A Phenotypic Based Target Screening Approach Delivers New Antitubercular CTP Synthetase Inhibitors

Abstract

Despite its great potential, the target-based approach has been mostly unsuccessful in tuberculosis drug discovery, while whole cell phenotypic screening has delivered several active compounds. However, for many of these hits, the cellular target has not yet been identified, thus preventing further target-based optimization of the compounds. In this context, the newly validated drug target CTP synthetase PyrG was exploited to assess a target-based approach of already known, but untargeted, antimycobacterial compounds. To this purpose the publically available GlaxoSmithKline antimycobacterial compound set was assayed, uncovering a series of 4-(pyridin-2-yl)thiazole derivatives which efficiently inhibit the Mycobacterium tuberculosis PyrG enzyme activity, one of them showing low activity against the human CTP synthetase. The three best compounds were ATP binding site competitive inhibitors, with K values ranging from 3 to 20 μM, but did not show any activity against a small panel of different prokaryotic and eukaryotic kinases, thus demonstrating specificity for the CTP synthetases. Metabolic labeling experiments demonstrated that the compounds directly interfere not only with CTP biosynthesis, but also with other CTP dependent biochemical pathways, such as lipid biosynthesis. Moreover, using a M. tuberculosis pyrG conditional knock-down strain, it was shown that the activity of two compounds is dependent on the intracellular concentration of the CTP synthetase. All these results strongly suggest a role of PyrG as a target of these compounds, thus strengthening the value of this kind of approach for the identification of new scaffolds for drug development.

Unpacking the Pathogen Box—An Open Source Tool for Fighting Neglected Tropical Disease

“…Importantly in this study, PyrG was validated as a target for M. tuberculosis . Identifying the potential of PyrG for M. tuberculosis , Chiarelli and co‐workers screened the phenotypically derived GlaxoSmithKline antimycobacterial compound set for potential inhibitors . From this screen, three 4‐(pyridine‐2‐yl)thiazole‐containing compounds, 295 , 296 and MMV676409 ( 297 ), showed the greatest inhibition of PyrG.…”

“…also identified a handful of antimycobacterial 2,4,6‐trisubstituted pyrimidines, MMV021013 ( 393 ), MMV688888 ( 394 ), MMV688122 ( 395 ) and MMV661713 ( 396 ), without indications of MOA . Compound 393 has also demonstrated promising antitrypanosomal activity, while compound 394 is structurally related to antiplasmodial compound 397 and moderate PyrG inhibitor 398 whose class are discussed above . Compounds 395 is structurally related to human ALK5 inhibitor 399 …”

A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK

“…PyrG was used for target-based screening of antitubercular compound libraries including the GlaxoSmithKline antimycobacterial compound set (GSK TB-set) 8 leading to the discovery of a series of 4-(pyridin-2-yl)thiazole derivatives as PyrG inhibitors 9 . However, except for one compound, these inhibitors also efficiently targeted the human CTP synthetase 9 .

Figure 1 The two prodrugs 7904688 and 797882 as well as 11426026, which is the active metabolite of 797882.

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Screening of Preselected Libraries Targeting Mycobacterium abscessus for Drug Discovery

“…The phenylalanine derivative MMV688845 (34), a CTP synthetase inhibitor, also showed a lower MIC 90 in MHII. The spiropiperidines GSK2200160A and GSK2200157A are also considered to be CTP synthetase inhibitors (34); however, some analogues of this compound class have been shown to target MmpL3 (35). Regardless of the mode of action, both analogues possess selective activity in MHII medium (concentration [c], Յ25 M).…”