Previous studies from our lab looked at the
Previous studies from our lab looked at the effect of interfering with crossover helix interactions in TS-DHFR. One study utilized peptide mimetics of the crossover helix to inhibit wild-type TS-DHFR noncompetitively, and with an IC of 230 μM. A second study used a virtual screening approach to identify small molecules capable of binding in a non-active site pocket below the crossover helix, adjacent to the TS domain. In the latter example, the PR619 receptor Flavin Mononucleotide (FMN) was also found to inhibit DHFR activity noncompetitively, though with an improved IC value of 55 μM. Here too, we demonstrate that DHFR activity can be inhibited by disrupting interactions between the crossover helix and DHFR with small molecules aimed at a novel non-active site pocket. Collectively, these efforts help underscore the importance of crossover helix interactions for DHFR catalysis. Additionally, our findings lend support to the use of virtual screening and structure-guided modeling approaches in the discovery of compounds targeting novel binding pockets. Computational modeling proved especially helpful in instructing the design of, compound , which is the first example of a covalent inhibitor designed to target a non-catalytic pocket in TS-DHFR. Targeting non-catalytic cysteine residues is a promising strategy for lead generation and optimization, and is yet to be fully explored in drug discovery efforts aimed at developing -specific inhibitors.
In conclusion, we have conducted a structure-based virtual screen of a novel non-active site pocket of TS-DHFR. Our screen identified compound , which inhibits DHFR by 52%, displays mixed noncompetitive inhibition with respect to dihydrofolate, and is not competitive with respect to NADPH. Furthermore, we conducted an SAR study utilizing derivatives of compound which led to the development of covalent compounds designed to target Cys44. Compound demonstrated time-dependent inhibition through covalent interaction via a disulfide bond and selectively inhibits DHFR, while mutation of Cys44 interferes with binding of with the non-active site pocket. Work is currently underway to obtain co-crystal structures in order to validate our modeling and obtain a better understanding of the interactions between compound and the proposed TS-DHFR non-active site pocket. The discovery of offers an excellent starting point for further lead optimization of covalent inhibitors to the DHFR allosteric site.
This work is supported by NIH Grant (AI083146) to K.S.A., and Training Grant (5T32AI007404-23) to D.J.C.
Introduction According to WHO 2017 report, tuberculosis (TB) is the ninth leading cause of death worldwide and the leading cause from a single infectious agent, ranking above HIV/AIDS. In 2016, there were an estimated 1.3 million TB deaths among HIV-negative people and an additional 374,000 deaths among HIV-positive people. Mycobacterium tuberculosis (Mtb), the causative agent of TB in humans, is a slow-growing acid-fast bacterium with a highly impermeable cell wall. Mtb is an opportunistic pathogen that is able to survive within macrophages in a latent form for decades and reactivates in immune compromised individuals such as those with a concurrent HIV infection.1, 2 The spread of multidrug-resistant (MDR) TB and the emergence of extensively drug-resistant (XDR) TB lead to revitalization of antitubercular drug discovery efforts.3, 4, 5 The discovery of bedaquiline (inhibitor of mitochondrial ATP synthase) and delamanid (inhibitor of mycolic acid biosynthesis) after a gap of more than 40 years offered some relief in treatment of MDR-TB but the two agents have certain pronounced issues like hERG toxicity and ADME issues.3, 6, 7 Also, very few chemical entities (TBA- 354 (nitroimidazole), PBTZ169 (benzothiazinone), and Q203 (imidazopyridine) are in phase I clinical studies. Therefore, there is a significant unmet medical need for safer, more effective TB drugs with different mechanisms.