IN VITRO EFFICACY OF NEW ANTIFOLATES AGAINST TRIMETHOPRIM-RESISTANT BACILLUS ANTHRACIS

Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, Oklahoma 74078, Basilea Pharmaceutica, Basel, Switzerland

^* To whom correspondence should be addressed. Email: bill.barrow{at}okstate.edu.

	Abstract

Bacillus anthracis is innately resistant to trimethoprim (TMP), a synthetic antifolate that selectively inhibits several bacterial dihydrofolate reductases (DHFR) but not human DHFR. Previously, we were able to confirm that TMP resistance in B. anthracis (MIC >2,048 µg/mL) is due to the lack of selectivity of TMP for the B. anthracis DHFR (2). In this investigation, twenty-four 2,4-diaminopyrimidine derivatives, representing a class with dihydrophthalazine side chains, were screened for their in vitro effect on B. anthracis Sterne and selectivity for the B. anthracis DHFR. Minimal inhibitory concentrations (MIC) were obtained using a colorimetric (alamarBlue^TM) broth microdilution assay. Purified recombinant human (rDHFR) and B. anthracis rDHFR, were used in a validated enzyme assay to determine IC₅₀ values and selectivity ratios for the derivatives. The MICs ranged from 12.8 to 128 µg/mL for all but nine for which the MIC was 128 µg/mL. The IC₅₀ values for B. anthracis rDHFR ranged from 46-600 nM, whereas the IC₅₀ values for human rDHFR were >16,000 nM. This is the first report on the in vitro inhibitory action for this class of antifolates against TMP resistant B. anthracis. The selective inhibition of B. anthracis rDHFR and the in vitro activity against B. anthracis demonstrate that members of this class of compounds have the potential to be developed into clinically important therapeutic choices for the treatment of infections by TMP-resistant bacteria such as B. anthracis.