A fragment-based approach to the identification of novel ketopantoate reductase inhibitors
thesisposted on 06.02.2017, 05:47 by Vom, Amelia
Pantothenate (Vitamin B5) is a key precursor to coenzyme A, an essential cofactor for fatty acid biosynthesis and other metabolic processes. The pathway for pantothenate biosynthesis is only present in microorganisms and plants, suggesting it may be a target for developing novel antimicrobial agents. Ketopantoate reductase (KPR), a 34 kDa protein, is a key enzyme on this pathway, catalysing the NADPH-dependent reduction of ketopantoate to pantoate. In this study, a fragment-based lead design (FBLD) approach was used to identify small molecule inhibitors of KPR. The expression and purification of labelled and unlabelled KPR was optimised to give high yields suitable for screening and kinetic studies. NMR based screening of 500 fragments using STD-NMR identified 47 hits that were validated by 1H/15N-HSQC NMR, resulting in 14 confirmed fragment hits, correlating to a hit rate of 3%. These hits were assessed for inhibitory activity, with 4 compounds showing an IC50 < 500 μM. The fragment hits were prioritised based on ligand efficiency (the quality of binding interactions between the fragment and target protein) and tractability for synthetic elaboration and derivatisation. Preliminary SAR was established for three series by testing simple analogues of the initial hit. Further analogues based on the scaffolds - sulfonamide, thienopyrrole and phenylthiophene were designed and synthesised to optimise binding to the active site and improve the potency. Binding locations of the fragment hits were identified using 1H/15N-HSQC NMR based on the assigned backbone resonances of E. coli KPR. This thesis also describes the risks of false positives in fragment-based identification of KPR inhibitors. The lead series were identified to be inhibiting through a non-specific mechanism, which was confirmed to be aggregation based, by comparison with literature studies of aggregating compounds. As a result, an NMR-based fragment screening approach and enzyme assay was developed to overcome many of the challenges posed by false positives. Post elimination of false positives, a number of fragment hits has been confirmed to inhibit KPR activity in an in vitro assay.