CRYSTAL STRUCTURE OF PNEUMOCYSTIS CARINII DIHYDROFOLATE REDUCTASE COFACTOR COMPLEX WITH TAB, A POTENT, PC-SELECTIVE ANTIFOLATE
N. Galitsky1, V. Cody1, D. Rak1, J.R. Luft1, W. Pangborn1, S.F. Queener2 M.F.G. Stevens3, C. Laughton3, and D. Chan3
1Hauptman-Woodward Medical
Research Institute, Inc., 73 High St., Buffalo, New York 14203,
USA
2Department of
Pharmacology and Toxicology, Indiana University, Indianapolis, IN
46202, USA.
3University of Nottingham, Nottingham,
UK NG7 2RD
Keywords: Dihydrofolate reductase,
Pneumocystis carinii, antifolate, selectivity, structure-based
drug design
To treat the pneumonia caused by opportunistic
infectious agents, a major cause of mortality among patients with
AIDS, the design of antifolate agents with selectivity against
dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) has
been the focus of many antifolate synthetic studies1. As part of
our program to study inhibitor-bound DHFR complexes, we report
the crystal structure determination of the ternary complex of
pcDHFR with NADPH and
2,4-diamino-5-[3-[2-(acetyloxy)ethyl]-3-benzyltriazen-1-yl]-4-chlorophenyl]-6-ethylpyrimi-
dine (TAB), one of the most potent and pcDHFR selective
antifolates reported. The IC50 for pcDHFR is 0.17 mM with a
selectivity index of 114 against rat liver DHFR2. Data were
collected at room temperature on an RaxisIIc area detector system
to 2.1A resolution for the monoclinic P21 crystals ( a = 37.481,
b = 43.137, c = 61.266 A,b = 94.74o) and refined to R = 0.204. Analysis
of the difference electron density indicate more than one
conformer for TAB in the active site of pcDHFR. The
2,4-diaminopyrimidine ring interacts with Glu-32 in the same
manner as other antifolates3 and the p-chlorophenyl ring torsion
angle is near 60o. There are two alternate conformers for the
3'-benzyltriazenyl group in which the benzyl and acetoxy groups
switch places. One conformer places the benzyl ring in a
hydrophobic pocket between Phe-36 and Phe-69, with the acetyloxy
group near Lys-30, and the second orientation positions the
benzyl group near Ile-33, Pro-66 and Phe-69, with the acetoxyl
group near Lys-37. While the benzyl ring occupies hydrophobic
pockets in both orientations, there are no direct hydrogen bonds
involving the keto function of the acetyl group, except through
water mediation. Modeling studies of the binding interactions in
the DHFR active site using molecular dynamics simulations were
also carried out. These data showed four low energy binding
models of which only two are in close agreement with this crystal
structure determination. Comparison of structures of both human
and pcDHFR suggest that the increased affinity for pcDHFR could
result from sequence changes in the active site region. For
example, interactions made by Lys-30, Ile-33, Lys-37 and Phe-69
with TAB in pcDHFR are replaced by Arg-28, Phe-31, Gln-35 and
Asn-64 in the human structure. These changes, if mimicked in the
rat liver enzyme, could play a key role in enhancing pcDHFR
selectivity of this antifolate.
(Supported by GM-51670 (VC) and N01-AI-35171
(SFQ)).
1. S.F. Queener, J. Med. Chem. 38, 4739-4759 (1995).
2. M.F.G. Stevens, K.S. Phillip, D.L. Rathbone, D.M. O'Shea, S.F. Queener, C.H. Schwalbe and P.A. Lambert, J. Med. Chem. 40, 1886-1893 (1997).
3. V. Cody, N. Galitsky, J.R. Luft, W. Pangborn, A. Gangjee, R. Devraj, S.F. Queener & R.L.
Blakley, Acta Cryst. D53, 638-649 (1997).