Discovery of selective inhibitors of the Clostridium difficile dehydroquinate dehydratase

Abstract

A vibrant and healthy gut flora is essential for preventing the proliferation of Clostridium difficile, a pathogenic bacterium that causes severe gastrointestinal symptoms. In fact, most C. difficile infections (CDIs) occur after broad-spectrum antibiotic treatment, which, by eradicating the commensal gut bacteria, allows its spores to proliferate. Hence, a C. difficile specific antibiotic that spares the gut flora would be highly beneficial in treating CDI. Towards this goal, we set out to discover small molecule inhibitors of the C. difficile enzyme dehydroquinate dehydratase (DHQD). DHQD is the 3(rd) of seven enzymes that compose the shikimate pathway, a metabolic pathway absent in humans, and is present in bacteria as two phylogenetically and mechanistically distinct types. Using a high-throughput screen we identified three compounds that inhibited the type I C. difficile DHQD but not the type II DHQD from Bacteroides thetaiotaomicron, a highly represented commensal gut bacterial species. Kinetic analysis revealed that the compounds inhibit the C. difficile enzyme with Ki values ranging from 10 to 20 µM. Unexpectedly, kinetic and biophysical studies demonstrate that inhibitors also exhibit selectivity between type I DHQDs, inhibiting the C. difficile but not the highly homologous Salmonella enterica DHQD. Therefore, the three identified compounds seem to be promising lead compounds for the development of C. difficile specific antibiotics.

Figure 1. The high-throughput screen assay used to identify the three cdDHQD inhibitors.

(A). The conversion of 3-dehydroquinate (DHQ) to 3-dehydroshikimate (DHS) by cdDHQD is coupled to saturating concentrations of two downstream enzymes in the shikimate pathway, shikimate dehydrogenase (SDH) and shikimate kinase (SK). The loss of NADPH fluorescence in the SDH-catalyzed step of the reaction provides a measurable, continuous readout for the assay. The presence of SK drives the reversible SDH reaction to completion and increases the dynamic range of the assay. (B) The structure of the three hit compounds and corresponding K_i values.

Figure 2. Fluorescence Thermal Shift demonstrates that Compounds 1–3 bind selectively to cdDHQD.

(A) The effect of 160 µM DHS/compounds 1–3 on the thermal stability of cdDHQD, seDHQD, and btDHQD as determined by FTS. The ΔT_m represents the difference in midpoint thermal denaturation temperature compared to the ligand-less control. (B) FTS experiments show ΔT_m of cdDHQD as a function of compound 1–3 concentration.

Figure 3. NMR demonstrates that Compounds 1–3 bind selectively to cdDHQD.

NMR titration of (A) compound 1, (B) compound 2, (C) compound 3 to cdDHQD. All three compounds demonstrate binding to cdDHQD with K_d values of ∼25 µM for compounds 1 & 2, and ∼65 µM for compound 3. (D) NMR titration of compound 3 to seDHQD reveals much reduced affinity, with a K_d of ∼400 µM binding. In (A) and (B), the red and black lines represent the results from two different resonances.

Figure 4. NMR competition experiments to characterize the binding site of compounds 1–3 on cdDHQD.

WaterLOGSY NMR competition experiment between compounds 1–3 and product DHS in the presence of cdDHQD. The reduced WaterLOGSY signal for DHS in the presence of the compounds indicate that the compounds compete with DHS for binding to cdDHQD.

Figure 5. STD NMR experiments map the binding of compounds 2 and 3 on cdDHQD.

The relative proximity of compounds’ carbons to cdDHQD atoms based on STD NMR data is shown.