Synthesis of a new compound class of anti-tuberculosis natural products

Abstract

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. It usually affects the lungs (pulmonary TB) but can also affect other parts of the body (extrapulmonary TB). According to the World Health Organisation (WHO) report in 2020, TB is one of the top ten leading causes of death worldwide and the main cause of death from infectious disease, ranked above HIV/AIDS. TB treatment is challenging and requires early diagnosis, effective chemotherapy regimens, and drug-resistance screening. Therefore, development of shorter and simpler drug regimens that are safe, suitable for joint tuberculosis/HIV treatment, and well tolerated is essential. Fragment-based drug discovery (FBDD) that screens small molecules with molecular masses of < 250 Da, has become a reliable approach for early stage drug development. It includes the elaboration of small molecules to produce potent ligands. In this project, the physiochemical properties of 39 TB drugs (both marketed and drugs in the development pipeline) and 1271 synthetic compounds reported in 40 publications from 2006 to early 2020 were analysed. A new TB space of physiochemical properties was proposed in this thesis that may provide more appropriate guidelines for design of anti-TB drugs. The putative TB space includes limited ranges for three physiochemical properties: MW ≤ 700, -4 ≤ cLog P ≤ 3, and 30 ≤ PSA ≤ 140 Å. This guideline was consequently used to analyse compounds synthesised in this project. One desirable fragment was selected for further elaboration based on the result of a previous screening of a fragment library against TB proteins by Native Mass Spectrometry. Different analogues of the desired fragments were synthesised with moderate to good yields and screened against two tuberculosis proteins (BirA and Rv3267) by Native Mass Spectrometry. The method is based on the observation and evaluation of protein-ligand complexes to a target protein. The hits were categorised as weak, medium, and strong binders according to the ratio of the protein-ligand peak to the total protein in the mass spectrum. SAR analysis revealed that variation of the substitutions affects the binding affinity. Activity of the hits were also tested in vitro against Mycobacterium smegmatis (M. smegmatis) strain. Screening of a library of 600 compounds against a TB protein (phenylalanyl t-RNA synthetase) was performed by Native Mass Spectrometry and hits were identified. For a specific target, observation of the peaks corresponding to one ligand bound to the protein indicates binding to the same site (competitive), while observation of the ternary complex peak shows that two ligands could bind to different sites of the protein simultaneously (non-competitive). In this screening, some ternary complexes were detected in the mass spectra where two compounds could bind to different binding sites of the protein at the same time. One hit (RQ202049) showed 100% activity in four concentrations of 25, 50, 100 and 300 μM in vitro against M. smegmatis that makes it a possible lead compound. In addition, a library of 1630 compounds (with molecular weight of more than 250 Da) and 330 fragments (with molecular weight of less than 250 Da) were screened against a cancer protein (DnaJ fusion of Protein Kinase A) and hits were identified. In some mass spectra, ternary complexes were also detected which means two compounds could bind to different binding sites (or domains) of the protein.