Isomerization of antimalarial drug WR99210 explains its inactivity in a commercial stock

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>WR99210, a former antimalarial drug candidate now widely used for the selection of <jats:italic>Plasmodium</jats:italic> transfectants, selectively targets the parasite dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing human <jats:italic>dhfr</jats:italic> have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds <jats:bold>1</jats:bold> and <jats:bold>2</jats:bold>). Compound <jats:bold>1</jats:bold> proved effective at low nanomolar concentrations against <jats:italic>Plasmodium falciparum</jats:italic> parasites, whereas compound <jats:bold>2</jats:bold> was ineffective even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of <jats:bold>1</jats:bold> and <jats:bold>2</jats:bold>; however, the compounds displayed differences by thin layer chromatography, reverse phase high performance liquid chromatography, and ultraviolet-visible spectroscopy, indicating important isomeric differences. Structural evaluations by <jats:sup>1</jats:sup>H, <jats:sup>13</jats:sup>C, and <jats:sup>15</jats:sup>N nuclear magnetic resonance spectroscopy confirmed <jats:bold>1</jats:bold> as WR99210 and <jats:bold>2</jats:bold> as an isomeric dihydrotriazine. Induced fit, computational docking models showed that <jats:bold>1</jats:bold> binds tightly and specifically in the <jats:italic>P. falciparum</jats:italic> DHFR active site whereas <jats:bold>2</jats:bold> fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of isomer <jats:bold>2</jats:bold>, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that can promote isomerization. Absorption spectroscopy may serve for assays of the unrearranged and rearranged triazines.</jats:p>