Puneet Saxena

2-Carboxyquinoxalines Kill Mycobacterium tuberculosis through Noncovalent Inhibition of DprE1

Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 μM, Ty38c is bactericidal and active against intracellular bacteria. To investigate its mechanism of action, we isolated mutants resistant to Ty38c and sequenced their genomes. Mutations were found in rv3405c, coding for the transcriptional repressor of the divergently expressed rv3406 gene. Biochemical studies clearly showed that Rv3406 decarboxylates Ty38c into its inactive keto metabolite. The actual target was then identified by isolating Ty38c-resistant mutants of an M. tuberculosis strain lacking rv3406. Here, mutations were found in dprE1, encoding the decaprenylphosphoryl-d-ribose oxidase DprE1, essential for biogenesis of the mycobacterial cell wall. Genetics, biochemical validation, and X-ray crystallography revealed Ty38c to be a noncovalent, noncompetitive DprE1 inhibitor. Structure-activity relationship studies generated a family of DprE1 inhibitors with a range of IC50s and bactericidal activity. Co-crystal structures of DprE1 in complex with eight different quinoxaline analogs provided a high-resolution interaction map of the active site of this extremely vulnerable target in M. tuberculosis.

Structure-Based Selectivity Optimization of Piperidine–Pteridine Derivatives as Potent Leishmania Pteridine Reductase Inhibitors

The upregulation of pteridine reductase (PTR1) is a major contributor to antifolate drug resistance in Leishmania spp., as it provides a salvage pathway that bypasses dihydrofolate reductase (DHFR) inhibition. The structure-based optimization of the PTR1 inhibitor methyl-1-[4-(2,4-diaminopteridin-6-ylmethylamino)benzoyl]piperidine-4-carboxylate (1) led to the synthesis of a focused compound library which showed significantly improved selectivity for the parasites folate-dependent enzyme. When used in combination with pyrimethamine, a DHFR inhibitor, a synergistic effect was observed for compound 5b. This work represents a step forward in the identification of effective antileishmania agents.

Optimization of Peptides That Target Human Thymidylate Synthase to Inhibit Ovarian Cancer Cell Growth

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The d-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.

Hotspots in an Obligate Homodimeric Anticancer Target. Structural and Functional Effects of Interfacial Mutations in Human Thymidylate Synthase

Human thymidylate synthase (hTS), a target for antiproliferative drugs, is an obligate homodimer. Single-point mutations to alanine at the monomer-monomer interface may enable the identification of specific residues that delineate sites for drugs aimed at perturbing the protein-protein interactions critical for activity. We computationally identified putative hotspot residues at the interface and designed mutants to perturb the intersubunit interaction. Dimer dissociation constants measured by a FRET-based assay range from 60 nM for wild-type hTS up to about 1 mM for single-point mutants and agree with computational predictions of the effects of these mutations. Mutations that are remote from the active site retain full or partial activity, although the substrate KM values were generally higher and the dimer was less stable. The lower dimer stability of the mutants can facilitate access to the dimer interface by small molecules and thereby aid the design of inhibitors that bind at the dimer interface.

Alanine mutants of the interface residues of human thymidylate synthase decode key features of the binding mode of allosteric anticancer peptides.

Allosteric peptide inhibitors of thymidylate synthase (hTS) bind to the dimer interface and stabilize the inactive form of the protein. Four interface residues were mutated to alanine, and interaction studies were employed to decode the key role of these residues in the peptide molecular recognition. This led to the identification of three crucial interface residues F59, L198, and Y202 that impart activity to the peptide inhibitors and suggest the binding area for further inhibitor design.

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit Trypanosoma brucei Pteridine Reductase in Support of Early-Stage Drug Discovery.

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained.

Computational Insights into ADAMTS4, ADAMTS5 and MMP13 Inhibitor Selectivity

The results obtained by means of Molecular Dynamics simulations and Multiway Explorative Data Analysis on ADAMTS4, ADAMTS5 and MMP13 complexed with Marimastat and two cis‐1(S)2(R)‐amino‐2‐indanol ligands suggest that determinant characteristics for ligand binding and selectivity among the three enzymes are to be found in the different protein conformation flexibility. Moreover, the role of the TS‐domain in the inhibitor binding to ADAMTS enzymes has been investigated for the first time in this work. The results obtained suggest that the influence of the TS‐domain on the S1′ loop fluctuations of ADAMTS4 and ADAMTS5 could be exploited for the design of therapeutics for chronic osteoarthritis diseases.

Conformational Propensity and Biological Studies of Proline Mutated LR Peptides Inhibiting Human Thymidylate Synthase and Ovarian Cancer Cell Growth

LR and [d-Gln4]LR peptides bind the monomer-monomer interface of human thymidylate synthase and inhibit cancer cell growth. Here, proline-mutated LR peptides were synthesized. Molecular dynamics calculations and circular dichroism spectra have provided a consistent picture of the conformational propensities of the [Pro n]-peptides. [Pro3]LR and [Pro4]LR show improved cell growth inhibition and similar intracellular protein modulation compared with LR. These represent a step forward to the identification of more rigid and metabolically stable peptides.