2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis: Structure–Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization

Abstract

Mycobacterium tuberculosis (MTb) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose MTb whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the ndh encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in MTb was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by ndhA in MTb. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial MTb SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors.

FAD/NADH Dependent Oxidoreductases: From Different Amino Acid Sequences to Similar Protein Shapes for Playing an Ancient Function

“…Currently, some of the described FAD/NADH-dependent oxidoreductases are considered in microbiology a crucial target of antibiotics or chemotherapeutics (i.e., those structurally related to RYL-552, a quinolinic derivative or to SL827, a sulphonamide derivative [10,48,[51][52][53]) because those enzymes play a fundamental role for the ATP synthesis and oxidative pathways in most microorganisms. The lack of NDH-2-orthologous enzymes in Mammalia was retained an important proof of the selective action of potential drug development [10,[54][55][56][57].…”

Oxidative Phosphorylation—an Update on a New, Essential Target Space for Drug Discovery in Mycobacterium tuberculosis

“…There is a proton-translocating type I NADH dehydrogenase (NDH-1) and two non-proton-translocating type II NADH dehydrogenases (NDH-2) [28]. NDH-2 has been chemically validated as a drug target in M. tb [28][29][30]. Unlike NDH-1, it is absent in the mammalian genome, which is an advantage to develop specific inhibitors.…”

“…A 2-mercapto-quinazolinones cluster of hits (1, 2, and 3) was identified in a screening of a commercial diversity library [30]. Compound 1 potently inhibiting M. tb growth in vitro in the low micromolar range without cytotoxicity against HepG2 cells [30].…”

“…A 2-mercapto-quinazolinones cluster of hits (1, 2, and 3) was identified in a screening of a commercial diversity library [30]. Compound 1 potently inhibiting M. tb growth in vitro in the low micromolar range without cytotoxicity against HepG2 cells [30]. Bioenergetic analyses conducted in cells in which the membrane potential was uncoupled from ATP production revealed a decrease in oxygen consumption rates (OCR) in response to the inhibitor, while IMVs showed mercapto-quinazolinonedependent inhibition of ATP production when NADH was the primary electron donor of the respiratory chain.…”

Synthesis of Functionalised Chromonyl‐pyrimidines and Their Potential as Antimycobacterial Agents

“…While effective pharmaceutical agents for TB exist, the increase in drug‐resistant strains of M. tuberculosis has triggered a need for the development of new TB drugs and drug targets for this pathogen. Several groups, including our own, have recently focused on classes of anti‐tubercular agents that function by inhibiting the respiratory type II NADH dehydrogenase (NDH‐II) . NDH‐II is an attractive M. tuberculosis drug target as it is absent from mammalian mitochondria yet is essential for mycobacterial growth .…”