W0015

Analysis of Human and Pneumocystis carinii Dihydrofolate Reductase Complexes With a Novel Tetrahydroquinazoline Antifolate. V. Cody, J.R. Luft and W. Pangborn, Hauptman-Woodward Medical Research Institute, Buffalo, NY 14203.

As part of a program to understand the role of active site residues on selectivity and specificity of inhibition of dihydrofolate reductase (DHFR), we report structural data for the first example of a tetrahydroquinazoline antifolate, (6R, 6S)-2,4-diamino-6-(1-indolinomethyl)-5,6,7,8-tetrahydro- quinazoline (18) (Gangjee et al, J. Med. Chem., 1995), crystallized as a ternary complex with NADPH with human (h) DHFR (1.9A resolution), and as a binary complex with Pneumocystis carinii (pc) DHFR (2.3A resolution). Compound 18 was synthesized and tested as a 6R, 6S racemic mixture and conformational restriction about the N10-C1’ bond was achieved by formation of an indoline ring system. DHFR inhibition data (IC50) showed a greater selectivity for Toxoplasma gondii (tg) DHFR than for pcDHFR. Structural analysis of the human ternary DHFR complex revealed preferential binding for the 6S-equitorial racemate with the indoline ring benzene moiety in contact with the conserved hydrophobic residues Leu-22 and Pro-61 (h numbering). These data also showed the indoline ring occupies an alternate conformation (75, 25% occupancy) that places the indoline ring near Phe-31. This orientation of the indoline ring differs from that observed for the trimethoxybenzyl ring of the parent compound, trimetrexate, in the binary complex with hDHFR. The binding orientation of 18 is similar in the pcDHFR binary complex that has an Ile at the equivalent position Phe-31. Analysis of the binding of the 6R racemate of 18 reveals that the indoline ring makes unfavorable contacts in the active and implies that the 6S racemate is biologically more active. Comparison of the h and pcDHFR structures shows that there is a change in sequence from Asp-21 (h) to Ser-24 (pc), as well as a shift in this loop conformation that makes the active site more constrained in the hDHFR structure. These data provide insight into the role of changes in sequence and conformation on inhibitor binding preferences that can be exploited in the design of novel antifolates. Supported in part by GM51670 (VC).