Theme 1: Target Discovery

Theme 1: Bacterial Target Discovery

Theme 1 concentrates on producing druggable targets that have been validated both in vitro and in vivo and that show bactericidal activity against dormant mycobacteria. Using genetic and chemical approaches within TB persistence models, the team works out the molecular basis of mycobacterial dormancy and pinpoints new bacterial targets needed for the survival of actively growing and dormant mycobacteria. The theme also assesses existing candidate targets and builds target-based whole-cell and biochemical assays that support lead finding and optimisation carried out under Theme 2. Alongside this, Theme 1 advances knowledge of mycobacterial biology and of how the host responds to infection.

Theme 1 Lead: A/Prof. Thomas Dick, Department of Microbiology, National University of Singapore (NUS), Singapore

Projects

DHFR inhibitors

Project lead: Wassihun Wedajo Aragaw

Dihydrofolate reductase (DHFR) is a central enzyme of the folate metabolic pathway, catalysing formation of the reduced folate cofactor required to synthesise thymidine, purines, methionine, glycine and serine. Despite being a well-studied target, no DHFR inhibitor has yet been developed for TB treatment. This project sets out to find potent and selective inhibitors of M. tuberculosis DHFR by screening a triazaspiroalkene scaffold library focused on DHFR.

Selected publication: 1,3,5-triazaspiro[5.5]undeca-2,4-dienes as selective Mycobacterium tuberculosis dihydrofolate reductase inhibitors with potent whole cell activity. Eur J Med Chem. 2018 Jan 20;144:262-276.

Pyrazinoic Acid and Pyrazinamide

Project lead: Pooja Gopal

Adding pyrazinamide (PZA) to TB therapy cut treatment duration from 9–12 months down to 6 months while keeping relapse rates low. Why PZA sterilises so effectively in patients was long unexplained, given its weak in vitro potency (MIC = 30–100 µg/mL). PZA is a prodrug converted to its active form, pyrazinoic acid (POA), by the bacterial amidase PncA and by the host. This project defined the mechanisms through which Mtb resists POA both in vitro and in vivo.

How does POA work? The team identified aspartate decarboxylase PanD, an enzyme needed for coenzyme A biosynthesis, as the first genetically, metabolomically and biophysically validated target of PZA. Current work aims to clarify the molecular details of PanD inhibition by POA, with the goal of producing a next-generation PZA with stronger treatment-shortening and lesion-sterilising properties.

Selected publications: Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD. ACS Infect Dis. 3(11):807-819. 2017 Nov 10. Pyrazinamide Resistance is Caused by Two Distinct Mechanisms: Prevention of Coenzyme A Depletion and Loss of Virulence Factor Synthesis. ACS Infect Dis. 2016 Sep 9;2(9):616-626.

Membrane-targeting cationic amphiphilic indole derivatives

Project lead: Li Ming

Drug resistance and mycobacterial persistence are the two central challenges in TB treatment. Because membrane composition and function are complex and membrane integrity is essential, membrane-targeting compounds tend to show a low propensity for spontaneous resistance mutations and can kill both actively replicating bacilli and non-replicating persisters. The indole scaffold, a benzene ring fused to a nitrogen-containing pyrrole ring, was previously found to have good antimycobacterial activity (Rao et al). Earlier indole work with A/Prof Go Mei Lin (Department of Pharmacy, NUS) established cationic amphiphiles as the structural signature that makes indoles potent and selective antimycobacterials acting through a membrane-targeting mechanism. This project explores a wider range of cationic amphiphilic indoles to identify more potent and more selective membrane-targeting agents with anti-resistance and anti-persistence properties and in vivo efficacy suitable for preclinical development.

Selected publications: Indolylalkyltriphenylphosphonium Analogues Are Membrane-Depolarizing Mycobactericidal Agents. ACS Med Chem Lett. 8:1165-1170. 2017 Oct 9. Indolyl Azaspiroketal Mannich Bases Are Potent Antimycobacterial Agents with Selective Membrane Permeabilizing Effects and in Vivo Activity. J Med Chem. 2018 Jun 25.

Gut microbiota targets lung TB

Project lead: Dereje Abate Negatu

The team recently found that indole propionic acid, a metabolite produced by the gut microbiota, has anti-TB activity in vitro and in mouse infection models, establishing for the first time a functional gut-microbiota–lung-TB axis (Negatu et al., 2018). Work now focuses on defining the antibacterial and host-directed mechanisms of action of this endogenously produced natural product, and on exploring how the gut microbiota influences TB disease.

Selected publication: Whole cell screen of fragment library identifies gut microbiota metabolite indole propionic acid as antitubercular. Antimicrob Agents Chemother. 2018 Feb 23;62(3). pii: e01571-17.

Mycobacterial ATP Synthase

Project lead: Jickky Palmae Sarathy

Bedaquiline, the great new hope for TB treatment, is limited by side effects linked to the drug's high hydrophobicity. A second-generation Bedaquiline is therefore needed that keeps the potency but is less toxic. Through a collaboration spanning microbiology and biophysics, this project tests a novel strategy: targeting the ε-subunit of mycobacterial ATP synthase rather than the drug's main target, the c-subunit of the same protein. Since the ε-subunit is not embedded in the lipid-rich mycobacterial membrane, unlike the c-subunit, targeting it could free the next generation of Bedaquiline from the need to be highly hydrophobic and so make it less toxic.

Theme 1 Collaborators

• Experimental Therapeutics Centre, A*STAR, Singapore — Jeffrey Hill, Anders Poulsen
• Department of Pharmacy, National University of Singapore, Singapore — Mei Lin Go, Brian Dymock
• Singapore Eye Research Institute, Singapore — Roger Beuerman, Shouping Liu
• Genome Institute of Singapore, A*STAR, Singapore — Swaine Chen
• School of Biological Sciences, Nanyang Technological University, Singapore — Gerhard Gruber
• SMART/MIT, Singapore — Peter Dedon
• National University Hospital, Singapore — Catherine Ong, Roland Jureen, Jeanette Teo
• Harvard University, MA, USA — Eric Rubin
• Public Health Research Institute, NJ, USA — Veronique Dartois

Research projects in Theme 1 are carried out in collaboration with Singapore's largest research BSL-3 Core Facility at the National University of Singapore, which houses a TB Research Laboratory licensed for work with a range of mycobacterial strains and animal models.

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