Selective peptide inhibitors of bifunctional thymidylate synthase‐dihydrofolate reductase from Toxoplasma gondii provide insights into domain–domain communication and allosteric regulation

Abstract

The bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) plays an essential role in DNA synthesis and is unique to several species of pathogenic protozoans, including the parasite Toxoplasma gondii. Infection by T. gondii causes the prevalent disease toxoplasmosis, for which TS-DHFR is a major therapeutic target. Here, we design peptides that target the dimer interface between the TS domains of bifunctional T. gondii TS-DHFR by mimicking bstrands at the interface, revealing a previously unknown allosteric target. The current study shows that these b-strand mimetic peptides bind to the apo-enzyme in a species-selective manner to inhibit both the TS and distal DHFR. Fluorescence spectroscopy was used to monitor conformational switching of the TS domain and demonstrate that these peptides induce a conformational change in the enzyme. Using structure-guided mutagenesis, nonconserved residues in the linker between TS and DHFR were identified that play a key role in domain-domain communication and in peptide inhibition of the DHFR domain. These studies validate allosteric inhibition of apo-TS, specifically at the TS-TS interface, as a potential target for novel, species-specific therapeutics for treating T. gondii parasitic infections and overcoming drug resistance.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea

“…Such enzymes can also be found in parasitic protists like trypanosomatids. One example is the bifunctional enzyme dihydrofolate reductase/thymidylate synthase (DHFR-TS), encoded by a single copy gene (dhfr-ts), that plays a role in the reduction of folate, a key cofactor for DNA synthesis [351][352][353]. It has an N-terminal DHFR domain and a C-terminal TS domain, with the two domains separated by a junction peptide of variable size.…”

Virtual screening reveals allosteric inhibitors of the Toxoplasma gondii thymidylate synthase–dihydrofolate reductase

“…7–8 Peptides that bind to the interface between the apo-dUMP TS domains of both Tg and human TS disrupt the organization of the TS/TS interface and thus reduce TS activity. 5, 9 Recent results suggest that the conformational changes that take place in unliganded human TS to allow for peptide binding could also occur TgTS. 9 We therefore reasoned that small drug-like molecules could bind at the TS/TS interface in T. gondii .…”

“…5, 9 Recent results suggest that the conformational changes that take place in unliganded human TS to allow for peptide binding could also occur TgTS. 9 We therefore reasoned that small drug-like molecules could bind at the TS/TS interface in T. gondii . However, the unliganded structure of TgTS-DHFR with a peptide inhibitor at the TS/TS interface has not yet been solved.…”

“…5a, 9 We therefore wished to determine whether the compounds tested displayed a preference for the apo-enzyme relative to the dUMP-bound enzyme. We followed up on compounds that displayed approximately 50% or greater inhibition at a concentration of 100 µM by testing for this kinetic phenotype.…”

Protein science “best paper” awards to Mark Landau and Brian Ziemba

“…Mark's award‐winning article was the result of probing the TS‐DHFR structure and function to aid the design of allosteric inhibitors with increased selectivity. This led to the discovery of peptide inhibitors, which mimic β‐strands at the enzyme's dimer interface, that bind specifically to the apo‐enzyme form of the parasitic TS‐DHFR and inhibit both TS and DHFR activity but do not affect human TS or DHFR.…”