4-Aminoquinolone Piperidine Amides: Noncovalent Inhibitors of DprE1 with Long Residence Time and Potent Antimycobacterial Activity

Abstract

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.

The 8-Pyrrole-Benzothiazinones Are Noncovalent Inhibitors of DprE1 from Mycobacterium tuberculosis

“…Of note, these compounds are the first non-nitro-benzothiazinones that demonstrate significant mycobacterial activity in vitro. Several noncovalent DprE1 inhibitors have been described recently (12,18,20,21,25), and these display MIC values close to those of PyrBTZ01 and PyrBTZ02 presented in this work. In particular, TCA1 (18) and the azaindoles (26) were reported to be efficacious in a mouse model of TB, although those compounds are less potent than BTZ043 or PBTZ169 in vitro (1,3).…”

“…PyrBTZ01 and PyrBTZ02 were then tested in the Amplex Red peroxidase-coupled enzymatic assay with recombinant wild-type M. tuberculosis DprE1 and were found to inhibit the enzyme with IC 50 values of 1.61 M (PyrBTZ01) and 7.34 M (PyrBTZ02) ( Table 2). The IC 50 values were higher than expected based on the relatively low MICs in M. tuberculosis and in comparison with values reported elsewhere for other noncovalent inhibitors such as the best aminoquinolones (IC 50 of 0.02 M and MIC of 0.39 M) (20) and pyrazolopyridones (IC 50 of 0.01 M and MIC of 0.1 M) (21). The favorable MICs observed for the PyrBTZs could be indicative of better uptake, easier access to DprE1, and accumulation in the bacterium, or even a secondary target in M. tuberculosis.…”

“…Genetic studies showed that PyrBTZ resistance in M. tuberculosis stemmed from mutations of the active site Cys387 residue, as was reported earlier for the nitro-BTZ counterparts. This was unexpected, since resistant mutants of M. tuberculosis generated against several other noncovalent DprE1 inhibitors, including TCA1 and carboxyquinoxalines, displayed mutations elsewhere, namely, at amino acid residues Gly15, Tyr314, and Leu386 (12,18,20). However, molecular docking studies show that PyrBTZ inhibitors occupy roughly the same positions as BTZ043 and PBTZ169 in DprE1, with the pyrrole group being close to and likely clashing with Cys387 (Fig.…”

Characterization of DprE1-Mediated Benzothiazinone Resistance in Mycobacterium tuberculosis

“…Covalent inhibitors such as BTZ, the nitroquinoxaline VI-9376 (12), and the nitroimidazole 377790 (13) are nitroaromatic compounds possessing the necessary nitro group required for covalent adduct formation at C387 on DprE1. Noncovalent inhibitors such as TCA1 (14), 1,4-azaindoles (15), pyrazolopyridones (16), 4-aminoquinolone piperidine amides (17), and Ty38c (18) block enzyme activity by forming hydrophobic, electrostatic, and van der Waals interactions with particular residues in the DprE1 active site.…”

The Future for Early-Stage Tuberculosis Drug Discovery

“…For example, BM212, AU1235, C215, THPP, Spiro, SQ109 and NITD-304 target mmpL3 despite having no structural similarity [33,44–48]. DprE1 is targeted by several compound classes including TCA1 [29], 1,4-azaindoles [49], 4-aminoquinoine piperidine amides and pyrazolopyridones [50,51], as well as novel BTZ analogs consisting of a sulfonamide, reverse-amide or ester [52]. Similarly, Q203 was identified in a screen of infected macrophages while imi-dazopyridines were found in a screen of ATP inhibitors under low oxygen conditions, and both compounds target QcrB, an important component of the electron transport chain [27,53].…”