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out, for example, using radiolabeled inhibitors or possibly by monitoring any UV-spectral shifts in the
case of aromatic compounds.
B. What We Need to Know before We can Start Structure-Based Drug Design
The review of known integrase inhibitors presented in the previous section demonstrates the paucity of
effective inhibitors reported in the literature. A limited number of structural types have been
investigated, focused heavily on compounds with aromatic ring systems and hydroxy substituents. Most
inhibitors reported to date are only moderately effective in in vitro assays, with IC 50 values residing in
the low μM range (see Table 1).
To build on this knowledge base, and to use known molecules as lead compounds for the development
of more effective inhibitors, it would be extremely valuable to obtain a high-resolution crystal structure
of an integrase-inhibitor complex. We and others are vigorously pursuing this goal. It is not clear if the
lack of success to date is because the inhibitors identified so far manifest their effects in in vitro assays
predominantly at the level of the DNA, or if some aspect of the structure of the HIV-1 integrase core
itself—for example, its mobility in certain regoins—prevents the formation of a tight complex. It is also
possible that binding is weaker to the HIV-1 core domain than to the full-length protein because of
missing enzyme-inhibitor contacts. Co-crystallization attempts would benefit from in vitro studies to
determine relative binding constants as a guide in selecting the most tightly bound inhibitors. It would
also be useful to obtain information on the effect of variables, such as Mn 2+ or Mg 2+, on binding
constants.
It may be futile at this stage to attempt to model the binding of known inhibitors to the catalytic core
domain of HIV-1 integrase in the absence of more complete information. It cannot a priori be assumed
that the site of action of all these inhibitors is the enzyme active site identified by the constellation of
conserved acidic residues. For example, certain very effective nonnucleoside inhibitors of HIV reverse
transcriptase bind not to the enzyme active site, but rather to a small pocket adjacent to it. There are no
obvious structural features of the integrase core—such as the deep trough surrounding active site
residues in the case of HIV protease [61,62]—that can be readily identified as a potential inhibitor
binding site. Furthermore, since part of the region defining the active site of HIV-1 integrase is
disordered in our crystal structure, this prevents a surface rendering of the region around the conserved
acidic residues. It also seems unlikely, given the known differences in active-site geometries between
the HIV and ASV integrase (see Section IV.B), that the structure of the related ASV integrase core
domain by itself would be particularly useful in this regard. As the active form of the enzyme
presumably binds divalent metal ions, it will be important to determine how or if the structure of HIV
integrase changes
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