Structure Guided Lead Generation for M. tuberculosis Thymidylate Kinase (Mtb TMK): Discovery of 3-Cyanopyridone and 1,6-Naphthyridin-2-one as Potent Inhibitors

Abstract

M. tuberculosis thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which we discovered two novel classes of inhibitors, 3-cyanopyridones and 1,6-naphthyridin-2-ones, respectively. We describe three cyanopyridone subseries that arose during our hit to lead campaign, along with cocrystal structures of representatives with Mtb TMK. Structure aided optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK. Fragment based lead generation, augmented by crystal structures and the SAR from the cyanopyridones, enabled us to drive the potency of our 1,6-naphthyridin-2-one fragment hit from 500 μM to 200 nM while simultaneously improving the ligand efficiency. Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity against M. tuberculosis. To the best of our knowledge, these compounds are the first reports of non-thymidine-like inhibitors of Mtb TMK.

Elaboration of a proprietary thymidylate kinase inhibitor motif towards anti-tuberculosis agents

“…s.,1H,4H,Ph),1H,Ph),4H,Ph) 7.69 (s,1H,11.18 (s,1H,NH). 13 5-methyl-1-(1-(3-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4(1H,3H)…”

<p>In silico Design and Synthesis of Tetrahydropyrimidinones and Tetrahydropyrimidinethiones as Potential Thymidylate Kinase Inhibitors Exerting Anti-TB Activity Against <em>Mycobacterium tuberculosis</em></p>

“…[18][19][20][21] Two novel classes, 3cyanopyridones and 1.6-naphthyridin-2-ones, were recently identified as MTB-TMK inhibitors via high-throughput screening and structural optimization to improve potency ( Figure 1). 22 These two scaffolds share a common substructure with thymidine, namely, the unsubstituted lactam functional group, which is important for the formation of hydrogen bonds with thymidylate kinase receptors.…”

Targeting tuberculosis using structure-guided fragment-based drug design

“…However, phenotypic screens have not produced a substantial number of hits [2]. Fragment-based approaches have been able to produce potent lead compounds in vitro, some of which show good antimycobacterial activity despite the small number of dedicated studies in TB drug discovery using this methodology [48,49,54]. The fragments provide good starting points for rational compound elaboration that explores the protein hotspots.…”

“…Target-focused drug discovery campaigns performed on this enzyme have identified some inhibitors but all of the compounds obtained were either thymidine monophosphate analogs or contained a thymidine moiety [53]. Naik and colleagues performed a fragment screen using NMR and a biochemical assay that led to identification of new scaffolds inhibiting TMK [54]. They prioritized a 3-cyanopyridone-containing fragment for chemical elaboration (Figure 4a,b).…”

“…The subsequent development of the fragment, supported by X-ray crystal structures of key compounds, allowed them to obtain nanomolar-potency lead compounds against this enzyme that retained high LE. These compounds differed from the substrate analogs and some proved to be active against M. tuberculosis [54] (Figure 4c-f). tuberculosis [57], with known high-affinity binders that show potent activity in vitro and in cell-based assays [58,59].…”