WO2016127003A9 - Bortezomib as an inhibitor of mycobacterial caseinolytic protease (clp) for treatment of tuberculosis - Google Patents

Abstract

Methods and compositions for the treatment of Mycobacterium tuberculosis comprising Bortezomib, CEP-18770 (Delanzomib), Ixazomib (MLN-2238), Ixazomib citrate (MLN-9708), MG-262 and related compounds, analogs and derivatives are disclosed herein.

Description

BORTEZOMIB AS AN INHIBITOR OF MYCOBACTERIAL CASEINOLYTIC PROTEASE (CLP) FOR TREATMENT OF TUBERCULOSIS

RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.

62/112,158, filed on February 4, 2015. This application also claims the benefit of U.S.

Provisional Application No. 62/113,067, filed on February 6, 2015. The entire teachings of both applications are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Mycobacterium tuberculosis (Mtb) is becoming a global health emergency that is rapidly worsening due to antibiotic resistance, resulting in nearly 2 million deaths annually, making it one of the leading causes of infectious disease mortality. It has been estimated that a third of all humans are infected with latent Mycobacterium tuberculosis (Mtb). Mtb has become increasingly resistant to available antibiotics, therefore, identifying new targets for drug development {i.e., enzymes that are essential for viability of Mtb) and developing selective inhibitors of their function is essential.

SUMMARY OF THE INVENTION

[0003] The invention relates to a novel whole-cell active anti-mycobacterial molecule Bortezomib, an inhibitor of mycobacterial caseinolytic protease (Clp) for the treatment of tuberculosis. The said drug is identified as a human 26S proteasome drug and a potent inhibitor of ClpPlP2 in mycobacteria activity by high throughput screen. Bortezomib blocks degradation of caseinolytic protease substrate WhiB l and further supports to exert its antibacterial activity via modulation of ClpPlP2. The invention demonstrates the feasibility of target mechanism-based whole cell screens, provides chemical validation of ClpPlP2 as target, and identifies a drug in clinical use as a new lead compound for tuberculosis.

[0004] Described herein are methods of treating tuberculosis, for example

Mycobacterium tuberculosis, comprising administering to a subject (e.g., a human, such as a patient) in need thereof an effective amount of a caseinolytic protease inhibitor, such as a Bortezomib, CEP- 18770 (delanzomib), MLN-2238, MLN-9708, MG-262, or any derivative thereof. In some embodiments, the caseinolytic protease is ClpPlP2. In some embodiments, the caseinolytic protease inhibitor MLN-9708. In some embodiments, the caseinolytic protease inhibitor is administered in combination with an aminoglycoside. The aminoglycoside can be amikacin, streptomycin, or a combination thereof.

[0005] Also described are methods of inhibiting mycobacterial caseinolytic protease in a patient suffering from tuberculosis comprising administering to a patient in need thereof an effective amount of Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, MG- 262, or any derivative thereof. In some embodiments, an aminoglycoside, such as amikacin, streptomycin, or a combination thereof, is also administered to the patient.

[0006] Also described are methods of blocking degradation of the substrate of ClpP121 comprising contacting ClpPlP2 with Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, MG-262, and all derivatives thereof. In some embodiments, the substrate is WhiBl .

[0007] Also described are methods of increasing sensitivity of Mycobacterium bacteria to an aminoglycoside in a subject who has Mycobacterium tuberculosis comprising

administering Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, MG-262, or any derivative thereof. In some embodiments, the aminoglycoside can be amikacin, streptomycin, or a combination thereof.

[0008] Also described are methods of inhibiting mycobacterial caseinolytic protease activity comprising contacting mycobacterial caseinolytic protease with Bortezomib, CEP- 18770 (delanzomib), MLN-2238, MLN-9708, MG-262, or any derivative thereof.

[0009] In some embodiments of the methods described herein, the methods further comprise administering to the patient one or more aminoglycoside(s). In some embodiments of the methods described herein, the methods further contacting ClpPlP2 with one or more aminoglycoside(s). In some embodiments, the aminoglycoside is amikacin, streptomycin, or a combination thereof. Other examples of aminoglycosides include gentamicin and tobramycin. In some embodiments, the aminoglycoside is a mistranslation-inducing aminoglycoside.

[0010] Also described herein are compositions for the treatment of Mycobacterium tuberculosis comprising an aminoglycoside and at least one caseinolytic protease inhibitor, e.g., a compound from the group consisting of Bortezomib, CEP-18770 (delanzomib), MLN- 2238, MLN-9708, MG-262, or any derivative thereof. In some embodiments, the aminoglycoside can be amikacin, streptomycin, or a combination thereof. [0011] The caseinolytic protease inhibitor and the aminoglycoside can be administered in appropriate dosages by any appropriate mode of administration. In some embodiments, streptomycin is administered daily as a single intramuscular injection 15 mg/kg (maximum 1 g). In some embodiments, the period of drug treatment of tuberculosis is a minimum of 1 year and up to 2 years. In some embodiments, amakacin, is administered 15 mg/kg (maximum 1 g) IM or IV every 24 hours for up to 2 years. In some embodiments, the MLN-9708 is administered orally.

[0012] In one embodiment, the dosage for Bortezomib is 1.3 mg/m². In one embodiment, it is administered as a bolus intravenous injection or subcutaneously. In one embodiment, it is administered twice weekly for two weeks (days 1, 4, 8, and 11) followed by a ten day rest period (days 12 through 21). The three week period can be considered a treatment cycle. In one embodiment, a minimum of 72 hours elapses between consecutive doses of bortezomib.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0014] The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.

[0015] FIGs. 1 A-C. Reporter strains and assays. FIG 1 A. Reporter assay principle.

Under undisturbed conditions, ClpPlP2 protease recognizes and degrades SsrA-tagged (YALAA) (SEQ ID NO: 1) GFP protein resulting in a low fluorescence level. In the presence of a ClpPlP2 inhibitor, GFP is not degraded. Its accumulation results in an increase in fluorescence. FIG. IB. M. smegmatis pTet-GFP-SsrA / pTet-GFP and assay activities. SsrA-tagged GFP (or untagged GFP) expression has been placed under the control of an anhydrotetracycline (ATc) inducible promoter (pTet). In the absence of ATc induction, the fluorescence signal remains basal with both SsrA-tagged and untagged GFP. In the presence of ATc, fluorescence is low in Smeg-pTet-GFP-SsrA due to GFP degradation, whereas the fluorescence increases in cultures expressing untagged GFP. FIG 1C. M. smegmatis p38- mRFP-SsrA / p38-mRFP and assay activities. Similarly, SsrA-tagged RFP (or untagged RFP) expression has been placed under the control of a constitutive promoter (p38). The fluorescence signal is low in Smeg-pTet-RFP-SsrA culture due to RFP degradation whereas it increases in culture expressing untagged RFP. Shown is the average of three independent experiments with error bars representing standard deviation. RFU, relative fluorescence unit.

[0016] FIG. 2. Scatter plot of primary hits from HTS. A library of half a million compounds was screened at a single concentration of 10 μΜ for inhibitors of ClpPlP2 activity using M. smegmatis carrying GFP-SsrA under the control of pTet (FIG. IB).

Compounds mean fluorescence is represented by the red line. A threshold of two times the standard deviation from the mean (2SD, pink line) was used as a cut-off for hit selection. RFU, relative fluorescence units.

[0017] FIG. 3. Screening cascade and work-up of hits.

[0018] FIGs. 4A-B. Growth inhibition activity of Bortezomib in bacteria with decreased and increased ClpPlP2 levels. FIG 4A. ClpPlP2 under-expression in M. smegmatis pTet(chromosome)-ClpPlP2. Bortezomib growth inhibition was assessed in a M. smegmatis strain in which the expression of chromosomal ClpPl and ClpP2 genes was placed under the control of a pTet promoter. Low, 1 μΜ, concentration of ATc inducer resulted in lower level of ClpP2 expression as compared to wild typeM. smegmatis culture, as confirmed on

Western blot depicted on the right. On the left, the effect of lower ClpPlP2 level on BZ susceptibility is shown. FIG 4B. ClpPlP2 over-expression in M. smegmatis pTet-ClpPlP2. This strain carries, in addition to a chromosomal copy, an episomal copy of ClpPlP2 placed under the control of an ATc inducible pTet promoter. High, 50 μΜ, concentration of inducer resulted in higher level of ClpP2 as compared to un-induced control, as confirmed on the Western blot depicted on the right. On the left, the effect of increased ClpPlP2 level on BZ susceptibility is shown. Anti-RpoB probing was carried out to confirm equal protein loading. Shown in the growth inhibition experiments are the averages of at three independent experiments with error bars representing standard deviation. The Western blots were carried three times showing the same results. One representative example is shown.

[0019] FIG. 5. Combination of Bortezomib and antibiotics. M. smegmatis wild type was treated with sub-inhibitory concentrations of Bortezomib (BZ, 1.5μΜ), amikacin (AK, 0.06μΜ), chloramphenicol (CM, 0.75uM), ciprofloxacin (CIPRO, 0.6μΜ) or rifampicin (RTF, 0.03 μΜ), independently or in combination as indicated. After 24 hours, growth was assessed via OD600 measurement. Shown is the average of three independent experiments with error bars representing standard deviation.

[0020] FIGs. 6A-C. Effect of Bortezomib on the level of the caseinolytic protease substrate WhiB l . FIG. 6A. Reporter strain principle. WhiBl is a substrate of ClpPlP2.

Under undisturbed conditions GFP-WhiB l is recognized, and degraded, resulting in a basal level of fluorescence. In presence of an inhibitor of ClpPlP2 degradation is reduced, GFP- WhiBl accumulates, resulting in an increase in fluorescence. FIG. 6B. WhiBl -GFP has been placed under the control of the pTet promoter and introduced episomally in M. smegmatis. M. smegmatis pTet-GFP-WhiB 1 was exposed to increasing concentrations of BZ for 6 hours, in presence or absence of the inducer (ATc), upon which fluorescence was measured. FIG. 6C M. smegmatis pTet-GFP was used as a control, demonstrating WhiBl dependence of fluorescence increase in B. Shown is the average of at three independent experiments with error bars representing standard deviation. RFU, relative fluorescence units.

[0021] FIGs. 7A-C. Correlation between ClpPlP2-dependent proteolytic- and growth inhibition potencies of structural derivatives of Bortezomib FIG. 7 A. Structures of BZ and derivatives. FIG. 7B. Inhibition of ClpPlP2 proteolytic activity. M. smegmatis p38-mRFP- SsrA was used as the reporter strain. FIG. 7C. Growth inhibition ofM. smegmatis WT.

Compounds were assessed in a dose-response manner up to ΙΟΟμΜ. Shown is the average of three independent experiments with error bars representing standard deviation. The same experiments were carried out in M. bovis p38-mRFP-SsrA (for RFU) and M. bovis WT (for growth inhibition) resulting in the same pattern observed forM. smegmatis: BZ and CEP- 18770 showed high RFU increase and strongest growth inhibition, MLN-2238 and MG-262 showed medium strong, and BZ-al and MG-132 showed no responses in both assays (data not shown). RFU, relative fluorescence units.

[0022] FIG. 8. Modeling of Bortezomib into one of the 7 ClpPl catalytic sites of

ClpPlP2. ClpPl is shown in red ribbon and ClpP2 in blue ribbon. The binding site residues are shown with grey carbon in thin stick while the catalytic triad Ser98-Hisl23-Aspl72 is shown in thick stick. Bortezomib is shown with plum carbon in thick stick. Hydrogen bonds between Bortezomib and ClpPlP2 are shown in purple dashed lines. The boronic acid of Bortezomib is covalently attached to the catalytic Serine. The boron is shown in green. [0022.1] FIG. 9. ClpP lP2 activity- and growth inhibition of prioritized hits. Shown are the structure, ClpP l P2 activity dose response in M. smegmatis p38-mRFP-SsrA and the growth inhibition dose response in M, smegmatis and M, hovis BCG. The experiments were carried out three times in showing the same results. One representative example is depicted, RFU, relative fluorescence units.

DETAILED DESCRIPTION OF THE INVENTION

[0023] A description of example embodiments of the invention follows.

[0024] Recent experimental evidence strongly suggests that proteasome inhibitors may indeed be beneficial in certain pathologies, such as in cancer, asthma, brain infarct, autoimmune encephalomyelitis and other infections.

[0025] It has been shown that Mtb expresses a proteasome core consisting of typical four heptameric rings stacked in a cylinder. Hence, proteasome inhibitors might be useful in the treatment of tuberculosis. However, the extensive conservation of proteasome structures militates against species selectivity of proteasome inhibitors.

[0026] Bortezomib, (VELCADE(R)), is the first approved therapeutic known to act as a potent and specific proteasome inhibitor. The present invention is directed to Bortezomib, a novel anti-tuberculosis lead compound and an inhibitor of mycobacterial caseinolytic protease (Clp), which blocks degradation of caseinolytic protease substrate WhiBl and exerts its antibacterial activity via modulation of ClpPlP2. There is a significant need for new treatments for tuberculosis.

[0027] A novel type of antibacterial screen, a target mechanism-based whole cell screen, was developed to combine the advantages of target- and whole cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpPlP2) activity was engineered allowing detection of inhibitors of this enzyme inside intact bacilli. A high throughput screen identified Bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpPlP2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that Bortezomib exerts its antimicrobial activity indeed via inhibition of ClpPlP2: Down / up modulation of the intracellular protease level resulted in hyper / hypo sensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpPlP2-specific substrate WhiB l accumulated upon exposure, and growth inhibition potencies of Bortezomib derivatives correlated with ClpPlP2 inhibition potencies. Furthermore, molecular modelling showed that the drug can bind into the catalytic sites of ClpPlP2. This work demonstrates the feasibility of target mechanism- based whole cell screens, provides chemical validation of ClpPlP2 as target, and identifies a drug in clinical use as a new lead compound for tuberculosis.

[0028] With 8.6 million new cases and 1.3 million deaths annually, tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a global infectious disease threat (1). Half a million new cases of multidrug resistant patients each year compound the situation. There is an urgent medical need for new drugs with new mechanism of action to control drug resistant disease (2). After the failure of the genomics-driven, biochemical screening-based antibacterial drug discovery strategy employed during the previous decade, the field moved largely back to classical whole cell approaches. Although empirical whole cell strategies delivered several candidates and a new TB drug, they suffer from limited productivity due to their 'black box' nature (3-5). The lack of target knowledge prevents the use of structure based design during lead finding and optimization and can result in generation of compounds with mechanism-based toxicity. Another pitfall of whole cell-based drug discovery is that compounds might be optimized for targets that are only required under in vitro culture conditions but are dispensable in vivo (6-8). Using isolated biochemical targets in screening campaigns on the other hand often result in identification of potent enzyme inhibitors lacking antibacterial activity, due to their inability to penetrate cell membranes (9). Engineering compounds to penetrate bacterial cell envelopes turned out to be challenging because the physico-chemical and structural rules that govern permeability through bacterial cell walls remain highly complex. The situation is even more challenging for mycobacteria because they have a two-membrane system: an outer membrane made up of tightly packed mycolic acids and an inner, more standard plasma membrane. The mycobacterial double membrane system represents a formidable permeability barrier. This argues for a screening strategy that includes screening targets inside the mycobacterial cell, using the double-membrane barrier as a filter. This strategy enables selection of hits that are not only able to bind to their molecular target but are also able to access it (10).

[0029] Target- or pathway -based whole cell screens have therefore been developed combining the advantages of target- and cell-based approaches to identify enzyme inhibitors with antibacterial activity (11, 12). These screens employ pathway-selective sensitization via antisense RNA or conditional gene expression (11, 13-17) in which reduced expression of the targeted gene results in an increased sensitivity to inhibitors acting on that target. Abrahams et al. used tetracycline-regulatable promoter elements to generate mycobacterial strains that conditionally express pantothenate synthetase (panC) and subsequently screened for compounds that display greater potency against PanC-depleted TB bacteria (13). Antisense strategies have been employed to reduce the expression of the chromosome partitioning protein ParA in M. smegmatis and compounds with higher anti-mycobacterial activity have been identified (18). Another type of pathway specific strategy makes use of strains that carry a reporter gene fused to a promoter that specifically responds to certain types of disturbances, such as 'cell wall synthesis stress' (19). The selective induction of the reporter signal enables screening for compounds that affect the pathway of interest. Applying this approach, Sequella screened a library withM. tuberculosis carrying the RV0341 gene promoter fused to a luciferase reporter gene. This screen identified SQ109 which is now evaluated in phase II clinical trials (20). A similar approach has led to the identification of thiophenes as a new class of anti-mycobacterials inhibiting mycolic acid biosynthesis (21). Both pathway- selective sensitization and stress-induced promoter assays provide means to identify hits that are whole cell active and pathway specific but may not provide information on the exact cellular target.

[0030] In this study we explore the feasibility of a novel type of target-based whole cell screen, a target mechanism-based whole cell approach in Mycobacterium. We selected the caseinolytic protease (ClpPlP2) as target and our aim was to identify whole cell active inhibitors of this enzyme, thus chemically validating ClpPlP2 as target for TB, and providing starting points for lead finding. Caseinolytic proteases are serine proteases found in a wide range of bacteria including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus (12, 22, 23). In contrast to site-specific proteases, caseinolytic proteases form a degradative complex involved in removal of partially synthesized and misfolded proteins. In addition to these proteome housekeeping functions, caseinolytic proteases are also involved in adaptive processes by selectively removing specific regulatory functions (24). The transcription factor WhiBl is the first regulatory function identified to be specifically degraded by mycobacterial caseinolytic protease (24). The caseinolytic protease complex is composed of catalytic protease subunits (ClpP) and regulatory subunits (ATPases). The regulatory subunits recognize substrates and provide the energy for unfolding of proteins that are to be degraded. The catalytic ClpP subunits form a degradative chamber in which proteolysis occurs. It was recently demonstrated that the proteolytic chamber of mycobacterial caseinolytic protease consists of two different subunits, ClpPl and ClpP2, which are both essential for growth of M. tuberculosis in culture and in a mouse model of TB (25, 26). Importantly, these genetic studies also suggest that the ClpPlP2 protease core represents a vulnerable target with cidal potential: reduced protein levels resulted in growth arrest and cell death, suggesting that a small molecule inhibitor of ClpPlP2 should be able to inhibit intracellular proteolytic activity to a degree that causes phenotypic consequences (25). The demonstration of genetic essentiality in vitro and in vivo, vulnerability, cidal potential, together with the demonstrated presence of ClpPlP2 in all clinical isolates, and - as protease - apparent druggability, makes mycobacterial caseinolytic protease an attractive target. Furthermore, genetic ClpPlP2 depletion experiments suggest that inhibitors may show synergy with mistranslation-inducing aminoglycosides, important second line drugs for TB, adding to the attractiveness of ClpPlP2 as target for tuberculosis drug development.

[0031] One function of caseinolytic proteases is the removal of aborted translation products. The tmRNA trans-translation system, a bacterial rescue system that frees ribosomes stuck during protein synthesis, tags partially synthesised proteins with a caseinolytic protease specific (SsrA) degradation peptide (27). SsrA-tagged proteins are recognized by the caseinolytic protease and degraded. We took advantage of this mechanism and used this caseinolytic protease-specific peptide degradation tag to develop a fluorescence-based synthetic phenotype to detect and measure intracellular ClpPlP2 inhibition. We carried out a high throughput screen, worked up the hit list with a series of secondary assays to

demonstrate on-target whole cell activity and identified the first caseinolytic protease inhibitor with antibacterial whole cell activity.

[0032] Here, we developed a novel type of anti-mycobacterial screen attempting to combine the advantages of target- and whole cell approaches: a target-mechanism based whole cell screen. The degradative caseinolytic protease ClpPlP2 was selected as target and a reporter strain with a synthetic phenotype was engineered that allowed detection of inhibitors via intracellular accumulation of green fluorescent protein. A 500,000 compound library was screened and the human proteasome inhibitor BZ was found to be positive in two

independent whole cell reporter assays measuring ClpPlP2 proteolytic activity. The compound showed growth inhibition and cidal activity in the screening strain M. smegmatis as well as in the tubercle bacillus M. bovis BCG, consistent with previous genetic depletion data of ClpPlP2. Six additional lines of evidence suggest that BZ exerts its antibacterial activity indeed via inhibition of the caseinolytic protease: i. Modulation of the intracellular ClpPlP2 level via genetic under- and over expression resulted in BZ hyper- and hypo sensitivity of the bacteria, ii. The drug potentiated the effect of aminoglycosides, pheno- copying ClpPlP2 hypomorphs. iii. BZ exposure resulted in the accumulation of the ClpPlP2- specific substrate WhiB l . iv. Whole cell growth-inhibition potencies of BZ derivatives correlated with inhibition potencies against ClpPlP2 activity, v. Replacement of the 'anti- protease' boronic acid warhead of BZ with an aldehyde resulted in an inactive compound in both the ClpPlP2 activity and growth inhibition assay, vi. Molecular modelling of

Bortezomib and its boronic acid derivatives showed that they can be covalently attached to ClpPlP2 catalytic sites.

[0033] This work has several implications. Firstly, it demonstrates the feasibility of target mechanism-based whole cell screens as a new approach to anti-mycobacterial drug discovery. After the drug discovery community moved back to rather inefficient black box whole cell strategies, new avenues that reconnect antibacterial discovery with modern genome biology are urgently needed (5). Target-mechanism based screens might be a useful complement for other ongoing activities employing pathways screens with hyper-sensitized bacterial strains and pathway-stress specific promoters.

[0034] Secondly, we provide chemical validation of mycobacterial ClpPlP2 as target for tuberculosis. With BZ we show for the first time that a small molecule inhibitor of ClpPlP2 can indeed inhibit growth and kill mycobacteria, demonstrating pharmacologically the vulnerability and cidal potential of ClpPlP2. It should be noted that lactone derivatives with anti-mycobacterial properties have been proposed to act through inhibition of the

caseinopolytic protease (lactone 4 and 7 in (38, 39)). However, these compounds displayed a dramatic disconnect between biochemical (mM) and growth inhibitory (μΜ) potency, suggesting that the antibacterial activity of these compounds is off-target, i.e. unrelated to the weak, biochemically observed, anti-ClpPlP2 activity. Indeed, lactones were neither positive in our ClpPlP2 activity reporter strain nor our ClpPlP2 under-expressing, hypersensitized strain (data not shown). It is interesting to note that several molecules, including

acyldepsipeptides and cyclomarin have been identified that appear to increase the

promiscuity of the caseinolytic protease complex and thus allow unspecific degradation of proteins (40, 41). Furthermore, lassomycin was recently identified to stimulate ATPase activity of a regulatory subunit of the caseinolytic protease while uncoupling it from the proteolytic activity of the complex (42). Our discovery of the first ClpPlP2-targeting whole cell active inhibitor adds to the growing list of caseinolytic protease modulators and shows that this proteolytic degradation machine represents an attractive multi-mechanism, multi- target complex for chemotherapeutic intervention. [0035] Thirdly, we identified BZ, a human proteasome-targeting anti-cancer drug in clinical use, as a new lead compound for tuberculosis. Interestingly, mycobacteria are one of the few prokaryotes possessing a mammalian-like proteasome (43). Whereas this function is dispensable (non-essential) in M. smegmatis (44), the proteasome is essential in the tubercle bacillus (45-47). This might explain why we see stronger antibacterial potency of BZ inM bovis BCG when compared withM. smegmatis: In the tubercle bacillus BZ might inhibit both ClpPlP2 and the tubercle bacillus' proteasome. Indeed BZ has been used in biochemical studies of theM. tuberculosis proteasome (48-50).

[0036] BZ is given intravenously and has a short half-life (34, 35). In addition to an unfavourable route of administration and poor pharmacokinetics, high costs and significant adverse effects including peripheral neuropathy, neutropenia and cytopenia (34, 35), obviously limit its direct use for tuberculosis. Second generation proteasome inhibitors, including orally bioavailable produgs are in development. It is worthwhile to mention that we tested one of the boronic acid ester produgs, MLL-9708 (51, 52), and found the compound to be active in both our ClpPlP2 reporter and growth inhibition assays (data not shown), indicating that mycobacteria can hydrolyse this prodrug to its biologically active boronic acid component. This suggest that introduction of oral bioavailability in a TB lead optimization program might be achievable. Considering the availability of in vitro assays for potency determination, the tools required for introducing selectivity, and the structural differences between the human proteasome and the mycobacterial caseinolytic protease (26, 32, 53, 54), BZ optimization appears to be an attractive opportunity.

[0037] In conclusion, our work demonstrates feasibility of target mechanism-based whole cell screens for anti-mycobacterial drug discovery, provides chemical validation of ClpPlP2 as target for TB, and identifies with BZ a new lead compound.

[0038] Material and methods

[0039] Bacterial strains, culture media and chemicals. M. smegmatis ( ATCC 700084 / mc2155) , M. bovis BCG (ATCC35734), wild-type strains and derived GFP and mRFP reporter strains were maintained in Middlebrook 7H9 media (Difco) supplemented with 0.5 % (v/v) glycerol, 0.05% (v/v) Tween 80 and 10 % (v/v) Middlebrook ADC (Albumin- Dextrose-Catalase) (Difco). When appropriate, hygromycin B (Roche) and

anhydrotetracycline (Acros Organic) was added. Enumeration of bacteria was performed by plating on Middlebrook 7hl0 (Difco) agar plates containing 0.5% (v/v) glycerol, and 10 % (v/v) Middlebrook OADC (Oleic acid-Albumin-Dextrose-Catalase) (Difco). Antibiotics were purchased from Sigma-Aldrich. Stock solutions of the compounds were prepared in 90% DMSO. The Experimental Therapeutic Centre chemical library was collected from various providers.

[0040] GFP and mRFP plasmids constructs and reporter strains. pTet-GFP plasmid comprise the wild-type allele of the GFP gene cloned downstream of the tetracycline inducible pTet. GFP was amplified from GFPmut3 wildtype DNA via PCR and subsequently recombined into the pTet vector using gateway recombination (Clontech) as previously described (55). The fusions GFP-SsrA was generated via amplification from the same template using primers pair

GGGGACAAGTTTGTACAAAAAAGCAGGCTGAAGGAGATATACATATGGCTAGCA AAGGAGAAGAAC (SEQ ID NO: 2) and

GGGGACCACTTTGTACAAGAAAGCTGGGTCGGCAGCGAGAGCG TAGTCG (SEQ ID NO: 3) and cloned into the same vectors to generate pTet-GFP-SsrA. pTET-GFP and pTet-GFP-SsrA, plasmids were electroporated separately into WT M. smegmatis to generate Smeg-pTet-GFP, Smeg-pTet-GFP-SsrA, strains respectively. pGMEH-p38-mRFP plasmid carries mCherry RFP cloned downstream of the p38 strong mycobacterial promoter (56). pGMEH-p38-mRFP-SsrAec carries the same construct including the E. coli SsrA tag fused to mRFP gene. Both plasmids were obtained from Addgene (#27058 and 27059) and electroporated into WT M. smegmatis to generate Smeg-p38-mRFP and Smeg-p38-mRFP- SsrA, respectively.

[0041] pTet-GFP-SsrA assay optimization and high throughput primary screen. Smeg- pTet-GFP and Smeg-pTet-GFP-SsrA pre-cultures were harvested at mid-log phase and diluted to OD600 0.2 in complete 7H9 media. Anhydrotetracycline was added when appropriate and the bacterial suspension was distributed in flat-bottom, dark, medium-binding 384-well plate (30 μΕΛνεΙΙ) (Greiner bio-one) and incubated at 37°C for 3 hours.

Fluorescence signal (RFU) was measured using Synergy HI microplate reader (Biotek) (excitation λ = 485 nm, emission λ = 520nm) with a 90 seconds shaking step prior to reading. OD normalization between each strain was verified by bacterial enumeration as mentioned above. DMSO tolerance was assessed by growing the strains in presence of increasing concentration of DMSO in 7H9 media and measuring effect on RFU as well as plating the cells and determining CFU. Following optimization of the primary screen assay, a high throughput format was validated following the same procedure. Prior to screening the complete 503 879 compound library, The Pharmakonl600 (1600 compounds) library was used in a validation run to assessed the performances indicators of the assay under high throughput conditions. We used Topotecan (Sigma-Aldrich) as a positive control for the inhibitor screen. Each compound was screened in duplicate at a final concentration of 10 μΜ. GFP signal was measured on a Safire II microplate reader (Tecan) with the same parameters as described above. Hits were defined as compounds that induce a GFP signal response higher than a cut-off value defined by the mean+2*SD and were submitted to a re-test in an identical assay. Auto-fluorescence of re-test positive hits was measured by dispensing 10 μΜ of each compounds (in 90% DMSO) in 384-well plate and measuring fluorescence signal with the same signal acquisition parameters as the primary screen. Auto-fluorescent compounds were filtered out. We next evaluated the GFP dose-response profile for all inhibitor hits and determined their respective GFP IC50. Briefly, all selected hits were tested in 3 -fold serial dilution GFP assay at a maximum concentration of 100 μΜ in a 96 well plate format of the GFP assay using M200 Pro plate reader (Tecan).

[0042] Constitutive p38-mRFP secondary assay. All 89 selected hits from the primary screen results were re-ordered and subjected to secondary screening assays from fresh- powder stocks (90% DMSO) using M. smegmatis strains carrying a constitutive mRFP reporter system. We first proceeded with optimization and validation of the secondary assay as described above. We then proceeded to re-screen selected hits using Smeg-p38-mRFP- SsrA. Pre-culture were harvested at mid-log phase, diluted to OD600 0.2 in complete 7H9 media and dispensed into 96-well plates (200μL/well) in presence of compounds. Smeg-p38- mRFP-SsrA alone was used as negative control whereas Smeg-p38-mRFP was used as positive control. Fluorescence signal acquisition was carried after 3 hours incubation using M200 Pro plate reader (Tecan). Red fluorescence was acquired under excitation/emission at λ= 587/630nm.

[0043] Turbidity-based growth inhibition assay. An inhibition assay was performed on selected hits to assess their inhibition potency. M. smegmatis strain WT pre-cultures were harvested at mid-log phase and diluted to OD600 0.05 in complete 7H9 media. Bacterial suspensions were then dispensed in 96-well plate (200 L/well, M. smegmatis) or in 24 well plates (1 ml/well, BCG) with the indicated compound concentration and incubated for 24 hours (M. smegmatis) or 5 days (BCG) at 37°C under shaking (100 rpm). Cells were manually resuspended and OD was measured at 600nm on M200Pro plate reader (Tecan). Positive control used ciprofloxacin at an MIC90 concentration of 0.6μΜ.

[0044] Aminglycosides potentiation assay. M. smegmatis WT (inoculum 0.01 OD600 in lmL of 7H9) was treated with sub-inhibitory concentrations of Bortezomib (BZ, 1.5μΜ), Amikacin (AK, 0.06μΜ), Chloramphenicol (CM, 0.75μΜ), Ciprofloxacin (CIPRO, 0.6μΜ) or Rifampicin (RTF, 0.03 μΜ), independently or in combination where indicated. After 24 hours, growth was assessed via OD600 measurement and growth inhibition was determined.

[0045] pTet-GFP-WhiB 1 assay. M. smegmatis pTet-GFP-WhiB 1 pre-culture was harvested at mid-log phase, diluted to OD600 0.2 in complete 7H9 media and dispensed into 96-well plates (200μΙL/well) in presence of ATc and BZ were indicated. M. smegmatis pTet- GFP was used as a control and assessed in similar conditions. Cells were incubated for 6 hours, manually resuspended and fluorescence signal was acquired as described above.

[0046] Protein purification and immunoblotting. Total protein lysates were prepared from equivalent cell numbers using bead beating. After probing with primary antibody,

visualization was performed using HRP-conjugated secondary antibodies (Invitrogen), and detection was performed using Western Lighting Plus-ECL (Perkin Elmer) according the manufacturer's protocol. In all cases, blots were probed with monoclonal anti-RpoB (Abeam) to ensure equivalent loading of samples.

[0047] Clp under- and over expression and GFP-WhiB l strains. The engineering of M. smegmatis pTet(Chromosome)-ClpPlP2 in which the native promoter of ClpPlP2 has been replaced by a tetracycline inducible promoter, and of M. smegmatis carrying an episomal copy of ClpPlP2 under the control of a tetracycline-inducible promoter (M. smegmatis pTet- ClpPlP2) in which the overexpression of ClpPlP2 can be induced by ATc have been previously described in (25). M. smegmatis pTet-GFP-WhiB 1 carrying an episomal copy of WhiBl gene fused to GFP and placed under the control of the pTet promoter has been described elsewhere (24).

[0048] Molecular Modeling. The M. tuberculosis ClpPlP2 X-ray structure of 4U0G was downloaded from the Protein Data Bank (54). Addition of hydrogens atoms, setting of protonation and tautomer states and hydrogen bond network optimization was done using the Protein Preparation Wizard in Maestro (Schrodinger Suite version 2014-2, Schrodinger, LLC: New York, NY, 2014). ClpPlP2 has 14 catalytic sites. The catalytic triad of the 7 ClpPl sites consist of residues Ser98-Hisl23-Aspl72 and those of the 7 ClpP2 sites are Serl 10-Hisl35- Asp 186. The conformation of Bortezomib from the Yeast 20S Proteasome X-ray structure 4FWD (32) was manually positioned in the ClpPlP2 catalytic sites in an orientation that allowed hydrogen bonding between both amide NH donors and both amide carbonyls of Bortezomib and protein backbone residues. These are Gly69, Ile71 and Leu 126 of the ClpPl sites and Gly81, Phe83 and Serl38 of the ClpP2 sites. The boronic acid was covalently attached to the catalytic serine, Ser98 in ClpPl and Serl 10 in ClpP2. One oxygen of the boronic acid group occupied the oxyanion hole hydrogen bonding with the backbone NH of Gly69 and Met99 in ClpPl and Gly81 and Alal 11 in ClpP2. The other boronic acid oxygen formed a salt bridge with the catalytic histidine Hisl23 of ClpPl and Hisl35 of ClpP2. This complex was minimized using the OPLS2005 force field, GBSA salvation model and 500 steps of Polak-Ribiere-Conjugate-Gradient method with MacroModel default settings (Schrodinger Suite version 2014-2, Schrodinger, LLC: New York, NY, 2014). All residues more than 12A from Bortezomib were constrained during the minimization. Delanzomib, MG-262 and Ixazomib (MLN-2238; Millennium) were also modelled into ClpPlP2 using the conformation of Bortezomib as a template and the complexes were minimized.

[0049] Bortezomib, Delanzomib (CEP- 18770, orally bioavailable), Ixazomib citrate (MLN-9708; Millennium) and Ixazomib (MLN2238; orally bioavailable), and MG262 are novel lead compounds for treatment of Tuberculosis. Further, each of these compounds, is a novel lead/target couple with ClpPlP2 for the initiation of medicinal chemistry. In addition, these compounds represent novel inhibitors of the target with whole-cell activity. Disclosed herein is the structure-activity relationship for these compounds as illustrated in the figures of this application, as well as their whole cell activity and target specificity. The means of assessing the target inhibition within the cell (via a target-based whole-cell assay) described herein can be employed to identify and develop new molecules that will be specific for bacteria (ClpPlP2 being the target) and not for human cells (proteasome).

[0050] As used herein, the terms "administering" and "introducing" are used

interchangeably and refer to the placement of a modulator of ClpPlP2 protease into a subject by a method or route which results in at least partial localization of such agents at a desired site, such as a site of aM tuberculosis bacterium, such that a desired effect(s) is produced.

[0051] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with M. tuberculosis infection. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of M. tuberculosis is reduced or halted. That is, "treatment" includes not just the improvement of symptoms, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the M. tuberculosis, stabilized (i.e., not worsening) state ofM. tuberculosis, delay or slowing of disorder progression, amelioration or palliation of M. tuberculosis, and remission (whether partial or total), whether detectable or undetectable. The term "treatment" ofM. tuberculosis also includes providing relief from the symptoms or side effects of M. tuberculosis.

[0052] As used herein, "pharmaceutically acceptable" refers to those compounds, compositions, and/or dosage forms which are suitable for administration to humans and animals without excessive toxicity, irritation, allergic response, or other problem or complications.

[0053] Also provided are methods of treating or preventing a Mycobacterium

tuberculosis (Mtb) infection in a subject with or at risk of developing a Mtb infection.

Subjects (e.g., humans, such as patients, or animals) with Mtb include subjects diagnosed with an Mtb infection. A subject at risk includes a subject with a known exposure or with a potential exposure to a Mtb source.

[0054] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, media, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of, a bispecific or multispecific polypeptide agent. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

[0055] The terms "decrease", "reduced", "reduction", "decrease" or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. For example, they can mean a decrease by at least about 5%-10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%>, or at least about 70%, or at least about 80%>, or at least about 90%) decrease or any decrease between 10-90%> as compared to a reference level.

[0056] Bortezomib is a compound having the formula [(lR)-3-methyl-l-[[(2S)-l-oxo-3- phenyl-2-[(pyrazinylcarbonyl) amino]propyl]amino]butyl]boronic acid.

[0057] Delanzomib (CEP-18770) is a compound having the formula ((R)-l-((2S,3R)-3- Hydroxy-2-(6-phenylpicolinamido)butanamido)-3-methylbutyl)boronic acid, also known as[(lR)-l-[[(2S,3R)-3-hydroxy-2-[(6-phenylpyridine-2-carbonyl)amino]butanoyl]amino]-3- methylbutyl]boronic acid.

[0058] Ixazomib (MLN-2238) is a compound having the formula (R)-(l-(2-(2,5- di chl orob enzami do)acetami do)-3 -methy lbuty l)boroni c aci d .

[0059] MLN-9708 is a compound having the formula 4-(carboxymethyl)-2-((R)-l-(2-

(2,5-dichlorobenzamido)acetamido)-3-methylbutyl)-6-oxo-l,3,2-dioxaborinane-4-carboxylic acid.

[0060] MG-262 is a compound having the formula [(lR)-3-methyl-l-[[(2S)-4-methyl-2- [[(2S)-4-methyl-2-

(phenylmethoxycarbonylamino)pentanoyl]amino]pentanoyl]amino]butyl]boronic acid.

[0061] MG-132 is a compound having the formula benzyl (S)-4-methyl-l-((S)-4-methyl- 1 -((S)-4-methyl- 1 -oxopentan-2-ylamino)- 1 -oxopentan-2-ylamino)- 1 -oxopentan-2- ylcarbamate.

[0062] As used herein and unless otherwise indicated, the term "bortezomib and derivatives thereof refers to a compound of formula

or a physiologically acceptable salt thereof,

wherein

W is (C₆-C₁₂)aryl, (5-12 atom)heteroaryl, or

with halo or (C₆-C₁₂)aryl;

X is (C₆-C₁₂)aryl, (C₁ to C₆)alkyl, hydroxyl, (5-12 atom)heteroaryl or H, further wherein alkyl and aryl are optionally substituted with H, deuterium, straight chained, branched or cycloalkyl (including (C₁ to C₆)alkyl), (C₁ to C₆)alkoxyl, (C2-C₂₀)alkenyl , or (C₂- C₂₀)alkynyl, (C₆-C₁₂)aryl, CO-(C₁-C₂₀)alkyl, CO-(C₂-C₂₀)alkenyl, CO-(C₂-C₂o)alkynyl, (C₆- C₁₂)aryl, (5-12 atom) heteroaryl, CO-(C₆-C₁₂)aryl, or CO-(5-12 atom)heteroaryl, CO- alkoxyalkyl, CO-aryloxyalkyl, sulfonyl, (C₁ to C₆)alkylsulfonyl, (C₆-C₁₂)arylsulfonyl, aralkylsulfonyl, halo or -CF₃, each optionally substituted with halo, -CF₃ or (C₁-C₆)alkyl; Y is (C₁ to C₆)alkyl; and

wherein— represents a point of attachment between two atoms.

[0063] In another embodiment, the term "bortezomib and derivatives thereof refers to a compound of formula:

or physiologically acceptable salt thereof, wherein

Rl, R2, R3, R4, and R5 each are independently H, deuterium, straight chained, branched or cycloalkyl (including (C₁ to C₆)alkyl), (C₁ to C₆)alkoxyl, (C2-C₂₀)alkenyl , or (C₂- C₂₀)alkynyl, (C₆-C₁₂)aryl, CO-(C₁-C₂₀)alkyl, CO-(C₂-C₂o)alkenyl, CO-(C₂-C₂o)alkynyl, (C₆- C₁₂)aryl, (5-12 atom) heteroaryl, CO-(C₆-C₁₂)aryl, or CO-(5-12 atom)heteroaryl, CO- alkoxyalkyl, CO-aryloxyalkyl, sulfonyl, (C₁ to C₆)alkylsulfonyl, (C₆-C₁₂)arylsulfonyl, aralkylsulfonyl, halo or -CF₃, optionally substituted with halo or (C₁-C₆)alkyl.

[0064] In another embodiment, the term "bortezomib and derivatives thereof refers to a compound, also referred to as MLN-9708, of formula:

[0065] All definitions of substituents set forth herein are further applicable to the use of the term in conjunction with another substituent.

[0066] The term "alkyl," as used herein, refers to both a saturated aliphatic branched or straight-chain monovalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(C₁-C₆) alkyl" means a radical having from 1-6 carbon atoms in a linear or branched arrangement. Examples of "(C₁-C₆) alkyl" include, for example, η-propyl, i-propyl, η-butyl, i-butyl, sec-butyl, t-butyl, η-pentyl, η-hexyl, 2-methylbutyl, 2-methylpentyl, 2-ethylbutyl, 3- methylpentyl, and 4-methylpentyl. Alkyl can be optionally substituted with halogen, -OH, oxo, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, (C₁-C₆) alkoxy(C₁-C4)alkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, carbocyclyl, nitro, cyano, amino, acylamino, or carbamyl, -C(O)O(C₁-C₁₀)alkyl, or -C(0)(C₁-C₁o)alkyl.

[0067] The term "cycloalkyl," as used herein, refers to saturated aliphatic cyclic hydrocarbon ring. Thus, "(C₃-C₈) cycloalkyl" means (3-8 membered) saturated aliphatic cyclic hydrocarbon ring. (C₃-C₈) cycloalkyl includes, but is not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, or cyclooctyl. Cycloalkyl can be optionally substituted in the same manner as alkyl, described above.

[0068] The term "alkenyl," as used herein, refers to a straight-chain or branched alkyl group having one or more carbon-carbon double bonds. Thus, "(C₂-C₆) alkenyl" means a radical having 2-6 carbon atoms in a linear or branched arrangement having one or more double bonds. Examples of alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and the like. The one or more carbon-carbon double bonds can be internal (such as in 2-butene) or terminal (such as in 1-butene).

[0069] The term "alkynyl," as used herein, refers to a straight-chain or branched alkyl group having one or more carbon-carbon triple bonds. Thus, "(C₂-C₆) alkynyl" means a radical having 2-6 carbon atoms in a linear or branched arrangement having one or more triple bonds. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. The one or more carbon-carbon triple bonds can be internal (such as in 2-butyne) or terminal (such as in 1-butyne).

[0070] The term "alkoxy", as used herein, refers to an "alkyl-O-" group, wherein alkyl is defined above. Examples of alkoxy group include methoxy or ethoxy groups.

[0071] The terms "halogen" or "halo," as used herein, refer to fluorine, chlorine, bromine or iodine.

[0072] The term "aryl," as used herein, refers to an aromatic monocyclic or polycyclic (e.g. bicyclic or tricyclic) carbocyclic ring system. Thus, "(C₆-C₁₈) aryl" is a 6-18 membered monocylic or polycyclic system. Aryl systems include optionally substituted groups such as phenyl, biphenyl, naphthyl, phenanthryl, anthracenyl, pyrenyl, fluoranthyl or fluorenyl. An aryl can be optionally substituted. Examples of suitable substituents on an aryl include halogen, hydroxyl, (C₁-C₁₂) alkyl, (C₂-C₆) alkenyl, (C₂-C₆) alkynyl, (C₁-C₆) haloalkyl, (C₁-C3) alkylamino, (C1-C3) dialkylamino (C₁-C₆) alkoxy, (C₆-C₁₈) aryloxy, (C₆-C₁₈) arylamino, (C₆- C₁₈) aryl, (C₆-C₁₈) haloaryl, (5-12 atom) heteroaryl, -NO₂, -CN, -OF₃ and oxo.

[0073] In some embodiments, a (C₆-C₁₈) aryl is phenyl, indenyl, naphthyl, azulenyl, heptalenyl, biphenyl, indacenyl, acenaphthylenyl, fluorenyl, phenalenyl, phenanthrenyl, anthracenyl, cyclopentacyclooctenyl or benzocyclooctenyl. In some embodiments, a (C₆-C₁₈) aryl is phenyl, naphthalene, anthracene, lH-phenalene, tetracene, and pentacene.

[0074] The term "heteroaryl," as used herein, refers aromatic groups containing one or more atoms is a heteroatom (O, S, or N). A heteroaryl group can be monocyclic or polycyclic, e.g., a monocyclic heteroaryl ring fused to one or more carbocyclic aromatic groups or other monocyclic heteroaryl groups. The heteroaryl groups of this invention can also include ring systems substituted with one or more oxo moieties. Examples of heteroaryl groups include, but are not limited to, thiophenyl, pyridinyl, pyridazinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, quinolyl, isoquinolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, purinyl, oxadiazolyl, thiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzotriazolyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, dihydroquinolyl, tetrahydroquinolyl, dihydroisoquinolyl,

tetrahydroisoquinolyl, benzofuryl, furopyridinyl, pyrolopyrimidinyl, and azaindolyl. [0075] In other embodiments, a 5-20-membered heteroaryl group is pyridyl, 1-oxo- pyridyl, furanyl, benzo[l,3]dioxolyl, benzo[l,4]dioxinyl, thienyl, pyrrolyl, oxazolyl, imidazolyl, thiazolyl, a isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, a triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, indolyl, tetrahydroindolyl, azaindolyl, imidazopyridyl, quinazolinyl, purinyl, pyrrolo[2,3]pyrimidinyl, pyrazolo[3,4]pyrimidinyl, imi dazo [ 1 , 2-a]py ri dy 1 , b enzothi eny 1.

[0076] The term "haloalkyl," as used herein, includes an alkyl substituted with one or more F, CI, Br, or I, wherein alkyl is defined above.

[0077] The term "haloaryl," as used herein, includes an aryl substituted with one or more F, CI, Br, or I, wherein aryl is defined above.

[0078] The term "hetero," as used herein, refers to the replacement of at least one carbon atom member in a ring system with at least one heteroatom selected from N, S, or O.

"Hetero" also refers to the replacement of at least one carbon atom member in a acyclic system. A hetero ring system or a hetero acyclic system may have 1, 2, or 3 carbon atom members replaced by a heteroatom.

[0079] The terms "heterocyclyl" or "heterocyclic," as used herein, refer to a saturated or unsaturated group having a single ring or multiple condensed rings, from 1 to 10 carbon atoms and from 1 to 4 heteroatoms selected from nitrogen, sulfur or oxygen. In fused ring systems, one or more of the rings can be aryl or heteroaryl, provided that the point of attachment is at the heterocyclyl. Heterocyclyl can be unsubstituted or substituted in accordance with cycloalkyl.

[0080] The term "oxo," as used herein, refers to =0. When an oxo group is a substituent on a carbon atom, they form a carbonyl group (C(O)).

[0081] — , as used herein, represents a point of attachment between two atoms.

[0082] , as used herein, represents both (R) and (S) stereochemical isomers. For example, the structure:

refers to:

EXAMPLE 1 - Reporter strain and assay development.

[0083] Previous work had shown that the caseinolytic protease is structurally and functionally conserved in the fast-growing and non-pathogenic mycobacterial model organism M. smegmatis (Raju, R. M., Unnikrishnan, M., Rubin, D. H., Knshnamoorthy, V., Kandror, O., Akopian, T. N., Goldberg, A. L., and Rubin, E. J. (2012) "Mycobacterium tuberculosis ClpPl and ClpP2 function together in protein degradation and are required for viability in vitro and during infection," PLoS Pathog 8, el002511). We took advantage of this finding and engineered aM. smegmatis screening strain that allows detection of inhibitors of intracellular ClpPlP2 activity via accumulation of SsrA-tagged green fluorescent protein (GFP). The underlying principle is that in the undisturbed state ClpPlP2 degrades SsrA-GFP to background fluorescence levels. An inhibitor of ClpPlP2 activity would block degradation of tagged GFP resulting in a gain of signal (FIG. 1 A). The engineered screening strain, M. smegmatis pTet-GFP-SsrA, carries an episomal SsrA-tagged GFP gene placed under the control of a tetracycline-inducible promoter (FIG. IB). A strain carrying an untagged episomal GFP gene under the control of the same tetracycline-inducible promoter (M. smegmatis pTet-GFP) was used as a control for GFP expression and, as a ClpPlP2 small molecule inhibitor as a positive control for the assay was not available, to provide an estimated upper value of fluorescence signal upon complete inhibition of SsrA- GFP degradation in the screening strain (FIG. IB).

[0084] We developed a reporter assay in 384-well plate format for High Throughput Screening (HTS). In the optimized assay format bacteria were seeded in 30 μΙ_, at low density (OD600=0.2, log phase) and incubated for 3 hours (to remain within one generation time) in presence or absence of 50 ng/mL of the inducer anhydrotetracycline (ATc) prior to GFP fluorescence signal measurement. FIG. IB shows that after 3h induction with ATc, the screening strain M. smegmatis pTet-GFP-SsrA showed low level background fluorescence, whereas high fluorescence levels were detected in the strain expressing the untagged version of GFP (M. smegmatis pTet-GFP). The assay was assessed in a pilot screen using a small collection of 1600 compounds (Pharmakon) to examine its robustness and reproducibility. The corresponding performance indicators were satisfactory with signal-to-noise ratio of 5.6+/-0.3, a Z' factor of 0.8+/-0.1, and a low hit rate of 0.5%.

[0085] High throughput screen: 1000 primary hits. A library of 503 879 compounds was screened at a single point concentration of 10 μΜ. Performance indicators were again satisfactory with a signal-to-noise ratio of 3.8+/-1 and a Z' factor value of 0.8+/-0.1. Using a cut-off of two times the standard deviation from the mean value of all compounds, 1033 primary hits were identified (0.2% hit rate) (FIG. 2). Auto-fluorescent compounds were eliminated and non/low-fluorescent hits (209) were subjected to a 10 points dose-response assay. Compounds that showed any type of dose response and were available as powders (89) were characterized further (FIG. 3).

[0086] Secondary ClpPlP2 activity-based assay: 3 survivors. To exclude false positive hits due to interference with the tetracycline-dependent pTet-GFP-SsrA reporter system of the screening strain we employed a second reporter system for ClpPlP2 activity in which both the promoter and the reporter were different from the system used in the primary screen: the SsrA-tagged mCherry Red Fluorescent Protein (mRFP) gene placed under the control of a constitutive p38 promoter (M. smegmatis p38-mRFP-SsrA, FIG. 1C). A strain carrying an untagged version of the reporter protein (M. smegmatis p38-mRFP) was used again as a positive control for signal acquisition (FIG. 1C). Using this assay, and compound solutions newly prepared from powder stocks, three hits were selected that induced a significant and dose-dependent increase in mRFP fluorescence (#52, #96, #100, Table 1).

[0087] Growth inhibition activity of potential ClpPlP2 inhibitors: 1 survivor. To determine whether any of the three candidate ClpPlP2 inhibitors showed antibacterial activity we carried out 8 point growth inhibition assays withM. smegmatis and turbidity as readout. Table 1 shows that, whereas compounds #52 and #96 did not display any growth inhibition activity up to a concentration of ΙΟΟμΜ, compound #100 showed an MIC50 of 4μΜ, comparable to a ClpPlP2 IC50 of 6μπι (Table 1). Determination of cidal activity of #100 showed that MBC90, the minimum bactericidal concentration that kills 90% of an initial inoculum, was 30μΜ.

[0088] The compounds were also tested for growth inhibition potency against the tubercle bacillus M. bov BCG and again compound #100 showed clear growth inhibition whereas the other two compounds did not inhibit growth up to ΙΟΟμΜ (Table 1). It is interesting to note that #100 was more potent against the tubercle bacillus (MIC50 = 0.3μΜ) compared to M. smegmatis (MIC50 = 4μΜ, see discussion). Determination of cidal activity of #100 showed that MBC90 forM. bovis BCG was with 0.75μΜ also accordingly lower.

[0089] The survivor: Bortezomib, a human proteasome inhibitor. The whole cell active, candidate ClpPlP2 protease inhibitor, compound #100 (Table 1) is the dipeptide-boronic acid Bortezomib (BZ, VELCADE, CYTOMIB). BZ is the first proteasome inhibitor approved by the US FDA for the treatment of newly diagnosed multiple myeloma and relapsed/refractory multiple myeloma and mantle cell lymphoma (28-30). The human proteasome, like bacterial caseinolytic protease, is a degradative protease complex involved in proteome housekeeping in man. The boronic acid warhead of BZ forms a covalent adduct to the catalytic hydroxyl group of threonine in the active site of the proteasome, resulting in enzyme dysfunction leading to cell-cycle arrest and apoptosis in cancer cells (31, 32).

EXAMPLE 2 - Growth inhibition activity of Bortezomib in bacteria with decreased and increased ClpPlP2 levels.

[0090] The identification of the proteasome (protease) inhibitor BZ as inhibitor in our cell-based ClpPlP2 proteolytic activity assay suggests that BZ might inhibit directly the catalytic protease subunits of the caseinolytic protease complex, ClpPlP2. To determine whether the growth inhibition effect of BZ is indeed due to interference with ClpPlP2 (and not some other, caseinolytic protease complex-related or -unrelated targets) we measured the effect of reducing and increasing intracellular ClpPlP2 levels on the growth inhibition activity of the compound. Reducing the level of ClpPlP2 is expected to increase sensitivity of the bacterium to the compound, whereas increasing the level is expected to decrease sensitivity of the cells.

[0091] To generate cultures of bacteria with lower and higher ClpPlP2 levels compared to the wild type, we employed two different M. smegmatis strains in which the ClpPl and ClpP2 genes are under control of a tetracycline dependent promoter. To generate M.

smegmatis with reduced ClpPlP2 level we employed M. smegmatis pTet(chromosome)- ClpPlP2 in which the expression of the native {i.e., chromosomal) ClpPlP2 genes was placed under the control of a tetracycline-dependent promoter (25). In this strain the level of ClpPlP2 can be modulated as a function of added concentrations of the inducer ATc. The Western blot analysis in FIG. 4A shows that under low (ΙμΜ) ATc concentration the

ClpPlP2 protease level was indeed reduced. Comparative growth inhibition experiments of low-level ClpPlP2 culture with wild type bacteria depicted in FIG. 4A show that reduction of ClpPlP2 protein level indeed resulted in a pronounced hyper-sensitization of the bacteria to BZ: MIC50 of BZ shifted down from 4μΜ to 0.5μΜ.

[0092] To generate M. smegmatis with increased ClpPlP2 level we employed M.

smegmatis pTet-ClpPlP2, a strain which carried in addition to the wild type chromosomal ClpPlP2 genes an episomal copy of ClpPlP2 under control of the same tetracycline- inducible promoter mentioned above (25). Addition of an appropriate high (50μΜ) ATc concentration increased the level of ClpPlP2 as shown in the Western blot analysis in FIG. 4B. FIG. 4B also shows that increase of ClpPlP2 level de-sensitized the bacteria: BZ's MIC50 shifted from 4μΜ to 20μΜ. Taken together, the ClpPlP2 under- and over expression results, showing an inverse correlation between candidate target level and antibacterial drug susceptibility, suggest that BZ exerts its growth inhibitory whole cell effect by targeting ClpPlP2.

[0093] In the growth inhibition experiment with M. smegmatis wild type only BZ/#100 showed an effect. Compounds #52 and #96, weakly positive in the reporter ClpPlP2 activity assays, did not show any antibacterial activity (Table 1). To determine whether antibacterial whole cell activity might be detectable in sensitized bacteria with reduced ClpPlP2 level, we determined the effect of compounds #52 and #96 against under-expressing cultures of M. smegmatis pTet(chromosome)-ClpPlP2. However, no effect on growth was observed up to concentration of 100 μΜ (data not shown). This suggests that #52 and #96 do not act via ClpPlP2. Whether these two compounds act via other, non-proteolytic components of the caseinolytic protease complex such as the regulatory ATPases, or whether their apparent reporter activity in the ClpPlP2 fluorescence assays is an artefact remains to be determined.

EXAMPLE 3 - Combination of Bortezomib with aminoglycosides.

[0094] Genetic depletion experiments showed previously that bacteria with reduced ClpPlP2 level display increased sensitivity to the aminoglycosides amikacin and

streptomycin, supporting the notion that mycobacterial ClpPlP2 is involved in the removal of mistranslated proteins as shown for other bacteria (25). This potentiation effect for protein synthesis inhibitors was specific for the mistranslation inducing aminoglycosides, e.g., amikacin and streptomycin, whereas no potentiation was observed for the ribosome stalling antibiotic chloramphenicol (33). If BZ is an authentic small molecule ClpPlP2 inhibitor the drug is expected to copy that phenotype. FIG. 5 shows that the predicted selective potentiation effect can be indeed observed. A combination of sub-inhibitory concentrations of BZ and amikacin or streptomycin caused complete growth inhibition whereas BZ had no potentiation effect on chloramphenicol. Taken together, the drug combination experiments show that BZ copies the selective aminoglycoside-hypersensitivity phenotype observed for ClpPlP2 under-expressing bacteria, consistent with BZ being an inhibitor of this protease.

EXAMPLE 4 - Effect of Bortezomib on the level of the caseinolytic protease substrate WhiBl.

[0095] Genetic experiments combined with quantitative proteomic and transcriptomic analyses identified recently the first specific protein substrate of ClpPlP2, the transcription factor WhiBl . Depletion of ClpPlP2 resulted in accumulation of this DNA binding protein (25). If BZ is an authentic ClpPlP2 inhibitor, exposure of the bacteria to the compound is expected to copy the effect of ClpPlP2 under-expression on WhiBl and result in an increase of WhiBl protein level. To determine the effect of BZ on the degradation of WhiB l we employed again aM. smegmatis reporter strain expressing tetracycline-inducible GFP, but with WhiBl as ClpPlP2-specific degradation 'tag' instead of the SsrA-tag used for the primary screen (M. smegmatis pTet-GFP-WhiBl, FIGS. 6A, 6B). FIG. 6B shows that BZ exposure increased fluorescence in a dose dependent manner, suggesting that the drug indeed inhibits ClpPlP2-dependent degradation of WhiBl resulting in accumulation of GFP-WhiBl . FIG. 6C shows that this effect of BZ on the GFP signal was WhiB l -dependent: BZ did not affect the fluorescence ofM. smegmatis culture carrying the same episomal pTet-GFP construct but with GFP lacking the WhiBl tag. Our results that BZ blocks degradation of the caseinolytic protease substrate WhiBl further supports the model that the drug exerts its antibacterial activity via modulation of ClpPlP2.

EXAMPLE 5 - Correlation between ClpPlP2-dependent proteolytic- and growth inhibition potencies of structural derivatives of Bortezomib.

[0096] Apparent dose-dependent ClpPlP2 protease inhibition in two strains carrying different ClpPlP2-activity -based SsrA reporter systems, expected susceptibility shifts in under- and over expressing ClpPlP2 strains, specific synergy with aminoglycosides and accumulation of the ClpPlP2 substrate WhiBl upon BZ exposure, are all supporting the notion that BZ inhibits ClpPlP2 and that it is via this interaction that the compound exerts its whole cell growth inhibitory activity.

[0097] A powerful independent method to show that a particular chemical scaffold exerts its whole cell growth inhibitory effect via modulation of a particular target is based on demonstrating a correlation between the two (whole cell vs enzyme) structure activity relationships. The concept is to identify structural derivatives of the scaffold that cover a range of enzyme inhibition activities (highly, medium and no potency) and determine whether the IC50s for the enzyme correlate with whole cell MIC50s. A positive correlation argues for an on-target effect. FIG. 7 A shows three Bortezomib derivatives CEP- 18770, MNL-2238 and MG-262. CEP-18770 and MNL-2238 are second generation proteasome inhibitors, and MG-262 is another boronate peptide showing activity against human proteasome (34-37). FIG. 7B shows that these three compounds show high (CEP-18770, same activity as BZ itself), and medium (MNL-2238, MG-262) inhibitory potencies in the cell-based fluorescent assay measuring ClpPlP2 proteolytic activity (M. smegmatis p38- mRFP-SsrA). FIG. 7C shows that the whole cell growth inhibitory activities of the compounds follow the same pattern: CEP-18770 shows the same potent growth inhibition as Bortezomib. The weaker inhibitors in the ClpPlP2 reporter assay, MNL-2238 and MG-262, also show weaker growth inhibition. The observed correlation between potency against ClpPlP2 and growth inhibition of BZ analogues suggests that the drug acts via the assumed target.

EXAMPLE 6 - Dependence of ClpPlP2 and growth inhibitory activity of Bortezomib on its boronic acid warhead.

[0098] The boronic acid warhead of the human proteasome inhibitors reacts covalently with the active site threonine hydroxyl moiety of the proteasome and is important for selectivity and potency of the compounds. For MG-262 for instance it has been shown that substitution of the boronic acid warhead with an aldehyde resulted in a 100-fold reduced activity against the proteasome (34, 35). Similarly, BZ was developed as a more potent analogue of its peptide aldehyde counterpart (36, 37). Assuming a similar, boronic acid- dependent mechanism for the inhibition of the mycobacterial ClpPlP2 serine proteases, the prediction is that removal of the warhead results in a simultaneous loss of activity of the compound in both the cellular ClpPlP2 activity and the growth inhibition assay. FIG. 7 A shows the aldehyde derivatives of BZ and MG-262, BZ-al and MG-132, respectively. FIG. 7B and 7C show that the substitution of boronic acid with aldehyde completely abrogated both enzyme- and growth-inhibition activity of the two compounds. These results show that the boronic acid warhead is essential for anti-ClpPlP2 proteolytic- and anti -bacterial activity, and indicate that Bortezomib inhibits ClpPlP2 via covalent modification of its active sites.

EXAMPLE 7 - Modeling of inhibitors with boronic acid warhead into M. tuberculosis ClpPlP2.

[0099] Both ClpPl and ClpP2 are heptamers and each has 7 catalytic sites. The boronic acid based inhibitors (FIG. 7) were modelled into the ClpPl and ClpP2 catalytic sites and covalently attached to the serine of the catalytic triad (Ser98 and Serl 10 in ClpPl and ClpP2, respectively). All the nitrogen and oxygen atoms of the inhibitor amide groups hydrogen bond with the protein backbone. One oxygen of the boronic acid occupies the oxyanion hole while the other forms a salt bridge to the catalytic histidine (Hisl23 and Hisl35 of ClpPl and ClpP2, respectively). This is shown in FIG. 8 for Bortezomib modelled into one of the ClpPl sites. The PI site is hydrophobic in both ClpPl and ClpP2 consistent with the hydrophobic side chain of the inhibitors. Neither ClpPl nor ClpP2 has a proper P2 site and the P2 side chain of the inhibitors makes poor contacts with the protein. Bortezomib, CEP-18770, and MNL-2238 modelled well into both ClpPl and ClpP2 sites. MG-262 has an extra residue compared to the other inhibitors and this compound did not model well due to the P3 side- chain clashing with the protein (data not shown). This is consistent with the observed enzyme inhibitory activities shown in FIG. 7B in which MG-262 was the least potent compound of the four.

[00100] FIG. 9. ClpP l P2 activity- and growth inhibition of prioritized hits. Shown are the structure, ClpP l P2 activity dose response in Af. smegmatis p38-mRFP-SsrA and the growth inhibition dose response in M. smegmatis and M. bovis BCG. The experiments were carried out three times in showing the same results. One representative example is depicted. RFU, relative fluorescence units.

[00101J The teachings of ail patents, published applications and references cited herein are incorporated by reference in their entirety.

[00102] While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

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Claims

CLAIMS What is claimed is:

1. A method of treating Mycobacterium tuberculosis comprising administering to a patient in need thereof an effective amount of a caseinolytic protease inhibitor in combination with an aminoglycoside.

2. The method of Claim 1 wherein the caseinolytic protease inhibitor is selected from the group consisting of Bortezomib, CEP-18770 (Delanzomib), MLN-2238, MLN- 9708, and MG-262.

3. The method of Claim 1 or 2 wherein the caseinolytic protease is ClpPlP2.

4. The method of Claim 1 or 2, wherein the aminoglycoside is amikacin, streptomycin, or a combination thereof.

5. The method of Claim 2, wherein the caseinolytic protease inhibitor is Bortezomib.

6. The method of Claim 2, wherein the caseinolytic protease inhibitor is CEP-18770.

7. A method of inhibiting mycobacterial caseinolytic protease in a patient suffering from tuberculosis comprising administering to the patient an effective amount of

Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, or MG-262.

8. The method of Claim 7 further comprising administering to the patient an

aminoglycoside.

9. A method of blocking degradation of the substrate of ClpP121 comprising contacting ClpPlP2 with Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, MG- 262, or any derivative thereof.

10. The method of claim 9 wherein the substrate is WhiB 1.

11. A composition for the treatment of Mycobacterium tuberculosis comprising an

aminoglycoside and at least one compound selected from the group consisting of Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, and MG-262.

12. A method of increasing sensitivity of Mycobacterium bacteria to an aminoglycoside in a patient suffering from Mycobacterium tuberculosis comprising administering Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, or MG-262.

13. The method of claim 12 wherein the aminoglycoside is amikacin, streptomycin, or a combination thereof.

14. The method of claim 12 wherein the aminoglycoside is a mistranslation-inducing aminoglycoside.

15. A method of inhibiting mycobacterial caseinolytic protease activity comprising

contacting mycobacterial caseinolytic protease with Bortezomib, CEP-18770

(delanzomib), MLN-2238, MLN-9708, or MG-262.

16. A method of treating Mycobacterium tuberculosis comprising administering to a patient in need thereof an effective amount of MLN-9708.

17. The method of Claim 16, wherein the MLN-9708 is administered orally.

18. A method of treating Mycobacterium tuberculosis comprising administering to a patient in need thereof an effective amount of Bortezomib, CEP-18770 (delanzomib), MLN-2238, MLN-9708, or MG-262.