Structural Studies on Bioactive Compounds. 30. Crystal Structure and Molecular Modeling Studies on the Pneumocystis carinii Dihydrofolate Reductase Cofactor Complex with TAB, a Highly Selective Antifolate^,,

Abstract

The crystal structure of the ternary complex of NADPH, the potent antifolate [2, 4-diamino-5-¿3-[3-(2-acetyloxyethyl)-3-benzyltriazen-1-yl]-4 -chloroph enyl¿-6-ethylpyrimidine] (TAB, 1) and Pneumocystis carinii dihydrofolate reductase (pcDHFR), refined to 2.1 A resolution, reveals that TAB binds similar to the antifolates trimethoprim and methotrexate. These data also reveal multiple conformations for the binding geometry of TAB with two preferred orientations of the acetyloxy and benzyl groups that results from a 180 degrees rotation about the N2-N3 triazenyl bond. The methyl of the acetyloxy and benzyl ring of TAB probes large hydrophobic regions of the p-aminobenzoyl folate binding pocket of the active site, in particular the region near Phe69, which is unique to the pcDHFR sequence. These results confirm prior molecular modeling investigations of the binding of TAB to pcDHFR that identified four low-energy binding geometries, two involving rotations about the terminal N(2)-N(3) triazenyl linkage and two involving atropisomerism about the pivotal pyrimethamine-phenyl bond. The primary differences in the molecular dynamics (MD) models and those observed in this crystal complex result from small conformational changes in active-site residues on energy minimization. However, two MD models place the acetyloxy and benzyl ring groups in a region of the active site between the cofactor-binding region and the p-aminobenzoyl folate pocket; an orientation never observed in any DHFR crystal structure to date. These conformers interact with solvent near the enzyme surface and are probably not observed due to the loss of specific hydrogen bonds with the enzyme. The high species pcDHFR selectivity of TAB could be the result of ligand flexibility that enables multiple binding orientations at the enzyme active site. Further modification of the acetyloxy region of TAB could increase its potency and selectivity for pcDHFR.

Crystal Structure of Bacillus anthracis Dihydrofolate Reductase with the Dihydrophthalazine-Based Trimethoprim Derivative RAB1 Provides a Structural Explanation of Potency and Selectivity

“…This allows RAB1 to more easily conform to the binding site. Ligand flexibility has previously been touted as a predictor of selectivity (17).…”

Pneumocystis carinii: Genetic Diversity and Cell Biology

“…Despite this primary sequence difference, both DHFR enzymes showed similar K M values for dihydrofolate and NADPH and similar inhibition profiles with a variety of antifolate inhibitors. The crystal structure of the interaction of purified recombinant DHFR with antifolate compounds has been resolved, and domains important in the inhibitory activity of the compound have been identified (Cody et al, 2000). Correlation of structure-activity predictions with results of inhibitory studies of a wide array of DHFR inhibitors should lead to more potent and selective inhibitors of P. carinii (and specifically P. carinii f. sp.…”

Isolation of Rat Dihydrofolate Reductase Gene and Characterization of Recombinant Enzyme

“…Since immunosuppressed patients and those with AIDS are severly affected by these pathogens, efforts have been focused on the design of antifolates that are selective against P. carinii DHFR (pc DHFR) and T. gondii (or tgDHFR) (12, 18, 22-24, 27, 28, 33, 42, 44, 48). In most of these studies, antifolate selectivity reported as a ratio of 50% inhibitory concentrations (IC 50 s) from two DHFR species was measured against crude DHFR preparations from rat liver (44,47,48).Evidence from sequence analysis and three-dimensional crystal structures of DHFR from many species shows that there is a high degree of conservation at the primary sequence and structural level among DHFRs (8,[10][11][12][13][14]17,19,29,34,36,37,41,43,50,53,55). However, kinetic and biochemical characterization data reveal differences in the mechanism of action that result in significant species specificity by selected inhibitors (15-17, 21, 25, 26, 31, 36, 39, 43).…”

“…Evidence from sequence analysis and three-dimensional crystal structures of DHFR from many species shows that there is a high degree of conservation at the primary sequence and structural level among DHFRs (8,[10][11][12][13][14]17,19,29,34,36,37,41,43,50,53,55). However, kinetic and biochemical characterization data reveal differences in the mechanism of action that result in significant species specificity by selected inhibitors (15-17, 21, 25, 26, 31, 36, 39, 43).…”

“…For this reason, DHFR has been studied extensively and many antifolates have been synthesized and tested as potential candidates for drugs (5, 19-24, 28, 30, 35, 44-48, 52). More recently, antifolates have been shown effective against such opportunistic infectious agents as Pneumocystis carinii and Toxoplasma gondii (1,6,9,10). Since immunosuppressed patients and those with AIDS are severly affected by these pathogens, efforts have been focused on the design of antifolates that are selective against P. carinii DHFR (pc DHFR) and T. gondii (or tgDHFR) (12, 18, 22-24, 27, 28, 33, 42, 44, 48).…”