Clostridium difficile DNA Polymerase IIIC: Basis for Activity of Anti-Bacterial Compounds

Abstract

Based on the finding that aerobic Gram-positive antibacterials that inhibit DNA polymerase IIIC (pol IIIC) were potent inhibitors of the growth of anaerobic Clostridium difficile (CD) strains, we chose to clone and express the gene for pol IIIC from this organism. The properties of the recombinant enzyme are similar to those of related pol IIICs from Gram-positive aerobes, e.g. B. subtilis. Inhibitors of the CD enzyme also inhibited B. subtilis pol IIIC, and were competitive with respect to the cognate substrate 2′-deoxyguanosine 5′-triphosphate (dGTP). Significantly, several of these inhibitors of the CD pol IIIC had potent activity against the growth of CD clinical isolates in culture.

DNA replication proteins as potential targets for antimicrobials in drug-resistant bacterial pathogens

“…One of these compounds, 362E, showed potent antimicrobial activity against C. difficile strains, similar to the first-line therapeutics metronidazole and vancomycin (MIC 90 of 4 mg/L) 91 , 92 . Limited in vitro tests indicated that 362E is inactive against certain other Gram-positive anaerobes, 91 but the impact of 362E on the gut microbiota has not been reported to date. 362E protected hamsters from death due to C. difficile similarly to vancomycin and, though recurrent CDI was observed in both treatment groups when animals were treated for 3 days, prolonged treatment with 362E resulted in a reduced recurrence rate 92 .…”

“…Many PolC inhibitors have a broad Gram-positive spectrum and are therefore likely to significantly affect the composition of the microbiota. However, the isolation and purification of the C. difficile PolC as well as polymerases from various other organisms enabled the identification of a series of novel 7-substituted DCBG inhibitors that showed improved potency and specificity against C. difficile 91 . One of these compounds, 362E, showed potent antimicrobial activity against C. difficile strains, similar to the first-line therapeutics metronidazole and vancomycin (MIC 90 of 4 mg/L) 91 , 92 .…”

“…However, the isolation and purification of the C. difficile PolC as well as polymerases from various other organisms enabled the identification of a series of novel 7-substituted DCBG inhibitors that showed improved potency and specificity against C. difficile 91 . One of these compounds, 362E, showed potent antimicrobial activity against C. difficile strains, similar to the first-line therapeutics metronidazole and vancomycin (MIC 90 of 4 mg/L) 91 , 92 . Limited in vitro tests indicated that 362E is inactive against certain other Gram-positive anaerobes, 91 but the impact of 362E on the gut microbiota has not been reported to date.…”

“…For instance, the DNA replication proteins in C. difficile are merely predicted through homology with proteins in other organisms (Table 1), but functional evidence is limited to C. difficile PolC so far 91 . It can be expected that the extensive biochemical and structural characterization of replisome proteins of drug-resistant pathogens might lead to the identification of unique features that can be used to design antimicrobials that specifically target this specific species.…”

“… oriC , chromosomal origin of replication.aPutative; most replication proteins of C. difficile are not characterized (apart from PolC) 91 b

These have been recently characterized 104

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