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structure of a candidate would complement the active site of PNP. We used computational chemistry to
estimate the strength of the attractive and repulsive forces between a candidate and the enzyme.
We synthesized only those candidates suggested by chemical intuition and computer simulation to have
high affinity for the target. Then we measured the inhibition of PNP and compared the proposed with the
actual fit. Because modeling programs and expert opinion are imperfect, certain compounds did not
meet expectations. After exploring the reasons for the successes and the failures, we returned to
interactive computer graphics to propose modifications that might increase the effectiveness of drug
candidates.
The resulting compounds were evaluated by determination of their IC 50 values (the inhibitor
concentration causing 50% inhibition of PNP) and by x-ray diffraction analysis using difference Fourier
maps. This iterative strategy—modeling, synthesis, and structural analysis—led us to a number of highly
potent compounds that tested well in whole cells and in animals.
D. Previously Known Inhibitors
At the beginning of our studies several PNP inhibitors had been reported with Ki values in the 10-6 to 10- 7 range, including 8-aminoguanine [11], 9-benzyl-8-aminoguanine [12], and 5'-iodo-9-deazainosine [13].
Acyclovir diphosphate had been shown to have a Ki near 10-8 if assayed at 1 mM phosphate rather than
the more frequently used value of 50 mM phosphate [14]. During our studies, the synthesis of 8-amino-
9(2-thienylmethyl)guanine was reported with a Ki of 6.7 × 10-8 M [15]. Figure 3 illustrates some of these
structures.
Despite the potential benefits of PNP inhibitors and the large number of PNP inhibitors that had been
synthesized, no compound had reached clinical trials. None of these compounds were potent enough to
be useful for therapy and also capable of crossing the cell membrane intact. Although potencies for the
best compounds had affinities 10–100 fold higher than the natural substrate (K m = 20 μM), it is expected
that T-cell immunotoxicity will only occur with very tight binding inhibitors (K i < 10 nM) due to the
high level of in vivo PNP activity and competition with substrate.
II. Crystallography
At the present time, x-ray crystallography is the preferred technique for obtaining the required atomic
resolution structural data. In the late 1970s when this project was first conceived, determining the
structure of a protein was far from routine. The x-ray structural determination occupied a team of
crystallographers led by Steven E. Ealick, then at the University of Alabama at Birmingham through
most of the 1980s.
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