Mycobacterial drug discovery

Abstract

Mycobacterium tuberculosis is the causative pathogen of the pulmonary disease tuberculosis. Despite the availability of effective treatment programs, there is a global pursuit of new anti-tubercular agents to respond to the developing threat of drug resistance, in addition to reducing the extensive duration of chemotherapy and any associated toxicity. The route to mycobacterial drug discovery can be considered from two directions: target-to-drug and drug-to-target. The former approach uses conventional methods including biochemical assays along with innovative computational screens, but is yet to yield any drug candidates to the clinic, with a high attrition rate owing to lack of whole cell activity. In the latter approach, compound libraries are screened for efficacy against the bacilli or model organisms, ensuring whole cell activity, but here subsequent target identification is the rate-limiting step. Advances in a variety of scientific fields have enabled the amalgamation of aspects of both approaches in the development of novel drug discovery tools, which are now primed to accelerate the discovery of novel hits and leads with known targets and whole cell activity. This review discusses these traditional and innovative techniques, which are widely used in the quest for new anti-tubercular compounds.

Fig. 1. Whole cell phenotypic screening and target identification. A) Drug-to-target drug discovery generally exploits whole cell phenotypic screening, which involves the incubation of a mid-log culture of mycobacteria with a library of compounds at a specified concentration (usually 10–20 μM), in a multi-well plate format to allow for high-throughput. Cell viability can be established by the microplate alamar blue assay, where a non-fluorescent resazurin (blue) is reduced to a fluorescent resorufin (pink) by living cells. B) Following the discovery of an anti-tubercular compound, various methods can be used to establish the target. These include spontaneous resistant mutant generation against the compound followed by (i) WGS to identify resistance conferring mutations, or (ii) changes in gene expression profiles, such as the up-regulation of the target gene. Alternatively, mass spectrometry can be exploited: (iii) in a chemoproteomics approach, inhibitor-bound matrices can be used to pull down competitively binding proteins from a cell lysate; (iv) metabolomic analysis of drug-treated cell culture can be used to identify changes to the metabolome, for example the reduction of a product from an inhibited pathway. In morphological profiling (v), cells are treated with drugs that have known and unknown modes of action and the morphological features analysed. Comparisons of the two groups can reveal target pathways. This list of target identification methods is not exhaustive.

Fig. 2. Drug candidates identified from whole cell HTS. The structures of A) delamanid, B) pretomanid, C) Q203, D) SQ109 and E) bedaquiline are shown, which are currently progressing through clinical trials.

Fig. 3. In silico drug discovery. Structure-based or ligand-based drug discovery involves the computational screening of a compound database against the protein target structure (e.g. active site) or the ligand (e.g. substrate, transition state or product analogues).

Fig. 4. Drug discovery techniques combining drug-to-target and target-to-drug approaches. A) Target-based whole-cell phenotypic screening. In this technique, a compound library is screened against cells over-expressing a target protein (from a mycobacterial vector). Assessing cell viability at a fixed drug concentration enables the comparison of drug sensitivities between strains with and without the over-expression of the target protein and can reveal inhibitor-target engagement. B) Target protein knockdown by CRISPRi. In this approach, target protein expression is reduced by interfering with the transcription of mRNA. Anhydrotetracycline (ATc) regulates the transcription of a small guide RNA (sgRNA) and a catalytically dead endonuclease, dCas9. The sgRNA contains a short complementary sequence to the target gene and a region for dCas9 recognition. A proto-spacer adjacent motif (PAM) is essential for dCas9 binding. The sgRNA–dCas9 complex anneals to the target sequence, adjacent to PAM, blocking transcription by RNA polymerase, and depleting the expression of the target protein. C) Target protein knockdown by protease degradation allows the reduction of a specific protein through targeted degradation. In this specific approach, a degradation tag (DAS) and a barcode are integrated onto the 3′ end of the target gene. ATc drives the expression of SspB, which recognises the DAS tag and shuttles the protein to a Clp protease. In a drug screen, a strain will exhibit increased inhibitor sensitivity if the degraded protein is the drug target. The specific strain can be detected from a mixed culture of hypomorphs through quantitative sequencing of the barcode.