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hydrophobic, composed of residues Ala 116, Phe 200, and Val 217. The phosphate binding site uses
residues Ser 33, Arg 84, His 86, and Ser 220 with the phosphate positioned for nucleophilic attack at C1'
of the nucleoside. The sugar binding site is mostly hydrophobic consisting of residues Tyr 88, Phe 200,
His 257 from one subunit and Phe 159 of the adjacent subunit. This hydrophobic pocket orients the
sugar to facilitate nucleophilic attack by phosphate and subsequent inversion of C1'.
C. Initial Inhibitor Complexes
In order to understand the interaction of inhibitors with the active site residues, the previously known
inhibitors were obtained and crystallographic analyses were carried out. The most important findings
were (1) 8-amino substituents enhance binding of guanines by forming hydrogen bonds with Thr 242
and possibly the carbonyl oxygen atom of Ala 116; (2) substitution by hydrophobic groups at the 9position
of a purine enhances binding through interaction with the hydrophobic region of the ribose
binding site; and (3) acyclovir diphosphate is a multisubstrate inhibitor with the acyclic spacer between
the purine N9 and the phosphate of near optimal length to accommodate these two binding sites. Based
on these results, a number of starting compounds were proposed that incorporated these and other
features predicted to enhance inhibitor binding.
III. Molecular Modeling
Structural information in combination with graphical methods for displaying accessible volume,
electrostatic potential, and hydrophobicity of the active site of the target macromolecule greatly
facilitates the drug design process. Accurate prediction of binding affinities and protein conformational
changes are currently not routinely possible, although significant advances are being made.
Proposed compounds were screened by modeling the enzyme-inhibitor complex using interactive
computer graphics. Macromodel [21] and AMBER [22] based molecular energetics were used along
with Monte Carlo/energy minimization techniques [23] to sample the conformational space available to
potential inhibitors docked into the PNP active site. Methods based on the work of Goodford [24]
employing custom software were also used. Qualitative evaluation of the enzyme-inhibitor complexes
by molecular graphics and semiquantitative evaluation of the interaction energies between the inhibitors
and the enzyme aided in the prioritization of compounds for chemical synthesis.
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