delivery vehicle may be utilized.
  • Liposomes depending upon the embodiment, are suitable for delivery of a non nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor in view of their structural and chemical properties.
  • NRTI non nucleoside reverse transcriptase inhibitor
  • DPP9 dipeptidyl peptidase 9
  • liposomes are spherical vesicles with a phospholipid bilayer membrane.
  • the lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • DPP9 dipeptidyl peptidase 9
  • Liposomes may be comprised of a variety of different types of phosolipids having varying hydrocarbon chain lengths.
  • Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE).
  • PA phosphatidic acid
  • PS phosphatidylserine
  • PI phosphatidylinositol
  • PG phosphatidylglycerol
  • DPG diphosphatidylglycerol
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • the fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated.
  • Suitable fatty acid chains include (common name presented in parentheses) n-dodecanoate (laurate), n- tretradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9, 12- octadecandienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linolenate
  • the two fatty acid chains of a phospholipid may be identical or different.
  • Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenyl PS, and the like.
  • the phospholipids may come from any natural source, and, as such, may comprise a mixture of phospholipids.
  • egg yolk is rich in PC
  • Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used. Mixtures of different phospholipids may result in liposome compositions having advantageous activity or stability of activity properties.
  • phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(1-(2,3-dioleolyoxy)propyl)-N,N,N- trimethyl ammonium chloride, 1 ,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchloarate, 3,3’-deheptyloxacarbocyanine iodide, 1 ,1’-dedodecyl-3,3,3’,3’- tetramethylindocarbocyanine perchloarate, 1 ,1’-dioleyl-3,3,3’,3’-tetramethylindo carbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1 ,1 ,-dilinoleyl-3,3,3’,3
  • Liposomes may optionally comprise sphingolipids, in which sphingosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes.
  • Liposomes may optionally contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.
  • Liposomes may further comprise a suitable solvent.
  • the solvent may be an organic solvent or an inorganic solvent.
  • Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.
  • Liposomes carrying a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in U.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561 , 4,755,388, 4,828,837, 4,925,661,
  • liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing.
  • the liposomes are formed by sonication.
  • the liposomes may be multilamellar, which have many layers like an onion, or unilamellar.
  • the liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar lipsomes.
  • liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compound, concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.
  • a composition of the disclosure may be delivered to a cell as a microemulsion.
  • Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and “oil.”
  • the "oil” in this case, is the supercritical fluid phase.
  • the surfactant rests at the oil- water interface.
  • Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art.
  • the aqueous microdomains suitable for use in the disclosure generally will have characteristic structural dimensions from about 5 nm to about 100 nm. Aggregates of this size are poor scatterers of visible light and hence, these solutions are optically clear.
  • microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates.
  • the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil.
  • the microemulsion structure is the lamellae. It comprises consecutive layers of water and oil separated by layers of surfactant.
  • the “oil” of microemulsions optimally comprises phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions.
  • the non nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor may be encapsulated in a microemulsion by any method generally known in the art.
  • a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor may be delivered in a dendritic macromolecule, or a dendrimer.
  • a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe.
  • the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer.
  • phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments. Any method generally known in the art may be utilized to make dendrimers and to encapsulate compositions of the disclosure therein.
  • dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape.
  • the final size of a dendrimer is typically controlled by the number of iterative steps used during synthesis. A variety of dendrimer sizes are suitable for use in the disclosure. Generally, the size of dendrimers may range from about 1 nm to about 100 nm.
  • a composition according to the disclosure may comprise one or more active agents in addition to the non-nucleoside reverse transcriptase inhibitor (NNRTI) and the dipeptidyl peptidase 9 (DPP9) inhibitor.
  • NRTI non-nucleoside reverse transcriptase inhibitor
  • DPP9 dipeptidyl peptidase 9
  • Anti-retroviral therapeutics consist of several class of drugs which may be used in combination with each other and with the non-nucleoside reverse transcriptase inhibitor (NNRTI) and the dipeptidyl peptidase 9 (DPP9) inhibitor as described herein. Use of these drugs in combination can be termed anti-retroviral therapy (ART), combination anti-retroviral therapy (cART) or highly active anti-retroviral therapy (FIAART).
  • Anti-retroviral therapeutics are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits.
  • Entry inhibitors interfere with binding, fusion and entry of H IV-1 to the host cell by blocking one of several targets.
  • Maraviroc and enfuvirtide are the two currently available agents in this class. Maraviroc works by targeting CCR5, a co-receptor located on human helper T-cells. Caution should be used when administering this drug however due to a possible shift in tropism which allows FI IV to target an alternative co-receptor such as CXCR4.
  • individuals may have a mutation in the CCR5 delta gene which results in a non-functional CCR5 co- receptor and in turn, a means of resistance or slow progression of the dis-ease.
  • Enfuvirtide is a peptide drug that must be injected and acts by interacting with the N-terminal heptad repeat of gp41 of HIV to form an inactive hetero six-helix bundle, there-fore preventing infection of host cells.
  • Nucleoside reverse transcriptase inhibitors and nucleotide reverse transcriptase inhibitors (NtRTI) are nucleoside and nucleotide analogues which inhibit re-verse transcription. HIV is an RNA virus and hence unable to become integrated into the DNA in the nucleus of the hu-man cell; it must be “reverse” transcribed into DNA. Since the conversion of RNA to DNA is not done in the mammalian cell it is performed by a viral protein which makes it a selective target for inhibition. NRTIs are chain terminators such that once incorporated, work by preventing other nucleosides from also being incorporated into the DNA chain because of the absence of a 3’ OH group. Both act as competitive substrate inhibitors. Examples of currently used NRTIs include zidovudine, abacavir, lamivudine, emtricitabine, and tenofovir.
  • Integrase inhibitors also known as integrase nuclear strand transfer inhibitors or INSTIs
  • integrase inhibitors inhibit the viral enzyme integrase, which is responsible for integration of viral DNA into the DNA of the infected cell.
  • integrase inhibitors There are several integrase inhibitors currently under clinical trial, and raltegravir became the first to receive FDA approval in October 2007.
  • Raltegravir has two metal binding groups that compete for substrate with two Mg 2+ ions at the metal binding site of integrase.
  • two other clinically approved integrase inhibitors are elvitegravir and dolutegravir.
  • Protease inhibitors block the viral protease enzyme necessary to produce mature virions upon budding from the host membrane. Particularly, these drugs prevent the cleavage of gag and gag/pol precursor proteins. Virus particles produced in the presence of protease inhibitors are defective and mostly non-infectious. Examples of HIV protease inhibitors are Lopinavir, Indinavir, Nelfinavir, Amprenavir and Ritonavir. Darunavir and atazanavir are currently recommended as first line therapy choices. Maturation inhibitors have a similar effect by binding to gag, but development of two experimental drugs in this class, Bevirimat and Vivecon, was halt-ed in 2010.
  • Second generation drugs have been developed that are effective against otherwise resistant HIV variants
  • Combinations of antiretrovirals create multiple obstacles to HIV replication to keep the number of offspring low and reduce the possibility of a superior mutation. If a mutation that conveys resistance to one of the drugs being taken arises, the other drugs continue to suppress reproduction of that mutation. With rare exceptions, no individual antiretroviral drug has been demonstrated to suppress an HIV infection for long; these agents must be taken in combinations in order to have a lasting effect. As a result, the standard of care is to use combinations of antiretroviral drugs. Combinations usually comprise of three drugs from at least two different classes. This three drug combination is commonly known as a triple cocktail. Combinations of antiretrovirals are subject to positive and negative synergies, which limits the number of useful combinations. Additionally, there are now several options that combine three drugs into one pill taken once daily.
  • the quantity of a pharmaceutical composition necessary to deliver a therapeutically effective dose can be determined by routine in vitro and in vivo methods, common in the art of drug testing. See, for example, D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages for various therapeutic entities are well known to those of skill in the art.
  • Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LDso (the dose lethal to 50% of the population) and the EDso, (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al.
  • Administration of a composition of the disclosure can occur as a single event or over a time course of treatment.
  • one or more of a nanoparticle composition can be administered daily, weekly, bi-weekly, or monthly.
  • the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
  • compositions of the disclosure can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent.
  • compositions as disclosed herein can be administered simultaneously with another agent, such as an antiviral, an antibiotic, or an anti inflammatory.
  • Simultaneous administration can occur through administration of separate compositions, each containing one or more active agents.
  • Simultaneous administration can occur through administration of one composition containing two or more active agents.
  • a composition as disclosed herein can be administered sequentially with an antiviral, an antibiotic, an anti-inflammatory, or another agent.
  • a composition of the disclosure can be administered before or after administration of an antiviral, an antibiotic, an anti-inflammatory, or another agent.
  • compositions comprising compounds as disclosed above, so as to facilitate administration and promote stability of the active agent.
  • a compound of this disclosure may be admixed with at least one pharmaceutically acceptable carrier or excipient resulting in a pharmaceutical composition which is capably and effectively administered (given) to a living subject, such as to a suitable subject (i.e. “a subject in need of treatment” or “a subject in need thereof”).
  • a suitable subject i.e. “a subject in need of treatment” or “a subject in need thereof”.
  • the subject may be a human or any other animal.
  • the present disclosure is based, at least in part, on the discovery that the induction of CARD8 inflammasome can clear residual HIV-1 in patients.
  • the CARD8 inflammasome senses HIV-1 protease activity. HIV-1 protease remains inactive in infected cells to avoid CARD8 recognition.
  • NRTI anti-HIV-1 non-nucleoside reverse transcriptase inhibitors
  • RSV rilpivirine
  • EVF efavirenz
  • HIV-1 persists in a latent form primarily in quiescent CD4+ T cells and tissue macrophages.
  • antiretroviral therapy does not cure HIV-1 infection and lifelong ART is required. Killing the latent viral reservoirs is required to achieve HIV-1 eradication.
  • a class of anti-HIV drugs such as EFV, ETR, and RPV activates HIV protease, which in turn activates CARD8 to kill the infected cells.
  • EFV HIV-1 -infected cells
  • RPV a class of anti-HIV drugs
  • EFV HIV-1 -infected cells
  • RPV activates HIV protease
  • CARD8 CARD8 to kill the infected cells.
  • Combining RPV with talabostat mesylate enhanced the killing effect (see e.g., Example 2).
  • Talabostat mesylateis is not in the same drug class as EFV and RPV.
  • NNRTIs which have been used to treat HIV-1 infection for more than two decades can facilitate CARD8 sensing by mediating premature intracellular activation of HIV-1 protease.
  • NNRTIs bind to HIV-1 RT and act as enhancers of Gag-Pol dimerization to activate Pol-embedded viral protease.
  • NNRTI-containing treatment regimens do not eliminate HIV-1 infection in patients because the viral latent reservoirs are rapidly established prior to treatment initiation, inclusion of NNRTIs without protease inhibitors in the initial ARV regimen may partially reduce the seeding of latent viral reservoirs.
  • inclusion of NNRTIs in HIV-1 cure strategies should facilitate the elimination of infected cells after viral latency reversal.
  • CARD8 is preferentially and highly expressed in blood and lymphoid tissues as well as in many hematopoietic-derived cells, suggesting that targeting the CARD8 inflammasome may be effective in lymphoid tissues, the most important anatomical sites for persistent HIV-1 infection.
  • the cell-killing IC50 of EFV and RPV is approximately 1-2 mM, which is about 100-fold higher than the infection-blocking IC50.
  • the plasma EFV concentration in patients receiving EFV- containing regimens (1-4 pg/ml or 3-12 pM) is within the therapeutic range for cell killing. This strategy may not be effective in tissues with markedly lower drug concentration such as central nervous system.
  • HIV-1 Pol that confer resistance to NNRTIs also abrogates NNRTI-triggered cell killing likely because the resistant viral variants can avoid drug binding.
  • identification of more potent chemical compounds that promote Gag-Pol dimerization regardless of viral inhibition is warranted.
  • the present disclosure encompasses methods to treat, prevent, or reduce the infectivity of a HIV in a subject in need thereof, the method generally comprises administering to the subject a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor.
  • the present disclosure provides methods of reducing the amount of HIV-infected cells, the methods generally comprises contacting HIV-infected cells with or administering to a subject having HIV-infected cells, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor.
  • the present disclosure provides methods of inducing pyroptosis in HIV-infected cells, the methods generally comprise contacting HIV-infected cells with or administering to a subject having HIV- infected cells, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor.
  • the present disclosure provides methods of sensitizing the CARD8 inflammasome to NNRTI-induced activation, the methods generally comprise contacting HIV-infected cells with or administering to a subject having HIV-infected cells, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor.
  • the present disclosure provides methods of treating a subject with NNRTI-resistance, the methods generally comprise administering to the subject a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor.
  • the present disclosure provides methods of reducing the amount of HIV reservoirs in a subject, the methods generally comprise administering to the subject, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor. Suitable compositions for use within the above methods are described herein for instance those described in Section II which is incorporated by reference into this section in its entirety.
  • compositions of the disclosure are useful to selectively kill HIV-infected cells.
  • selectively killing one or more HIV-infected cells is meant a composition of the disclosure does not appreciably kill non-HIV-infected cells at the same concentration.
  • the median lethal dose or LDso of the composition in non-HIV-infected cells may be about 2 to about 50 times higher than the LDso of the composition in HIV-infected cells.
  • the LD50 is the concentration of composition required to kill half the cells in the cell sample.
  • the LD50 of the composition in non-HIV-infected cells may be greater than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 times higher than the LD50 of the composition in HIV-infected cells.
  • the LD50 of the composition in non-HIV-infected cells may be greater than about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 times higher than the LD50 of the composition in HIV-infected cells.
  • the LD50 of the composition in non-HIV-infected cells may be greater than 50 times higher than the LD50 of the composition in HIV-infected cells.
  • the LD50 of the composition in non-HIV-infected cells is about 2 to about 10 times higher than the LD50 of the composition in HIV-infected cells. In an exemplary embodiment, the LD50 of the composition in non-HIV-infected cells is about 3 to about 6 times higher than the LD50 of the composition in HIV-infected cells.
  • HIV-infected cells Method to detect HIV-infected cells are known in the art, for example, a human immunodeficiency virus (HIV) test detects HIV antigens, or the genetic material (DNA or RNA) of HIV in a biological sample (e.g., blood or another type of sample). This can show if an HIV infection is present (HIV-positive). Suitable detection methods include enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and indirect fluorescent antibody (IFA). In some embodiments, the HIV-infected cell is a CD4+ T cell.
  • ELISA enzyme-linked immunosorbent assay
  • PCR polymerase chain reaction
  • IFA indirect fluorescent antibody
  • the HIV-infected cell is a CD4+ T cell.
  • one or more HIV-infected cells are detected in a sample.
  • a sample may be a cell sample, blood sample, a tissue sample, or a biopsy sample from a subject.
  • a method of the disclosure may comprise measuring cel! death of H!V-infected cells.
  • Methods of measuring cel! death are known in the art.
  • cell death may be measured by Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay,
  • cell death is due to induction of pyroptosis.
  • Pyroptosis is a highly inflammatory form of lytic programmed cell death that occurs most frequently upon infection with intracellular pathogens and is likely to form part of the antimicrobial response. Pyroptosis is initiated by formation of a large supramolecular complex termed the inflammasome (also known as a pyroptosome) upon intracellular danger signals. The inflammasome activates a different set of caspases as compared to apoptosis, for example, caspase-1/4/5 in humans and caspase-11 in mice.
  • TUNEL terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments
  • ISEL in situ end labeling
  • a therapeutically effective amount of a composition of the disclosure may be administered to a subject.
  • Administration is performed using standard effective techniques, including peripherally (i.e. not by administration into the central nervous system) or locally to the central nervous system.
  • Peripheral administration includes but is not limited to intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • Local administration, including directly into the central nervous system (CNS) includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation.
  • compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like are used as appropriate.
  • pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like are used as appropriate.
  • Remington's Pharmaceutical Sciences Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners.
  • a therapeutically effective amount of a composition of the disclosure is administered to a subject.
  • a “therapeutically effective amount” is an amount of the therapeutic composition sufficient to produce a measurable response (e.g., cell death of HIV-infected cells, an improvement in symptoms associated with HIV).
  • Actual dosage levels of active ingredients in a therapeutic composition of the disclosure can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject.
  • the selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, HIV-related disease or condition, HIV infection, the symptoms, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.
  • the frequency of dosing may be daily or once, twice, three times or more per week or per month, as needed as to effectively treat the symptoms.
  • the timing of ad-ministration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case.
  • Treatment could begin immediately, such as at the site of the injury as administered by emergency medical personnel.
  • Treatment could begin in a hospital or clinic itself, or at a later time after discharge from the hospital or after being seen in an outpatient clinic.
  • Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments.
  • Typical dosage levels can be determined and optimized using standard clinical techniques and will be dependent on the mode of administration.
  • a subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal.
  • the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc.
  • the subject may be a livestock animal.
  • suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas.
  • the subject may be a companion animal.
  • companion animals may include pets such as dogs, cats, rabbits, and birds.
  • the subject may be a zoological animal.
  • a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears.
  • the subject is a human.
  • the subject may be a subject at risk of developing a HIV infection.
  • a skilled artisan would be able to determine a subject at risk of developing a HIV infection.
  • a subject may be determined to be at risk of developing a HIV infection based on patient history, clinical presentation, a diagnostic exams, and/or HIV exposure.
  • the disclosure provides a method (also referred to herein as a "screening assay") for identifying inhibitors, i.e. , candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which downregulate DPP9, for example, DPP9 nucleic acid expression, DPP9 protein expression or DPP9 activity.
  • DPP9 inhibitor may directly or indirectly downregulate DPP9.
  • Screening assays may also be used to identify molecules that modulate DPP9 nucleic acid expression, DPP9 protein expression or DPP9 activity.
  • DPP9 inhibits the activation of the CARD8 inflammasome to prevent pyroptosis.
  • pyroptosis, cell viability, or caspase-1 may be measured as an indication of DPP9 activity or expression.
  • Pyroptosis and cell viability may be measured using methods standard in the art as described above.
  • Caspase-1 may be measured using methods to detect protein expression as described herein.
  • DPP9 nucleic acid expression or protein expression may be measured as an indication of DPP9 activity or expression. Methods to detect DPP9 nucleic acid or protein expression are standard in the art.
  • the disclosure provides assays for screening candidate or test compounds which bind to or modulate the activity or expression of DPP9.
  • the test compounds of the present disclosure can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).
  • Libraries of compounds may be presented in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature 354:82- 84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci.
  • an assay is one in which cells are contacted with a test compound and the ability of the test compound to downregulate DPP9 is determined. Determining the ability of the test compound to downregulate DPP9 may be accomplished, for example, by detecting DPP9 protein expression. Numerous methods for detecting protein are known in the art and are contemplated according to the disclosure, see Section II. Specifically, an immunoblot may be used to detect DPP9 protein. Alternatively, determining the ability of the test compound to downregulate DPP9 may be accomplished, for example, by measuring cell death or pyroptosis. Methods of measuring cell death or pyroptosis are known in the art, see the below examples.
  • Another method for determining the ability of the test compound to downregulate DPP9 may be accomplished by a reporter assay for DPP9 expression.
  • DPP9 protein expression may be fused to a reporter protein such that DPP9 expression may be monitored by measuring the expression of the reporter protein.
  • reporter proteins may include a fluorescent protein, luciferase, alkaline phosphatase, beta-galactosidase, beta-lactamase, horseradish peroxidase, and variants thereof.
  • determining the ability of the test compound to downregulate DPP9 may be accomplished, for example, by detecting DPP9 nucleic acid expression. Methods of measuring nucleic acid expression are known in the art, see Section II.
  • DPP9 mRNA may be detected via standard methods. Determining the ability of the test compound to down-regulate DPP9 may be accomplished, for example, by determining the ability of DPP9 to inhibit the activation of caspase 1. Methods for detecting caspase 1 are known in the art. For example, methods to detect protein expression may be used to detect caspase 1.
  • an assay is one in which DPP9 is contacted with a test compound and the ability of the test compound to bind to DPP9 is determined. Determining the ability of the test compound to bind to DPP9 may be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to DPP9 may be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 125 l, 35 S, 14 C, or 3 H , either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • an assay of the present disclosure is a cell-free assay comprising contacting DPP9 with a test compound and determining the ability of the test compound to bind to DPP9. Binding of the test compound to DPP9 may be determined either directly or indirectly.
  • a competitive binding assay includes contacting DPP9 with a compound known to bind DPP9 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with DPP9, wherein determining the ability of the test compound to interact with DPP9 comprises determining the ability of the test compound to preferentially bind to DPP9 as compared to the known binding compound.
  • an assay is a cell-free assay comprising contacting DPP9 with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of DPP9. Determining the ability of the test compound to modulate the activity of DPP9 can be accomplished, for example, by determining the ability of DPP9 to bind to or interact with a DPP9 target molecule (e.g., CARD8). In an alternative embodiment, determining the ability of the test compound to modulate the activity of DPP9 can be accomplished by determining the ability of DPP9 to further modulate a DPP9 target molecule.
  • a "target molecule" is a molecule with which DPP9 binds or interacts in nature.
  • modulators of DPP9 expression are identified in a method in which a cell is contacted with a candidate compound and the expression of the DPP9 promoter, mRNA or protein in the cell is determined.
  • the level of expression of DPP9 mRNA or protein in the presence of the candidate compound is compared to the level of expression of DPP9 mRNA or protein in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of DPP9 expression based on this comparison. For example, when expression of DPP9 mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of DPP9 mRNA or protein expression.
  • the candidate compound when expression of DPP9 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of DPP9 mRNA or protein expression.
  • the level of DPP9 mRNA or protein expression in the cells can be deter-mined by methods described herein for detecting DPP9 mRNA or protein.
  • the activity of the DPP9 promoter can be assayed by linking the DPP9 promoter to a reporter gene such as luciferase, secreted alkaline phosphatase, or beta-galactosidase and introducing the resulting construct into an appropriate vector, transfecting a host cell line, and measuring the activity of the reporter gene in response to test compounds.
  • kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein.
  • the different components of the composition can be packaged in separate containers and admixed immediately before use.
  • Components include, but are not limited to a non-nucleoside reverse transcriptase (NNRT) inhibiting agent or a DPP9 inhibiting agent or assays to screen for such agents.
  • NRT non-nucleoside reverse transcriptase
  • DPP9 DPP9 inhibiting agent or assays to screen for such agents.
  • Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may, for example, comprise metal or plastic foil such as a blister pack.
  • Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
  • Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, and the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, and the like.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
  • HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to target immutable components are needed to clear HIV-1 infection.
  • the present example provides the CARD8 inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells prior to viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1 - infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation.
  • HIV-1 an inflammasome sensor of HIV-1 , which holds promise as a strategy for clearance of persistent HIV-1 infection.
  • the adaptive immune system recognizes cognate epitopes based on amino acid sequences and associated conformations that are subject to mutation. Retroviruses such as human immunodeficiency virus type 1 (HIV-1) accumulate mutations at high rates due to the lack of proofreading activity of their reverse transcriptase coupled with high levels of virus replication in vivo. The within-host diversity of HIV-1 allows rapid selection of antibody and T cell escape variants. In patients who received antiretroviral therapy (ART), HIV-1 persists in a latent form primarily in quiescent CD4+ T cells and possibly tissue macrophages.
  • HIV-1 persists in a latent form primarily in quiescent CD4+ T cells and possibly tissue macrophages.
  • Immune escape variants achieved in the latent viral reservoirs present one of the major obstacles to HIV-1 eradication.
  • host cells persistently infected with HIV-1 are long-lived and resistant to virus- and immune-mediated apoptotic cell death.
  • the immune system could (1 ) sense the functions of an essential HIV-1 protein including those with viral enzymatic activities, which are highly immutable and (2) mediate robust target cell killing independent of apoptosis.
  • the host immune system utilizes germ line-encoded pattern-recognition receptors (PRRs) to detect microbial products.
  • PRRs germ line-encoded pattern-recognition receptors
  • a set of intracellular PRRs characterized by the presence of a caspase recruitment domain (CARD) or a pyrin (PYD) domain co-oligomerize with pro- caspase-1 and form high-molecular weight inflammasome complexes upon sensing of their cognate ligands. Inflammasome assembly in response to cytoplasmic microbial or danger signals leads to caspase-1 (CASP1) activation and pyroptosis, an inflammatory form of programmed cell death.
  • caspase-1 caspase-1
  • NLRPIb Murine nucleotide-binding domain leucine-rich repeat pyrin domain-containing 1b
  • NLRPIb can be activated via direct proteolysis of its N-terminus by Bacillus anthracis lethal factor protease (LF). Briefly, N-terminal cleavage of murine NLRPIb creates a neo-N- terminus, which is ubiquitylated by the N-end-rule pathway and targeted for proteasome degradation. Due to the break in the polypeptide chain by FUND auto-processing, the C- terminal bioactive subunit is liberated from the proteasome complex and can initiate CASP1 -dependent inflammasome assembly. Recently, two studies on human NLRP1 inflammasome reported that it senses viral infection in epithelial barrier tissues and its activation is also proteasome-dependent.
  • LF Bacillus anthracis lethal factor protease
  • HIV-1 infection can be sensed by innate sensors associated with inflammasome activation in CD4+ T cells, with the exception of the bystander effect triggered by abortive HIV-1 transcripts.
  • the bystander cell death mechanism only applies to quiescent, lymphoid-resident CD4+ T cells that are non-perm issive to HIV-1. It does not apply to the cells productively or latently infected with HIV-1, and thus has no impact on persistently infected cells.
  • the CARD8 inflammasome senses HIV-1 protease activity:
  • the caspase recruitment domain-containing protein 8 (CARD8) has been implicated in inflammasome activation and pyroptosis of CD4 + T cells and macrophages.
  • a key question is whether CARD8 is an inflammasome sensor, and if so, which pathogens physiologically activate it.
  • the C-terminus of CARD8 protein contains a “function-to-find” domain (FUND) followed by a CARD domain.
  • FUND function-to-find domain
  • Human CARD8 shares structural similarity with murine NLRPIb and also undergoes autoprocessing, as evidenced by the detection of both the full length and the autoprocessed N- and C-terminal fragments by immunoblotting (FIG. 1A).
  • CARD8 is a sensor for HIV-1 protease
  • HEK293T cells were co-transfected with an HIV-1 reporter vector (pNL4-3- GFP) and human CARD8 vector with an N-terminal HA tag (HA-CARD8).
  • the N- terminal tag added 43 amino acids to CARD8 protein.
  • HIV-1 protease activation requires dimerization of the Gag-Pol polyprotein, which occurs during or soon after the viral budding process. Overexpression of HIV-1 Gag-Pol polyprotein in transfected cells resulted in its intracellular dimerization and premature protease activation.
  • CARD8 was not cleaved by the viral vector when the viral protease was inactivated by a single mutation (D25H), as evidenced by the lack of HIV-1 Gag (p55) cleavage.
  • various mutations were introduced to the viral vector. Cleavage of CARD8 was not affected when mutations were introduced to any viral genes except pol but was blocked by an HIV-1 protease inhibitor lopinavir (LPV).
  • CASP1- and pro-ILip-expressing plasmids were co-transfected together with pNL4-3-GFP into HEK293T cells. Although the level of endogenous expression of CARD8 in HEK293T cells was not sufficient for the detection of N-terminal cleavage, it was sufficient to trigger the downstream signaling cascade upon HIV-1 protease cleavage, as evidenced by the processing of pro-IL-1 b into mature IL-1 b (FIG. 1C).
  • NRTI non nucleoside reverse transcriptase inhibitors
  • RPV rilpivirine
  • EVF efavirenz
  • NTP nevirapine
  • proteasome inhibitors MG132 or bortezomib were added together with RPV into transfected cells.
  • the baseline level of IL-1 b-r17 was not affected by proteasome inhibitors because the inhibitors were added 24 hours post transfection.
  • RPV-induced processing of pro-IL-1 b but not HIV-1 Gag cleavage was blocked by bortezomib and MG132 (FIG. 1E), which excluded the possibility that MG132 and bortezomib directly affected HIV-1 protease activity.
  • HIV-1 protease cleaves the N-terminus of CARD8: To examine whether CARD8 was directly cleaved by HIV-1 protease, HA-CARD8 S297A was immunoprecipitated and incubated with purified and lysed HIV-1 particles. When the viral protease was functional, two additional bands were visualized using a CARD8 C- terminus antibody (FIG. 1F), which confirmed the two cleavage sites recognized by the viral protease. RPV did not further enhance CARD8 cleavage because the lysed HIV-1 particles already contained mature viral protease.
  • a truncated CARD8 A21 ⁇ 70 was first generated which did not contain the first cleavage site.
  • CARD8 A21 ⁇ 70 was cleaved at the second site (FIG. 2A), but was unable to trigger inflammasome activation (FIG. 2B).
  • FIG. 2C To identify the first cleavage site, a set of truncated CARD8 proteins were tested and found that CARD8 A51 60 was not cleaved (FIG. 2C). Next, it was found that F59 and F60 were likely to be at the P1 and PT positions, respectively (FIG. 2D).
  • the H IV-1 protease prefers to have large hydrophobic amino acids flanking the scissile bond. Phenylalanine is the most common residue at the P1 position, and its presence at both P1 and PT positions improved the cleavage rate of FI IV-1 protease. It was further demonstrated that the first cleavage site was required for the CARD8 inflammasome activation by FI IV-1 protease (FIG. 2E). Thus, FI IV-1 protease can cleave the N-terminus of CARD8 and that activation of the viral protease can lead to CARD8 inflammasome activation in the FIEK293T transfection system.
  • HI V- 1 triggers CARD8-depedent pyroptosis of infected macrophages: Since CARD8 is expressed in FI IV-1 target cells, including primary CD4 + T cells and macrophages, the next question was whether induction of premature intracellular activation of FI IV-1 protease could trigger CARD8-dependent pyroptosis of infected cells.
  • FI IV-1 -infected (GFP + ) macrophages rapidly underwent pyroptotic cell death as evidenced by membrane swelling and rupture (FIG. 3), as well as secretion of IL-1 b (FIG. 3E).
  • MG132 and bortezomib or an N-end-rule pathway inhibitor bestatin methyl ester were added together with RPV to FI IV-1 -infected macrophages.
  • Me-Bs effectively blocked RPV-mediated killing of infected macrophages and secretion of IL-1 b (FIG. 3).
  • FI IV-1 -infected TFIP-1 cells were also susceptible to NNRTI-triggered killing (FIG. 4).
  • HIV-1 triggers CARD8-depedent pyroptosis of infected CD4+ T cells:
  • NNRTIs can induce killing of HIV-1 -infected CD4 + T cells through an unknown mechanism. It was hypothesized that the cell killing observed in those studies was due to NNRTI-triggered HIV-1 protease activation which led to CARD8 inflammasome activation. Since resting CD4 + T cells are the most well characterized cellular reservoirs for HIV-1 , the expression levels of key components of the CARD8 inflammasome were examined in different subsets of primary CD4 + T cells.
  • CARD8 is expressed in both activated and unstimulated blood CD4 + T cells, as well as in memory and naive CD4 + T cells in lymphoid tissues (FIG. 5A). Both activated (FIG. 5B) and unstimulated CD4 + T cells were susceptible to HIV-1 protease-triggered cell death when treated with NNRTIs including EFV, RPV, and Etravirine (ETR), but not NVP. Since several HIV-1 proteins can induce death of primary CD4 + T cells, we produced different reporter viruses carrying mutations in vif, vpr, vpu, env, and nef by transfecting HEK293T cells with different viral plasmids.
  • NNRTI-induced CASP1 activation and pyroptosis of HIV-1 -infected primary CD4 + T cells was abrogated when CARD8 was knocked out (FIG. 5).
  • a CASP1 knockout or knockdown in primary CD4 + T cells also conferred resistance to HIV-1 protease-mediated pyroptosis (FIG. 5J).
  • FOG. 5J HIV-1 protease-mediated pyroptosis
  • NNRTI-mediated killing of primary CD4 + T cells infected with HIVBaL was also CARD8- dependent.
  • Activation of the CARD8 inflammasome clears latent HI V- 1 in patient CD4+ T cells: To determine whether HIV-1 protease function in activating the CARD8 inflammasome is conserved, a panel of HIV-1 virus isolates from chronically infected individuals of subtypes A, B, C, and D were tested. Subtype B is the dominant subtype in Europe and North America, whereas A, C, and D are more prevalent worldwide. T-20 and Raltegravir (RAL) were used to completely block new infection but had no killing effect or cellular toxicity. The addition of EFV and RPV but not NVP effectively cleared primary CD4 + T cells infected with all HIV-1 subtypes.
  • CARD8 is a sensor for HIV-1 protease activity to trigger inflammasome activation and pyroptosis of infected cells.
  • CARD8 and NLRP1 inflammasomes share similar mechanisms of activation, which involves their N-terminal cleavage by microbial proteases, followed by proteasome-mediated release of the bioactive C-terminal fragment to trigger inflammasome assembly and CASP1 activation.
  • HIV-1 protease cleaves CARD8 at two different sites.
  • Cleavage of the unstructured N- terminus but not the FUND domain leads to inflammasome activation. It was also observed that a deletion (D51-60) or mutations (F59A or F60A) of the first cleavage site increased the cleavage efficiency at the second site (FIG. 2C), suggesting a competition between the two sites. Cleavage of HIV-1 Gag and Gag-Pol by the viral protease is a sequential process regulated by the rate of cleavage at individual site. Since CARD8 activation requires a cleavage within the unstructured N-terminus, the cleavage site preference may influence the CARD8 inflammasome activation.
  • NNRTIs which have been used to treat HIV-1 infection for more than two decades can facilitate CARD8 sensing by mediating premature intracellular activation of HIV-1 protease.
  • NNRTIs bind to HIV-1 RT and act as enhancers of Gag- Pol dimerization to activate Pol-embedded viral protease. Additional investigations are needed to better understand the mechanism of the NNRTI-mediated Gag-Pol dimerization process.
  • NNRTI-containing treatment regimens cannot eliminate HIV-1 infection in patients because the viral latent reservoirs are rapidly established prior to treatment initiation, inclusion of NNRTIs without protease inhibitors in the initial ARV regimen may partially reduce the seeding of latent viral reservoirs.
  • inclusion of NNRTIs in HIV-1 cure strategies should facilitate the elimination of infected cells after viral latency reversal.
  • CARD8 is preferentially and highly expressed in blood and lymphoid tissues as well as in many hematopoietic-derived cells, suggesting that targeting the CARD8 inflammasome may be effective in lymphoid tissues, the most important anatomical sites for persistent HIV-1 infection.
  • the cell-killing IC50 of EFV and RPV is approximately 1-2 mM (FIG. 2C), which is about 100- fold higher than the infection-blocking IC50.
  • the plasma EFV concentration in patients receiving EFV-containing regiments (1-4 pg/ml or 3-12 mM) is within the therapeutic range for cell killing. This strategy is unlikely to be effective in tissues with markedly lower drug concentration such as central nervous system.
  • H IV-1 Pol that confer resistance to NNRTIs also abrogate NNRTI-triggered cell killing likely because the resistant viral variants can avoid drug binding.
  • the identification of more potent chemical compounds that promote Gag-Pol dimerization regardless of viral inhibition is warranted.
  • NRTIs Non-nucleoside reverse transcriptase inhibitors
  • NNRTIs Non-nucleoside reverse transcriptase inhibitors
  • the present example provides that this sensitization can be done through chemical inhibition of the CARD8 negative regulator DPP9.
  • Val-boroPro can act synergistically with NNRTIs to increase their efficacy in killing HIV-1 -infected cells. It is also shown that VbP is able to partially overcome issues with NNRTI resistance and is capable of killing infected cells without the presence of NNRTIs. This offers a promising strategy for enhancing NNRTI efficacy in elimination of HIV-1 reservoirs in patients.
  • the inflammasome is a multi-protein complex that is assembled upon sensing of their cognate ligand.
  • Caspase-1 (CASP1 ) is the key effector for the inflammasome, and its active form can cleave Gasdermin D leading to pyroptosis.
  • PRRs pattern-recognition receptors
  • CARD caspase recruitment domain
  • PYD Pyrin domain
  • CARD8C CARD8 C-terminus
  • HIV-1 protease was found to cleave CARD8 on the N-terminal subunit, which allows proteasomal degradation of the N-terminal fragment thereby freeing the C-terminal fragment.
  • the C-terminal fragment in high enough concentrations, can then activate CASP1 and induce pyroptosis.
  • freed C- terminal fragment may also be sequestered by the CARD8 negative regulator DPP9 which can inhibit pyroptosis efficiency.
  • HIV-1 protease is not typically functional intracellularly before budding and it must be activated by other methods to be properly sensed by the CARD8 inflammasome.
  • NNRTIs non-nucleoside reverse transcriptase inhibitors
  • NNRTIs at micromolar concentrations drive Gag-Pol dimerization and intracellular protease activation which cleaves CARD8 leading to pyroptosis of HIV-1 infected cells.
  • Strategies for enhancement of cell killing potency of these drugs are needed for efficient clearance of HIV-1 -infected cells in vivo.
  • NNRTIs induce death of HIV-1 -infected cells in a dose-dependent manner : While NNRTI pharmacodynamics have been heavily studied for their ability to inhibit HIV-1 reverse transcription, they have yet to be studied in the context of their ability to activate the CARD8 inflammasome. To determine the in vitro pharmacodynamics of NNRTIs in CD4 + T cells, an HIV-1 reporter virus (pNL4-3- pol) was used to infect primary blood CD4 + T cells isolated from three independent healthy donors.
  • Efavirenz Efavirenz
  • RPV Rilpivirine
  • ETR Etravirine
  • DOR Doravirine
  • NNP Nevirapine
  • NNRTI high affinity for binding human serum proteins in vivo.
  • CD4 + T cells were cultured with the presence of 50% human serum and show stark shifts in the dose response curves for both EFV and RPV (FIG. 7F and FIG. 7G). With the presence of human serum, the dose response curve for RPV is shifted out of clinical concentration recommendations whereas EFV maintains some efficacy with the presence of human serum.
  • EFV is less affected by the presence of human serum as RPV as evidenced by a smaller log fold change in the ECso (FIG. 7H).
  • DPP9 inhibition sensitizes the CARD8 Inflammasome to NNRTI- induced pyroptosis: DPP9 can bind to CARD8 first as a heterodimer with one copy of the full length CARD8 protein, then as a heterotrimer by catching a freed CARD8C14.
  • DPP9 As the C-terminal fragment is responsible for inflammasome activation, DPP9’s ability to catch CARD8C inhibits CARD8-induced pyroptosis. Overcoming DPP9 inhibition therefore should increase the rate of CARD8 inflammasome activation and sensitize the inflammasome to sensing HIV-1 protease activity. It was recently reported that VbP is able to bind to the DPP9-CARD8 heterodimer and prevent heterotrimer formation hence increasing intracellular CARD8C concentrations. Additionally, VbP has another mechanism of action where it can induce N-terminal degradation of CARD8 which is also able to activate the inflammasome, although the direct mechanism of action has yet to be elucidated.
  • VbP VbP
  • NNRTI induced killing of HIV-1 infected CD4 + T cells was enhanced upon treatment with VbP (FIG. 8B).
  • This enhancement of NNRTIs was shown to be dose-dependent upon increasing concentrations of VbP.
  • the EC50 had log fold change shifts up to -1.1 for both EFV and RPV (FIG. 8C). This has the potential to overcome the EC50 shift due to the presence of human serum and demonstrates that DPP9 inhibition is essential for CARD8 inflammasome activation in vivo.
  • VbP Due to VbP’s ability to inhibit the capture of CARD8C by DPP9, it was hypothesized that this relationship would be synergistic in nature. To further understand this complex relationship, SynergyFinder2.0 was used to identify whether this relationship was additive or synergistic. The combination treatment of VbP with EFV or RPV was found to be synergistic by four synergistic modeling methods: HSA, BLISS, Loewe, and ZIP (FIG. 8D). To evaluate the non-specific killing, VbP in both HIV-1 - infected and uninfected cells was tested.
  • VbP at concentrations lower than 3.33 mM has no significant toxicity in uninfected CD4 + T cells, indicating this mechanism of cell killing is specific to HIV-1 (FIG. 8E). Additionally, VbP is able to induce low levels of cell killing of HIV-1 infected cells (22% and 13% for 3.33 pM and 1.11 pM respectively) (FIG. 8B).
  • VbP enhancement of NNRTI-induced cell killing To first begin understanding the dynamics of VbP enhancement of NNRTI induced cell killing, the killing in CD4 + T cells upon combination or single treatment across time was analyzed. Upon treatment with EFV at physiologically relevant concentration, the killing of HIV-1 -infected cells became more rapid and robust with the presence of VbP (FIG. 9A). It was previously shown that inhibition of the CARD8C capture by DPP9 was a rapid response, which was hypothesized is the main contributor to rapid enhancement of NNRTI induced cell killing. There is a second phase of cell killing between 6 and 24 hours before the maximal killing plateaus for both EFV and combination treatments.
  • VbP is known to bind to both DPP9 and DPP8, knock-down of DPP8 does not ablate NNRTI enhancement suggesting a DPP9 specific mechanism of action.
  • primary CD4 + T cells were infected, and the cells treated concurrently with a dye that specifically stains the active form of CASP1.
  • FIG. 9F addition of VbP to EFV showed increased CASP1 activation in HIV-1 -infected CD4 + T cells which is not the case for uninfected cells.
  • VbP alone at low concentrations ( ⁇ 1 mM) shows significant CASP1 activation specifically in HIV-1 -infected cells, suggesting that it relies upon the presence of HIV-1 to help induce the CARD8 inflammasome. This underscores that while VbP is able to activate the CARD8 inflammasome and cause issues with cytotoxicity, lower concentrations of VbP do not have the same issues and are specific to killing HIV-1 infected cells and enhancing NNRTI-mediated pyroptosis. The killing and enhancement by VbP are dependent on CASP1 was also shown (FIG. 9G).
  • VbP sensitization of the CARD8 inflammasome can overcome NNRTI resistance: As HIV-1 has a high mutation rate, the circulating pool of HIV-1 strains shows distinct genetic variation across clades. The initial work demonstrating protease cleavage of CARD8 demonstrated that proteases from all clades can induce the CARD8 inflammasome, albeit at varying levels of efficiency. This poses a significant barrier for implementation in the clinic as not every patient will have a viral reservoir that is highly sensitive to NNRTI-induced killing. Therefore, the example aimed to evaluate this combination strategy against clinical HIV-1 isolates from clades A, B, C, and D. Briefly, CD4 + T cells were infected until 10-20% infection was reached, where cells were then treated with EFV or combination along with entry inhibitor T-20 and integrase inhibitor Raltegravir to prevent further rounds of replication. As can be seen in FIG.
  • NNRTI resistance associated mutations As NNRTI RAMs show significant shifts in the ECso values for blocking reverse transcriptase activity, therefore, to first understand if these RAMs would also show resistance to NNRTI-induced CARD8 inflammasome activation was investigated.
  • RAMs were introduced into our HIV-1 reporter virus (pNL4-3-pol) via site-directed mutagenesis. RAMs were chosen for key regions on HIV-1 reverse transcriptase which can be seen in FIG. 10B.
  • NNRTI RAMs differ in the level of resistance that they confer. Strong NNRTI RAMs may confer complete resistance to NNRTI-mediated killing whereas others may simply show reduced efficacy. This may help in the classification of viral strains that may respond to NNRTI treatment alone versus those that require VbP enhancement for their function. These NNRTI RAMs were then tested for their killing efficiency upon addition of VbP.
  • VbP Enhances Clearance of HIV-1 -infected cells in humanized mice: Flumanized mice were used to test the ability of NNRTIs to induce pyroptosis of HIV-1- infected CD4+ T cells in vivo.
  • the MISTRG-6-15 mouse was developed through knock-ins of human cytokine coding genes including m-csf, il-3, gm-csf, sirp-ct, thpo, il-6 and il-15.
  • Primary CD4+ T cells infected with pNL4-3-pol were transfused into mice.
  • HIV-infected CD4+ T cells were treated with 3mM EFV alone or in combination with another DPP8/9 inhibitor named 1G244. Around 50% HIV-infected cells were cleared by EFV alone. The EFV-mediated cell killing was increased to 80%, 85%, and 90% with the presence of 123nM, 370nM, and 1000nM 1G244, respectively. 1G244 alone had limited cell killing effect. Therefore, both VbP and 1G244 can enhance NNRTI-triggered pyroptosis of HIV-infected cells (FIG. 12).
  • NNRTIs offer a promising strategy for eradication of HIV-1 latent reservoirs, improving their in vivo cell killing potency is essential to the treatment efficacy.
  • This example proves that sensitization of the CARD8 inflammasome through DPP9 inhibition can reduce the threshold and provide more effective clearance of HIV-1 infected cells for clinically relevant scenarios.
  • DPP9 inhibition through chemical means such as with VbP can induce targeted cell killing on their own which varies across viral strains with NNRTI RAMs.
  • VbP there may be varying levels of intrinsic Gag-Pol dimerization of these strains which can be enhanced by VbP, which are not due to NNRTIs due to the lack of dimerization induced from NNRTI binding.
  • NNRTI resistance may prove to be a significant barrier in implementation of NNRTIs for a shock and kill approach, they are not insurmountable when the CARD8 inflammasome is sensitized through DPP9 inhibition.
  • Plasmids To generate plasmids for these viruses, mutations were introduced into the pNL4-3-GFP vector (AIDS Reagent Program #111100), which contains an enhanced green fluorescent protein (EGFP) inserted into env. L40C- CRISPR.EFS.PAC (Addgene #89393) and SGL40C-H1.EFS.RFP657 (Addgene #69148) vectors were used for sgRNA delivery via lentivirus. CRISPR/Cas9 guide RNAs were selected using the CCTop selection tool (43). pLKO.Ipuro (Addgene #8453) was used for gene knowckdown via lentivirus vectors. Site-directed mutagenesis to obtain NNRTI RAM’s was done using PCR primers on the NL4-3-Pol plasmid and were confirmed by sequencing.
  • EGFP enhanced green fluorescent protein
  • HEK293T CRL-3216
  • THP-1 cells THP-1 cells
  • DMEM fetal bovine serum
  • FBS heat- inactivated fetal bovine serum
  • penicillin 1 U/ml penicillin
  • streptomycin Gibco
  • CD4+ T cells from blood were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) using the BioLegend human CD4+ T cell isolation kit (BioLegend #480010).
  • CD4+ T cells were co-stimulated with plate-bound CD3 antibody (Biolegend #300333) with media containing soluble CD28 (Biolegend #302943) antibody and 20 ng/ml IL-2 (Biolegend #589106) for 3 days.
  • Fluman serum containing media comprised 50% human serum obtained from Gemini Bio (#100-110) with 10% FBS, 1U/ml_ penicillin, 100mg/ml_ streptomycin, and 40% RPMI 1640 medium.
  • MTS CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) from Promega was used (Promega #G3580).
  • Uninfected CD4+ T cells or THP-1 cells were treated with EFV, RPV, VbP, or DMSO for two days prior to addition of MTS reagent, MTS reading was done following manufacturer’s protocol.
  • Preparation of HIV-1 and lentivirus stocks Reporter viruses were packaged by co-transfecting HEK293T cells with viral vectors, packaging vector pC- Help (44), and pVSV-G (Addgene #8454). To expand clinical HIV-1 isolates, CD8- depleted PHA-stimulated PBMCs were infected with the international HIV-1 isolates (AIDS reagent program #11412). Culture supernatant was collected after 6-9 days and filtered prior to use.
  • Lentiviruses for knockdown or knockout were also packaged in HEK293T cells by co-transfecting pVSV-G, psPAX2 (Addgene #12260), and sgRNA or shRNA using Lipofectamine 2000 (Thermo Fisher). Lenti-X Concentrator (TaKaRa #631232) was used to concentrate supernatant containing virus.
  • THP-1 cells with gene knockout or knockdown The sgRNA and shRNA sequences can be found in Table S2 and were verified by sequencing. THP-1 cells were transduced with sgRNA or shRNA lentiviruses via spin inoculation for 2 hours at 1200g at 25°C. Cells were then selected with puromycin (1 pg/ml) for 5-7 days prior to infection with HIV-1 reporter virus NL4-3-AVif-Vpr. Immunoblotting was performed to confirm knockout or knockdown efficiency. The controls for knockout cells were transduced with a Cas9-expressing lentiviral vector without sgRNA.
  • HIV-1 infection and cell killing HIV-1 p24 ELISA was used to verify viral stock concentration (XpressBio #XB-1000). HIV-1 reporter virus infection was performed at a multiplicity of infection (MOI) of 10 and 0.1 for clinical isolates. Infection was again performed by spin inoculation (1 ,200g) for 2 hours at 25 °C.
  • Antiretrovirals (ARVs) were obtained from the NIH AIDS Research and Reference Reagent Program: rilpivirine (RPV), efavirenz (EFV), etravirine (ETR), nevirapine (NVP), T-20, and raltegravir (RAL).
  • intracellular HIV-1 -p24 staining was performed using the Cytofix/CytopermTM kit (BD #554714) using anti-HIV-1 p24-PE antibody (#6604667, 1:1000 dilution) purchased from Beckman Coulter.
  • the FLICA660 Caspasel staining reagents were purchased from ImmunoChemistry Technologies (#9122).
  • Percent infection GFP+ or p24+ was determined by flow cytometry (BD LSRFortessa, BD FACSCanto, or BD accuri c6 plus). Flow cytometry data were analyzed by Flowjo software.
  • mice Humanized mice The generation of knock-in mice encoding human MCSF, GMCSF, IL3, SIRPA, THPO, IL6, and IL15 in a 129xBALB/c (N3) genetic background was performed using Velocigene technology by Regeneron Pharmaceuticals. Mice were bred to a Rag2 /_ IL2rg /_ background with homozygous knockin to generate the MISTRG-6-15 mouse colony MCSF h/h !L3 h/h SIRPA h/h THPO h/h IL6 h/h IL15 h/h RAG ⁇ IL2rg nu " .
  • Transfusion experiments were conducted using primary CD4+ T cells isolated from healthy donors as previously described that were infected with pNL4-3-pol prior to IV transfusion of 5-10 million cells per mouse.
  • VbP was injected via IV injection and 60 pg/mouse doses were diluted in DMSO.
  • FIG. 11A IP injections of EFV were also diluted in DMSO prior to injection. All other cohorts received an IP injection of EFV diluted in vehicle (0% DMSO, 40% PEG300, 5% Tween-80, and 45% saline). Controls received the respective EFV diluent from the same cohort. Blood was collected at 6 and 24 hours (24 and 72 for multi-dose cohort) and lung tissues were collected at 24 hours post treatment.
  • CD4+ T cells were collected from lung tissues that were homogenized to achieve single cell suspension and analyzed via flow cytometry.

Abstract

The present disclosure relates to the seminal discovery of compositions and methods of using a non-nucleoside reverse transcriptase inhibitor and DPP9 inhibitor to effectively kill latent HIV-infected cells thereby reducing HIV reservoirs in a subject. Specifically, the present disclosure relates to the synergistic killing effects of sensitizing the CARD8 inflammasome to non-nucleoside reverse transcriptase inhibitor activation through the inhibition of DPP9.

Description

COMPOSITIONS AND METHODS FOR TARGETING CARD8
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority of US provisional application number 65/155,124, filed March 1, 2021, which is hereby incorporated by reference in its entirety.
GOVERNMENT SUPPORT STATEMENT
[0002] This invention was made with government support under AM 25065 awarded by the National Institutes of Health. The government has certain rights in the invention.
FIELD OF THE TECHNOLOGY
[0003] The present disclosure encompasses compositions and methods for the treatment of subjects infected with retroviruses. Specifically, aspects of the present disclosure are directed to compositions and methods for inducing pyroptosis of HIV-infected cells.
BACKGROUND
[0004] HIV-1, the causative agent of AIDS, infects 35 million people worldwide and nearly 1.2 million persons in the United States. The advent of antiretroviral therapy (ART) has dramatically reduced morbidity and mortality for HIV- infected individuals with access to healthcare in resource-rich countries. However, despite years of potent therapy, eradication of infection is not achieved with conventional treatment strategies, necessitating lifelong ART. Moreover, accumulating data suggest that “non-AIDS” cardiovascular, renal and hepatic diseases are amplified by HIV infection, and the immune system may exhibit premature senescence even among patients with viral suppression. Although enormous progress has been made to provide ART in resource limited settings, there are huge economic, political and operational challenges to reach the goal of universal access to lifelong treatment. These realities have created a pronounced interest in developing strategies to eradicate HIV-1 in infected individuals.
[0005] It has been shown that despite long periods of suppressive antiretroviral therapy, a population of latently infected cells persists which is capable of producing replication competent virus upon ex vivo activation. These cells are comprised chiefly of resting memory CD4+ T lymphocytes and cells of the monocyte- macrophage lineage are believed to harbor latent virus as well. Rebounding plasma HIV RNA levels following discontinuation of antiretroviral therapy even after prolonged suppression of plasma viremia confirm the in vivo significance of this reservoir. In one of the largest studies to examine the half-life of the circulating CD4+ reservoir involving 62 HIV-infected adults with up to 7 years of viral suppression on HAART, frequencies of latently-infected T cells remained between 0.03-3 infectious units per million resting CD4+ T cells, with no significant decline over time. The total reservoir size was estimated to be 106 cells with a half-life of 44.2 months, yielding an estimated reservoir eradication time of 73.2 years, suggesting that clearance is not achieved spontaneously on a relevant time scale.
[0006] Elimination of the HIV-1 latent reservoir is critical to achieving HIV- 1 eradication in vivo. The “shock and kill” strategy is currently the most widely discussed approach to eliminating the viral reservoir. In this approach, drugs are administered to reverse HIV-1 latency and induce viral production, ultimately resulting in the death of infected cells by direct viral cytopathic effects or immune-mediated clearance. Latency reversing agents are administered during suppressive antiretroviral therapy (ART), thereby preventing reactivated virus from replenishing the reservoir through infection of new cells. Although the theory behind this approach is intuitive and elegant, to date, the shock and kill strategy has only achieved limited success. Clinical trials involving latency reversing agents (LRAs) such as vorinostat and disulfiram have failed to demonstrate significant reduction in reservoir size, although transient elevation in viral RNA has been observed. Accordingly, in vitro experiments have revealed that the majority of existing LRAs exert weak effects on HIV-1 transcription and reactivation. The future success of shock and kill will depend on our capacity to design highly efficacious new LRAs and/or adjuvant therapies to boost the reactivation potential of existing LRAs.
SUMMARY OF THE DISCLOSURE
[0007] In one embodiment, the present disclosure provides methods to reduce the number of HIV-infected cells in a subject or cell population by administering to the subject a therapeutically effective amount or contacting the cell population with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
[0008] In another embodiment, the present disclosure provides methods to induce pyroptosis in a HIV-infected cell by contacting the HIV-infected cell with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
[0009] In still another embodiment, the present disclosure provides methods to sensitize the CARD8 inflammasome to non-nucleoside reverse transcriptase inhibitor-induced activation in a HIV-infected cell by contacting the HIV infected cell or administering to a subject having an HIV-infected cell an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
[0010] In yet another embodiment, the present disclosure provides method of treating a subject with a HIV-infection by administering to the subject a therapeutically effective amount of a corn-position comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
[0011 ] In still yet another embodiment, the present disclosure provides methods of treating a subject with a NNRTI resistant HIV-infection by administering to the subject a therapeutically effective amount of a composition comprising a non nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
[0012] In another embodiment, the present disclosure provides methods to reduce HIV reservoirs in a subject with an HIV-infection by administering to the subject a therapeutically effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor. [0013] In still another embodiment, the present disclosure provides methods of activating HIV-1 protease activity and inhibiting DPP9 activity in an HIV- infected cell by contacting the cell with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
[0014] In each of the above embodiments, the NNRTI can be selected from nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), etravirine (ETR), or rilpivirine (RPV), or a combination thereof. In some aspects, the NNRTI is selected from rilpivirine (RPV) or efavirenz (EFV) or a combination thereof.
[0015] In each of the above embodiments, the DPP9 inhibitor can be selected from talabostat, talabostat mesylate, or 1G244.
[0016] In each of the above embodiments, the NNRTI is administered in an amount effective to induce premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of H I V-1 -infected cells.
[0017] In each of the above embodiments, the DPP9 inhibitor is administered in an amount effective to positively regulate CARD8 function, which triggers CARD8 inflammasome activation and rapid killing of virus-infected cells.
[0018] In each of the above embodiments, the combination of the NNRTI and DPP9 inhibitor is administered in an amount effective to induce the clearance of latent HIV-1 in a HIV-infected CD4+ T cell after viral reactivation.
[0019] In each of the above embodiments, the combination of the NNRTI and DPP9 inhibitor is administered in an amount effective to induce clearance of persistent HIV-1 infection.
[0020] In each of the above embodiments, the combination of the NNRTI and DPP9 inhibitor is administered to blood, lymphoid tissues, or hematopoietic-derived cells, simultaneously or separately.
[0021] In each of the above embodiments, the subject has been or is currently undergoing suppressive ART.
[0022] In an embodiment, the present disclosure provides methods of screening for CARD8 activating agents by contacting a test agent or compound with primary CD4+ T cells infected with clinical viral isolates; and detecting CARD8- dependent caspase-1 activation and/or pyroptosis by intracellular p24 staining.
[0023] In another embodiment, the present disclosure provides methods for measuring the clearance of HIV-1 , by obtaining blood CD4+ T cells from a subject under suppressive ART to measure the size of viral reservoirs; and detecting viral replication after test agent or test compound treatment.
BRIEF DESCRIPTION OF THE FIGURES
[0024] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0025] FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, FIG. 1E and FIG. 1F depicts The CARD8 inflammasome senses HIV-1 protease activity. FIG. 1A shows domain architecture of the CARD8 protein. CARD8 undergoes autoproteolytic processing in the FUND domain at position 296, generating the N-terminal ZU5 and C-terminal UPA- CARD fragments that remain associated non-covalently. Two HIV-1 protease cleavage sites are in the non-structured N-terminus and ZU5 domain, respectively. FIG. 1B shows HIV-1 protease cleaves the N-terminus of CARD8. HEK293T cells were co transfected with HA-CARD8-expressing plasmid together with pNL4-3-GFP or PR- D25H. Cells were collected 24 hours after transfection. Anti-HA, anti-CARD8-N, anti- CARD8-C, and anti-p24 antibodies were used sequentially on the same blot. Cleaved fragments are denoted with red asterisks. FIG. 1C shows HIV-1 protease triggers CARD8 inflammasome activation. Control or CARD8- KO HEK293T cells were co transfected with plasmids encoding CASP1, pro-IL-1 b, and pNL4-3-GFP. RPV and LPV were added immediately after transfection. FIG. 1D shows autoproteolytic processing is required for HIV-1 protease triggered CARD8 inflammasome activation. CARD8- KO HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and CARD8 or CARD8S297A, together with pNL4-3-GFP. LPV was added immediately after transfection. FIG. 1E shows HIV-1 protease triggered CARD8 inflammasome activation is proteasome-dependent. HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and pNL4-3-GFP. Indicated drugs were added 24 hours after transfection. Cells were collected 6 hours after drug treatment. In FIG. 1B to FIG. 1E, cell lysates were evaluated by immunoblotting. FIG. 1F shows HIV-1 protease cleaves CARD8 in vitro. HA-tagged CARD8 was immunoprecipitated and incubated with lysed HIV-1 particles with the presence of indicated drugs. The eluate was collected for immunoblotting. Full-length CARD8 (CARD8-FL), N terminal CARD8 (CARD8-N), freed N terminus (free Nt), freed C terminus (free Ct). Data are representative of three or more independent experiments..
[0026] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D and FIG. 2E depicts HIV-1 protease cleaves the N-terminus of CARD8. FIG. 2A shows CARD8A21 70 is cleaved at the second site. HEK293T cells were transfected with plasmids encoding CARD8 or CARD8A21 70, and pNL4-3-GFP or PR-D25H. FIG. 2B shows cleavage in the ZU5 domain does not activate the CARD8 inflammasome. CARD8- KO HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and WT or mutant CARD8, together with pNL4-3-GFP. FIG. 2C and FIG. 2D show mapping of the first cleavage site. HEK293T cells were transfected with plasmids encoding truncated or mutated CARD8, and pNL4-3-GFP. FIG. 2E shows cleavage in the N-terminus is required to activate the CARD8 inflammasome. CARD8- KO HEK293T cells were co-transfected with plasmids encoding CASP1, pro-IL-1 b, and WT or mutant CARD8, together with pNL4-3-GFP. Cell lysates were evaluated by immunoblotting. Data are representative of three or more independent experiments. Empty vector (EV).
[0027] FIG. 3A, FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G,
FIG. 3H and FIG. 3I depict HIV-1 protease triggers pyroptosis of infected macrophages upon NNRTI treatment. FIG. 3A to FIG. 3E show HIV-1 protease activation by NNRTIs induced rapid pyroptosis of infected monocyte-derived macrophages (MDMs). MDMs were infected with replication-competent HIVNL4-3/BaL. On day 4, RAL and T-20 were added to block new infection. Cells were then treated with RPV, EFV, LPV, or combinations for 24 hours or as indicated. GFP+ cells were detected by flow cytometry. DMSO controls were used to determine cell killing by NNRTIs (FIG. 3A to FIG. 3C). Representative images of infected MDMs were taken at 0 and 3 hours post RPV treatment. Scale bars represent 100 pm (FIG. 3D). Culture supernatant was collected after NNRTI treatment for IL-1 b ELISA (FIG. 3E). FIG. 3F and FIG. 3G show pyroptosis of H I V-1 -infected MDMs is CASP1 -dependent. MDMs were infected and treated as described above. Cleavage of pro-CASP1 and cleaved CASP1 (p10 and p20) in infected MDMs after RPV treatment for one hour (FIG. 3F). Infected MDMs were pre treated with CASP1 inhibitor VX-765 or pan-Caspase inhibitor Z-VAD-FMK for 3 hours then treated with RPV for 4 hours. Flow cytometry analysis was performed to determine cell killing (FIG. 3G). FIG. 3H and FIG. 3I shows HIV-1 protease mediated inflammasome activation is proteasome-dependent. MDMs were infected and treated as described above. Infected MDMs were pretreated with proteasome inhibitors MG132, bortezomib, or Me-Bs for 30 min and then treated with RPV for 4 hours. Cell killing was determined by flow cytometry (FIG. 3H). Culture supernatant from (FIG. 3H) was collected for IL-1 b measurement by ELISA (FIG. 3I). In FIG. 3B, FIG. 3G, and FIG.
3H, P-values were calculated using one-way ANOVA and Dunnett's test. In FIG.
3C, FIG. 3E, and FIG. 3I, P-values were calculated using two-way ANOVA and Tukey’s multiple comparison tests. *P< 0.05, ****P<0.0001. In each graph, n³ 3. Error bars show mean values with SEM. Data are representative of three or more independent experiments.
[0028] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D and FIG. 4E depict CARD8 inflammasome is required for pyroptosis of HIV-1 -infected THP-1 cells. FIG.
4A and FIG. 4B show NNRTIs induce death of HIV-1 -infected THP-1 cells. THP-1 cells were infected with VSV-G pseudotyped HIV-1 reporter virus NL4-3-GFP. On day 4 post infection, cells were treated with NNRTIs for another 2 days before flow cytometry analysis. FIG. 4C to FIG. 4E show NNRTI-triggered cell death is CARD8 inflammasome-dependent. Bulk populations of knockout THP-1 cells were used. Knockouts of CARD8, ASC, CASP1 or NLRP3 in THP-1 cells were confirmed by immunoblotting (C). Infected THP-1 cells were pre-treated with LPS (100 ng/ml) for 3 hours before RPV treatment. GFP expression was analyzed by flow cytometry 24 hours post RPV treatment. Data were normalized to the control group (FIG. 4D). Culture supernatant was collected 48 hours post RPV treatment for IL-1 b detection (FIG. 4E).
In FIG. 4B and FIG. 4D, P-values were calculated using one-way ANOVA and Dunnett's test. In FIG. 4E, P-values were calculated using two-way ANOVA and Tukey’s multiple comparison tests. **P<0.01, ****P<0.0001. In each bar graph, n= 3. Error bars show mean values with SEM. Data are representative of three or more independent experiments.
[0029] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E, FIG. 5F, FIG. 5G,
FIG. 5H, FIG. 5I and FIG. 5J depict HIV-1 Protease induces CARD8 inflammasome activation and subsequent cell death in CD4+ T cells. FIG. 5A shows the analysis of CARD8, caspase 1, and caspase 3 expression levels in unstimulated (U) and activated (FIG. 5A) primary CD4+ T cells by immunoblotting. FIG. 5B and FIG. 5C show HIV-1 protease activation by NNRTIs leads to killing of infected primary CD4+ T cells. Activated primary CD4+ T cells were infected with HIV-1 reporter virus NL4-3-Pol (FIG. 5B) or different HIV-1 reporter viruses (FIG. 5C) for 3 days before treatment with RPV or indicated NNRTIs. Cells were analyzed for GFP expression by flow cytometry 48 hours post NNRTI treatment. FIG. 5D and FIG. 5E show HIV-1 protease activation by NNRTIs induced CASP1 activation in infected primary CD4+ T cells. Activated primary CD4+ T cells were infected with the NL4-3-Pol. On day 3 post infection, cells were treated with EFV, RPV, LPV or combinations for 3 hours before staining for active CASP1. FIG. 5F shows chemical inhibition of caspase-1 blocks NNTRI-induced cell death. Activated primary CD4+ T cells were infected with the NL4-3-Pol. On day 3 post infection, cells were pre-treated with CASP1 inhibitor VX765 or pan-caspase inhibitor Z- VAD-FMK for 3 hours before adding RPV. GFP expression was measured by flow cytometry. FIG. 5G shows knockout of CARD8 in primary CD4+ T cells was confirmed by immunoblotting. FIG. 5H to FIG. 5G show the CARD8 inflammasome is required for pyroptosis of HIV-1 -infected primary CD4+ T cells. CARD8-, ASC- or CASP1 -knockout primary CD4+ T cells were co-stimulated and then infected with NL4-3-Pol. On day 3 post infection, cells were treated with RPV. Staining of active CASP1 3 hours post RPV treatment (FIG. 5H). GFP expression was measure by flow cytometry 24 hours after RPV treatment (FIG. 5I and FIG. 5J). In FIG. 5C, FIG. 5E, FIG. 5I, and FIG. 5J, P- values were calculated using one-way ANOVA and Dunnett's test. In F and H, P-values were calculated using two-way ANOVA and Tukey’s multiple comparison tests. **P<0.01 , ***P<0.001 , and ****P<0.0001. In each bar graph, n>3. Error bars show mean values with SEM. Data are representative of five or more independent experiments.
[0030] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D and FIG. 6E depict the Induction of CARD8 inflammasome activation clears infection of clinical HIV-1 isolates. FIG. 6A shows killing of primary CD4+ T cells infected with different subtypes of clinical HIV-1 isolates. Activated CD4+ T cells were infected with a panel of international HIV-1 isolates. RAL and T-20 were added with or without EFV, RPV or NVP on day 6 post infection. Viral infection was measured by intracellular p24 staining on day 8 and normalized to the DMSO controls. FIG. 6B shows killing of infected primary CD4+ T cells is proteasome-dependent. Activated primary CD4+ T cells were infected with different subtypes of clinical HIV-1 isolates. RAL, T-20, and RPV were added with or without bortezomib on day 6 post infection. Viral infection was measured by intracellular p24 staining 6 hours after drug treatment. FIG. 6C shows the CARD8 inflammasome is required for the killing of infected primary CD4+ T cells. Activated CARD8- KO or control primary CD4+ T cells were infected with different subtypes of clinical HIV-1 isolates.
RAL, T-20, and RPV were added on day 6 post infection. Viral infection was measured by intracellular p24 staining 24 hours after drug treatment. In FIG. 6A to FIG. 6C, n= 3. Error bars show mean values with SEM. FIG. 6D shows the scheme of shock and kill strategy and quantitative viral outgrowth assay (QVOA) using patient CD4+ T cells. FIG. 6E shows the clearance of latent HIV-1 by RPV treatment. Frequency of latent HIV-1 was determined by QVOA. Red: ARV for 2 days and RPV at 2.5 mM. Blue: ARV for 3 days and RPV at 5 pM. Open circle: no detectable HIV-1 infection by p24 ELISA.
In FIG. 6B and FIG. 6C, P-values were calculated using multiple t test. In FIG. 6E, P- value was calculated by ratio paired t test. *P<0.01 , **P<0.01 , ***P<0.001 , and ****PO .001.
[0031] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D, FIG. 7E, FIG. 7F, FIG. 7G and FIG. 7H show NNRTIs induce death of HIV-1 -infected cells in a dose-dependent manner. FIG. 7A is a depiction of how CD4+ T Cells were treated with NNRTI’s and assayed for killing. FIG. 7B is a dose response curves for various NNRTI’s in successive three-fold dilutions in three healthy donor CD4+ T cells isolated from PBMC. EC50 Values for EFV, RPV, and ETR, are as follows: 266.1 nM, 87.8nM, and 786.6nM respectively. ETR and DOR did not provide sufficient killing for EC50 calculation. FIG.
7B is a dose response curves for THP-1 infected with NL4-3-GFP and treated with EFV, RPV, or DOR as in panel A. ECso for EFV is 2128nM and 438 for RPV. FIG. 7D is a dose response curves for killing of NL4-3-Avif-vpr infected THP-1 CARD8KO or Cas9 transduced control cells treated with EFV, RPV, or NVP. The ability of EFV and RPV to kill infected cells is CARD8 dependent regardless of concentration. E) EFV based killing and activation of the CARD8 inflammasome is dependent upon CASP1 as evidenced by knockout of CASP1 in NL4-3-Avif-vpr infected THP-1 cells with CRISPR/Cas9. (**** = p<.0001 by two-way ANOVA with Sidak’s multiple comparison test). FIG. 7F and FIG.
7G show treatment of CD4+ T cells with EFV and RPV with or without the presence of 50% human serum in the culture media (NL4-3-pol). FIG. 7G shows the log fold change increase in EC50 due to the presence of human serum.
[0032] FIG. 8A, FIG. 8B, FIG. 8C, FIG. 8D and FIG. 8E show DPP9 inhibition sensitizes the CARD8 Inflammasome to NNRTI-induced pyroptosis. FIG. 8A is a graphical depiction of CARD8 inflammasome activation and sensitization through VBP. CARD8 first undergoes autoprocessing of its FUND domain leaving two non- covalently bound fragments (N-terminal and C-terminal). HIV-1 protease, after premature activation due to Gag-Pol dimerization through NNRTIs, can cleave the N- terminal fragment leading to proteasomal degradation. The released C-terminal fragment can either activate the inflammasome through CASP1 or be caught by DPP9. VBP inhibits DPP9 and allows more freed C-terminal fragment to activate the inflammasome. FIG. 8B is a dose response curves for three donors of primary CD4+ T cells treated with EFV or RPV in combination with VBP. The green highlighted area denotes the drug plasma concentration range. Zero NNRTI concentration values were plotted at 10-1 nM concentration to allow log-transformation (****= p .0001 by two-way ANOVA with Tukey’s multiple comparison test). FIG. 8C shows log fold changes in EC50 due to varying VBP concentrations are plotted for EFV (left) and RPV (right). FIG.
8D shows VBP and EFV combination treatment denotes a synergistic relationship. Four independent synergy calculation methods from SynergyFinder2.0 (Bliss, HSA, Loewe, and ZIP) were used. FIG. 8E shows VBP toxicity in CD4+ T Cells as denoted by the heatmap of MTS assay results of three separate donors of primary CD4+ T Cells treated for two days with EFV and/or VBP.
[0033] FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D, FIG. 9E, FIG. 9F and FIG. 9G show characterization of VBP enhancement of NNRTI-induced cell killing. FIG. 9A is a time course treatment of HIV-1 -infected primary CD4+ T cells treated with DMSO, EFV ( 5pM), VBP (.5mM), or combination. Killing of infected cells plateaus after 48 hours in all conditions. FIG. 9B shows fold change enhancement of combination treatment in comparison to EFV alone treatment from panel A. FIG. 9C and FIG. 9D show time course treatment and fold change enhancement of H I V-1 -infected THP-1 cells treated with DMSO, EFV (1 mM), VBP (.5mM), or combination. FIG. 9E shows NNRTI-based killing and VBP enhancement is specific to the CARD8 inflammasome. FIG. 9F shows CASP1 activation by NNRTIs and VBP. Cells were simultaneously treated with EFV and VBP and stained with CASP1 staining dye. FIG. 9G shows CASP1 is required for the cell killing by NNRTIs and VBP (** = p<.01 , *** = p<.001 , **** = p .0001 by one-way ANOVA with Tukey’s multiple comparison test).
[0034] FIG. 10A, FIG. 10B, FIG. 10C, FIG. 10D, FIG. 10E and FIG. 10F show VBP sensitization of the CARD8 inflammasome can overcome NNRTI resistance. FIG. 10A shows killing and enhancement of clinical isolates from Clades A, B, C, and D. One Donor of primary CD4+ T Cells were treated with DMSO, EFV (.5mM), or EFV with VBP ( 5pM) in the presence of media containing T-20 (1 mM), Raltegravir (1 pM). Each strain showed significant enhancement upon addition of VBP (Two-way ANOVA with Tukey’s multiple comparison test). FIG. 10B is a graphical depiction of the location of NNRTI RAMS. FIG. 10C shows NNRTI RAMS show diminished capacity for killing HIV infected primary CD4+ T Cells from one donor upon treatment with 5pM of EFV with respective fold change from no RAM control shown in panel FIG. 10D (Two-way ANOVA with Tukey’s multiple comparison test). FIG. 10E shows upon VBP treatment, these same mutants show significantly enhanced killing with increasing VBP concentrations (One-way ANOVA with Tukey’s multiple comparison test). Additionally, some RAM containing viruses have increased levels of killing efficiency as depicted by the fold change of killing by 1 mM VBP treatment from no RAM virus in panel FIG.
10F (Two-way ANOVA with Dunnett’s multiple comparison test). * = p<.05, ** = p<.01 , *** = p<.001 , **** = pc.0001.
[0035] FIG. 11 A. FIG. 11B, FIG. 11C, FIG. 11D, FIG. 11E, FIG. 11F and FIG. 11G show VbP enhances clearance of HIV-1 infected cells in humanized mice. Primary CD4+ T cells were infected with NL4-3-Pol. 3 days post infection; these cells were infused into mice (5-10 million cells per mouse). EFV and VbP were provided by IP and IV injection, respectively. FIG. 11A shows killing of HIV-1 -infected human CD4+
T cells from blood of transfused mice at 6hr and 24hr post treatment with EFV. Mice were either treated with DMSO or varying doses of EFV (0.1 mg, 0.25 mg, or 0.5 mg per mouse). FIG. 11 A, FIG. 11B, FIG. 11C and FIG. 11 D show killing of infected human CD4+ T Cells in blood (FIG. 11B) and lung tissues (FIG. 11C and FIG. 11D) at 24hr post treatment with 60 pg VbP, 0.5 mg EFV, or combo. FIG. 11E shows cross cohort comparison of four independent cohorts of mice treated with EFV or combo as described above. CD4+ T cells from four healthy blood donors were used. FIG. 11 F and FIG. 11G show killing of infected human CD4+ T cells in blood (FIG. 11E) or lung (FIG. 11 F) with single dose or multidose combination treatment regimens. The multi dose regimen group received 0.5 mg EFV injections on days 0, 1, and 2, and 60 pg VbP injections on days 0 and 2. The single-dose regimen received both EFV and VbP only on day 0. Error bars show mean values with SEM. One-way ANOVA with Dunnett’s multiple comparison test (a, d), orTukey’s multiple comparison test (b, c, e, and f), or Holm- Sidak's multiple comparisons test (g). * p<0.05, **p<0.01 , *** p<0.001 , **** p O.0001.
[0036] FIG. 12 shows DPP8/9 inhibitor 1G244 enhances EFV-triggered killing of HIV-infected cells. Primary CD4+ T cells were infected with a HIV reporter virus. 1G244 at indicated concentration (nM) with or without 3mM EFV was added to the culture on day 3 post infection. Cell killing was measured by FACS 2 days post drug treatment. DETAILED DESCRIPTION
[0037] Among the various aspects of the disclosure is the provision of using compositions and methods comprising a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor to kill HIV infected cells. As described in greater detail herein, it has been discovered that that HIV-infected cells (e.g., CD4+ T cells) are sensitized to NNRTI mediated pyroptosis through the inhibition of DPP9. Applicant has unexpectedly found DPP9 inhibition with NNRTIs act synergistically to increase their efficacy in killing HIV-infected cells. In addition, DPP9 inhibitor-mediated sensitization of the CARD8 inflammasome in HIV-infected cells is shown to overcome NNRTI resistance. Additional aspects of the disclosure are described below.
I. Definitions
[0038] So that the present disclosure may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the disclosure pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present disclosure without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present disclosure, the following terminology will be used in accordance with the definitions set out below.
[0039] Concentrations, amounts, and other numerical data may be expressed or presented herein in a range format. It is to be understood that such a range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. As an illustration, a numerical range of “about 2 to about 50” should be interpreted to include not only the explicitly recited values of 2 to 50, but also include all individual values and subranges within the indicated range. Thus, included in this numerical range are individual values such as 2, 2.4, 3, 3.7, 4, 5.5, 10, 10.1 , 14, 15, 15.98, 20, 20.13, 23, 25.06, 30, 35.1, 38.0, 40, 44, 44.6, 45, 48, and sub-ranges such as from 1 -3, from 2-4, from 5-10, from 5-20, from 5-25, from 5-30, from 5-35, from 5-40, from 5-50, from 2-10, from 2-20, from 2-30, from 2-40, from 2-50, etc. This same principle applies to ranges reciting only one numerical value as a minimum or a maximum. Furthermore, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.
[0040] The term “about,” as used herein, refers to variation of in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, distance, and amount. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations, which can be up to ± 5%, but can also be ± 4%, 3%,
2%, 1%, etc. Whether or not modified by the term “about,” the claims include equivalents to the quantities.
[0041] In this disclosure, “comprises,” “comprising,” “containing,” and “having” and the like can have the meaning ascribed to them in U.S. Patent Law and can mean “includes,” “including,” and the like, and are generally interpreted to be open ended terms. The terms “consisting of” or “consists of” are closed terms, and include only the components, structures, steps, or the like specifically listed in conjunction with such terms, as well as that which is in accordance with U.S. Patent law. “Consisting essentially of” or “consists essentially of” have the meaning generally ascribed to them by U.S. Patent law. In particular, such terms are generally closed terms, with the exception of allowing inclusion of additional items, materials, components, steps, or elements, that do not materially affect the basic and novel characteristics or function of the item(s) used in connection therewith. For example, trace elements present in a composition, but not affecting the composition’s nature or characteristics would be permissible if present under the “consisting essentially of” language, even though not expressly recited in a list of items following such terminology. In this specification when using an open ended term, like “comprising” or “including,” it is understood that direct support should be afforded also to “consisting essentially of” language as well as “consisting of” language as if stated explicitly and vice versa.
[0042] As used herein, a “biological sample” refers to a sample of tissue, cells, or fluid isolated from a subject, including but not limited to, for example, blood, buffy coat, plasma, serum, immune cells (e.g., macrophages), sputa, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, organs, biopsies and also samples of in vitro cell culture constituents, including, but not limited to, conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components. As used herein, the term “blood sample” refers to a biological sample derived from blood, preferably peripheral (or circulating) blood. The blood sample can be whole blood, plasma or serum, although plasma is typically preferred.
[0043] The terms “treat,” "treating," or "treatment" as used herein, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disease/disorder. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, a delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease, condition, or disorder as well as those prone to have the disease, condition or disorder or those in which the disease, condition or disorder is to be prevented.
[0044] As used herein, the terms “effective amount” or “therapeutically effective amount” of a drug used to treat a disease is an amount that can reduce the severity of a disease, reduce the severity of one or more symptoms associated with the disease or its treatment, or delay the onset of more serious symptoms or a more serious disease that can occur with some frequency following the treated condition. An “effective amount” may be determined empirically and in a routine manner, in relation to the stated purpose.
[0045] Retroviruses are a family of enveloped viruses that replicate in a host cell through the process of reverse transcription. A retrovirus is a single-stranded positive sense RNA virus with a DNA intermediate and, as an obligate parasite, targets a host cell. Once inside the host cell cyto-plasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern. This new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as part of its own genome, translating and transcribing the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus. It is difficult to detect the virus until it has infected the host. At that point, the infection will persist indefinitely.
[0046] Examples of human retroviruses identified include HTLV 1 (T-cell leukaemias/lymphomas, Tropical spastic paraparesis), HTLV 2 (no known pathology), HIV 1 & 2 - AIDS and HTLV-3 and HTLV-4 (no known pathology). All of the human retorviruses infect T cells. HTLV-1 is known to cause a type of cancer called adult T- cell leukemia/lymphoma and a demyelinating disease called HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-2 is a virus closely related to HTLV-I and shares approximately 70% genomic homology with HTLV-I. HTLV-3 and HTLV-4 are two new human retroviruses recently identified which are related to HTLV1 and 2.
[0047] The human immunodeficiency virus (HIV) causes the acquired immunodeficiency syndrome (AIDS), a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. Without treatment, average survival time after infection with HIV is estimated to be 9 to 11 years, depending on the HIV subtype. [0048] HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including apoptosis of uninfected bystander cells, direct viral killing of infected cells, and killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells. When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections.
[0049] HIV establishes a persistent infection in its host and only causes death many years later. Most individuals experience a primary infection resulting in a febrile illness about 2-4 weeks after exposure. This illness coincides with seroconversion. The symptoms are similar to those of glandular fever, namely fever, sore throat, night sweats, lymphadenopathy, diarrhea. Following the primary infection, the patient enters a stage of clinical latency. During this time the patient feels fine, but they are infectious as they have ongoing viral replication and have HIV antibodies in their blood. As the CD4 counts drop, there is a gradual onset of a variety of prodromal disorders, such as weight loss, fever, persistent lymphadenopathy, oral candidiasis and diarrhea. These symptoms precede the progression to AIDS.
[0050] The human immunodeficiency virus (HIV) causes the acquired immunodeficiency syndrome (AIDS), a condition in humans in which progressive failure of the immune system allows life-threatening opportunistic infections and cancers to thrive. Without treatment, average survival time after infection with HIV is estimated to be 9 to 11 years, depending on the HIV subtype.
[0051] HIV infects vital cells in the human immune system such as helper T cells (specifically CD4+ T cells), macrophages, and dendritic cells. HIV infection leads to low levels of CD4+ T cells through a number of mechanisms, including apoptosis of uninfected bystander cells, direct viral killing of infected cells, and killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells. When CD4+ T cell numbers decline below a critical level, cell-mediated immunity is lost, and the body becomes progressively more susceptible to opportunistic infections. [0052] HIV establishes a persistent infection in its host and only causes death many years later. Most individuals experience a primary infection resulting in a febrile illness about 2-4 weeks after exposure. This illness coincides with sero conversion. The symptoms are similar to those of glandular fever, namely fever, sore throat, night sweats, lymphadenopathy, diarrhea. Following the primary infection, the patient enters a stage of clinical latency. During this time the patient feels fine, but they are infectious as they have on-going viral replication and have HIV antibodies in their blood. As the CD4 counts drop, there is a gradual onset of a variety of prodromal disorders, such as weight loss, fever, persistent lymphadenopathy, oral candidiasis and diarrhea. These symptoms precede the progression to AIDS.
[0053] Discussed below are components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules of the compound are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
[0054] Various aspects of the disclosure are described in further detail in the following sections.
II. COMPOSITIONS
[0055] In some embodiments, the present disclosure provides a composition comprising a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor. In one aspect, a composition of the disclosure comprises Efavirnez and a DPP9 inhibitor. In one aspect, a composition of the disclosure comprises Rilpivirine and a DPP9 inhibitor. In still another aspect, a composition of the disclosure comprises a NNRTI and talbostat (Val-boroPro) or 1G244.
[0056] A composition of the disclosure may optionally comprise one or more additional drugs or therapeutically active agent in addition to the non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor. A composition of the disclosure may further comprise a pharmaceutically acceptable excipient, carrier or diluent. Further, a composition of the disclosure may contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (substances of the present disclosure may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents or antioxidants.
(a) Non-Nucleoside reverse transcriptase inhibitors (NNRTI)
[0057] Non-nucleoside reverse transcriptase inhibitor is an antiretroviral (ARV) HIV drug class. Non-Nucleoside reverse transcriptase inhibitors (NNRTI) can inhibit HIV reverse transcriptase (an HIV enzyme) by binding to an allosteric site of the enzyme; NNRTIs can act as non-competitive inhibitors of reverse transcriptase. NNRTIs affect the handling of substrate (nucleotides) by reverse transcriptase by binding near the active site. HIV uses reverse transcriptase to convert its RNA into DNA (reverse transcription). Blocking reverse transcriptase and reverse transcription prevents HIV from replicating. NNRTIs can be further classified into 1st generation and second generation NNRTIs. 1st generation NNRTIs include nevirapine and efavirenz. Second generation NNRTIs are etravirine and rilpivirine. HIV-2 is naturally resistant to NNRTIs.
[0058] Non-limiting examples of suitable NNRTIs include efavirenz, nevirapine, etravirine, doravirine, nevirapine, delavirdine, and rilpivirine. In some embodiments, a NNRTI of the disclosure is selected from the group consisting of efavirenz, rilpivirine, and etravirine. In one aspect, a NNRTI of the disclosure is not nevirapine. In another aspect, of the disclosure is not doravirine. In some embodiments, a NNRTI according to the disclosure drive Gag-Pol dimerization and intracellular protease activation which cleaves CARD8 and leads to activation of the CARD8 inflammasome.
(b) Dipeptidyl Peptidase 9 (DPP9) inhibitor
[0059] Dipeptidyl Peptidase 9 (HGNC: 18648 NCBI Entrez Gene: 91039 Ensembl: ENSG00000142002 OMIM®: 608258 UniProtKB/Swiss-Prot: Q86TI2) is encoded by the DPP9 gene (chr19:4,675,224-4,724,673). This gene encodes a protein that is a member of the S9B family in clan SC of the serine proteases. The protein has been shown to have post-proline dipeptidyl aminopeptidase activity, cleaving Xaa-Pro dipeptides from the N-termini of proteins. Although the activity of this protein is similar to that of dipeptidyl peptidase 4 (DPP4), it does not appear to be membrane bound. In general, dipeptidyl peptidases appear to be involved in the regulation of the activity of their substrates and have been linked to a variety of diseases including type 2 diabetes, obesity and cancer. Several transcript variants of this gene have been described but not fully characterized. A "DPP9 polynucleotide", within the meaning of the disclosure, shall be understood as being a nucleic acid molecule selected from a group consisting of (i) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 1. A "DPP9 polypeptide", within the meaning of the disclosure, shall be understood as being a polypeptide selected from a group consisting of (i) polypeptides having the sequence of SEQ ID NO: 1 , (ii) polypeptides comprising the sequence of SEQ ID NO: 1, (iii) polypeptides encoded by DPP9 polynucleotides; and (iv) polypeptides which show at least 99%, 98%, 95%, 90%, or 80% homology with a polypeptide of (i), or (iii); wherein said polypeptide has DPP9 activity. [0060] As used herein, a “DPP9 inhibitor” is any compound that is capable of reducing DPP9 activity and/or expression. "Inhibitor" is any substance which retards or prevents a chemical or physiological reaction or response. Common inhibitors include but are not limited to antisense molecules, antibodies, and antagonists. A compound with the ability to inhibit DPP9 may include, without limitation, a compound, a drug, a small molecule, a peptide, a nucleic acid molecule, a protein, an antibody, and combinations thereof. A nucleic acid molecule may be an antisense oligonucleotide, a ribozyme, a small nuclear RNA (snRNA), a long noncoding RNA (LncRNA), or a nucleic acid molecule which forms triple helical structures.
[0061] For example, DPP9 nucleic acid expression, DPP9 protein expression, or DPP9 activity may be measured as described in more detail below and in the Examples.
[0062] In a specific embodiment, a DPP9 inhibitor may be Talabostat (Val- boroPro, PT100) or Talabostat mesylate. Thus, a DPP9 inhibitor compound of the disclosure is a compound of formula (I):
Figure imgf000022_0001
Formula (I)
[0063] In another embodiment, a DPP9 inhibitor may be 1 G244. Thus, a DPP9 inhibitor compound of the disclosure is a compound of formula (II):
Figure imgf000022_0002
Formula (II)
[0064] In another embodiment, a DPP9 inhibitor may be SLRFLYEG (SEQ ID NO: 3) described in Ross et al. (PNAS 115(7) 2018; Structures and mechanism of dipeptidyl peptidases 8 and 9, important players in cellular homeostasis and cancer) incorporated herein by reference in its entirety. Additional DPP9 inhibitors useful according to the present disclosure include those disclosed in WO 2005106487; WO 2014068023; WO2014127747; US 20050215784; WO 2011113895, US 20110218142; US 2008/0293618; WO 2005/106487 describes antibodies and siRNAfor modulating DPP9 activity; the disclosures of which are herein incorporated by reference in their entirety.
[0065] Dosages of a compound that inhibits DPP9 can vary between wide limits, depending upon the disease or disorder to be treated and/or the age and condition of the subject to be treated. In an embodiment where a composition comprising a compound that inhibits DPP9 is contacted with a sample, the concentration of the compound that inhibits DPP9 may be from about 1 mM to about 40 pM. Alternatively, the concentration of the compound that inhibits DPP9 may be from about 5 pM to about 25 pM. For example, the concentration of the compound that inhibits DPP9 may be about 1 , about 2.5 about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 12, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 30, about 35, or about 40 pM. Additionally, the concentration of the compound that inhibits DPP9 may be greater than 40 pM. For example, the concentration of the compound that inhibits DPP9 may be about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 or about 100 pM. In certain embodiments, the concentration of the compound that inhibits DPP9 may be from about 1 pM to about 10 pM, from about 10 pM to about 20 pM, from about 20 pM to about 30 pM, or from about 30 pM to about 40 pM. In a specific embodiment, the concentration of the compound that inhibits DPP9 may be from about 1 pM to about 10 pM.
[0066] In an embodiment where the composition comprising a compound that inhibits DPP9 is administered to a subject, the dose of the compound that modulates DPP9 may be from about 0.1 mg/kg to about 500 mg/kg. For example, the dose of the compound that inhibits DPP9 may be about 0.1 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, or about 25 mg/kg. Alternatively, the dose of the compound that inhibits DPP9 may be about 25 mg/kg, about 50 mg/kg, about 75 mg/kg, about 100 mg/kg, about 125 mg/kg, about 150 mg/kg, about 175 mg/kg, about 200 mg/kg, about 225 mg/kg, or about 250 mg/kg. Additionally, the dose of the compound that inhibits DPP9 may be about 300 mg/kg, about 325 mg/kg, about 350 mg/kg, about 375 mg/kg, about 400 mg/kg, about 425 mg/kg, about 450 mg/kg, about 475 mg/kg or about 500 mg/kg.
/. DPP9 nucleic acid expression
[0067] In an embodiment, DPP9 nucleic acid expression may be measured to identify a compound that inhibits DPP9. For example, when DPP9 nucleic acid expression is decreased in the presence of a compound relative to an untreated control, the compound inhibits DPP9. In a specific embodiment, DPP9 mRNA may be measured to identify a compound that inhibits DPP9.
[0068] Methods for assessing an amount of nucleic acid expression in cells are well known in the art, and all suitable methods for assessing an amount of nucleic acid expression known to one of skill in the art are contemplated within the scope of the disclosure. The term “amount of nucleic acid expression” or “level of nucleic acid expression” as used herein refers to a measurable level of expression of the nucleic acids, such as, without limitation, the level of messenger RNA (mRNA) transcript expressed or a specific variant or other portion of the mRNA, the enzymatic or other activities of the nucleic acids, and the level of a specific metabolite. The term “nucleic acid” includes DNA and RNA and can be either double stranded or single stranded. Non-limiting examples of suitable methods to assess an amount of nucleic acid expression may include arrays, such as microarrays, PCR, such as RT-PCR (including quantitative RT-PCR), nuclease protection assays and Northern blot analyses. In a specific embodiment, determining the amount of expression of a target nucleic acid comprises, in part, measuring the level of target nucleic acid mRNA expression.
[0069] In one embodiment, the amount of nucleic acid expression may be determined by using an array, such as a microarray. Methods of using a nucleic acid microarray are well and widely known in the art. For example, a nucleic acid probe that is complementary or hybridizable to an expression product of a target gene may be used in the array. The term “hybridize” or “hybridizable” refers to the sequence specific non-covalent binding interaction with a complementary nucleic acid. In a preferred embodiment, the hybridization is under high stringency conditions. Appropriate stringency conditions which promote hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6. The term “probe” as used herein refers to a nucleic acid sequence that will hybridize to a nucleic acid target sequence. In one example, the probe hybridizes to an RNA product of the nucleic acid or a nucleic acid sequence complementary thereof. The length of probe depends on the hybridization conditions and the sequences of the probe and nucleic acid target sequence. In one embodiment, the probe is at least 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 400, 500 or more nucleotides in length.
[0070] In another embodiment, the amount of nucleic acid expression may be determined using PCR. Methods of PCR are well and widely known in the art, and may include quantitative PCR, semi-quantitative PCR, multiplex PCR, or any combination thereof. Specifically, the amount of nucleic acid expression may be determined using quantitative RT-PCR. Methods of performing quantitative RT-PCR are common in the art. In such an embodiment, the primers used for quantitative RT-PCR may comprise a forward and reverse primer for a target gene. The term “primer” as used herein refers to a nucleic acid sequence, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand is induced (e.g. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon factors, including temperature, sequences of the primer and the methods used. A primer typically contains 15-25 or more nucleotides, although it can contain less or more. The factors involved in determining the appropriate length of primer are readily known to one of ordinary skill in the art. [0071] The amount of nucleic acid expression may be measured by measuring an entire mRNA transcript for a nucleic acid sequence, or measuring a portion of the mRNA transcript for a nucleic acid sequence. For instance, if a nucleic acid array is utilized to measure the amount of mRNA expression, the array may comprise a probe for a portion of the mRNA of the nucleic acid sequence of interest, or the array may comprise a probe for the full mRNA of the nucleic acid sequence of interest. Similarly, in a PCR reaction, the primers may be designed to amplify the entire cDNA sequence of the nucleic acid sequence of interest, or a portion of the cDNA sequence. One of skill in the art will recognize that there is more than one set of primers that may be used to amplify either the entire cDNA or a portion of the cDNA for a nucleic acid sequence of interest. Methods of designing primers are known in the art. Methods of extracting RNA from a biological sample are known in the art.
[0072] The level of expression may or may not be normalized to the level of a control nucleic acid. Such a control nucleic acid should not specifically hybridize with an mRNA nucleotide sequence of the disclosure. This allows comparisons between assays that are performed on different occasions.
//. DPP9 protein expression
[0073] In another embodiment, DPP9 protein expression may be measured to identify a compound that inhibits DPP9. For example, when DPP9 protein expression is decreased in the presence of a compound relative to an untreated control, the compound inhibits DPP9. In a specific embodiment, DPP9 protein expression may be measured using immunoblot. In another specific embodiment, DPP9 protein expression may be measured using immunofluorescence staining.
[0074] Methods for assessing an amount of protein expression are well known in the art, and all suitable methods for assessing an amount of protein expression known to one of skill in the art are contemplated within the scope of the disclosure. Non-limiting examples of suitable methods to assess an amount of protein expression may include epitope binding agent-based methods and mass spectrometry based methods. [0075] In some embodiments, the method to assess an amount of protein expression is mass spectrometry. By exploiting the intrinsic properties of mass and charge, mass spectrometry (MS) can resolve and confidently identify a wide variety of complex compounds, including proteins. Traditional quantitative MS has used electrospray ionization (ESI) followed by tandem MS (MS/MS) (Chen et al. , 2001 ;
Zhong et al., 2001 ; Wu et al., 2000) while newer quantitative methods are being developed using matrix assisted laser desorption/ionization (MALDI) followed by time of flight (TOF) MS (Bucknall et al., 2002; Mirgorodskaya et al., 2000; Gobom et al., 2000). In accordance with the present disclosure, one can use mass spectrometry to look for the level of protein encoded from a target nucleic acid of the disclosure.
[0076] In some embodiments, the method to assess an amount of protein expression is an epitope binding agent-based method. As used herein, the term “epitope binding agent” refers to an antibody, an aptamer, a nucleic acid, an oligonucleic acid, an amino acid, a peptide, a polypeptide, a protein, a lipid, a metabolite, a small molecule, or a fragment thereof that recognizes and is capable of binding to a target gene protein. Nucleic acids may include RNA, DNA, and naturally occurring or synthetically created derivative.
[0077] As used herein, the term “antibody” generally means a polypeptide or protein that recognizes and can bind to an epitope of an antigen. An antibody, as used herein, may be a complete antibody as understood in the art, i.e. , consisting of two heavy chains and two light chains, or may be any antibody-like molecule that has an antigen binding region, and includes, but is not limited to, antibody fragments such as Fab’, Fab, F(ab’)2, single domain antibodies, Fv, and single chain Fv. The term antibody also refers to a polyclonal antibody, a monoclonal antibody, a chimeric antibody and a humanized antibody. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. Means for preparing and characterizing antibodies are also well known in the art (See, e.g. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; herein incorporated by reference in its entirety). [0078] As used herein, the term “aptamer” refers to a polynucleotide, generally a RNA or DNA that has a useful biological activity in terms of biochemical activity, molecular recognition or binding attributes. Usually, an aptamer has a molecular activity such as binging to a target molecule at a specific epitope (region). It is generally accepted that an aptamer, which is specific in it binding to a polypeptide, may be synthesized and/or identified by in vitro evolution methods. Means for preparing and characterizing aptamers, including by in vitro evolution methods, are well known in the art (See, e.g. US 7,939,313; herein incorporated by reference in its entirety).
[0079] In general, an epitope binding agent-based method of assessing an amount of protein expression comprises contacting a sample comprising a polypeptide with an epitope binding agent specific for the polypeptide under conditions effective to allow for formation of a complex between the epitope binding agent and the polypeptide. Epitope binding agent-based methods may occur in solution, or the epitope binding agent or sample may be immobilized on a solid surface. Non-limiting examples of suitable surfaces include microtitre plates, test tubes, beads, resins, and other polymers.
[0080] An epitope binding agent may be attached to the substrate in a wide variety of ways, as will be appreciated by those in the art. The epitope binding agent may either be synthesized first, with subsequent attachment to the substrate, or may be directly synthesized on the substrate. The substrate and the epitope binding agent may be derivatized with chemical functional groups for subsequent attachment of the two. For example, the substrate may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the epitope binding agent may be attached directly using the functional groups or indirectly using linkers.
[0081] The epitope binding agent may also be attached to the substrate non-covalently. For example, a biotinylated epitope binding agent may be prepared, which may bind to surfaces covalently coated with streptavidin, resulting in attachment. Alternatively, an epitope binding agent may be synthesized on the surface using techniques such as photopolymerization and photolithography. Additional methods of attaching epitope binding agents to solid surfaces and methods of synthesizing biomolecules on substrates are well known in the art, i.e. VLSIPS technology from Affymetrix (e.g., see U.S. Pat. No. 6,566,495, and Rockett and Dix, Xenobiotica 30(2): 155-177, both of which are hereby incorporated by reference in their entirety).
[0082] Contacting the sample with an epitope binding agent under effective conditions for a period of time sufficient to allow formation of a complex generally involves adding the epitope binding agent composition to the sample and incubating the mixture for a period of time long enough for the epitope binding agent to bind to any antigen present. After this time, the complex will be washed and the complex may be detected by any method well known in the art. Methods of detecting the epitope binding agent-polypeptide complex are generally based on the detection of a label or marker. The term “label”, as used herein, refers to any substance attached to an epitope binding agent, or other substrate material, in which the substance is detectable by a detection method. Non-limiting examples of suitable labels include luminescent molecules, chemiluminescent molecules, fluorochromes, fluorescent quenching agents, colored molecules, radioisotopes, scintillants, biotin, avidin, stretpavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni2+, Flag tags, myc tags, heavy metals, and enzymes (including alkaline phosphatase, peroxidase, and luciferase). Methods of detecting an epitope binding agent-polypeptide complex based on the detection of a label or marker are well known in the art.
[0083] In some embodiments, an epitope binding agent-based method is an immunoassay. Immunoassays can be run in a number of different formats. Generally speaking, immunoassays can be divided into two categories: competitive immmunoassays and non-competitive immunoassays. In a competitive immunoassay, an unlabeled analyte in a sample competes with labeled analyte to bind an antibody. Unbound analyte is washed away and the bound analyte is measured. In a non competitive immunoassay, the antibody is labeled, not the analyte. Non-competitive immunoassays may use one antibody (e.g. the capture antibody is labeled) or more than one antibody (e.g. at least one capture antibody which is unlabeled and at least one “capping” or detection antibody which is labeled.) Suitable labels are described above.
[0084] In some embodiments, the epitope binding agent-based method is an ELISA. In other embodiments, the epitope binding agent-based method is a radioimmunoassay. In still other embodiments, the epitope binding agent-based method is an immunoblot or Western blot. In alternative embodiments, the epitope binding agent-based method is an array. In another embodiment, the epitope binding agent-based method is flow cytometry. In different embodiments, the epitope binding agent-based method is immunohistochemistry (IHC) or immunofluorescence. IHC uses an antibody (or fluorescently labeled antibody in the case of immunofluorescence) to detect and quantify antigens in intact tissue samples. The tissue samples may be fresh- frozen and/or formalin-fixed, paraffin-embedded (or plastic-embedded) tissue blocks prepared for study by IHC or immunofluorescence. Methods of preparing tissue block for study by IHC or immunofluorescence, as well as methods of performing IHC or immunofluorescence are well known in the art. iii. DPP9 activity
[0085] In an embodiment, DPP9 activity may be measured to identify a compound that inhibits DPP9. DPP9 is a protease and therefore its ability to cleave peptides can be a measure of DPP9 activity. Methods for measuring protease activity are standard in the art and also described below. For example, the ability of DPP9 to bind to CARD8 can be measured as a determination of DPP9 activity, where a compound which disrupts the ability of DPP9 to bind with CARD8 is an inhibitor of DPP9 activity.
[0086] In another embodiment, HIV-infected cell viability may be measured as an indication of DPP9 activity. Cell viability may be measured using methods standard in the art as described below in the Examples. For example, when cell viability of a HIV-infected cells is decreased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
[0087] In still another embodiment, caspases may be measured as an indication of DPP9 activity. Specifically, caspase 1 may be measured as an indication of DPP9 activity. Inhibition of DPP9, leads to CASP1 activation in HIV-infected cells. For example, when CASP1 activation in HIV-infected cells is increased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
[0088] In still yet another embodiment, CARD8 inflammasome activation may be measured as an indication of DPP9 activity. Inhibition of DPP9, leads to CARD8 inflammasome activation in HIV-infected cells. For example, when CARD8 inflammasome activation in HIV-infected cells is increased in the presence of a compound relative to an untreated control, the compound inhibits DPP9.
(c) components of the composition
[0089] The present disclosure also provides pharmaceutical compositions. The pharmaceutical composition comprises a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor, as active ingredients, and at least one pharmaceutically acceptable excipient.
[0090] The pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant, or a coloring agent. The amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science.
[0091] In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e. , plastically deformable) or abrasively brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate. [0092] In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.
[0093] In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non-limiting example, the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.
[0094] In still another embodiment, the excipient may be a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).
[0095] In various embodiments, the excipient may be a pH modifier. By way of non-limiting example, the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.
[0096] In a further embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non- effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.
[0097] In yet another embodiment, the excipient may be a dispersant or dispersing enhancing agent. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.
[0098] In another alternate embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.
[0099] In a further embodiment, the excipient may be a lubricant. Non limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate or stearic acid.
[00100] In still a further embodiment, the excipient may be a coloring agent. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).
[00101 ] The weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1 % or less of the total weight of the composition.
[00102] The composition can be formulated into various dosage forms and administered topically by a number of different means that will deliver a therapeutically effective amount of the active ingredients. Such compositions administered topically in dosage unit formulations may contain conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. Formulation of drugs is discussed in, for example, Gennaro, A.
R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980).
[00103] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules. In such solid dosage forms, the active ingredient is ordinarily combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[00104] For parenteral administration (including subcutaneous, intradermal, intravenous, intramuscular, intra-articular and intraperitoneal), the preparation may be an aqueous or an oil-based solution. Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as etheylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil.
[00105] The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets. [00106] For topical (e.g., transdermal or transmucosal) administration, penetrants appropriate to the barrier to be permeated are generally included in the preparation. Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In some embodiments, the pharmaceutical composition is applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Transmucosal administration may be accomplished through the use of nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal administration may be via ointments, salves, gels, patches, or creams as generally known in the art.
[00107] In certain embodiments, a composition comprising a non nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells (e.g., HIV-infected cells), to increase the stability of the composition, or to minimize potential toxicity of the composition. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering a composition of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers and other phospholipid-containing systems. Methods of incorporating compositions into delivery vehicles are known in the art.
[00108] In one alternative embodiment, a liposome delivery vehicle may be utilized. Liposomes, depending upon the embodiment, are suitable for delivery of a non nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor in view of their structural and chemical properties. Generally speaking, liposomes are spherical vesicles with a phospholipid bilayer membrane. The lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells. In this manner, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor may be selectively delivered to a cell by encapsulation in a liposome that fuses with the targeted cell’s membrane.
[00109] Liposomes may be comprised of a variety of different types of phosolipids having varying hydrocarbon chain lengths. Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated. Suitable fatty acid chains include (common name presented in parentheses) n-dodecanoate (laurate), n- tretradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9, 12- octadecandienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linolenate), and all cis-5,8,11 ,14-eicosatetraenoate (arachidonate). The two fatty acid chains of a phospholipid may be identical or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenyl PS, and the like.
[00110] The phospholipids may come from any natural source, and, as such, may comprise a mixture of phospholipids. For example, egg yolk is rich in PC,
PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brain or spinal cord is enriched in PS. Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used. Mixtures of different phospholipids may result in liposome compositions having advantageous activity or stability of activity properties. The above mentioned phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(1-(2,3-dioleolyoxy)propyl)-N,N,N- trimethyl ammonium chloride, 1 ,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchloarate, 3,3’-deheptyloxacarbocyanine iodide, 1 ,1’-dedodecyl-3,3,3’,3’- tetramethylindocarbocyanine perchloarate, 1 ,1’-dioleyl-3,3,3’,3’-tetramethylindo carbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1 ,1 ,-dilinoleyl-3,3,3’,3’-tetramethylindocarbocyanine perchloarate.
[00111 ] Liposomes may optionally comprise sphingolipids, in which sphingosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes. Liposomes may optionally contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.
[00112] Liposomes may further comprise a suitable solvent. The solvent may be an organic solvent or an inorganic solvent. Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.
[00113] Liposomes carrying a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in U.S. Pat. Nos. 4,241,046, 4,394,448, 4,529,561 , 4,755,388, 4,828,837, 4,925,661,
4,954,345, 4,957,735, 5,043,164, 5,064,655, 5,077,211 and 5,264,618, the disclosures of which are hereby incorporated by reference in their entirety. For example, liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing. In a preferred embodiment the liposomes are formed by sonication. The liposomes may be multilamellar, which have many layers like an onion, or unilamellar. The liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar lipsomes.
[00114] As would be apparent to one of ordinary skill, all of the parameters that govern liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of methionine compound, concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.
[00115] In another embodiment, a composition of the disclosure may be delivered to a cell as a microemulsion. Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and “oil." The "oil" in this case, is the supercritical fluid phase. The surfactant rests at the oil- water interface. Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art. The aqueous microdomains suitable for use in the disclosure generally will have characteristic structural dimensions from about 5 nm to about 100 nm. Aggregates of this size are poor scatterers of visible light and hence, these solutions are optically clear. As will be appreciated by a skilled artisan, microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates. In one embodiment, the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil. In an alternative embodiment, the microemulsion structure is the lamellae. It comprises consecutive layers of water and oil separated by layers of surfactant. The “oil” of microemulsions optimally comprises phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions. The non nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor may be encapsulated in a microemulsion by any method generally known in the art.
[00116] In yet another embodiment, a non-nucleoside reverse transcriptase inhibitor (NNRTI) and a dipeptidyl peptidase 9 (DPP9) inhibitor may be delivered in a dendritic macromolecule, or a dendrimer. Generally speaking, a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe. Generally, the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer. Alternatively, phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments. Any method generally known in the art may be utilized to make dendrimers and to encapsulate compositions of the disclosure therein. For example, dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape. Furthermore, the final size of a dendrimer is typically controlled by the number of iterative steps used during synthesis. A variety of dendrimer sizes are suitable for use in the disclosure. Generally, the size of dendrimers may range from about 1 nm to about 100 nm.
[00117] A composition according to the disclosure may comprise one or more active agents in addition to the non-nucleoside reverse transcriptase inhibitor (NNRTI) and the dipeptidyl peptidase 9 (DPP9) inhibitor. Anti-retroviral therapeutics consist of several class of drugs which may be used in combination with each other and with the non-nucleoside reverse transcriptase inhibitor (NNRTI) and the dipeptidyl peptidase 9 (DPP9) inhibitor as described herein. Use of these drugs in combination can be termed anti-retroviral therapy (ART), combination anti-retroviral therapy (cART) or highly active anti-retroviral therapy (FIAART). Anti-retroviral therapeutics are broadly classified by the phase of the retrovirus life-cycle that the drug inhibits.
[00118] Entry inhibitors (or fusion inhibitors) interfere with binding, fusion and entry of H IV-1 to the host cell by blocking one of several targets. Maraviroc and enfuvirtide are the two currently available agents in this class. Maraviroc works by targeting CCR5, a co-receptor located on human helper T-cells. Caution should be used when administering this drug however due to a possible shift in tropism which allows FI IV to target an alternative co-receptor such as CXCR4. In rare cases, individuals may have a mutation in the CCR5 delta gene which results in a non-functional CCR5 co- receptor and in turn, a means of resistance or slow progression of the dis-ease.
However as mentioned previously, this can be overcome if an HIV variant that targets CXCR4 becomes dominant. To prevent fusion of the virus with the host membrane, enfuvirtide can be used. Enfuvirtide is a peptide drug that must be injected and acts by interacting with the N-terminal heptad repeat of gp41 of HIV to form an inactive hetero six-helix bundle, there-fore preventing infection of host cells.
[00119] Nucleoside reverse transcriptase inhibitors (NRTI) and nucleotide reverse transcriptase inhibitors (NtRTI) are nucleoside and nucleotide analogues which inhibit re-verse transcription. HIV is an RNA virus and hence unable to become integrated into the DNA in the nucleus of the hu-man cell; it must be “reverse” transcribed into DNA. Since the conversion of RNA to DNA is not done in the mammalian cell it is performed by a viral protein which makes it a selective target for inhibition. NRTIs are chain terminators such that once incorporated, work by preventing other nucleosides from also being incorporated into the DNA chain because of the absence of a 3’ OH group. Both act as competitive substrate inhibitors. Examples of currently used NRTIs include zidovudine, abacavir, lamivudine, emtricitabine, and tenofovir.
[00120] Integrase inhibitors (also known as integrase nuclear strand transfer inhibitors or INSTIs) inhibit the viral enzyme integrase, which is responsible for integration of viral DNA into the DNA of the infected cell. There are several integrase inhibitors currently under clinical trial, and raltegravir became the first to receive FDA approval in October 2007. Raltegravir has two metal binding groups that compete for substrate with two Mg2+ ions at the metal binding site of integrase. Recently, two other clinically approved integrase inhibitors are elvitegravir and dolutegravir.
[00121] Protease inhibitors block the viral protease enzyme necessary to produce mature virions upon budding from the host membrane. Particularly, these drugs prevent the cleavage of gag and gag/pol precursor proteins. Virus particles produced in the presence of protease inhibitors are defective and mostly non-infectious. Examples of HIV protease inhibitors are Lopinavir, Indinavir, Nelfinavir, Amprenavir and Ritonavir. Darunavir and atazanavir are currently recommended as first line therapy choices. Maturation inhibitors have a similar effect by binding to gag, but development of two experimental drugs in this class, Bevirimat and Vivecon, was halt-ed in 2010.
Resistance to some protease inhibitors is high. Second generation drugs have been developed that are effective against otherwise resistant HIV variants
[00122] Combinations of antiretrovirals create multiple obstacles to HIV replication to keep the number of offspring low and reduce the possibility of a superior mutation. If a mutation that conveys resistance to one of the drugs being taken arises, the other drugs continue to suppress reproduction of that mutation. With rare exceptions, no individual antiretroviral drug has been demonstrated to suppress an HIV infection for long; these agents must be taken in combinations in order to have a lasting effect. As a result, the standard of care is to use combinations of antiretroviral drugs. Combinations usually comprise of three drugs from at least two different classes. This three drug combination is commonly known as a triple cocktail. Combinations of antiretrovirals are subject to positive and negative synergies, which limits the number of useful combinations. Additionally, there are now several options that combine three drugs into one pill taken once daily.
Brand Name Drug Combination
Combivir zidovudine + lamivudine
Kaletra lopinavir + ritonavir Trizivir abacavir + zidovudine + lamivudine
Epzicom /Kivexa abacavir/lamivudine
Truvada tenofovir/emtricitabine
Atripla efavirenz + tenofovir/emtricitabine
Complera rilpivirine + tenofovir/emtricitabine
Stribild elvitegravir + cobicistat + tenofovir/emtricitabine
Triumeq dolutegravir + abacavir/lamivudine
[00123] The quantity of a pharmaceutical composition necessary to deliver a therapeutically effective dose can be determined by routine in vitro and in vivo methods, common in the art of drug testing. See, for example, D. B. Budman, A. H. Calvert, E. K. Rowinsky (editors). Handbook of Anticancer Drug Development, LWW, 2003. Therapeutically effective dosages for various therapeutic entities are well known to those of skill in the art.
[00124] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LDso (the dose lethal to 50% of the population) and the EDso, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.
[00125] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Bio-pharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.
[00126] Administration of a composition of the disclosure can occur as a single event or over a time course of treatment. For example, one or more of a nanoparticle composition can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
[00127] Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for an FI IV infection. A compositions of the disclosure can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent. For example, compositions as disclosed herein can be administered simultaneously with another agent, such as an antiviral, an antibiotic, or an anti inflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more active agents. Simultaneous administration can occur through administration of one composition containing two or more active agents. A composition as disclosed herein can be administered sequentially with an antiviral, an antibiotic, an anti-inflammatory, or another agent. For example, a composition of the disclosure can be administered before or after administration of an antiviral, an antibiotic, an anti-inflammatory, or another agent.
[00128] The present disclosure encompasses pharmaceutical compositions comprising compounds as disclosed above, so as to facilitate administration and promote stability of the active agent. For example, a compound of this disclosure may be admixed with at least one pharmaceutically acceptable carrier or excipient resulting in a pharmaceutical composition which is capably and effectively administered (given) to a living subject, such as to a suitable subject (i.e. “a subject in need of treatment” or “a subject in need thereof”). For the purposes of the aspects and embodiments of the disclosure, the subject may be a human or any other animal.
III. METHODS
[00129] The present disclosure is based, at least in part, on the discovery that the induction of CARD8 inflammasome can clear residual HIV-1 in patients. Applicant discovered the CARD8 inflammasome senses HIV-1 protease activity. HIV-1 protease remains inactive in infected cells to avoid CARD8 recognition. As shown herein, some clinically approved anti-HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTI) such as rilpivirine (RPV) and efavirenz (EFV) unexpectedly induce premature HIV-1 protease activation, which triggers CARD8 inflammasome activation and rapid killing of virus-infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation.
[00130] In HIV-1 patients who received antiretroviral therapy (ART), HIV-1 persists in a latent form primarily in quiescent CD4+ T cells and tissue macrophages. Thus, antiretroviral therapy (ART) does not cure HIV-1 infection and lifelong ART is required. Killing the latent viral reservoirs is required to achieve HIV-1 eradication.
[00131] Although many immunotherapeutic strategies for HIV-1 reservoir elimination have been tested in human clinical trials, immune escape HIV-1 variants achieved in the latent reservoirs present one of the major obstacles to HIV-1 eradication. The disclosed study identifies a novel and effective strategy for clearance of HIV-1 reservoirs, because the CARD8 inflammasome recognizes the immutable viral protease function.
[00132] Described herein are the mechanisms by which some anti-HIV drugs such as EFV and RPV have a “side job” or secondary activity to kill HIV-infected cells, an important step to curing HIV infection. These drugs are already used in clinics to treat HIV infection. Surprisingly, Applicant found inhibition of DPP9 works synergistically with NNRTIs to enhance the cell killing effect on HIV-infected cells. Thus, the present disclosure provides methods of using the combination of NNRTIs and DPP9 inhibitor for an HIV cure. For example, in HIV-1 -infected cells, a class of anti-HIV drugs such as EFV, ETR, and RPV activates HIV protease, which in turn activates CARD8 to kill the infected cells. Combining RPV with talabostat mesylate enhanced the killing effect (see e.g., Example 2). Talabostat mesylateis is not in the same drug class as EFV and RPV.
[00133] Surprisingly, some NNRTIs which have been used to treat HIV-1 infection for more than two decades can facilitate CARD8 sensing by mediating premature intracellular activation of HIV-1 protease. NNRTIs bind to HIV-1 RT and act as enhancers of Gag-Pol dimerization to activate Pol-embedded viral protease. Although NNRTI-containing treatment regimens do not eliminate HIV-1 infection in patients because the viral latent reservoirs are rapidly established prior to treatment initiation, inclusion of NNRTIs without protease inhibitors in the initial ARV regimen may partially reduce the seeding of latent viral reservoirs. In addition, inclusion of NNRTIs in HIV-1 cure strategies should facilitate the elimination of infected cells after viral latency reversal. Intriguingly, CARD8 is preferentially and highly expressed in blood and lymphoid tissues as well as in many hematopoietic-derived cells, suggesting that targeting the CARD8 inflammasome may be effective in lymphoid tissues, the most important anatomical sites for persistent HIV-1 infection. Notably, the cell-killing IC50 of EFV and RPV is approximately 1-2 mM, which is about 100-fold higher than the infection-blocking IC50. The plasma EFV concentration in patients receiving EFV- containing regimens (1-4 pg/ml or 3-12 pM) is within the therapeutic range for cell killing. This strategy may not be effective in tissues with markedly lower drug concentration such as central nervous system. Importantly, HIV-1 Pol that confer resistance to NNRTIs also abrogates NNRTI-triggered cell killing likely because the resistant viral variants can avoid drug binding. Thus, the identification of more potent chemical compounds that promote Gag-Pol dimerization regardless of viral inhibition is warranted. Taken together, this work reveals a mechanism of innate sensing of HIV-1 infection that has immediate implications for HIV-1 cure strategies.
[00134] The present disclosure encompasses methods to treat, prevent, or reduce the infectivity of a HIV in a subject in need thereof, the method generally comprises administering to the subject a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor. In another embodiment, the present disclosure provides methods of reducing the amount of HIV-infected cells, the methods generally comprises contacting HIV-infected cells with or administering to a subject having HIV-infected cells, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor. In still another embodiment, the present disclosure provides methods of inducing pyroptosis in HIV-infected cells, the methods generally comprise contacting HIV-infected cells with or administering to a subject having HIV- infected cells, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor. In yet another embodiment, the present disclosure provides methods of sensitizing the CARD8 inflammasome to NNRTI-induced activation, the methods generally comprise contacting HIV-infected cells with or administering to a subject having HIV-infected cells, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor. In still yet another embodiment, the present disclosure provides methods of treating a subject with NNRTI-resistance, the methods generally comprise administering to the subject a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor. In still another embodiment, the present disclosure provides methods of reducing the amount of HIV reservoirs in a subject, the methods generally comprise administering to the subject, a composition comprising a therapeutically effective amount of a NNRTI and a DPP9 inhibitor. Suitable compositions for use within the above methods are described herein for instance those described in Section II which is incorporated by reference into this section in its entirety.
[00135] As disclosed herein, the compositions of the disclosure are useful to selectively kill HIV-infected cells. By selectively killing one or more HIV-infected cells is meant a composition of the disclosure does not appreciably kill non-HIV-infected cells at the same concentration. Accordingly, the median lethal dose or LDso of the composition in non-HIV-infected cells may be about 2 to about 50 times higher than the LDso of the composition in HIV-infected cells. As used herein, the LD50 is the concentration of composition required to kill half the cells in the cell sample. For example, the LD50 of the composition in non-HIV-infected cells may be greater than about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9 or about 10 times higher than the LD50 of the composition in HIV-infected cells. Alternatively, the LD50 of the composition in non-HIV-infected cells may be greater than about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 times higher than the LD50 of the composition in HIV-infected cells. Additionally, the LD50 of the composition in non-HIV-infected cells may be greater than 50 times higher than the LD50 of the composition in HIV-infected cells. In certain embodiments, the LD50 of the composition in non-HIV-infected cells is about 2 to about 10 times higher than the LD50 of the composition in HIV-infected cells. In an exemplary embodiment, the LD50 of the composition in non-HIV-infected cells is about 3 to about 6 times higher than the LD50 of the composition in HIV-infected cells.
[00136] Method to detect HIV-infected cells are known in the art, for example, a human immunodeficiency virus (HIV) test detects HIV antigens, or the genetic material (DNA or RNA) of HIV in a biological sample (e.g., blood or another type of sample). This can show if an HIV infection is present (HIV-positive). Suitable detection methods include enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) and indirect fluorescent antibody (IFA). In some embodiments, the HIV-infected cell is a CD4+ T cell.
[00137] In certain embodiments, one or more HIV-infected cells are detected in a sample. A sample may be a cell sample, blood sample, a tissue sample, or a biopsy sample from a subject.
[00138] In an aspect, a method of the disclosure may comprise measuring cel! death of H!V-infected cells. Methods of measuring cel! death are known in the art. For example, cell death may be measured by Giemsa staining, trypan blue exclusion, acridine orange/ethidium bromide (AO/EB) double staining for fluorescence microscopy and flow cytometry, propidium iodide (PI) staining, annexin V assay, TUNEL assay,
DNA ladder, LDH activity, and MΪT assay. In a preferred embodiment, cell death is due to induction of pyroptosis. Pyroptosis is a highly inflammatory form of lytic programmed cell death that occurs most frequently upon infection with intracellular pathogens and is likely to form part of the antimicrobial response. Pyroptosis is initiated by formation of a large supramolecular complex termed the inflammasome (also known as a pyroptosome) upon intracellular danger signals. The inflammasome activates a different set of caspases as compared to apoptosis, for example, caspase-1/4/5 in humans and caspase-11 in mice. These caspases contribute to the maturation and activation of several proinf!ammatory cytokines and pore-forming protein gasdermins. Formation of pores causes ceil membrane rupture and release of cytokines, as well as various damage-associated molecular pattern (DAMP) molecules such as HMGB-1 , ATP and DNA, out of the cell. These molecules recruit more immune cells and further perpetuate the inflammatory cascade in the tissue. Biochemical methods include DNA laddering, lactate dehydrogenase enzyme release, and MTT/XTT enzyme activity. Additionally, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling of DNA fragments (TUNEL) and in situ end labeling (ISEL) techniques are used, which when used in conjunction with standard flow cytometric staining methods yield informative data relating cell death to various cellular parameters, including cell cycle and cell phenotype. See Loo and Rillema, Methods Cell Biol. 1998;57:251-64, which is incorporated herein by reference, for a review of these methods. In an embodiment, cell death due to pyroptosis may be measured by the induction of caspase-1.
(a) administration
[00139] In certain aspects, a therapeutically effective amount of a composition of the disclosure may be administered to a subject. Administration is performed using standard effective techniques, including peripherally (i.e. not by administration into the central nervous system) or locally to the central nervous system. Peripheral administration includes but is not limited to intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. Local administration, including directly into the central nervous system (CNS) includes but is not limited to via a lumbar, intraventricular or intraparenchymal catheter or using a surgically implanted controlled release formulation.
[00140] Pharmaceutical compositions for effective administration are deliberately designed to be appropriate for the selected mode of administration, and pharmaceutically acceptable excipients such as compatible dispersing agents, buffers, surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and the like are used as appropriate. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton Pa., 16Ed ISBN: 0-912734-04-3, latest edition, incorporated herein by reference in its entirety, provides a compendium of formulation techniques as are generally known to practitioners. [00141] For therapeutic applications, a therapeutically effective amount of a composition of the disclosure is administered to a subject. A “therapeutically effective amount” is an amount of the therapeutic composition sufficient to produce a measurable response (e.g., cell death of HIV-infected cells, an improvement in symptoms associated with HIV). Actual dosage levels of active ingredients in a therapeutic composition of the disclosure can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject. The selected dosage level will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, HIV-related disease or condition, HIV infection, the symptoms, and the physical condition and prior medical history of the subject being treated. In some embodiments, a minimal dose is administered, and dose is escalated in the absence of dose-limiting toxicity. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art of medicine.
[00142] The frequency of dosing may be daily or once, twice, three times or more per week or per month, as needed as to effectively treat the symptoms. The timing of ad-ministration of the treatment relative to the disease itself and duration of treatment will be determined by the circumstances surrounding the case. Treatment could begin immediately, such as at the site of the injury as administered by emergency medical personnel. Treatment could begin in a hospital or clinic itself, or at a later time after discharge from the hospital or after being seen in an outpatient clinic. Duration of treatment could range from a single dose administered on a one-time basis to a life-long course of therapeutic treatments.
[00143] Typical dosage levels can be determined and optimized using standard clinical techniques and will be dependent on the mode of administration.
(b) subject
[00144] A subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject may be a livestock animal. Non-limiting examples of suitable livestock animals may include pigs, cows, horses, goats, sheep, llamas and alpacas. In still another embodiment, the subject may be a companion animal. Non-limiting examples of companion animals may include pets such as dogs, cats, rabbits, and birds. In yet another embodiment, the subject may be a zoological animal. As used herein, a “zoological animal” refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a preferred embodiment, the subject is a human.
[0100] Still further, the subject may be a subject at risk of developing a HIV infection. A skilled artisan would be able to determine a subject at risk of developing a HIV infection. For example, a subject may be determined to be at risk of developing a HIV infection based on patient history, clinical presentation, a diagnostic exams, and/or HIV exposure.
(c) screening assays
[0101] The disclosure provides a method (also referred to herein as a "screening assay") for identifying inhibitors, i.e. , candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which downregulate DPP9, for example, DPP9 nucleic acid expression, DPP9 protein expression or DPP9 activity. A DPP9 inhibitor may directly or indirectly downregulate DPP9.
[0102] Screening assays may also be used to identify molecules that modulate DPP9 nucleic acid expression, DPP9 protein expression or DPP9 activity. For example, DPP9 inhibits the activation of the CARD8 inflammasome to prevent pyroptosis. Accordingly, pyroptosis, cell viability, or caspase-1 may be measured as an indication of DPP9 activity or expression. Pyroptosis and cell viability may be measured using methods standard in the art as described above. Caspase-1 may be measured using methods to detect protein expression as described herein. Alternatively, DPP9 nucleic acid expression or protein expression may be measured as an indication of DPP9 activity or expression. Methods to detect DPP9 nucleic acid or protein expression are standard in the art. For instance, as described herein. [0103] In one embodiment, the disclosure provides assays for screening candidate or test compounds which bind to or modulate the activity or expression of DPP9. The test compounds of the present disclosure can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233.
[0104] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991) Nature 354:82- 84), chips (Fodor (1993) Nature 364:555-556), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386_,390; Devlin (1990) Science 249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).
[0105] In one embodiment, an assay is one in which cells are contacted with a test compound and the ability of the test compound to downregulate DPP9 is determined. Determining the ability of the test compound to downregulate DPP9 may be accomplished, for example, by detecting DPP9 protein expression. Numerous methods for detecting protein are known in the art and are contemplated according to the disclosure, see Section II. Specifically, an immunoblot may be used to detect DPP9 protein. Alternatively, determining the ability of the test compound to downregulate DPP9 may be accomplished, for example, by measuring cell death or pyroptosis. Methods of measuring cell death or pyroptosis are known in the art, see the below examples. Another method for determining the ability of the test compound to downregulate DPP9 may be accomplished by a reporter assay for DPP9 expression. For example, DPP9 protein expression may be fused to a reporter protein such that DPP9 expression may be monitored by measuring the expression of the reporter protein. By way of example, reporter proteins may include a fluorescent protein, luciferase, alkaline phosphatase, beta-galactosidase, beta-lactamase, horseradish peroxidase, and variants thereof. In another embodiment, determining the ability of the test compound to downregulate DPP9 may be accomplished, for example, by detecting DPP9 nucleic acid expression. Methods of measuring nucleic acid expression are known in the art, see Section II. Specifically, DPP9 mRNA may be detected via standard methods. Determining the ability of the test compound to down-regulate DPP9 may be accomplished, for example, by determining the ability of DPP9 to inhibit the activation of caspase 1. Methods for detecting caspase 1 are known in the art. For example, methods to detect protein expression may be used to detect caspase 1.
[0106] In another embodiment, an assay is one in which DPP9 is contacted with a test compound and the ability of the test compound to bind to DPP9 is determined. Determining the ability of the test compound to bind to DPP9 may be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to DPP9 may be determined by detecting the labeled compound in a complex. For example, test compounds can be labeled with 125l, 35S, 14C, or 3H , either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
[0107] In yet another embodiment, an assay of the present disclosure is a cell-free assay comprising contacting DPP9 with a test compound and determining the ability of the test compound to bind to DPP9. Binding of the test compound to DPP9 may be determined either directly or indirectly. In one embodiment, a competitive binding assay includes contacting DPP9 with a compound known to bind DPP9 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with DPP9, wherein determining the ability of the test compound to interact with DPP9 comprises determining the ability of the test compound to preferentially bind to DPP9 as compared to the known binding compound.
[0108] In another embodiment, an assay is a cell-free assay comprising contacting DPP9 with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of DPP9. Determining the ability of the test compound to modulate the activity of DPP9 can be accomplished, for example, by determining the ability of DPP9 to bind to or interact with a DPP9 target molecule (e.g., CARD8). In an alternative embodiment, determining the ability of the test compound to modulate the activity of DPP9 can be accomplished by determining the ability of DPP9 to further modulate a DPP9 target molecule. As used herein, a "target molecule" is a molecule with which DPP9 binds or interacts in nature.
[0109] In another embodiment, modulators of DPP9 expression are identified in a method in which a cell is contacted with a candidate compound and the expression of the DPP9 promoter, mRNA or protein in the cell is determined. The level of expression of DPP9 mRNA or protein in the presence of the candidate compound is compared to the level of expression of DPP9 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of DPP9 expression based on this comparison. For example, when expression of DPP9 mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of DPP9 mRNA or protein expression. Alternatively, when expression of DPP9 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of DPP9 mRNA or protein expression. The level of DPP9 mRNA or protein expression in the cells can be deter-mined by methods described herein for detecting DPP9 mRNA or protein. The activity of the DPP9 promoter can be assayed by linking the DPP9 promoter to a reporter gene such as luciferase, secreted alkaline phosphatase, or beta-galactosidase and introducing the resulting construct into an appropriate vector, transfecting a host cell line, and measuring the activity of the reporter gene in response to test compounds.
[0110] This disclosure further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein. IV. KITS
[0111] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to a non-nucleoside reverse transcriptase (NNRT) inhibiting agent or a DPP9 inhibiting agent or assays to screen for such agents. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
[0112] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal, or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.
[0113] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or another substrate, and/or may be supplied as an electronic-readable medium or video. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
EXAMPLES
[0114] The following examples are included to demonstrate preferred embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Example 1. CARD8 is an inflammasome sensor for HIV-1 protease activity
[0115] HIV-1 has high mutation rates and exists as mutant swarms within the host. Rapid evolution of HIV-1 allows the virus to outpace the host immune system, leading to viral persistence. Approaches to target immutable components are needed to clear HIV-1 infection. Here, the present example provides the CARD8 inflammasome senses HIV-1 protease activity. HIV-1 can evade CARD8 sensing because its protease remains inactive in infected cells prior to viral budding. Premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1 - infected cells. This strategy led to the clearance of latent HIV-1 in patient CD4+ T cells after viral reactivation. Thus, the present example identifies CARD8 as an inflammasome sensor of HIV-1 , which holds promise as a strategy for clearance of persistent HIV-1 infection. [0116] The adaptive immune system recognizes cognate epitopes based on amino acid sequences and associated conformations that are subject to mutation. Retroviruses such as human immunodeficiency virus type 1 (HIV-1) accumulate mutations at high rates due to the lack of proofreading activity of their reverse transcriptase coupled with high levels of virus replication in vivo. The within-host diversity of HIV-1 allows rapid selection of antibody and T cell escape variants. In patients who received antiretroviral therapy (ART), HIV-1 persists in a latent form primarily in quiescent CD4+ T cells and possibly tissue macrophages. Immune escape variants achieved in the latent viral reservoirs present one of the major obstacles to HIV-1 eradication. In addition, host cells persistently infected with HIV-1 are long-lived and resistant to virus- and immune-mediated apoptotic cell death. In the present example, it was evaluated whether the immune system could (1 ) sense the functions of an essential HIV-1 protein including those with viral enzymatic activities, which are highly immutable and (2) mediate robust target cell killing independent of apoptosis. The host immune system utilizes germ line-encoded pattern-recognition receptors (PRRs) to detect microbial products. A set of intracellular PRRs characterized by the presence of a caspase recruitment domain (CARD) or a pyrin (PYD) domain co-oligomerize with pro- caspase-1 and form high-molecular weight inflammasome complexes upon sensing of their cognate ligands. Inflammasome assembly in response to cytoplasmic microbial or danger signals leads to caspase-1 (CASP1) activation and pyroptosis, an inflammatory form of programmed cell death. Murine nucleotide-binding domain leucine-rich repeat pyrin domain-containing 1b (NLRPIb) contains the FUND and CARD domains. NLRPIb can be activated via direct proteolysis of its N-terminus by Bacillus anthracis lethal factor protease (LF). Briefly, N-terminal cleavage of murine NLRPIb creates a neo-N- terminus, which is ubiquitylated by the N-end-rule pathway and targeted for proteasome degradation. Due to the break in the polypeptide chain by FUND auto-processing, the C- terminal bioactive subunit is liberated from the proteasome complex and can initiate CASP1 -dependent inflammasome assembly. Recently, two studies on human NLRP1 inflammasome reported that it senses viral infection in epithelial barrier tissues and its activation is also proteasome-dependent. There is no evidence that HIV-1 infection can be sensed by innate sensors associated with inflammasome activation in CD4+ T cells, with the exception of the bystander effect triggered by abortive HIV-1 transcripts. The bystander cell death mechanism only applies to quiescent, lymphoid-resident CD4+ T cells that are non-perm issive to HIV-1. It does not apply to the cells productively or latently infected with HIV-1, and thus has no impact on persistently infected cells.
[0117] The CARD8 inflammasome senses HIV-1 protease activity: The caspase recruitment domain-containing protein 8 (CARD8) has been implicated in inflammasome activation and pyroptosis of CD4+ T cells and macrophages. A key question is whether CARD8 is an inflammasome sensor, and if so, which pathogens physiologically activate it. The C-terminus of CARD8 protein contains a “function-to-find” domain (FUND) followed by a CARD domain. Human CARD8 shares structural similarity with murine NLRPIb and also undergoes autoprocessing, as evidenced by the detection of both the full length and the autoprocessed N- and C-terminal fragments by immunoblotting (FIG. 1A). To determine whether CARD8 is a sensor for HIV-1 protease, HEK293T cells were co-transfected with an HIV-1 reporter vector (pNL4-3- GFP) and human CARD8 vector with an N-terminal HA tag (HA-CARD8). The N- terminal tag added 43 amino acids to CARD8 protein. HIV-1 protease activation requires dimerization of the Gag-Pol polyprotein, which occurs during or soon after the viral budding process. Overexpression of HIV-1 Gag-Pol polyprotein in transfected cells resulted in its intracellular dimerization and premature protease activation. When the HIV-1 vector was co-transfected with HA-CARD8, two smaller N-terminal fragments were detected by an HA antibody, and one of them was also detected by a CARD8 N- terminus antibody (FIG. 1B). In addition, two neo-C-terminal fragments were detected by a CARD8 C-terminus antibody. These results suggested that the HA-tagged N- terminus was cleaved by HIV-1 protease. The first cleavage site is in the unstructured N-terminus, and the second site is likely in the ZU5 domain. By contrast, CARD8 was not cleaved by the viral vector when the viral protease was inactivated by a single mutation (D25H), as evidenced by the lack of HIV-1 Gag (p55) cleavage. To evaluate whether other viral proteins were required for CARD8 cleavage, various mutations were introduced to the viral vector. Cleavage of CARD8 was not affected when mutations were introduced to any viral genes except pol but was blocked by an HIV-1 protease inhibitor lopinavir (LPV). To test whether N-terminal cleavage of CARD8 by HIV-1 protease was sufficient to assemble a functional inflammasome complex, CASP1- and pro-ILip-expressing plasmids were co-transfected together with pNL4-3-GFP into HEK293T cells. Although the level of endogenous expression of CARD8 in HEK293T cells was not sufficient for the detection of N-terminal cleavage, it was sufficient to trigger the downstream signaling cascade upon HIV-1 protease cleavage, as evidenced by the processing of pro-IL-1 b into mature IL-1 b (FIG. 1C). Some HIV-1 -specific non nucleoside reverse transcriptase inhibitors (NNRTI) such as rilpivirine (RPV) and efavirenz (EFV), but not nevirapine (NVP), can bind to HIV-1 Pol and enhance intracellular Gag-Pol polyprotein dimerization, which causes premature protease activation. As expected, IL-1 b processing, as well as HIV-1 Gag processing, was enhanced by RPV but blocked by LPV (FIG. 1C). By contrast, processing of pro-IL-1 b was blocked in CARD8- KO HEK293T cells (FIG. 1C). The autoproteolytic-deficient HA- CARD8S297A was not able to rescue inflammasome activation in CARD8- KO cells (FIG. 1D), suggesting that proteasome degradation of the N-terminus was required to release the bioactive UPA-CARD fragment. Thus, proteasome inhibitors MG132 or bortezomib were added together with RPV into transfected cells. The baseline level of IL-1 b-r17 was not affected by proteasome inhibitors because the inhibitors were added 24 hours post transfection. RPV-induced processing of pro-IL-1 b but not HIV-1 Gag cleavage was blocked by bortezomib and MG132 (FIG. 1E), which excluded the possibility that MG132 and bortezomib directly affected HIV-1 protease activity.
[0118] HIV-1 protease cleaves the N-terminus of CARD8: To examine whether CARD8 was directly cleaved by HIV-1 protease, HA-CARD8S297A was immunoprecipitated and incubated with purified and lysed HIV-1 particles. When the viral protease was functional, two additional bands were visualized using a CARD8 C- terminus antibody (FIG. 1F), which confirmed the two cleavage sites recognized by the viral protease. RPV did not further enhance CARD8 cleavage because the lysed HIV-1 particles already contained mature viral protease. To determine whether one or both cleavage sites were needed to activate CARD8, a truncated CARD8A21·70 was first generated which did not contain the first cleavage site. CARD8A21·70 was cleaved at the second site (FIG. 2A), but was unable to trigger inflammasome activation (FIG. 2B). To identify the first cleavage site, a set of truncated CARD8 proteins were tested and found that CARD8A51 60 was not cleaved (FIG. 2C). Next, it was found that F59 and F60 were likely to be at the P1 and PT positions, respectively (FIG. 2D). The H IV-1 protease prefers to have large hydrophobic amino acids flanking the scissile bond. Phenylalanine is the most common residue at the P1 position, and its presence at both P1 and PT positions improved the cleavage rate of FI IV-1 protease. It was further demonstrated that the first cleavage site was required for the CARD8 inflammasome activation by FI IV-1 protease (FIG. 2E). Thus, FI IV-1 protease can cleave the N-terminus of CARD8 and that activation of the viral protease can lead to CARD8 inflammasome activation in the FIEK293T transfection system.
[0119] HI V- 1 triggers CARD8-depedent pyroptosis of infected macrophages: Since CARD8 is expressed in FI IV-1 target cells, including primary CD4+ T cells and macrophages, the next question was whether induction of premature intracellular activation of FI IV-1 protease could trigger CARD8-dependent pyroptosis of infected cells. When treated with EFV or RPV, FI IV-1 -infected (GFP+) macrophages rapidly underwent pyroptotic cell death as evidenced by membrane swelling and rupture (FIG. 3), as well as secretion of IL-1 b (FIG. 3E). The cell death triggered by EFV or RPV was rapid and dose-dependent and completely blocked by LPV (FIG. 3). CASP1 activation was evidenced by detection of its active subunits p10 and p20 (FIG. 3F). In addition, the CASP1 -specific inhibitor VX765 and pan-caspase inhibitor Z-VAD-FMK were tested. Although most of the infected macrophages were killed by RPV, blocking of CASP1 activity reduced the loss of infected cells to 20% (FIG. 3G). The N-end-rule pathway mediates proteasome degradation of mouse NLRPI b. To test whether it was required for CARD8 activation, MG132 and bortezomib or an N-end-rule pathway inhibitor bestatin methyl ester (Me-Bs) was added together with RPV to FI IV-1 -infected macrophages. MG132, bortezomib, and Me-Bs effectively blocked RPV-mediated killing of infected macrophages and secretion of IL-1 b (FIG. 3). FI IV-1 -infected TFIP-1 cells were also susceptible to NNRTI-triggered killing (FIG. 4). As chemical inhibitors often have off-target effects, it was confirmed that the CARD8 inflammasome was responsible for HIV-1 sensing by generating bulk or single clone CARD8-, ASC-, CASP1- and A/LRP3-knockout THP-1 cells (FIG. 4C). Deletion of CARD8 or CASP1, but not ASC or NLRP3, inhibited HIV-1 protease-mediated pyroptosis and IL-1 b secretion (FIG. 4), consistent with the inhibitor experiments. This finding also confirms studies showing that CARD8 can form an ASC-independent inflammasome complex.
[0120] HIV-1 triggers CARD8-depedent pyroptosis of infected CD4+ T cells: Several studies have reported that NNRTIs can induce killing of HIV-1 -infected CD4+ T cells through an unknown mechanism. It was hypothesized that the cell killing observed in those studies was due to NNRTI-triggered HIV-1 protease activation which led to CARD8 inflammasome activation. Since resting CD4+ T cells are the most well characterized cellular reservoirs for HIV-1 , the expression levels of key components of the CARD8 inflammasome were examined in different subsets of primary CD4+ T cells. CARD8 is expressed in both activated and unstimulated blood CD4+ T cells, as well as in memory and naive CD4+ T cells in lymphoid tissues (FIG. 5A). Both activated (FIG. 5B) and unstimulated CD4+ T cells were susceptible to HIV-1 protease-triggered cell death when treated with NNRTIs including EFV, RPV, and Etravirine (ETR), but not NVP. Since several HIV-1 proteins can induce death of primary CD4+ T cells, we produced different reporter viruses carrying mutations in vif, vpr, vpu, env, and nef by transfecting HEK293T cells with different viral plasmids. Reporter viruses without a functional protease (AGag-Pol and PR-D25H) or deficient in Gag-Pol dimerization (RT- L234A and RT-W401 A) were unable to trigger cell death. None of the other viral proteins were required for cell killing (FIG. 5C). CASP1 activation was induced by EFV or RPV but blocked by LPV (FIG. 5). Both VX765 and Z-VAD-FMK blocked killing of HIV-1 -infected CD4+ T cells (FIG. 5F). Similar to infected macrophages, HIV-1 protease triggered CASP1 activation and cell death was also blocked by MG132, bortezomib, and Me-Bs. NNRTI-induced CASP1 activation and pyroptosis of HIV-1 -infected primary CD4+ T cells was abrogated when CARD8 was knocked out (FIG. 5). Similarly, a CASP1 knockout or knockdown in primary CD4+ T cells also conferred resistance to HIV-1 protease-mediated pyroptosis (FIG. 5J). In addition to the pseudotyped reporter virus, we also observed cell killing with a clinical isolate HIVBaL. Since HIVBaL is a replication-competent virus, all classes of antiretroviral drugs blocked viral replication; NNRTIs could further reduce viral infection by clearing cells already infected with HIV-1. NNRTI-mediated killing of primary CD4+ T cells infected with HIVBaL was also CARD8- dependent.
[0121] Activation of the CARD8 inflammasome clears latent HI V- 1 in patient CD4+ T cells: To determine whether HIV-1 protease function in activating the CARD8 inflammasome is conserved, a panel of HIV-1 virus isolates from chronically infected individuals of subtypes A, B, C, and D were tested. Subtype B is the dominant subtype in Europe and North America, whereas A, C, and D are more prevalent worldwide. T-20 and Raltegravir (RAL) were used to completely block new infection but had no killing effect or cellular toxicity. The addition of EFV and RPV but not NVP effectively cleared primary CD4+ T cells infected with all HIV-1 subtypes. Additionally, the killing efficiency did not correlate with viral replication fitness (FIG. 6A), suggesting that the enzymatic activity of HIV-1 protease with regard to CARD8 activation is well conserved across major HIV-1 subtypes. Next, it was confirmed that EFV and RPV treatment induced CARD8-dependent caspase-1 activation and pyroptosis of primary CD4+ T cells infected with clinical viral isolates (FIG. 6). To test whether strategies involving targeted activation of the CARD8 inflammasome could be used for the clearance of latent HIV-1 , blood CD4+ T cells from were obtained patients under suppressive ART to measure the size of viral reservoirs. The control antiretroviral (ARV) combination containing T-20, RAL, and NVP had no killing effect. The median IUPM in control and RPV groups was 2.61 and 0.16, respectively, suggesting a rapid clearance of 93.9% of the latent HIV-1 reservoirs (FIG. 6). Notably, three out of eight patient samples had no detectable viral replication after RPV treatment. In our “shock and kill” assay, cells were only treated with RPV for the first 2-3 days. It is possible that the residual viruses in the RPV group came from delayed virus reactivation which occurred after removal of RPV.
[0122] Due to rapid viral evolution, it is very difficult for the host immune system to control HIV-1 infection and clear residual viral reservoirs without targeting immutable components of the virus. This example provides that CARD8 is a sensor for HIV-1 protease activity to trigger inflammasome activation and pyroptosis of infected cells. This work demonstrates that the CARD8 and NLRP1 inflammasomes share similar mechanisms of activation, which involves their N-terminal cleavage by microbial proteases, followed by proteasome-mediated release of the bioactive C-terminal fragment to trigger inflammasome assembly and CASP1 activation. Interestingly, HIV-1 protease cleaves CARD8 at two different sites. Cleavage of the unstructured N- terminus but not the FUND domain leads to inflammasome activation. It was also observed that a deletion (D51-60) or mutations (F59A or F60A) of the first cleavage site increased the cleavage efficiency at the second site (FIG. 2C), suggesting a competition between the two sites. Cleavage of HIV-1 Gag and Gag-Pol by the viral protease is a sequential process regulated by the rate of cleavage at individual site. Since CARD8 activation requires a cleavage within the unstructured N-terminus, the cleavage site preference may influence the CARD8 inflammasome activation. In HIV-1 - infected cells, the CARD8 inflammasome cannot detect the virus because the viral protease remains inactive as a subunit of unprocessed viral Gag-Pol polyprotein. Surprisingly, some NNRTIs which have been used to treat HIV-1 infection for more than two decades can facilitate CARD8 sensing by mediating premature intracellular activation of HIV-1 protease. NNRTIs bind to HIV-1 RT and act as enhancers of Gag- Pol dimerization to activate Pol-embedded viral protease. Additional investigations are needed to better understand the mechanism of the NNRTI-mediated Gag-Pol dimerization process. Although NNRTI-containing treatment regimens cannot eliminate HIV-1 infection in patients because the viral latent reservoirs are rapidly established prior to treatment initiation, inclusion of NNRTIs without protease inhibitors in the initial ARV regimen may partially reduce the seeding of latent viral reservoirs. In addition, inclusion of NNRTIs in HIV-1 cure strategies should facilitate the elimination of infected cells after viral latency reversal. Intriguingly, CARD8 is preferentially and highly expressed in blood and lymphoid tissues as well as in many hematopoietic-derived cells, suggesting that targeting the CARD8 inflammasome may be effective in lymphoid tissues, the most important anatomical sites for persistent HIV-1 infection. Notably, the cell-killing IC50 of EFV and RPV is approximately 1-2 mM (FIG. 2C), which is about 100- fold higher than the infection-blocking IC50. The plasma EFV concentration in patients receiving EFV-containing regiments (1-4 pg/ml or 3-12 mM) is within the therapeutic range for cell killing. This strategy is unlikely to be effective in tissues with markedly lower drug concentration such as central nervous system. Importantly, H IV-1 Pol that confer resistance to NNRTIs also abrogate NNRTI-triggered cell killing likely because the resistant viral variants can avoid drug binding. Thus, the identification of more potent chemical compounds that promote Gag-Pol dimerization regardless of viral inhibition is warranted. Taken together, this work reveals a mechanism of innate sensing of H IV-1 infection that has immediate implications for H IV-1 cure strategies. Example 2. CARD8 inflammasome sensitization through DPP9 inhibition enhances NNRTI-triggered killing of HIV-1 -infected cells
[0123] Non-nucleoside reverse transcriptase inhibitors (NNRTIs) induce pyroptosis of HIV-1 infected CD4+ T cells through induction of intracellular viral protease activation, which then activates the CARD8 inflammasome. Due to high concentrations of NNRTIs being required for efficient CARD8 activation and elimination of HIV-1 - infected cells, it is important to elucidate ways to sensitize the CARD8 inflammasome to NNRTI-induced activation. The present example provides that this sensitization can be done through chemical inhibition of the CARD8 negative regulator DPP9. DPP9 inhibitor Val-boroPro (VbP) can act synergistically with NNRTIs to increase their efficacy in killing HIV-1 -infected cells. It is also shown that VbP is able to partially overcome issues with NNRTI resistance and is capable of killing infected cells without the presence of NNRTIs. This offers a promising strategy for enhancing NNRTI efficacy in elimination of HIV-1 reservoirs in patients.
[0124] Despite the enhancement of combined antiretroviral therapy (cART) that allows people living with HIV-1 (PLWH) to have an undetectable viral load, there have only been two documented cases of complete remission from HIV-1 infection. This clearly indicates the need for novel therapeutics for HIV-1 cure strategies. The primary hurdle in eradicating HIV-1 is the seeding of the latent reservoir which occurs quickly after infection primarily in activated CD4+ T cells that transition to resting memory cells and possibly in tissue macrophages. These cells can self-replenish and evade all immune responses due to HIV-1 transcriptional inactivity. However, in these latently infected cells, the integrated virus is still able to reactivate upon stimulation and spread infection. This poses a significant barrier to HIV-1 eradication as current antiretroviral therapies prevent viral replication but do not remove the latent reservoir. One of the main strategies to eliminate the HIV-1 reservoir is through the “shock and kill” approach. This strategy utilizes latency reversal agents (LRAs) to reactivate the latent reservoir (shock) and then induce targeted cell death of infected cells (kill). Optimal efficiency is needed for both steps of this strategy, but we recently reported that the inflammasome sensor caspase recruitment domain 8 (CARD8) is able to sense intracellular HIV-1 protease activity and induce targeted cell killing of HIV-1 infected cells.
[0125] The inflammasome is a multi-protein complex that is assembled upon sensing of their cognate ligand. Caspase-1 (CASP1 ) is the key effector for the inflammasome, and its active form can cleave Gasdermin D leading to pyroptosis.
There are numerous pattern-recognition receptors (PRRs) that have been shown to activate the inflammasome and are characterized by either having a caspase recruitment domain (CARD) or a Pyrin domain (PYD) which can then in turn activate CASP1 . Recent studies demonstrated that one such PRR, CARD8, triggered the CASP1 activation and pyroptosis in human CD4+ T cells when cells were treated with the known Dipeptidyl Peptidase 9 (DPP9) inhibitor Val-boroPro (VbP). More recently, CARD8 was shown to sense intracellular HIV-1 protease activity. CARD8 C-terminus (CARD8C) contains two key domains: the function-to-find domain (FUND) and a CARD domain. Full-length CARD8 undergoes autoprocessing at the FUND domain leaving two non-covalently associated subunits. HIV-1 protease was found to cleave CARD8 on the N-terminal subunit, which allows proteasomal degradation of the N-terminal fragment thereby freeing the C-terminal fragment. The C-terminal fragment, in high enough concentrations, can then activate CASP1 and induce pyroptosis. However, freed C- terminal fragment may also be sequestered by the CARD8 negative regulator DPP9 which can inhibit pyroptosis efficiency. [0126] HIV-1 protease is not typically functional intracellularly before budding and it must be activated by other methods to be properly sensed by the CARD8 inflammasome. Premature intracellular protease activation can be achieved through the usage of non-nucleoside reverse transcriptase inhibitors (NNRTIs). This strategy offers benefits over other immune-based kill strategies that often rely upon recognition of the highly variable HIV-1 epitopes due to HIV-1 protease being less tolerant to mutation. This is due to the critical need for the virus to maintain its enzymatic activities. Several reports have shown that NNRTIs, such as Efavirenz (EFV) and Rilpivirine (RPV), can induce HIV-1 protease-dependent killing of infected CD4+ T cells, which is due to activation of the CARD8 inflammasome. It is clear that NNRTIs at micromolar concentrations drive Gag-Pol dimerization and intracellular protease activation which cleaves CARD8 leading to pyroptosis of HIV-1 infected cells. Strategies for enhancement of cell killing potency of these drugs are needed for efficient clearance of HIV-1 -infected cells in vivo.
[0127] NNRTIs induce death of HIV-1 -infected cells in a dose-dependent manner : While NNRTI pharmacodynamics have been heavily studied for their ability to inhibit HIV-1 reverse transcription, they have yet to be studied in the context of their ability to activate the CARD8 inflammasome. To determine the in vitro pharmacodynamics of NNRTIs in CD4+ T cells, an HIV-1 reporter virus (pNL4-3- pol) was used to infect primary blood CD4+ T cells isolated from three independent healthy donors. Infected cells were then treated with Efavirenz (EFV), Rilpivirine (RPV), Etravirine (ETR), Doravirine (DOR), Nevirapine (NVP) in serial three-fold dilutions to assess the ECso of killing for each NNRTI (FIG. 7A). EFV, RPV, and ETR were able to induce robust cell killing at triple-digit nanomolar to low micromolar concentrations, whereas Doravirine and Nevirapine were ineffective at inducing cell death (FIG. 7B). As macrophages are also key cellular targets for HIV-1 , and were shown to have a functional CARD8 inflammasome, a similar dose-dependent relationship was also demonstrated (FIG. 7C). This relationship was due to CARD8 inflammasome activation as demonstrated by ablation of killing in CARD8-KO or CASP1-KO THP-1 cells. (FIG.
7D and FIG. 7E). [0128] The translatability of an NNRTI-based strategy for killing of HIV-1 - infected cells is met with several barriers that can potentially reduce NNRTI efficacy in vivo. One key barrier to implementation is NNRTI’s high affinity for binding human serum proteins in vivo. To assess this effect, CD4+ T cells were cultured with the presence of 50% human serum and show stark shifts in the dose response curves for both EFV and RPV (FIG. 7F and FIG. 7G). With the presence of human serum, the dose response curve for RPV is shifted out of clinical concentration recommendations whereas EFV maintains some efficacy with the presence of human serum. Additionally, EFV is less affected by the presence of human serum as RPV as evidenced by a smaller log fold change in the ECso (FIG. 7H). These data suggest that EFV offers a distinct benefit over RPV for use in NNRTI based shock and kill strategies due to its higher plasma concentration and intracellular concentration. However, the efficacy of EFV is reduced with the presence of human serum which calls for the elucidation of strategies that could either increase intracellular NNRTI concentrations or sensitize the CARD8 inflammasome to NNRTI-based killing.
[0129] DPP9 inhibition sensitizes the CARD8 Inflammasome to NNRTI- induced pyroptosis: DPP9 can bind to CARD8 first as a heterodimer with one copy of the full length CARD8 protein, then as a heterotrimer by catching a freed CARD8C14.
As the C-terminal fragment is responsible for inflammasome activation, DPP9’s ability to catch CARD8C inhibits CARD8-induced pyroptosis. Overcoming DPP9 inhibition therefore should increase the rate of CARD8 inflammasome activation and sensitize the inflammasome to sensing HIV-1 protease activity. It was recently reported that VbP is able to bind to the DPP9-CARD8 heterodimer and prevent heterotrimer formation hence increasing intracellular CARD8C concentrations. Additionally, VbP has another mechanism of action where it can induce N-terminal degradation of CARD8 which is also able to activate the inflammasome, although the direct mechanism of action has yet to be elucidated. Therefore, VbP’s ability to inhibit DPP9 and act synergistically with NNRTI-based killing due to sensitization of the CARD8 inflammasome was investigated (FIG. 8A). [0130] NNRTI induced killing of HIV-1 infected CD4+ T cells was enhanced upon treatment with VbP (FIG. 8B). This enhancement of NNRTIs was shown to be dose-dependent upon increasing concentrations of VbP. Upon addition of VbP, the EC50 had log fold change shifts up to -1.1 for both EFV and RPV (FIG. 8C). This has the potential to overcome the EC50 shift due to the presence of human serum and demonstrates that DPP9 inhibition is essential for CARD8 inflammasome activation in vivo. Due to VbP’s ability to inhibit the capture of CARD8C by DPP9, it was hypothesized that this relationship would be synergistic in nature. To further understand this complex relationship, SynergyFinder2.0 was used to identify whether this relationship was additive or synergistic. The combination treatment of VbP with EFV or RPV was found to be synergistic by four synergistic modeling methods: HSA, BLISS, Loewe, and ZIP (FIG. 8D). To evaluate the non-specific killing, VbP in both HIV-1 - infected and uninfected cells was tested. VbP at concentrations lower than 3.33 mM has no significant toxicity in uninfected CD4+ T cells, indicating this mechanism of cell killing is specific to HIV-1 (FIG. 8E). Additionally, VbP is able to induce low levels of cell killing of HIV-1 infected cells (22% and 13% for 3.33 pM and 1.11 pM respectively) (FIG. 8B).
It was hypothesized that this may be due to low levels of spontaneous intracellular dimerization of gag-pol in infected cells which is insufficient to induce killing but becomes sufficient upon sensitization of the CARD8 inflammasome by VbP.
[0131] Characterization of VbP enhancement of NNRTI-induced cell killing: To first begin understanding the dynamics of VbP enhancement of NNRTI induced cell killing, the killing in CD4+ T cells upon combination or single treatment across time was analyzed. Upon treatment with EFV at physiologically relevant concentration, the killing of HIV-1 -infected cells became more rapid and robust with the presence of VbP (FIG. 9A). It was previously shown that inhibition of the CARD8C capture by DPP9 was a rapid response, which was hypothesized is the main contributor to rapid enhancement of NNRTI induced cell killing. There is a second phase of cell killing between 6 and 24 hours before the maximal killing plateaus for both EFV and combination treatments. When looking at the cellular killing by combination treatment in comparison to EFV alone, the fold change enhancement remains relatively consistent indicating a rapid but uniform enhancement across time (FIG. 9B). In VbP only group, killing was not found until 48 hours post treatment. This slow and low level killing could either be due to the levels of CARD8C generated by inefficient spontaneous Gag-Pol dimerization now being sufficient with DPP9 inhibition or N-terminal degradation of CARD8 directly induced by VbP adding to the pool of HIV-1 PR-cleaved CARD8 fragments thereby inducing the inflammasome activation. This experiment was repeated for THP-1 cells and demonstrated similar results to CD4+ T Cells (FIG. 9C and FIG. 9D).
[0132] To eliminate the possibility that VbP-based enhancement of NNRTIs is due to an unknown mechanism of cell death, CARD8-KO THP-1 cells were used and tested combination treatment in comparison to Cas9 control cells. Upon combination treatment in CARD8-KO cells, all killing was abolished when treated with EFV alone and the combination (FIG. 9E). This clearly shows that any additional killing by the incorporation of VbP to NNRTI treatment is dependent upon CARD8 for its mechanism of action. As NLRP1 is also known to bind to DPP9 which can be released by VbP, NLRP1-KO THP-1 cells were generated and showed that enhancement was still present, which exclude the possibility that VbP enhancement is dependent on or regulated by NLRP1. Additionally, VbP is known to bind to both DPP9 and DPP8, knock-down of DPP8 does not ablate NNRTI enhancement suggesting a DPP9 specific mechanism of action. To ensure that the downstream components of the CARD8 inflammasome were demonstrating enhanced activation, primary CD4+ T cells were infected, and the cells treated concurrently with a dye that specifically stains the active form of CASP1. As can be seen in FIG. 9F, addition of VbP to EFV showed increased CASP1 activation in HIV-1 -infected CD4+ T cells which is not the case for uninfected cells. Additionally, VbP alone at low concentrations (<1 mM) shows significant CASP1 activation specifically in HIV-1 -infected cells, suggesting that it relies upon the presence of HIV-1 to help induce the CARD8 inflammasome. This underscores that while VbP is able to activate the CARD8 inflammasome and cause issues with cytotoxicity, lower concentrations of VbP do not have the same issues and are specific to killing HIV-1 infected cells and enhancing NNRTI-mediated pyroptosis. The killing and enhancement by VbP are dependent on CASP1 was also shown (FIG. 9G). [0133] VbP sensitization of the CARD8 inflammasome can overcome NNRTI resistance: As HIV-1 has a high mutation rate, the circulating pool of HIV-1 strains shows distinct genetic variation across clades. The initial work demonstrating protease cleavage of CARD8 demonstrated that proteases from all clades can induce the CARD8 inflammasome, albeit at varying levels of efficiency. This poses a significant barrier for implementation in the clinic as not every patient will have a viral reservoir that is highly sensitive to NNRTI-induced killing. Therefore, the example aimed to evaluate this combination strategy against clinical HIV-1 isolates from clades A, B, C, and D. Briefly, CD4+ T cells were infected until 10-20% infection was reached, where cells were then treated with EFV or combination along with entry inhibitor T-20 and integrase inhibitor Raltegravir to prevent further rounds of replication. As can be seen in FIG.
10A each strain demonstrated enhancement upon addition of 0.5 mM of VbP.
[0134] A major concern for implementation of NNRTIs in a “shock and kill” approach is the presence of NNRTI resistance associated mutations (RAMs). As NNRTI RAMs show significant shifts in the ECso values for blocking reverse transcriptase activity, therefore, to first understand if these RAMs would also show resistance to NNRTI-induced CARD8 inflammasome activation was investigated. RAMs were introduced into our HIV-1 reporter virus (pNL4-3-pol) via site-directed mutagenesis. RAMs were chosen for key regions on HIV-1 reverse transcriptase which can be seen in FIG. 10B. A set of RAMs were selected for initial testing (V90I, K103N, Y181C, and Y188L) and reductions in killing efficiency compared to the control virus can be seen for all four mutants tested (FIG. 10C). Although V90I and Y181C did not show statistically significant reductions, p-values in comparison to control were .051 and .053 respectively. Alternatively, K103N and Y188L showed stark, statistically significant reductions in killing efficiency and the fold changes from control can be seen in FIG. 10D. As previously documented for blocking reverse transcriptase activity, NNRTI RAMs differ in the level of resistance that they confer. Strong NNRTI RAMs may confer complete resistance to NNRTI-mediated killing whereas others may simply show reduced efficacy. This may help in the classification of viral strains that may respond to NNRTI treatment alone versus those that require VbP enhancement for their function. These NNRTI RAMs were then tested for their killing efficiency upon addition of VbP.
[0135] While all RAMs tested showed increased rates of killing upon combination treatment, two distinct stories again arise when comparing RAMs with reduced efficacy in comparison to those with complete resistance (FIG. 10E). The NNRTI RAMs that showed reduced efficacy, V90I and Y181C, demonstrated enhancement with VbP that surpassed the killing of the non-mutant control with EFV alone indicating a significant rescue of killing efficacy. The mutants that conferred near- complete resistance, K103N and Y188L, demonstrated significant increases in killing efficiency with combination treatment compared to EFV alone. Flowever, this enhancement was not significantly different from VbP alone treated samples. As can be seen in FIG. 10F some viruses are more susceptible to killing by VbP alone such as these mutants that confer complete resistance to NNRTIs.
[0136] VbP Enhances Clearance of HIV-1 -infected cells in humanized mice: Flumanized mice were used to test the ability of NNRTIs to induce pyroptosis of HIV-1- infected CD4+ T cells in vivo. The MISTRG-6-15 mouse was developed through knock-ins of human cytokine coding genes including m-csf, il-3, gm-csf, sirp-ct, thpo, il-6 and il-15. Primary CD4+ T cells infected with pNL4-3-pol were transfused into mice.
EFV alone at 20mg/kg (0.5mg per mouse) had modest but significant effects at killing HIV infected cells at 6 and 24 hours post treatment (FIG. 11 A). Since the dose used here was comparable to the 600 mg dose for patients, it clearly indicates the need for an enhancement strategy, such as with VbP, for NNRTI efficacy in vivo. Therefore, this experiment was repeated with groups treated either with 60 pg VbP/mouse, 0.5 mg EFV/mouse, or combination. VbP treatment alone showed comparable efficacy in killing HIV-1 -infected cells as EFV alone which was greatly enhanced when the two treatments were combined as evidenced by reductions in infected cells in blood (FIG. 11B). This phenotype held in lung tissue CD4+ T cells (FIG. 11C and FIG. 11D). This experiment was repeated in four independent mouse cohorts infused with CD4+ T cells from different blood donors and the therapeutic effects of EFV, VbP, and combo were consistent across all cohorts (FIG. 11E). [0137] To test if this strategy could further clear HIV-1 -infected cells in vivo, a multi-dose was tested regiment in comparison to a single combination dose. As can be seen in FIG. 11 F, there was a significant increase in killing efficiency in the blood of the multi-dose group in comparison to single dose with near significant improvement (p=0.08) in the lungs (FIG. 11G). Our single combination strategy was tested in four separate cohorts with similar results, indicating that this is a highly reproducible phenotype with significant enhancement in the presence of VbP. A separate cohort was also tested using IV injection and showed that there was no significant difference between modes of injection at 6 or 24 hours. Additionally, it is noted that there were no reductions in human CD4+ T cell counts in comparison to control, indicating that there was no significant in vivo human CD4+ T cell cytotoxicity due to treatment with EFV or VbP. Taken together, these results prove the ability of this combination strategy to clear HIV-1 -infected cells in vivo despite barriers such as low intracellular NNRTI concentrations.
[0138] HIV-infected CD4+ T cells were treated with 3mM EFV alone or in combination with another DPP8/9 inhibitor named 1G244. Around 50% HIV-infected cells were cleared by EFV alone. The EFV-mediated cell killing was increased to 80%, 85%, and 90% with the presence of 123nM, 370nM, and 1000nM 1G244, respectively. 1G244 alone had limited cell killing effect. Therefore, both VbP and 1G244 can enhance NNRTI-triggered pyroptosis of HIV-infected cells (FIG. 12).
[0139] Since NNRTIs offer a promising strategy for eradication of HIV-1 latent reservoirs, improving their in vivo cell killing potency is essential to the treatment efficacy. This example proves that sensitization of the CARD8 inflammasome through DPP9 inhibition can reduce the threshold and provide more effective clearance of HIV-1 infected cells for clinically relevant scenarios. Additionally, it shows that DPP9 inhibition through chemical means such as with VbP can induce targeted cell killing on their own which varies across viral strains with NNRTI RAMs. Thus, there may be varying levels of intrinsic Gag-Pol dimerization of these strains which can be enhanced by VbP, which are not due to NNRTIs due to the lack of dimerization induced from NNRTI binding. This suggests that viral strains that confer near-complete resistance to NNRTIs may still be sensitive to targeted killing through the sensitization of the CARD8 inflammasome. Taken together, these data prove that although NNRTI resistance may prove to be a significant barrier in implementation of NNRTIs for a shock and kill approach, they are not insurmountable when the CARD8 inflammasome is sensitized through DPP9 inhibition.
Materials and Methods:
[0140] Plasmids: To generate plasmids for these viruses, mutations were introduced into the pNL4-3-GFP vector (AIDS Reagent Program #111100), which contains an enhanced green fluorescent protein (EGFP) inserted into env. L40C- CRISPR.EFS.PAC (Addgene #89393) and SGL40C-H1.EFS.RFP657 (Addgene #69148) vectors were used for sgRNA delivery via lentivirus. CRISPR/Cas9 guide RNAs were selected using the CCTop selection tool (43). pLKO.Ipuro (Addgene #8453) was used for gene knowckdown via lentivirus vectors. Site-directed mutagenesis to obtain NNRTI RAM’s was done using PCR primers on the NL4-3-Pol plasmid and were confirmed by sequencing.
[0141] Cell Culture: HEK293T (CRL-3216) and THP-1 cells (TIB-202) were ordered from ATCC and cultured in DMEM or RPMI 1640 medium with 10% heat- inactivated fetal bovine serum (FBS), 1 U/ml penicillin, and 100 mg/ml streptomycin (Gibco). CD4+ T cells from blood were isolated from healthy donor peripheral blood mononuclear cells (PBMCs) using the BioLegend human CD4+ T cell isolation kit (BioLegend #480010). Purified CD4+ T cells were co-stimulated with plate-bound CD3 antibody (Biolegend #300333) with media containing soluble CD28 (Biolegend #302943) antibody and 20 ng/ml IL-2 (Biolegend #589106) for 3 days. Fluman serum containing media comprised 50% human serum obtained from Gemini Bio (#100-110) with 10% FBS, 1U/ml_ penicillin, 100mg/ml_ streptomycin, and 40% RPMI 1640 medium. For MTS assays the CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) from Promega was used (Promega #G3580). Uninfected CD4+ T cells or THP-1 cells were treated with EFV, RPV, VbP, or DMSO for two days prior to addition of MTS reagent, MTS reading was done following manufacturer’s protocol. [0142] Preparation of HIV-1 and lentivirus stocks: Reporter viruses were packaged by co-transfecting HEK293T cells with viral vectors, packaging vector pC- Help (44), and pVSV-G (Addgene #8454). To expand clinical HIV-1 isolates, CD8- depleted PHA-stimulated PBMCs were infected with the international HIV-1 isolates (AIDS reagent program #11412). Culture supernatant was collected after 6-9 days and filtered prior to use. Lentiviruses for knockdown or knockout were also packaged in HEK293T cells by co-transfecting pVSV-G, psPAX2 (Addgene #12260), and sgRNA or shRNA using Lipofectamine 2000 (Thermo Fisher). Lenti-X Concentrator (TaKaRa #631232) was used to concentrate supernatant containing virus.
[0143] Generation of THP-1 cells with gene knockout or knockdown: The sgRNA and shRNA sequences can be found in Table S2 and were verified by sequencing. THP-1 cells were transduced with sgRNA or shRNA lentiviruses via spin inoculation for 2 hours at 1200g at 25°C. Cells were then selected with puromycin (1 pg/ml) for 5-7 days prior to infection with HIV-1 reporter virus NL4-3-AVif-Vpr. Immunoblotting was performed to confirm knockout or knockdown efficiency. The controls for knockout cells were transduced with a Cas9-expressing lentiviral vector without sgRNA.
[0144] HIV-1 infection and cell killing: HIV-1 p24 ELISA was used to verify viral stock concentration (XpressBio #XB-1000). HIV-1 reporter virus infection was performed at a multiplicity of infection (MOI) of 10 and 0.1 for clinical isolates. Infection was again performed by spin inoculation (1 ,200g) for 2 hours at 25 °C. Antiretrovirals (ARVs) were obtained from the NIH AIDS Research and Reference Reagent Program: rilpivirine (RPV), efavirenz (EFV), etravirine (ETR), nevirapine (NVP), T-20, and raltegravir (RAL). Doravirine (DOR), Val-boroPro (VbP), along with additional EFV and RPV, were obtained from Selleck chem(#S6492, #S8455, #S4685, and # S7303). NNRTIs alone or in combination with VbP were added to HIV-11 -infected cells 3-4 days post infection. For dose response curves NNRTI’s were serially diluted 3-fold prior to addition to infected cells. For GFP-reporter viruses, infection was determined by flow cytometry. For clinical isolates, intracellular HIV-1 -p24 staining was performed using the Cytofix/CytopermTM kit (BD #554714) using anti-HIV-1 p24-PE antibody (#6604667, 1:1000 dilution) purchased from Beckman Coulter. The FLICA660 Caspasel staining reagents were purchased from ImmunoChemistry Technologies (#9122). Percent infection (GFP+ or p24+) was determined by flow cytometry (BD LSRFortessa, BD FACSCanto, or BD accuri c6 plus). Flow cytometry data were analyzed by Flowjo software.
[0145] Percent killing, log fold change in EC50, and fold change of enhancement were calculated as follows:
Figure imgf000074_0001
Humanized mice The generation of knock-in mice encoding human MCSF, GMCSF, IL3, SIRPA, THPO, IL6, and IL15 in a 129xBALB/c (N3) genetic background was performed using Velocigene technology by Regeneron Pharmaceuticals. Mice were bred to a Rag2/_ IL2rg /_ background with homozygous knockin to generate the MISTRG-6-15 mouse colony MCSFh/h !L3h/h SIRPAh/h THPOh/h IL6h/h IL15h/h RAG~ IL2rgnu" .
Transfusion experiments were conducted using primary CD4+ T cells isolated from healthy donors as previously described that were infected with pNL4-3-pol prior to IV transfusion of 5-10 million cells per mouse. VbP was injected via IV injection and 60 pg/mouse doses were diluted in DMSO. FIG. 11A IP injections of EFV were also diluted in DMSO prior to injection. All other cohorts received an IP injection of EFV diluted in vehicle (0% DMSO, 40% PEG300, 5% Tween-80, and 45% saline). Controls received the respective EFV diluent from the same cohort. Blood was collected at 6 and 24 hours (24 and 72 for multi-dose cohort) and lung tissues were collected at 24 hours post treatment. CD4+ T cells were collected from lung tissues that were homogenized to achieve single cell suspension and analyzed via flow cytometry.

Claims

CLAIMS What is claimed is:
1. A method of reducing the number of HIV-infected cells in a subject in need thereof, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
2. A method of inducing pyroptosis in a HIV-infected cell, the method comprising: contacting the HIV-infected cell with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
3. A method to sensitize the CARD8 inflammasome to non-nucleoside reverse transcriptase inhibitor-induced activation in an HIV-infected cell, the method comprising: contacting the HIV infected cell or administering to a subject having an HIV-infected cell an effective amount of a composition comprising a non nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
4. A method of treating a subject with an HIV-infection, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
5. A method of treating a subject with a NNRTI resistant HIV-infection, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
6. A method of reducing HIV reservoirs in a subject with an HIV-infection, the method comprising: administering to the subject a therapeutically effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
7. A method of activating HIV-1 protease activity and inhibiting DPP9 activity in an HIV-infected cell, the method comprising: contacting the cell with an effective amount of a composition comprising a non-nucleoside reverse transcriptase inhibitor and a dipeptidyl peptidase 9 (DPP9) inhibitor.
8. The method of any one of claims 1 to 7, wherein the NNRTI is selected from nevirapine (NVP), delavirdine (DLV), efavirenz (EFV), etravirine (ETR), or rilpivirine (RPV), or a combination thereof.
9. The method of any one of claims 1 to 7, wherein the NNRTI is selected from rilpivirine (RPV) or efavirenz (EFV) or a combination thereof.
10. The method of any one of claims 1 to 7, wherein the DPP9 inhibitor is selected from talabostat, talabostat mesylate, or 1G244.
11. The method of any one of the preceding claims, wherein the NNRTI is administered in an amount effective to induce premature intracellular activation of the viral protease triggered CARD8 inflammasome-mediated pyroptosis of HIV-1 - infected cells.
12. The method of any one of the preceding claims, wherein the DPP9 inhibitor is administered in an amount effective to positively regulate CARD8 function, which triggers CARD8 inflammasome activation and rapid killing of virus-infected cells.
13. The method of any one of the preceding claims, wherein the combination of the NNRTI and DPP9 inhibitor is administered in an amount effective to induce the clearance of latent HIV-1 in a HIV-infected CD4+ T cell after viral reactivation.
14. The method of any one of the preceding claims, wherein the combination of the NNRTI and DPP9 inhibitor is administered in an amount effective to induce clearance of persistent HIV-1 infection.
15. The method of any one of the preceding claims, wherein the combination of the NNRTI and DPP9 inhibitor is administered to blood, lymphoid tissues, or hematopoietic-derived cells, simultaneously or separately.
16. The method of any one of the preceding claims, wherein the subject has been or is currently undergoing suppressive ART.
17. A method for screening for CARD8 activating agents comprising: contacting a test agent or compound to primary CD4+ T cells infected with clinical viral isolates; and detecting CARD8-dependent caspase-1 activation and/or pyroptosis by intracellular p24 staining.
18. A method for measuring the clearance of HIV-1, comprising: obtaining blood CD4+ T cells from a subject under suppressive ART to measure the size of viral reservoirs; and detecting viral replication after test agent or test compound treatment.
PCT/US2022/018333 2021-03-01 2022-03-01 Compositions and methods for targeting card8 WO2022187241A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163155124P 2021-03-01 2021-03-01
US63/155,124 2021-03-01

Publications (1)

Publication Number Publication Date
WO2022187241A1 true WO2022187241A1 (en) 2022-09-09

Family

ID=83154449

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/018333 WO2022187241A1 (en) 2021-03-01 2022-03-01 Compositions and methods for targeting card8

Country Status (1)

Country Link
WO (1) WO2022187241A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030212044A1 (en) * 1997-05-07 2003-11-13 Huber Brigitte T. Treatment of HIV
WO2007127204A2 (en) * 2006-04-24 2007-11-08 Dara Biosciences, Inc. Methods and compositions relating to immunostimulation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030212044A1 (en) * 1997-05-07 2003-11-13 Huber Brigitte T. Treatment of HIV
WO2007127204A2 (en) * 2006-04-24 2007-11-08 Dara Biosciences, Inc. Methods and compositions relating to immunostimulation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CLARK KOLIN, WANG QIANKUN, SHAN LIANG: "CARD8 inflammasome sensitization through DPP9 inhibition enhances NNRTI-triggered killing of HIV-1-infected cells", BIORXIV, 1 September 2021 (2021-09-01), pages 1 - 23, XP055968286, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2021.09.01.458624v1.full.pdf> [retrieved on 20220424] *
GRISWOLD ANDREW R., BALL DANIEL P., BHATTACHARJEE ABIR, CHUI ASHLEY J., RAO SAHANA D., TAABAZUING CORNELIUS Y., BACHOVCHIN DANIEL : "DPP9's Enzymatic Activity and Not Its Binding to CARD8 Inhibits Inflammasome Activation", ACS CHEMICAL BIOLOGY, vol. 14, no. 11, 17 September 2019 (2019-09-17), pages 2424 - 2429, XP055968261 *
JOHNSON DARREN C., OKONDO MARIAN C., ORTH ELIZABETH L., RAO SAHANA D., HUANG HSIN-CHE, BALL DANIEL P., BACHOVCHIN DANIEL A.: "DPP8/9 inhibitors activate the CARD8 inflammasome in resting lymphocytes", CELL DEATH & DISEASE, vol. 11, no. 628, 14 August 2020 (2020-08-14), pages 1 - 10, XP055968272 *
WANG QIANKUN, GAO HONGBO, CLARK KOLIN M., MUGISHA CHRISTIAN SHEMA, DAVIS KEANU, TANG JACK P., HARLAN GRAY H., DESELM CARL J., PRES: "CARD8 is an inflammasome sensor for HIV-1 protease activity", SCIENCE, vol. 371, no. 6535, 4 February 2021 (2021-02-04), pages 1 - 19, XP055968258, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8029496/pdf/nihms-1680675.pdf> [retrieved on 20220425] *

Similar Documents

Publication Publication Date Title
Campbell et al. Hormonally active vitamin D3 (1α, 25-dihydroxycholecalciferol) triggers autophagy in human macrophages that inhibits HIV-1 infection
Doitsh et al. Abortive HIV infection mediates CD4 T cell depletion and inflammation in human lymphoid tissue
Kim et al. Semen-mediated enhancement of HIV infection is donor-dependent and correlates with the levels of SEVI
Kuhl et al. Tetherin and its viral antagonists
Bauby et al. TIP47 is Required for the Production of Infectious HIV‐1 Particles from Primary Macrophages
Agostini et al. Inhibition of non canonical HIV-1 Tat secretion through the cellular Na+, K+-ATPase blocks HIV-1 infection
US20080118494A1 (en) Compositions For Detection Of Latent Hiv Reactivation And Methods Of Using The Same
Rajashekar et al. Modulating HIV-1 envelope glycoprotein conformation to decrease the HIV-1 reservoir
WO2020169707A1 (en) Foxo1 inhibitor for use in the treatment of latent virus infection
US10765664B2 (en) Treatment of infectious diseases
Campbell et al. CD4+ T cell-mimicking nanoparticles encapsulating DIABLO/SMAC mimetics broadly neutralize HIV-1 and selectively kill HIV-1-infected cells
Soare et al. P2X1 selective antagonists block HIV-1 infection through inhibition of envelope conformation-dependent fusion
Ji et al. Newly synthesized AIFM1 determines the hypersensitivity of T lymphocytes to STING activation-induced cell apoptosis
US20040127422A1 (en) Combination therapy for treatment of HIV
WO2012118599A1 (en) C-abl tyrosine kinase inhibitors useful for inhibiting filovirus replication
US20040132161A1 (en) Methods and compositions for increasing CD4lymphocyte immune responsiveness
CN111920806A (en) Application of sunitinib malate in preparation of drugs for inhibiting coronaviruses
WO2022187241A1 (en) Compositions and methods for targeting card8
US20220249518A1 (en) Use of ouabain antagonists to inhibit viral infection
Secchi et al. Combination of the CCL5-derived peptide R4. 0 with different HIV-1 blockers reveals wide target compatibility and synergic cobinding to CCR5
US20230088583A1 (en) Inhibitors of mhc-i nef downmodulation for treating hiv
US9320773B2 (en) Compounds and therapeutic applications related to inhibition of dendritic cell immunoreceptor (DCIR) activity and signaling events
Oeyen Closing the gates: The interaction between enveloped viruses and cellular membranes as antiviral strategy
WO2014060442A1 (en) Vcp inhibitor for use in the prevention of virus infection
US20030232738A1 (en) Method and composition for increasing CD4+ T lymphocyte immune responsiveness

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22763895

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22763895

Country of ref document: EP

Kind code of ref document: A1