WO2017040370A1 - Inhibiteurs à petites molécules de la réplication du vih-1 - Google Patents

Inhibiteurs à petites molécules de la réplication du vih-1 Download PDF

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WO2017040370A1
WO2017040370A1 PCT/US2016/049199 US2016049199W WO2017040370A1 WO 2017040370 A1 WO2017040370 A1 WO 2017040370A1 US 2016049199 W US2016049199 W US 2016049199W WO 2017040370 A1 WO2017040370 A1 WO 2017040370A1
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alkyl
ebselen
ctd
membered
hiv
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Susana T. VALENTE
Suzie THENIN-HOUSSIER
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The Scripps Research Institute
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    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole

Definitions

  • the HIV-1 mature conical core consists of 1 ,500 capsid (CA) monomers assembled into 250 hexamers and 12 pentamers.
  • CA capsid
  • the capsid plays essential roles during the viral life cycle, e.g. the precise uncoating process of the capsid is tightly associated with reverse transcription [1 , 2], and capsid assembly and maturation are essential for viral particle integrity and infectivity [3, 4].
  • Antiretroviral therapy (ART) for HIV treatment consists of a "cocktail” or a combination of at least three drugs drawn from over 26 FDA-approved drugs, which fall in the categories of non- nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (Pis), integrase inhibitors (INIs), fusion inhibitors (FIs) and entry inhibitors. None of these drugs directly targets the assembly of the viral capsid protein, and given that the precise assembly of the HIV-1 capsid protein is crucial for HIV-1 replication, it represents an attractive target for drug development.
  • NRTIs non- nucleoside reverse transcriptase inhibitors
  • NRTIs nucleoside reverse transcriptase inhibitors
  • Pro protease inhibitors
  • IPIs integrase inhibitors
  • FIs fusion inhibitors
  • the viral capsid is synthetized from a 55 kDa Gag precursor, composed of three folded proteins [matrix (MA), capsid (CA) and nucleocapsid (NC)] and 3 small peptides [spacer peptides 1 and 2 (SP1 and SP2) and p6].
  • the Gag polyprotein plays an important role in membrane binding and in Gag-Gag lattice interaction in immature virions.
  • HIV maturation is initiated by proteolytic cleavage of Gag by the viral protease, resulting in the separation of CA from other functional domains of Gag, followed by the assembly of mature fullerene-like conical capsid.
  • the viral capsid plays a critical role in both early and late events in the HIV-1 life cycle.
  • fusion of the virus and target cell membranes triggers disassembly or uncoating of the conical capsid, which promotes completion of reverse transcription and synthesis of viral cDNA [1 , 2, 5].
  • uncoating process is not completely understood, it seems that the HIV- 1 capsid's stability and integrity during the early stages of infection is essential for efficient reverse transcription and infectivity. Mutations in CA that affect its stability compromise HIV- 1 uncoating and infection [6-9].
  • two cellular restriction factors have been shown to inhibit HIV-1 infection by targeting the viral capsid during the uncoating process.
  • Tripartite motif 5-alpha (TRIM5a) promotes a rapid and premature disassembly of viral capsids, thereby abrogating productive reverse transcription [10- 13], while the interferon-inducible MxB protein prevents uncoating by stabilizing the HIV- 1 core during infection, limiting integration of viral DNA [14-16].
  • capsid assembly and maturation have been shown to be essential for the formation of infectious viral particles, and mutagenesis studies have shown that mutations in capsid are detrimental for HIV-1 assembly and particle release [8, 17-23].
  • the CA protein consists of an independently folded N-terminal domain (NTD; 1 -145 residue) and a C-terminal domain (CTD; 151 -231 residue), connected by a 5-residue flexible linker.
  • NTD N-terminal domain
  • CTD C-terminal domain
  • the structures of the full-length capsid [24-28], NTD [29] and CTD domains [30-32] have been studied by crystallography, cryo-electron microscopy and NMR.
  • the NTD domain is composed of 7 a-helices (CA helices 1 - 7), while the smaller CTD domain is composed of a short 3 io helix followed by an extended strand and 4 a-helices (CA helices 8-1 1).
  • HIV-1 CA dimerizes with a dissociation constant (Kd) of 18 ⁇ . This dimerization is mainly dependent on Trpl 84 and Metl 85 residues in Helix 9 of the CTD [31]. Mutation of these residues interferes with CA assembly in vitro [27, 33] and abolishes viral infectivity [31 , 34]. This interface is therefore required for efficient assembly of both the mature and immature capsid lattice [35, 36].
  • the HIV viral capsid protein is emerging as an interesting target for the development of new antiviral agents (reviewed in [37] ).
  • Several screens have been conducted to identify small- molecule compounds or peptides inhibiting HIV- 1 infection, i.e., virtual screens [38-40], assays based on fluorescence-based assembly with CA-NC fusion proteins [41 -43], cell-based single- cycle HIV infection [44], replicative infection [45], phage display against CA and CA-NC [46], peptide mimicking CTD helix 9 sequence [47] and more recently, bimolecular fluorescence complementation (BiFC) using CA-CTD protein [48].
  • BiFC bimolecular fluorescence complementation
  • HIV-1 capsid protein is a target of interest for the development of antiretro viral therapeutics as it is of crucial structural importance for HIV-1 replication. This protein plays essential roles in both early (uncoating) and late (capsid assembly and maturation) events of the HIV-1 life cycle.
  • HTS-TR-FRET time-resolved fluorescence resonance energy transfer based high- throughput screening assay
  • Ebselen presents an anti-HIV-1 activity in single- and multiple-round of infection in permissive cell lines as well as in primary peripheral blood mononuclear cells. This compound acts at an early, post-entry stage of the HIV-1 replication cycle, by targeting a step prior to reverse transcription. It impairs the uncoating process by stabilizing the conical capsid core.
  • the invention provides, in various embodiments, a method for the inhibition of assembly and disassembly of HIV-1 capsid, comprising contacting a cell or a population of cells infected with HIV-1 and an effective amount or concentration of a compound of formula (I)
  • ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X;
  • ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;
  • X is Se, Se(O), S, S(O), or S(0) 2 ;
  • Z is N or CR
  • R is independently at each occurrence H or (Cl-C4)alkyl
  • nl 0, 1 , 2, 3, or 4
  • n2 0, 1 , 2, 3, or 4
  • or a pharmaceutically acceptable salt thereof
  • the invention provides a method of treatment or inhibition of an HIV-1 infection in a human, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I).
  • a compound suitable for carrying out a method of the invention is a compound of formula (IA)
  • X is Se, Se(O), S, S(O) or S(0) 2 ;
  • ring directly bonded to group X can further comprise one or two nitrogen atoms therewithin;
  • Y is CR or N
  • Z is N or CR
  • R is independently at each occurrence H or (Cl-C4)alkyl
  • the invention provides a method of treatment or inhibition of an HIV-1 infection in a human, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (IA).
  • FIG. 1 CTD/CTD dimerization.
  • A Schematic representation of TR-FRET assay.
  • B Schematic representation of CTD protein constructs.
  • C Analysis of purified CTD proteins by Coomassie blue staining and western-blot using anti-p24 antibody.
  • D ELISA assay revealing GST-CTD/CTD-Flag interaction. The background controls in the assay contained buffer only, GST-CTD and CTD-Flag alone and GST incubated with CTD-Flag protein.
  • E ELISA assay revealing inhibition of CTD dimerization by untagged CTD. A range of untagged CTD protein was added to constant concentrations of GST-CTD and CTD-Flag.
  • the background controls in the assay contained buffer only, GST-CTD, CTD-Flag and untagged CTD alone. The data shown were representative of three independent experiments with triplicate data points in each experiment.
  • TR-FRET Time-resolved fluorescence-resonance energy transfer assay revealing GST-CTD/CTD-Flag dimerization.
  • the background controls in the assay contained GST-CTD alone, anti-GST and anti-Flag antibodies alone and GST incubated with CTD-Flag protein. Untagged CTD at 100 ⁇ concentration was used as inhibitor of CTD/CTD
  • FIG. 1 LOPAC pilot screen.
  • A Summary of 1536-well format assay protocol for TR-FRET assay used to screen the LOPAC library.
  • B Primary results screen. Each black plot represents one compound screened in triplicate. Red plots (series at around 100 on "% Response" y-axis ) correspond to GST-CTD alone and represent the "no CTD dimerization” baseline. Green plots (series at around 0 to -10 on y-axis) correspond to GST-CTD and CTD-Flag interaction and represent the "100% dimerization" baseline.
  • FIG. 3 Activity of Ebselen on HIV-1 replication.
  • A HeLa-CD4-LacZ cells were infected with NL4.3 replicative virus in the presence of increasing concentrations of Ebselen for 72 hrs. Beta-galactosidase activity was determined by quantitative CPRG assay.
  • B Viral supernatants from Hela-CD4 infection were assayed for their p24 antigen content using a sandwich ELISA kit.
  • C Viral supernatants from PBMCs infection were assayed for their p24 antigen content using a sandwich ELISA kit.
  • D Cell viability MTT assay on HeLa-CD4 cells treated with increasing concentration of Ebselen for 72 hrs.
  • E Cell viability MTT assay of PBMCs treated with increasing concentration of Ebselen for 4 days.
  • Ebselen induces local structural changes in CA-CTD.
  • A-B 2D ['H- ⁇ NJ-HSQC spectra of CA-CTD with 1.2- (A) or 2.4-fold (B) molar excess of Ebselen.
  • C-D 2D [ ⁇ - 15 ⁇ ]- HSQC spectra of CA-CTD W184A/M185A (WAMA) with 1.2- (C) or 2.4-fold (D) molar excess of Ebselen. Peak assignments were based on published HSQC chemical shifts for CA-CTD [58] and the WAMA mutant [59].
  • E Circular dichroism of CA-CTD with or without 2.4x Ebselen.
  • F Tryptophan fluorescence quenching assay of CA-CTD or lysozyme as negative control, titrated with Ebselen or Raltegravir. Control is black in gray-scale, experiment is light in gray scale.
  • Ebselen covalently binds to both CA-CTD Cys residues.
  • A Representation of frequencies of C198 and C218 residues from 2890 sequences from 8 different clades (A, B, C, D, F, G, CRF01_AE and CRF01_AG) (HIV sequence database alignment) using the WebLogo application (http://weblogo.berkeley.edu).
  • B ESI-MS analysis of CA-CTD incubated with different molar ratios of Ebselen (Ebs).
  • C ESI-MS analysis of oxidized CA-CTD species (intramolecular disulfide bridge, or higher-order cysteine oxidations) treated with different molar ratios of Ebselen.
  • FIG. 1 Model of CA-CTD (Ebselen) 2 binding.
  • Ribbon model of CA-CTD monomer (PDB ID: 2BUO) rendered in PyMOL 11 Hybrid, with Cys 198 and Cys218 (magenta) linked via selenylsulfide bonds to two molecules of Ebselen.
  • the ligands are shown as sticks, with C, N, O, Se colored in green, blue, red and yellow, respectively.
  • Figure 8 Residues affected by Ebselen in I5 N-CA-CTD.
  • A HSQC peak shift with respect to CA-CTD (black) and WAMA (red) apo after adding 1.2: 1 molar excess of Ebselen.
  • B-C HIV capsid CTD structure (PDB 2BUO) rendered in PyMOL as a ribbon
  • Residue color scheme reflects peak shifts that are > +2 SD (red sticks), between +1 to +2 SD (orange) and within +1 SD from the mean peak perturbation (light orange). Residues Cys 198 and Cys 218 are shown as magenta sticks.
  • FIG. 9 Ebselen covalent linkage results in CTD dimer dissociation.
  • A Overlay of CA- CTD (black) and WAMA (red) HSQC spectra, where peaks that are substantially broadened in WAMA are labeled in CA-CTD; peaks that are mutated to alanine, namely W184 and Ml 85, are also labeled.
  • B Ribbon diagram of CA-CTD (PDB 2BUO) where peaks that are perturbed after adding 2.4: 1 Ebselen, but not by 1.2: 1 Ebselen are colored in red; residues in the dimerization domain are rendered as sticks. Cys 198 and Cys 218 are shown as magenta sticks.
  • FIG. 10 Mechanism of action of Ebselen.
  • A-C Impact of Ebselen on early replication events
  • A Activity of Ebselen on HIV-1 single-round infection in HeLa-CD4-LacZ cells. Beta- galactosidase activity was determined by quantitative CPRG assay.
  • B Effect of Ebselen on reverse-transcription products and HIV-1 integration. Early and late RT products were determined by qPCR, and provirus integration was quantified by Alu-PCR followed by a qPCR.
  • C Impact of Ebselen on HIV-1 capsid stability by fate of capsid assay.
  • D Impact of Ebselen on in vitro capsid assembly.
  • E-G Impact of Ebselen on late viral replication events.
  • E Effect of Ebselen on Tat-mediated transactivation in Hela-CD4-LTR-luc. Luciferase activity was measured 48 hrs post-transfection of Tat-Flag.
  • F Activity of the indicated drugs on Gag expression and maturation in 293T cells transfected with pNL4.3. Gag expression quantified by western blot with corresponding densitometry analysis (bottom).
  • G Impact of Ebselen on infectivity of the viral particles produced in (F) in infection of the reporter Hela-CD4-LTR-Luc cells. Luciferase activity measured 48 h post-infection.
  • FIG. 11 Impact of Ebselen on MoMuLV, SIV, HCV and Influenza viruses.
  • A Impact of Ebselen on MoMuLV retrovirus in TE671 cells infected with VSV-G-MoMuLV-GFP and VSV- G-NL4.3-GFP viruses. The percentage of GFP positive cells was determined by flow cytometry.
  • B Impact of Ebselen on SIV infection in CEM174 cells infected with SIVmac239 virus.
  • C Impact of Ebselen on HCV virus by replication and infectivity assays in Huh-7.5 cells containing an HCV reporter virus (pSG-Rluc-2a-neo-NS3-5B JFH1) by measuring luciferase activity (left) or infected with a cell-culture adapted strain of JCl (JCl . l) by determining the TCID 5 o/mL using an anti-NS5A antibody.
  • D Impact of Ebselen on Influenza virus (H1N1) in Hela-CD4 cells determined by flow cytometry using a monoclonal antibody to H1N1.
  • Ebselen inhibits HIV-1 primary isolates. Inhibitory activity of Ebselen against diverse primary isolates and drug resistant strains was determined in TZM-bl cells in presence of 10 ⁇ Ebselen.
  • Ebselen analogs Table 3. Structure and activity of Ebselen analogs. Maximal % inhibitory activity of Ebselen and analogs was measured in TR-FRET assay at a final concentration of 10 ⁇ . IC 5 o of analogs was determined by a dose-response inhibition of CTD dimerization in TR-FRET assay. EC50 of Ebselen, 2pyr-Ebselen and Ebselen oxide was determined in HIV-1 single-round infection in Hela-CD4-LTR-LacZ cells. CC50 were determined by an MTT assay on HeLa-CD4-LTR-LacZ cells for a period of 48 hrs.
  • the invention is directed, in various embodiments, to methods for interfering with assembly and disassembly of the capsid for the HIV-1 virus, through interference with the key step of dimerization and further associations of capsid CA protein.
  • the compound covalently binds to the C-terminal domain of the viral capsid, domain that plays an essential role in the formation of the intact viral capsid structure, necessary for the formation of an infectious viral particle.
  • the invention is consequently directed to methods for the inhibition and the treatment of HIV-1 infection in humans.
  • the invention provides, in various embodiments, a method for the inhibition of assembly and disassembly of HIV-1 capsid, comprising contacting a cell or a population of cells infected with HIV-1 and an effective amount or concentration of a compound of formula (I)
  • ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X;
  • ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;
  • X is Se, Se(O), S, S(O), or S(0) 2 ;
  • Z is N or CR
  • R is independently at each occurrence H or (Cl-C4)alkyl
  • a compound suitable for carrying out this method is a compound of formula (IA)
  • X is Se, Se(O), S, S(O) or S(0) 2 ;
  • ring directly bonded to group X can further comprise one or two nitrogen atoms therewithin;
  • Y is CR or N
  • Z is N or CR
  • a specific compound suitable for carrying out this method can be of any one of the following formulas:
  • the invention provides a method of treatment or inhibition of an HIV-1 infection in a human, comprising administering to a patient afflicted therewith an effective dose of a compound of formula (I)
  • ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X;
  • ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;
  • X is Se, Se(O), S, S(O), or S(0) 2 ;
  • Z is N or CR
  • R is independently at each occurrence H or (Cl-C4)alkyl
  • a compound suitable for carrying out this method is a compound of formula (IA)
  • X is Se, Se(O), S, S(O) or S(0) 2 ;
  • ring directly bonded to group X can further comprise one or two nitrogen atoms therewithin;
  • Y is CR or N
  • Z is N or CR
  • nl 0, 1 , 2, 3, or 4
  • n2 0, 1 , 2, 3, or 4;
  • a specific compound suitable for carrying out this method can be of any one of the following formulas:
  • the invention further rovides a medical use of a compound of formula (I)
  • ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X;
  • ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;
  • X is Se, Se(O), S, S(O), or S(0) 2 ;
  • Z is N or CR
  • R is independently at each occurrence H or (Cl-C4)alkyl
  • a compound of formula (I) can be used for the preparation of a medicament for the treatment or inhibition of HIV-1 infection in a human patient.
  • the compound can be of formula (IA)
  • X is Se, Se(O), S, S(O) or S(0) 2 ;
  • ring directly bonded to group X can further comprise one or two nitrogen atoms therewithin;
  • Y is CR or N
  • Z is N or CR
  • R is independently at each occurrence H or (Cl-C4)alkyl
  • nl 0, 1 , 2, 3, or 4
  • n2 0, 1 , 2, 3, or 4;
  • the invention further provides a compound of formula (I)
  • ring A is a 5- or 6-membered aryl or heteroaryl ring fused to the ring comprising atom X;
  • ring B is a 5- or 6-membered aryl or heteroaryl, wherein atom Z is bonded to a carbon atom thereof;
  • X is Se, Se(O), S, S(O), or S(0) 2 ;
  • Z is N or CR
  • R is independently at each occurrence H or (Cl -C4)alkyl
  • nl 0, 1 , 2, 3, or 4
  • n2 0, 1 , 2, 3, or 4
  • or a pharmaceutically acceptable salt thereof wherein the compound is not ebselen.
  • the invention provides a compound of formula (IA)
  • X is Se, Se(O), S, S(O) or S(0) 2 ;
  • ring directly bonded to group X can further comprise one or two nitrogen atoms therewithin;
  • Y is CR or N
  • Z is N or CR
  • R is independently at each occurrence H or (Cl-C4)alkyl
  • the invention further provides a compound of any of the following formulas:
  • the CTD domain of CA is responsible for capsid dimerization, and Trpl 84 and Metl 85 residues in helix 9 of the CTD mediate this interaction [31].
  • a peptide mimicking the helix 9 sequence, CAC-1 was shown to inhibit CTD-CTD dimerization [47], and a stabilized version of this peptide, NYAD-201 , displayed inhibitory activity on HIV-1 replication and inhibited in vitro capsid assembly [52].
  • HIV-1 CTD proteins were expressed as fusion proteins with either GST in N-terminal (GST-CTD) or Flag in C-terminal (CTD-Flag), both tagged with 6 histidines in N-terminal (Fig. IB), Free CTD domain was expressed and used as control for inhibition of CTD dimerization.
  • GST-CTD GST in N-terminal
  • CTD-Flag Flag in C-terminal
  • Fig. IB Free CTD domain was expressed and used as control for inhibition of CTD dimerization.
  • the proteins were produced in E.coli and purified by affinity chromatography on a Ni-NTA agarose column. The identity and homogeneity of the proteins were verified by SDS-PAGE followed by Coomassie blue staining and western-blot, revealing a major expected band at 37 kDa for GST-CTD, 12 kDa for CTD-Flag and 1 1 kDa for CTD (Fig. 1C).
  • CTD dimerization was assessed by TR-FRET using the donor europium (Eu) cryptate- conjugated anti-GST and the acceptor allophycocyanin (XL-665)-conjugated anti-Flag antibodies.
  • emission at 665 mm would reflect the interaction of CTD proteins.
  • a strong and specific absorbance was obtained between GST-CTD and CTD-Flag, while the interaction between free GST and CTD-Flag gave a background signal (Fig. IF).
  • the CTD dimerization was inhibited by a range of untagged CTD concentrations with an EC50 of 1.07 ⁇ (Fig. 1G). This assay presented excellent statistics with a Z' score > 0.7 and a signal-to- background ratio of 3.5, allowing to proceed with screening of the LOP AC.
  • the HTS-TR-FRET assay was miniaturized and implemented in a 1536-well plate format following the protocol in Figure 2A.
  • the LOPAC library composed of 1,280 pharmacologically active approved small molecules, was screened in triplicate at a concentration of 10 ⁇ . The results were summarized as a scatterplot with black plots representing individual compounds of the LOPAC library. GST-CTD protein alone was used as high control corresponding to "100% inhibition" (red plots) while GST-CTD/CTD-Flag proteins were used as low control
  • Ebselen is expected to bind to capsid
  • concentration of Ebselen used in these assays did not impact cell viability as Ebselen displayed a half-maximal cytotoxic concentration (CC 50 ) of 25.4 ⁇ 2.9 ⁇ in HeLa-CD4-LTR-LacZ (Fig. 3D) and > 30 ⁇ in PBMCs (Fig. 3E).
  • CC 50 half-maximal cytotoxic concentration
  • Fig. 3D HeLa-CD4-LTR-LacZ
  • Fig. 3E PBMCs
  • TZM-bl are Hela-CD4 cells that express the receptor (CD4) and the two coreceptors (CXCR4 and CCR5) of HIV-1, and express the ⁇ -galactosidase and luciferase genes under control of the HIV- 1 promoter.
  • Ebselen displayed between 86.2 and 98.6% inhibitory activity against all viruses (Table 1).
  • Ebselen is an organoselenium compound that mimics glutathione peroxidase activity (review in [61]). Previous studies show that this compound forms a covalent bond between its selenium atom and thiols in cysteine residues by forming a selenylsulfide (-Se-S-) linkage [62-64], HIV-1 capsid contains 2 cysteine residues, Cysl98 and Cys218 (full-length CA number), both located in the CTD. These cysteines residues are highly conserved across HIV-1 subtypes with 99.96% and 99.76% conservation for CI 98 and C218, respectively (Fig. 5 A).
  • CA-CTD became preferentially double labeled with two molecules of Ebselen even at a compound-to-protein molar ratio of 1 : 1.
  • Ebselen to CA-CTD ratios only the doubly modified CA-CTD was detected.
  • CA-CTD protein isoforms with intramolecular disulfide-linked (Cys-S-S-Cys) and oxidized Cys residues (Cys-S0 2 H) were also observed, but not the ion species equivalent to the corresponding Ebselen-linked oxidized CA-CTD (Fig. 5C).
  • Ebselen The binding of Ebselen to CTD is specific as Omeprazole [66], Methyl-3,4-dephostatin [62] and 3-Bromo- 1 , 1 ,1 - trifluoroacetone (BTFA) [67], three cysteine-binding compounds, have no effect on HIV-1 replication and display less or no activity on CTD dimerization inhibition compared to Ebselen (Table 2).
  • Omeprazole [66]
  • Methyl-3,4-dephostatin Methyl-3,4-dephostatin
  • BTFA 3-Bromo- 1 , 1 ,1 - trifluoroacetone
  • Capsid inhibitors as well as restriction factors targeting the viral capsid can inhibit HIV-1 replication at different steps of the viral life cycle, such as post-entry events (uncoating), capsid assembly or capsid maturation.
  • post-entry events uncoating
  • capsid assembly capsid maturation.
  • VSV-G pseudotyped virus VSV-G-NL4-3
  • Efavirenz displayed an EC50 of 1.22 ⁇ 0.22 nM, while Lopinavir, as expected, was not active in this assay (Fig. 10A).
  • Fig. 10A The viral DNA species, such as the early and late reverse transcription (RT) products, via quantitative PCR (qPCR).
  • qPCR quantitative PCR
  • proviral integration by Alu-PCR followed by qPCR (Fig. 10B).
  • Ebselen decreased early and late RT products in a dose-dependent manner, and as a result, inhibited the levels of integrated proviral DNA.
  • Raltegravir (IN) and Lopinavir (PI) had no impact on RT activity, and as expected Raltegravir inhibited proviral integration.
  • Ebselen was similar to that observed for the RT inhibitor Efavirenz (NNRTI), which suggests Ebselen may affect the reverse transcription process by negatively impacting the uncoating process.
  • NRTI RT inhibitor Efavirenz
  • Ebselen may affect the reverse transcription process by negatively impacting the uncoating process.
  • This assay monitors the uncoating process by measuring the relative level of soluble (disassembled CA) vs pelletable (fullerene core) capsid during HIV-1 infection [69].
  • Figure IOC Ebselen increased the amount of pelletable CA compared to DMSO control, suggesting a stabilization of the mature capsid, similar to what is observed for the cellular restriction factor MxB [14].
  • NTD inhibitor PF74 decreased the amount of pelletable CA protein during infection by destabilizing the mature capsid, as previously observed [70].
  • the stabilization effect observed in presence of Ebselen was also shown in in vitro capsid multimerization assay. While the addition of CTD and CAI (capsid assembly inhibitor) peptide inhibited capsid assembly [33, 71], addition of increasing concentration of Ebselen resulted in a dose-dependent increase in the rate of CA multimerization. This same phenomenon was previously observed with the NTD capsid inhibitor PF74 [49].
  • Raltegravir had no impact on HIV-1 capsid assembly (Fig. 10D).
  • the Gag protein expression and maturation was similar in presence of Ebselen, Raltegravir (IN) or DMSO control, while as expected, two protease inhibitors (Ritonavir and Lopinavir) affected the maturation process (Fig. 10F).
  • the viral particles produced in presence of Ebselen and Raltegravir showed similar infectivity to the virions produced in DMSO control, while Ritonavir and Lopinavir abolished the infectivity of the virions by interfering with the maturation process (Fig. 10G).
  • Ebselen forms a selenylsulfide (-Se-S-) linkage with cysteine residues.
  • S-Ebselen sulfur
  • O-Ebselen oxygen
  • C-Ebselen carbon
  • Ebselen oxide selenoxide group
  • Ebselen modification to Ebselen oxide or 2pyr-Ebselen slightly improved its potency.
  • Ebselen inhibited MoMuLV and HIV-1 with an EC 50 of 3.87 ⁇ and 1.75 ⁇ , respectively (Fig. 1 1A), and SIV with an EC 50 of 4.2 ⁇ (Fig. 1 IB).
  • SIVmac239 was expected since SIV CTD sequence is relatively conserved to HIV-1 and also contains the two cysteine residues, Cysl 98 and Cys218 [75], However, little homology exists between MoMuLV and HIV-1 capsids.
  • MoMuLV capsid contains a single cysteine residue at position 55 and a unique carboxy terminus containing charge-rich residues (arginine and glutamic acid), critical for proper viral assembly [76, 77]. These results suggest that the binding of Ebselen to capsid may also have a conformational component.
  • HCV Hepatitis C
  • Influenza viruses The ability of Ebselen to inhibit HCV infection was evaluated by replication and infectivity assays in Huh-7.5 cells containing an HCV reporter virus (pSG-Rluc-2a-neo- NS3-5B JFH1) or infected with a cell-culture adapted strain of JC1 (JC1.1). These cells were treated with two different concentrations of Ebselen, Raltegravir, BMS-052 (an active compound targeting the HCV NS5A protein, which disrupts both viral replication and assembly) or DMSO.
  • HCV reporter virus pSG-Rluc-2a-neo- NS3-5B JFH1
  • JC1.1 cell-culture adapted strain of JC1
  • Ebselen While the HCV specific BMS-052 compound was shown to inhibit HCV replication and infectivity, Ebselen, as well as Raltegravir, presented no effect on HCV infection (Fig. 1 1C).
  • the impact of Ebselen on Influenza virus was evaluated using H1N1 strain. The virus was mixed with Ebselen, Raltgravir or with DMSO as control and used to infect 293T cells. After 16 hrs post-infection, cells were fixed, stained with an anti-hemagglutinin antibody and the percentage of positive cells was determined by flow cytometry. Ebselen and Raltegravir, displayed no inhibition of H1N1 infection (Fig. 1 ID). These results suggest Ebselen to be rather specific for retroviruses.
  • the inhibitory activity of Ebselen could partly be due to cellular toxicity [78].
  • Ebselen was initially found as an inhibitor of dimerization in TR-FRET assays using only the CTD of capsid. In this context it is also a possibility that aggregation might have lead to a loss in fluorescence intensity. Interestingly, Ebselen inhibits diverse retroviruses but has no impact on non-related viruses such as HCV and influenza viruses. In addition, the selenium atom in Ebselen was shown to be important for its activity. However, Ebselen has been identified in several biological assays, suggesting lack of selectivity (reviewed in [61]), therefore chemical innovation around the Ebselen pharmacophore, while retaining the key selenium atom, will be necessary for increasing specificity for retroviral inhibition.
  • Ebselen demonstrated clinically relevant reduction in temporary threshold shift (TTS) induced by loud sound exposure and the doses of Ebselen administrated (200, 400 and 600 mg twice daily for 4 days) were well tolerated. Therefore, despite its lack of specificity, Ebselen is safely tolerated [81].
  • TTS temporary threshold shift
  • This study is a proof-of-concept employing a TR-FRET based HTS platform to identify a small molecule that targets the viral capsid.
  • the identification of Ebselen as an inhibitor of HIV-1 replication further highlights the validity of the HIV-1 capsid as a promising drug target.
  • this study lays the groundwork for future drug design involving covalently-linked capsid inhibitors that target HIV-1 replication.
  • HeLa-CD4 cells expressing ⁇ -galactosidase (HeLa-CD4-LTR-LacZ) or Firefly luciferase (HeLa-CD4-LTR-Luc) genes under the control of HIV-1 LTR promoter were provided by Dr. Uriel Hazan, Universite de Cachan, France.
  • HEK293T and the human rhabdomyosarcoma cell line TE671 were obtained from the American Type Culture Collection (ATCC).
  • Human hepatocarcinoma cell line Huh7.5 cells were provided by Dr. Charles Rice of Rockefeller University.
  • Human peripheral blood mononuclear cells were obtained from the blood of healthy seronegative donors.
  • HeLa, HeLa-CD4-LTR-Luc, HeLa-CD4-LTR-LacZ, HEK293T and TE671 cells were cultured in DMEM supplemented with 5% FBS, L-glutamine (292 ⁇ g/mL) and antibiotics (100 units/mL penicillin and 100 ⁇ g/mL streptomycin) at 37°C and 5% C0 2 .
  • Huh-7.5 cells were cultures in DMEM supplemented with 10% FBS, 1% penicillin/streptomycin antibiotics and 1% of non-essential amino acids (NEAA).
  • the Library of Pharmacologically Active compounds (LOPAC or LOPAC ® 1280), which contains 1280 compounds representing all major target classes, was purchased from Sigma- Aldrich (St. Louis, MO). The compounds were screened at a final concentration of 10 ⁇ . Ebselen was purchased from Adipogen Corporation (#AG-CR 1-0031) and Ebselen oxide from Cayman Chemical (#10012298). The 2pyr- and 3pyr-Ebselen analogs were synthesized at Wroclaw University of Technology, Tru, with purity and stability assessed at St. John's University, NY. Other Ebselen analogs, S-, O- and C-Ebselen, were provided by Dr. Barbara Slusher, Johns Hopkins University School of Medicine, Baltimore, MD.
  • cysteine's binding compounds Omeprazole (Afla-Aesar #AAJ62860-03), Methyl-3,4-dephostatin (Sigma #M9440) and 3-Bromo-l,l ,l-trifluoroacetone (BTFA- Sigma #18545) were purchased.
  • CTD sequence was also cloned into pET42b deleted for GST to generate CTD-His protein (called untagged CTD), used as inhibitor of GST-CTD/CTD-Flag interaction (these constructs were provided by Dr. Massimo Caputi).
  • the expression and purification of the recombinant untagged CTD, GST-CTD and CTD- Flag were performed as follows, pET42b and pT7-Flag plasmids encoding CTD, GST-CTD and CTD-Flag proteins were transformed into Escherichia coli BL21(DE3) cells (Biolabs) and grown at 37°C in Luria Bertani (LB) medium until the culture reached an optical density at 600 nm of 0.6-0.8. Expression was induced by adding 1 mM IPTG and incubation was continued for 3 hrs.
  • GST-CTD and GST proteins diluted in PBS were coated onto a high- binding 96-well plate overnight at 4°C.
  • the uncoated proteins at each step were washed out with 0.05 % Tween 20 in PBS (PBS-T).
  • Wells were saturated with 5 % milk-PBS for 1 hr at RT and a range of concentrations of CTD-Flag protein (0 to 500 nM in 100 ⁇ ,) was added for 1 hr at RT in 5 % milk-PBS.
  • Rabbit anti-Flag antibody (Rockland, cat # 600-401-384) was added at a dilution of 1 : 1000 in 5 % milk-PBS and incubated for 1 hr at RT.
  • anti-rabbit IgG-HRP antibodies catalog# 170-5047 were added at a dilution of 1 : 10000 in 0.5 % milk-PBS and incubated for 1 hr at RT. Color development was assessed using TMB super-sensitive HRP substrate (Immunochemistry Technologies). The reaction was stopped by adding TMB STOP solution (Immunochemistry Technologies) and absorbance was measured at 450 nm. Inhibition of dimerization was assessed as done previously (GST-CTD 250 nM) by adding increasing concentrations of untagged CTD (0 to 25 ⁇ ) to constant concentration of CTD-Flag (500 nM). CTD inhibition EC 5 o was calculated via Prism software using log (inhibitor) vs response, at 3 parameters.
  • TR- FRET time-resolved fluorescence resonance energy transfer
  • GST-CTD and CTD-Flag 200 nM each were mixed together with EU-conjugated anti-GST antibody (1.8 ng/well; Cisbio, cat # 61 GSTKLB) and XL-665-conjugated anti-Flag antibody (20 ng/well; Cisbio, cat # 61FG2XLB), and incubated overnight at RT. Interaction between the two proteins and illumination with a 320 nm beam resulted in transfer of fluorescence energy and emission at 665 nm. Untagged CTD was used as control of inhibition of GST-CTD/CTD-Flag dimerization.
  • the assay was further miniaturized to a 1536-well plate format. All concentrations were retained, yet the volume was scaled down to 5 uL.
  • the proteins and the test compounds (10 ⁇ ) were mixed together and incubated for 20 min at RT then the 2 antibody-tagged fluorophores were added. The mixture was incubated for 5 hrs at RT before reading the emission at 665 nm.
  • the TR-FRET signal was calculated as (665 nm/620 nm)* 10,000.
  • HCV core was used as a counterscreen [60]. All plates were analyzed and passed QC if their Z' was greater than 0.5. All data was archived into the Scripps drug discovery database and activity was determined based on high and low controls. The inhibitory activity of Ebselen and its analogs was further evaluated by TR-FRET in a 384-well plate format at a 10 ⁇ final concentration.
  • NMR data were collected on a 700 MHz Bruker NMR instrument equipped with a QCI cryoprobe.
  • CA-CTD and WAMA were prepared in 25 mM phosphate (pH 6.5), 100 mM NaCl, 0.02% NaN 3 , 10% D 2 0, with or without 5 mM DTT to a final concentration of 1 mM and 330 ⁇ , respectively.
  • 2D ['H, 15 N]-HSQC spectra were acquired at 298 K, with or without 1.2-fold or 2.4-fold molar excess of Ebselen prepared as 50 or 100 mM stocks in DMSO-d6. All samples contain the same amount of DMSO-d6 (2%). Chemical shifts were assigned using previously published CA-CTD [58] and WAMA HSQC peak assignments [59]. Data were processed using Bruker Topspin 3.0 and analyzed with NMRViewJ (OneMoon Scientific, Inc.).
  • CA-CTD and negative control protein, lysozyme were prepared at 5 ⁇ and 2.5 ⁇ respectively in the same buffer for NMR, with or without 5 mM DTT.
  • L-Trp was prepared at 25 ⁇ , with or without DTT.
  • Serial dilutions (1 : 1) of Ebselen and negative control ligand, Raltegravir were prepared in DMSO and added to the protein or L-Trp samples to final concentrations ranging from 25 nM to 50 ⁇ . Samples were plated in duplicate at 12 concentration points in a 96-well black quartz microplate (Hellma) and read using SpectraMax M5e at excitation and emission wavelengths of 280 nm and 335 nm, respectively.
  • the molecular mass of CA-CTD was measured on a LTQ XL linear ion trap mass spectrometer (Thermo-Fisher Scientific) connected to a liquid chromatography (LC) system.
  • the CA-CTD was reacted with different molar ratios of Ebselen and subsequently desalted and diluted to 4 ⁇ .
  • the resulting protein was acidified using 0.1% formic acid (FA) and separated onto a C8 column employing a 0% - 100% Acetonitrile gradient.
  • the mass spectra were acquired in the positive ion mode. Mass-to-charge ratios were extracted from the raw data, deconvoluted and deisotoped with the MaqTran software.
  • MTT mitochondrial metabolic activity
  • HeLa-CD4-LTR-lacZ cells were plated at 5xl0 4 cells per well in a 96-well plate. The next day, cells were infected with replicative NL4.3 in presence of increasing concentration of Ebselen, Efavirenz, Lopinavir or DMSO control in a total volume of 200 ⁇ .
  • lysis buffer 60 mM Na 2 HP0 4 , 40 mM NaH 2 P0 4 , 10 mM KC1, 10 mM MgS0 4 , 2.5 mM EDTA, 50 mM ⁇ -mercaptoethanol, 0.125%Nonidet P-40
  • CPRG chlorophenol red-B-D-galactopyranoside
  • the cell extracts were incubated in a reaction buffer (0.9 M phosphate buffer [pH 7.4], 9 mM MgCl 2 , 1 1 mM ⁇ -mercaptoethanol, 7 mM CPRG) until a red color developed and measured at 572 nm. Experiments were performed in triplicate.
  • a reaction buffer 0.9 M phosphate buffer [pH 7.4], 9 mM MgCl 2 , 1 1 mM ⁇ -mercaptoethanol, 7 mM CPRG
  • VSV-G-NL4.3 pseudotyped viruses were generated by co-transfecting 3x10 6 293T with 2 ⁇ g of VSV-G envelope (pMDG) and 2 ⁇ g of defective pNL4.3-eGFP using 7 ⁇ ms!T-LTl transfection reagent (Mirus).
  • pNL4.3-eGFP is based on pNL4.3-R-E- (AIDS Reagent) and corresponds to the entire HIV-1 genome with 2 frameshifts in vpr and env, and eGFP cloned in nef using Xhol and NotI restriction sites. The culture supernatant was collected 48 hrs and 72 hrs post-transfection, filtered, aliquoted and stored at -80°C.
  • the titer was determined in TE671 cells by Flow cytometry analysis. HeLa-CD4-LTR-lacZ were seeded at lxl 0 4 cells per well in a 96- well plate. The next day, cells were infected with VSV-G-NL4.3-eGFP viruses in presence of increasing concentration of Ebselen, Efavirenz, Lopinavir or DMSO as control. Forty-eight hours post-infection, ⁇ -galactosidase was measured by CPRG as previously described.
  • HeLa-CD4 cells were plated at 2x10 5 cells per well in a 6-well plate. Twenty-four hours later, cells were infected with VSV-G-NL4.3 pseudotyped viruses in presence of Ebselen, Efavirenz, Raltegrvir, Lopinavir or DMSO. Heat-inactivated virus for 10 min at 95°C, was used as control. Eighteen hours post-infection, genomic DNAs (gDNA) were prepared using the DNeasy Blood and Tissue Kit (Qiagen), following the manufacter's protocol, and subject to quantitative PCR (qPCR) using Sensifast Sybr mix (Bioline).
  • the early viral DNA products were amplified using the primer pair (PI) 5'-GGC TAA CTA GGG AAC CCA CTG-3' and (P2) 5'- CTG CTA GAG ATT TTC CAC ACT GAC-3', and the late viral DNA products were amplified using the primer pair (P3) 5'-TGT GTG CCC GTC TGT TGT G-3' and (P4) 5'-GAG TCC TGC GTC GAG G-3 ' [16].
  • the number of integrated pro viruses was quantified by Alu-Gag PCR followed by a nested qPCR [83].
  • the Alu-PCR was run with the following conditions: initial denaturation of 2 min at 94°C, then 20 cycles of amplification of 20 s at 94°C, 10 s at 50°C, and 3.5 min at 65°C, and a final elongation step of 7 min at 65°C.
  • Primers sequences were as follow: (Alu) 5' -TCC CAG CTA CTG GGG AGG CTG AGG-3'; (gag) 5'-CCT GTG TCA GCT GCT TG-3'; nested q-PCR done with primers PI and P2.
  • the qPCR amplification was run as follow: 3 min at 95°C, then 40 cycles of 5 s at 95°C, 20 s at 60°C, and 8 s at 68°C.
  • PBMCs were isolated from buffy coat as previously described [72]. Briefly, 25 mL of total blood diluted 2 fold in PBS were carefully laid over 14 mL of Ficoll-paque and centrifuged at 2000 rpm for 25 min at 20°C (brakes off). PBMCs ring cell was carefully removed and transferred to a new 50 mL tube. Enough PBS was added to the PBMCs to fill up to 50 mL and centrifuged at 1500 rpm for 10 min at 20°C (brakes on). The cell pellet was washed twice with PBS and finally recovered in 30 mL of RPMI 1640 supplemented with 10% serum, 1%
  • PBMCs were counted and plated at lxl 0 6 cells/mL.
  • PHA phytohaemagglutinin
  • IL-2 Interleukin 2
  • PHA activated PBMCs were centrifuged and recovered at lxlO 6 cells per well in a 6-well plate, and infected with NL4.3 in presence of increasing concentration of Ebselen for 6 hrs. Cells were washed 3 times in PBS and resuspended in 2 mL complete RPMI (+ 20 U/mL IL2) containing drugs. Four days post infection, the supernatant was recovered for p24 ELISA assay.
  • HeLa-CD4 cells were plated at 2xl0 6 cells in a 10 cm plate. Twenty-four hours later, cells were infected with NL4.3-eGFP virus for 30 min at 4°C to allow viral attachment to cells, and then were moved to 37°C for 16 hrs in presence of 20 ⁇ Ebselen, 6 ⁇ PF74 or DMSO. Cells were washed 3 times with ice-cold PBS, and then detached with 1 mL of pronase (7.0 mg/mL prepared in DMEM) for 5 min at room temperature.
  • pronase 7.0 mg/mL prepared in DMEM
  • Ebselen on CA assembly was measured by monitoring the turbidity at 350 nm as previously described [38]. Briefly, 75 ⁇ of NaCl solution (2 mL of 5M NaCl mixed with 1 mL of 200 mM sodium phosphate, pH8) containing increasing concentration of Ebselen, PF-74, Raltegravir (used as negative control) or DMSO were placed into a 96-well plate. To initiate the assembly reaction, 25 ⁇ of purified P24-His protein (100 ⁇ ; 25 ⁇ final) was added. Turbidity was monitored every 30 s for 20 m.
  • VSV-G-MoMuLV-pseudotyped viruses were generated by co-transfecting 5xl 0 6 293T in 15 cm 2 plates with a combination of 2 ⁇ g of VSV-G envelope (pMDG), 2 ⁇ g of MoMuLV Gag- Pol plasmid (pHit60) and 4 ⁇ g of pCNCG (MLV genome encoding a CMV-driven eGFP) using J ms!T-LTl transfection reagent (Mirus). The culture supernatant was collected at 48 hrs and 72 hrs post-transfection, filtered, aliquoted and stored at -80°C. The titer was determined in TE671 cells by Flow cytometer analysis.
  • Infectivity assays were assessed in TE671 plated at 7.5x10 4 cells per well in a 12-well plate. The day after, cells were infected with VSV-G- MoMuLV-pseudotyped viruses in complete media containing polybrene (5 ⁇ g/mL), in the presence of increasing concentrations of Ebselen. Two days later, cells were washed, pelleted and then GFP-positive cells were analyzed using a flow cytometer (Accuri Sampler).
  • CEM174 cells were infected with SIVmac239 for 6 hours, then washed and plated at 5xl 0 5 cells per well in a 6- well plate. Cells were treated with increasing concentrations of Ebselen or DMSO for 72 hours. The impact of Ebselen on SIV replication was assessed by p27 ELISA on culture supernatant.
  • the impact of Ebselen on HCV infection was evaluated by replication assay and infectivity assay.
  • the replication assay was monitored in Huh-7.5 cells containing pSG-Rluc-2a- neo-NS3-5B JFHl (subtype 2a). 2.5x10 5 PSG-Rluc-2a-neo-JFHl replicon cells were seeded per well in a 24-well plate. The next day, cells were treated with Ebselen (5 and 10 ⁇ ), Raltegravir (1 ⁇ ), BMS-052 Active compound (10 nM), or DMSO control.
  • the BMS-790052 Active compound targets the HCV NS5A protein, and disrupts both viral replication and assembly.
  • cells were treated with Ebselen (5 and 10 ⁇ ), Raltegravir (1 ⁇ ), BMS-052 Active compound (10 nM), or DMSO control.
  • Ebselen 5 and 10 ⁇
  • Raltegravir (1 ⁇ )
  • BMS-052 Active compound 10 nM
  • DMSO control DMSO control.
  • cells were treated with a cell- culture adapted strain of JC 1 ( JC 1.1 ; subtype 2a) at an MOI of 0.5 for 5 hrs, then culture supernatant was replaced by fresh media.
  • the viral supernantant were harvested, serially diluted and subsequently used to infect naive Huh-7.5 cells seeded 24 hrs before in a 96-well plate coated with poly-l-lysine at a density of 8x l 0 4 cells per well.
  • titers were analyzed using immunohistochemistry to detect infected cells by 9E10 anti-NS5A antibody (Provided by Dr. Charles Rice of Rockefeller University) staining as described [84].
  • the unbounded antibodies at each step were washed out 2X with PBS and IX with PBS + 0.1 % saponin.
  • Cells were fixed with 200 iL cold methanol for 10 min at room temperature, washed 2X with PBS and incubated in 50 ⁇ /well Blocking Buffer (BSA 1%, skim milk 0.2% in IX PBS, and 1 % saponin) for lhr at RT.
  • Blocking buffer was removed and endogenous peroxidase was blocked via incubation with PBS + 0.3% hydrogen peroxide (H 2 0 2 ) for 5 min at RT.
  • Cells were washed and then incubated for 1 hr at RT in 50 ⁇ / ⁇ of purified anti-NS5A 9E10 monoclonal at a 1 : 10000 dilution of a 1 mg/mL stock in PBS + 0.1% saponin.
  • an anti-mouse Ig Cat# MP-7402
  • an anti-mouse Ig Cat# MP-7402
  • the reaction was developed using DAB chromatogen substrate (Vector Labs) for 5-20 min at RT in 50 ⁇ according to the manufacturer's instructions. The reaction was stopped by washing 2X with PBS and positive wells were counted via microscope. 50% Tissue Culture Infectious Dose/mL (TCID 50 /mL) was determined using the Reed and Muench calculator as described previously [84].
  • Influenza A virus (H1N1 , A / Virginia / ATCC1 / 2009, ATCC VR-1736) produced in MDCK cells cultured in serum-free OptiPRO medium (Life Technologies), was mixed with Ebselen (1 and 10 ⁇ ), Raltegravir (1 ⁇ ), Aleuria Aurantia Lectin (100 and 300 nM) or DMSO control, and used to infect Hela-CD4 cells at 37°C. After one hr the culture supematants were removed and cells were further grown in fresh DMEM supplemented with 10% FBS until the next day.
  • Infected cells were then trypsinized, fixed with 1% paraformaldehyde in PBS, permeabilized in 0.1% saponin in PBS containing 2% goat serum and stained with a mouse monoclonal antibody recognizing H1N1/H2N2 (clone C179, Takara), followed by an Alexa647- conjugated goat anti-mouse antibody (Jackson Immunoresearch). Cells were then washed, fixed in 2% paraformaldehyde in PBS, and analyzed by flow cytometry (BD Biosciences C6 Accuri cytometer).
  • Fricke, T., et al., MxB binds to the HIV-1 core and prevents the uncoating process of HIV-1. Retrovirology, 2014. 11: p. 68.
  • Curreli, F., et al. Virtual screening based identification of novel small-molecule inhibitors targeted to the HIV-1 capsid. Bioorg Med Chem, 2011. 19(1): p. 77-90. Goudreau, N., et al., Novel inhibitor binding site discovery on HIV-1 capsid N-terminal domain by NMR and X-ray crystallography. ACS Chem Biol, 2013. 8(5): p. 1074-82. Lemke, C.T., et al., Distinct effects of two HIV-1 capsid assembly inhibitor families that bind the same site within the N-terminal domain of the viral CA protein. J Virol, 2012. 86(12): p. 6643-55.
  • HIV capsid is a tractable target for small molecule therapeutic intervention.

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Abstract

La présente invention concerne un essai de criblage à haut débit par transfert d'énergie de fluorescence par résonance à résolution temporelle (HTS-TR-FRET), permettant d'identifier des inhibiteurs de la dimérisation de capsides, à l'aide du domaine C-terminal (CTD) d'un capside du VIH -1. Ce dosage a été utilisé pour cribler la bibliothèque de composés pharmacologiquement actifs, composée de 1 280 médicaments actifs in vivo, et a identifié l'Ebselen, un composé d'organosélénium, comme inhibiteur puissant de la dimérisation du CTD d'un capside du VIH-1. Une analyse spectroscopique par résonance magnétique nucléaire (RMN) a confirmé l'interaction directe de l'Ebselen avec le CTD du capside du VIH-1 et la dissociation du dimère lorsque l'Ebselen se trouve en une proportion en excès molaire d'un facteur deux. La spectrométrie de masse à ionisation par électronébulisation (ESI-MS) révèle que l'Ebselen se lie de manière covalente au CTD du capside du VIH-1, probablement par l'intermédiaire d'une liaison de sélénylsulfure avec la Cys 198 et la Cys 218. Ce composé présente une activité anti-VIH au cours d'un ou plusieurs cycles d'infection de lignées cellulaires permissives ainsi que dans des cellules mononucléaires du sang périphérique primaire.
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Citations (5)

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WO1999049860A1 (fr) * 1998-03-30 1999-10-07 The Endowment For Research In Human Biology, Inc. Agents et procedes de modulation du transfert du zinc a l'aide de metallothioneine
US20090176776A1 (en) * 2005-10-21 2009-07-09 University Of Alabama At Birmingham Small molecule inhibitors of hiv-1 capsid assembly
US20100227899A1 (en) * 2008-03-05 2010-09-09 Blase Christopher Billack Process for the treatment of bacterial infections using 2-phenyl-1,2-benzisoselenazol-3(2H)-one 1-oxide
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