Development of Mycobacterium tuberculosis Whole Cell Screening

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Development of Mycobacterium tuberculosis Whole Cell Screening Hits as Potential Antituberculosis Agents Christopher B. Cooper J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400381v • Publication Date (Web): 08 Aug 2013 Downloaded from http://pubs.acs.org on August 20, 2013

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Journal of Medicinal Chemistry

Development of Mycobacterium tuberculosis Whole Cell Screening Hits as Potential Antituberculosis Agents Christopher B. Cooper*

Global Alliance for TB Drug Development (TB Alliance), 40 Wall Street, 24th Floor, New York, NY 10005

“O Death! The poor man’s dearest friend— The kindest and the best! Welcome the hour, my aged limbs Are laid with thee at rest! The great, the wealthy, fear thy blow, From pomp and pleasure torn! But, oh! A blessed relief to those That weary-laden mourn.”

From: “Man was made to mourn. A Dirge,” by Scottish poet and tuberculosis victim, Robert Burns

Abstract The global pandemic of drug sensitive tuberculosis (TB) as well as the increasing threat from various multidrug resistant forms of TB drives the quest for newer, safer, more effective TB treatment options. The general lack of success in progressing novel chemical matter from high throughput screens of Mycobacterium tuberculosis (M.tb) biochemical targets has prompted resurgence in interest and efforts in prosecuting mycobacterial phenotypic screens. Whole cell active compounds identified from such screens offer significant intrinsic advantages over biochemical screening hits, and derivatives of many of these have proven invaluable in helping to fill the current TB drug development pipeline. Modern techniques for “de-orphaning” such screening hits (i.e. determining their specific biological mechanism of action) offer the possibility of ultimately identifying improved next generation chemical series by screening these essential, pharmacologically validated biochemical targets as well.

Introduction Tuberculosis (TB) represents an enduring, deadly infectious disease for all of mankind. Nearly two billion people are currently infected with TB with a staggering 13.7 million active cases worldwide (see Figure 11). The World Health Organization (WHO) estimates that nearly 1.5 million people die from TB each year with the overwhelming majority of these from developing portions of the world2. In recent years, the significance of the disease has increased dramatically as TB is also the major

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cause of death among patients co-infected with HIV. In addition, new drug-resistant strains of TB have emerged which are exceedingly difficult to treat successfully. Every year, nearly half a million new cases of multidrug resistant tuberculosis (MDR-TB) are estimated to occur3. Extremely drug resistant TB (XDR-TB) is fatal in a large proportion of cases4.

For those suffering from drug sensitive (DS) tuberculosis, the standard treatment involves an oral, four-drug combination (isoniazid, rifampin, pyrazinamide, and ethambutol) given daily over a period of 6 to 9 months. Under optimal conditions, this treatment will cure ~85-90% of drug-sensitive TB patients if the treatment regimen is strictly adhered to1. Each of these drugs, however, exhibits a range of deleterious side-effects including gastrointestinal inflammation/pain, liver toxicity, and neurological/behavioural manifestations5. In many circumstances, the requirement to remain on this regimen for a prolonged period of time is wholly unacceptable, and patients abandon their treatments before sterilization is fully complete. This then leads to the emergence of various forms of drug resistant (DR) TB, including multidrug resistant, extremely drug resistant, and the recently diagnosed totally drug resistant (TDR) TB6. Not surprisingly, treatment options for patients with these forms of TB are even more limited, with much longer treatment periods (12-18 months or longer) requiring second-line, injectable agents which can only be administered in hospital/clinic settings. The incidence rate of TDR TB, while numerically still a very small percentage, underscores the genuine urgency to address this significant unmet medical need7.

Sources of new antitubercular agents Target-based screens. With the transcription of the M. tuberculosis genome in 19988, efforts have continued unabated to identify and interrogate specific, essential TB biochemical targets. The construction of rapid and robust high throughput assays against such targets could lead to the identification of novel chemical hits for elaboration ultimately into “drug-like” chemical series of interest. The underlying problem with this target-based approach remains the translation of potent,


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selective in vitro biochemical activity into whole cell, antimycobacterial activity. Efforts to enhance binding affinity and specificity can lead to molecules with unsatisfactory physicochemical properties for uptake by M.tb and/or exclusion by innate xenobiotic efflux mechanisms.

A range of target-based screens has been employed in recent years for early TB drug discovery. One example is the identification of inhibitors of the protein tyrosine phosphatases PtpA and PtpB. For these two targets, a fragment-based approach was adopted in order to find low molecular weight inhibitors as chemical “starting points” for further elaboration and development. From this screen, compounds 1 and 2 were found to inhibit PtpA with Ki values of 1.4 and 1.6 µM, respectively9,10 (see Figure 2). Compound 3 was found to bind to PtpB with a Ki of 1.3 µM with subsequent structurebased “lead hopping” resulting in the identification of analogue 4 which bound to this enzyme with a Ki value of 0.69 µM11, 12.

Phenotypic screens. Despite numerous biochemical target screening campaigns (by multiple large pharmaceutical organizations), success in identifying attractive, advanceable chemical series for infectious disease indications has been limited13. As a result, there is a pronounced trend toward returning to the phenotypic screening strategies which have proven successful in the past14. It is important to recognize that all current TB drugs were either identified by or derived from M.tb (or surrogate) whole cell screening “hits”. A recent increase in commitment, resources, and access to large pharmaceutical company infrastructures for TB drug discovery has provided expanded opportunities for high throughput whole cell screens of pharma collections against M.tb (or surrogate mycobacteria). In addition, genomic/proteomic mechanisms now exist to help “deorphan” whole cell actives, and thus elucidate new, viable, pharmacologically validated M.tb targets. Work in this field can be further supported by computational/structure-based drug design (SBDD) efforts to help identify improved, next generation agents.



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The success of this whole cell screening approach may be best illustrated with the discovery of the diarylquinoline TMC207 (bedaquiline 5, see Figure 3)15. Discovered by researchers at Tibotec (Janssen), this compound was prepared by medicinal chemistry optimization of a lead originally identified from a corporate collection screening campaign against M. smegmatis, and was subsequently developed as an antituberculosis agent. It is highly active against both drug sensitive and drug resistant strains of M. tuberculosis, and was recently approved by the Food and Drug Administration (FDA) for MDR TB16. Similar success has been seen with the development of the nitroimidazoles PA-824 617 and OPC-67683 (Delamanid) 718, and the diamine SQ-109 819 which are also in Phase II clinical trials. Other recent examples of novel active series in early discovery phase include the benzothiazinones (BTZ) 920 identified from the NM4TB program, and the 2aminothiazole-4-carboxylates 10a-10b (ATC) compounds from the TBD-UK consortium21. Additionally, a number of recent screening campaigns carried out with M. tuberculosis H37Rv have led to the identification of a large number of potential new lead compounds with indications of both whole cell antituberculosis activity and selectivity against representative eukaryotic cell lines22,23. One such example was the identification of macrolide derivatives 11 as reported by Falzari et al.24. Through the support of the Bill and Melinda Gates Foundation (BMGF), efforts are also underway to conduct M.tb phenotypic screens under “persistent” conditions (i.e. low oxygen, low nutrient, high reactive oxygen species (ROS), NO exposure, etc.). In such a manner, it is envisioned that novel chemotypes may be identified which could prove effective against non-replicating, persistent M.tb subpopulations25.

GSK antimycobacterial phenotypic

screen: hit

identification. Recently, researchers at

GlaxoSmithKline (GSK) conducted a large (~2M compound) phenotypic screening campaign in hopes of identifying novel antimycobacterial chemical series for further TB pursuit26. Like several other


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large pharmaceutical companies, GSK opted to use M. bovis BCG as an M. tuberculosis surrogate for primary screening purposes. They initially selected and tested a representative compound collection subset of ~20,000 compounds to confirm the use of M. bovis BCG as a predictor for M.tb activity overall. Compounds belonging to this subset were characterized by good membrane permeability, GSK available stocks, and reasonable overall “drug-like” properties. Compounds were assayed against both M. bovis BCG (“BCG”) and M. tuberculosis H37Rv (“H37Rv”) in two independent experiments at a single 10 µM concentration. Similar hit rates were found in both experiments: 127 primary hits were identified as inhibitors of BCG growth, and 112 hits as inhibitors of H37Rv. Retesting both sets of hits confirmed 88 of the compounds detected in the BCG screen (69%) and 73 of the primary hits from H37Rv (65%) as active.

The GSK team then evaluated the full 2M compound collection against BCG with an initial cut-off of >50% growth inhibition at a single, 10 µM concentration. This resulted in the identification of ~62,000 primary hits.

A series of in silico filtering (removing known antibacterials) and

physicochemical property clustering operations reduced this original number to ~15,000 hits. As depicted in Figure 4 (courtesy of L. Ballell, GSK), these hits were further interrogated to determine their minimum inhibitory concentration (MIC) values vs. M. bovis BCG, as well as their HepG2 cytotoxicity potential (IC50’s). Only those compounds which demonstrated M. bovis BCG MIC’s 50 were taken forward for M.tb MIC determinations. In this manner, this set of 850 “BCG” hits was reduced to 177 “M.tb” hits which had M.tb MIC95’s 50.

GSK antimycobacterial phenotypic screen: hit characterization and expansion. To further elucidate the lead progression potential of the antitubercular hit list, the GSK team selected a range of chemical clusters based on their intrinsic, in vitro antimycobacterial activities (M.tb H37Rv MIC’s < 1 µM), and the number of structural analogues represented within the full hit list. This provided seven



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chemical clusters (see Figure 5), representatives of which were selected for further characterization. Biological and physicochemical profiling consisted of MIC determination against various M. tuberculosis strains, characterization against intracellular growth, activity profiling against an internal panel of eight different bacteria for early assessment of the potential for wide-spectrum activity, aqueous solubility measurement, evaluation of cytotoxicity, artificial membrane permeability studies, cytochrome P450 (CYP) inhibition profiling against a representative panel of isoforms,27 and early in vitro metabolic studies to establish the stability of the hit structures in mouse and human microsomal fractions (CLint and t½ values). Working with the TB Alliance, GSK remains actively engaged in advancing two of these series (THPP’s and Spiros) through lead optimization medicinal chemistry.

Future prospects “De-orphaning” whole cell screening hits. Despite the genuine success of identifying active chemical matter through antimycobacterial phenotypic screens, the conversion of such hits into progressible chemical series and ultimately into clinical candidates is hampered by the lack of knowledge regarding the specific biochemical target(s) which such species inhibit and/or upregulate. Recent advances in M.tb genomics/proteomics can provide opportunities to “de-orphan” such screening hits (or advanced analogues derived from initial hits) and thus: (a) aid in further SAR development for a given chemical series, or (b) help identify new, essential, druggable M.tb targets of interest. These new targets can then be exploited further through subsequent dedicated high throughput screens to identify entirely different chemical starting points.

Furthermore, coupling target

identification work with structure-based drug design (SBDD) efforts may facilitate the synthesis of novel, improved compounds/chemical series against such pharmacologically-validated targets of interest.


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The determination of the precise mechanism(s) of action (MOA) of whole cell screening actives remains challenging. The inherent slow cycle time of M.tb, and the diminished viability of mutants lacking essential targets create significant difficulties in the generation of M.tb compound-specific resistant mutants. Additionally, the surfeit of “non-MOA” targets which have recently been identified (e.g. efflux pump substrates, growth media-specific targets, etc.) further complicates this process28. Experience indicates that target identification efforts for pleiotropic TB compounds – while exceedingly effective as therapeutic agents – are increasingly complex and difficult. Finally, the use of M.tb surrogates, while technically more convenient, can lead to the identification of false M.tb targets (i.e. wide range of target binding affinity differences observed between mycobacteria, and even among different M.tb strains29). Despite these challenges, efforts continue on a global level at multiple universities and institutes to: (a) generate and sequence M.tb mutants following increased exposures to novel phenotypic screening hit materials, (b) establish their precise biochemical target(s), and (c) confirm such on-target activities through the generation of knockout (KO), knockdown (KD), and over-expresser (OE) M.tb strains.

Coupling SBDD with M.tb target validation/new lead discovery efforts. Despite world-class diligent efforts by many hundreds of researchers over the past four decades, there are still very few pharmacologically validated TB drug targets to work on. The BMGF TB Drug Accelerator program is attempting to improve the current “barren landscape” by expanding the list of validated M.tb drug targets25. In concert with this genomic/proteomic work, the University of Toronto-based Structural Genomics Consortium (SGC) has proposed to use structure-guided methods to design and generate novel antitubercular drug leads de novo30. Their strategy is to express and purify milligram quantities of such validated protein targets and characterize the interactions with their corresponding phenotypic screening hits (or advanced analogues derived from the original hits). In cases where binding is confirmed, they hope to co-crystallize the target with its bound inhibitor. Furthermore, the SGC plans to expand attractive hit molecules into progressible compound series through



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medicinal chemistry. These new compounds would then be assessed in protein- and cell-based assays to generate data for structure-activity relationship (SAR) analysis.

Additionally, those

analogues with promising SAR and physicochemical properties would be co-crystallized with their targets, and, with co-structures available, an iterative lead optimization cycle would ensue comprising structure-guided design of new molecules, target assays to confirm and characterize target inhibition, cell-based assays to characterize potency of anti-M.tb activity, in vitro ADMET assessment, and finally rodent PK/efficacy testing. The goal of this exercise will be the generation of antitubercular lead molecules which meet the criteria for early drug discovery leads, and which demonstrate proof of concept efficacy in vivo. Further lead optimization of such series may ultimately produce “next generation” antitubercular preclinical candidates.

Conclusions The search for new TB drugs remains a challenging albeit vitally important task. With target-based screening approaches offering few tangible successes, the global TB community has predominantly adopted whole cell screening methods for the primary source of novel, tractable sources of lead molecules/series.

New whole cell screening campaigns conducted under various simulated

physiological conditions should reveal novel chemotypes which are active under chronic, persistent conditions. Improved technologies for de-orphaning phenotypic screening hits hold promise for rigorous evaluations of M.tb-specific biochemical targets (or pathways) through future target-based screens.

Finally, the large-scale preparation of M.tb-specific protein targets may permit the

inclusion of structure-guided drug design approaches to create entirely novel chemical matter for future TB drug development.

Acknowledgements. The author wishes to thank the members of the TB Alliance research staff for their helpful comments and advice in reviewing this paper. In addition, the author would like to


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acknowledge Lluis Ballell, Modesto Remuinan, and David Barros from GSK (Tres Cantos) for their collective assistance in preparing this manuscript.

Autobiography Christopher B. Cooper received his Ph.D. in Organic Chemistry with Professor Paul A. Wender in 1988. From 1988 through 1998, he was engaged in medicinal chemistry research at Pfizer, Inc., at both their Groton, Connecticut, and Sandwich, England, sites. In 1998, Chris joined Bristol-Myers Squibb, Inc., in Princeton, New Jersey, to launch the Lead Synthesis Group spearheading the design, development, and synthesis of novel, drug-like medicinal chemistry arrays. In 2009, he came to the Global Alliance for TB Drug Development (TB Alliance) where he is currently Senior Director, Chemistry, overseeing the Alliance’s global discovery and pre-clinical development chemistry activities. Chris has published over 50 peer-reviewed papers, and is co-inventor on 19 U.S. patents.

Corresponding Author Information: Email: [email protected] Phone: (646) 616-8634

Abbreviations: ADMET: Absorption, distribution, metabolism, excretion, and toxicity ATC: Amino-4-thiazole carboxylic acid BMGF: Bill and Melinda Gates Foundation BTZ: Benzothiazones CLint: Intrinsic clearance CYP: Cytochrome P450 DR: Drug resistant



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DS: Drug sensitive FDA: Food and Drug Administration GSK: GlaxoSmithKline HepG2: HepG2 human liver carcinoma cell line HIV: Human immunodeficiency virus KD: Knockdown KO: Knockout M.bovis BCG: Mycobacterium bovis Bacille de Calmette et Guérin MDR-TB: Multidrug resistant TB MIC: Minimum inhibitory concentration MOA: Mechanism of action M.tb: Mycobacterium tuberculosis NM4TB: New Medicines for TB PK: Pharmacokinetics PtpA: Protein tyrosine phosphatase A PtpB B: Protein tyrosine phosphatase B ROS: Reactive oxygen species SBDD: Structure-based drug design SGC: Structural Genomics Consortium TB: Tuberculosis TDR TB: Totally drug resistant TB THPP: Tetrahydropyrrolopyrimidine WHO: World Health Organization XDR-TB: Extremely drug resistant TB


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25.

Bill

&

Melinda

Gates

Foundation

-

Tuberculosis

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(http://www.gatesfoundation.org/What-We-Do/Global-Health/Tuberculosis), 2013. 26.

Ballell, L.; Bates, R.H.; Young, R.J.; Alvarez-Gomez, D.; Alvarez-Ruiz, E.; Barroso, V.; Blanco, D.; Crespo, B.; Escribano, J.; González, R.; Lozano, S.; Huss, S.; Santos-Villarejo, A.; MartínPlaza, J.J.; Mendoza, A.; Rebollo-Lopez, M.J.; Remuiñan-Blanco, M.; Lavandera, J.L.; PérezHerran, E.; Gamo-Benito, F.J.; García-Bustos, J.F.; Barros, D.; Castro, J.P.; Cammack, N. Fueling Open-Source Drug Discovery: 177 Small-Molecule Leads against Tuberculosis. ChemMedChem 2013, 8, 313-321.

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Kajbaf, M.; Longhi, R.; Montanari, D.; Vinco, F.; Rigo, M.; Fontana, S.; Read, K.D. A Comparative Study of the CYP450 Inhibition Potential of Marketed Drugs Using Two Fluorescence Based Assay Platforms Routinely Used in the Pharmaceutical Industry. Drug Metab. Lett. 2011, 5, 30-39.

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Sharma, U. Current possibilities and unresolved issues of drug target validation in Mycobacterium tuberculosis. Expert Opinion on Drug Discovery 2011, 6(11), 1171-1186, and references cited therein.

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(a) James, J.N.; Hasan, Z.-u.; Ioerger, T.R.; Brown, A.C.; Personne, Y.; Carroll, P.; Ikeh, M.; Tilston-Lunel, N.L.; Palavecino, C.; Sacchettini, J.C.; Parish, T. Deletion of SenX3–RegX3, a key two-component regulatory system of Mycobacterium smegmatis, results in growth defects under phosphate-limiting conditions. Microbiology 2012, 158(11), 2724-2731; (b) Agarwal, N.; Pareek, M.; Thakur, P.; Pathak, V. Functional characterization of EngA(MS), a P-loop GTPase of Mycobacterium smegmatis. PLoS ONE 2012, 7(4): e34571.

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Edwards, A., Univ. Toronto Structural Genomics Consortium (personal communications).


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Figure 2


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Figure 4.


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Figure 5.


Development of Mycobacterium tuberculosis Whole Cell Screening

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