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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 74078,¹ Basilea Pharmaceutica, Basel, Switzerland²

Received 11 May 2007/ Returned for modification 16 July 2007/ Accepted 29 August 2007

Bacillus anthracis is innately resistant to trimethoprim (TMP), a synthetic antifolate that selectively inhibits several bacterial dihydrofolate reductases (DHFRs) 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 (E. W. Barrow, P. C. Bourne, and W. W. Barrow, Antimicrob. Agents Chemother. 48:4643-4649, 2004). In this investigation, 24 2,4-diaminopyrimidine derivatives, representing a class of compounds with dihydrophthalazine side chains, were screened for their in vitro effects on B. anthracis Sterne and their selectivities for the B. anthracis DHFR. MICs were obtained by a colorimetric (Alamar blue) broth microdilution assay. Purified human recombinant DHFR (rDHFR) and B. anthracis rDHFR were used in a validated enzyme assay to determine the 50% inhibitory concentrations (IC₅₀s) and the selectivity ratios of the derivatives. The MICs ranged from 12.8 to 128 µg/ml for all but nine compounds, for which the MICs were 128 µg/ml. The IC₅₀ values for B. anthracis rDHFR ranged from 46 to 600 nM, whereas the IC₅₀ values for human rDHFR were >16,000 nM. This is the first report on the in vitro inhibitory actions of this class of antifolates against TMP-resistant B. anthracis isolates. 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 caused by TMP-resistant bacteria, such as B. anthracis.

Published ahead of print on 17 September 2007.