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The vinylogous urea, NSC727447, was proposed to allosterically inhibit ribonuclease H (RNase H) activity of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) by interacting with the thumb subdomain of its non-catalytic p51 subunit. Proximity of the p51 thumb to the p66 RNase H domain implied that inhibitor binding altered active site geometry, whereas protein foot printing suggested a contribution from α-helix I residues Cys-280 and Lys-281. To more thoroughly characterize the vinylogous urea binding site, horizontal alanine scanning mutagenesis between p51 residues Lys-275 and Thr- 286 (comprising α-helix I and portions of the neighboring αH/αI andαI/αJ connecting loops) was combined with a limited vertical scan of Cys-280. A contribution from Cys-280 was strengthened by our observation that all substitutions at this position rendered selectively mutated, reconstituted p66/p51 heterodimers∼45-fold less sensitive to inhibition. An∼19-fold reduced IC50 for p50 mutant T286A coupled with a 2-8-fold increased IC50 when intervening residues were substituted supports our original proposal of p51 α-helix I as the vinylogous urea binding site. In contrast to these allosteric inhibitors, mutant enzymes retained equivalent sensitivity to the natural product α-hydroxytropolone inhibitor manicol, which x-ray crystallography has demonstrated functions by chelating divalent metal at the p66 RNase H active site. Finally, reduced DNA strand-transfer activity together with increased vinylogous urea sensitivity of p66/p51 heterodimers containing short p51 C-terminal deletions suggests an additional role for the p51 C terminus in nucleic acid binding that is compromised by inhibitor binding. [ABSTRACT FROM AUTHOR]

Abstract: Human nuclear uracil DNA glycosylase (UNG2) and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) are the primary enzymes that prevent the incorporation and accumulation of deoxyuridine in genomic DNA. These enzymes are desirable targets for small molecule inhibitors given their roles in a wide range of biological processes ranging from chromosomal rearrangements that lead to cancer, viral DNA replication, and the formation of toxic DNA strand breaks during anticancer drug therapy. To accelerate the discovery of such inhibitors, we have developed a high-throughput approach for directed library synthesis and screening. In this efficient technology, a uracil-aldehyde ligand is covalently tethered to one position of a trivalent alkyloxyamine linker via an oxime linkage, and then the vacant linker positions are derivatized with a library of aldehydes. The resulting triskelion oximes were directly screened for inhibitory activity and the most potent of these showed micromolar binding affinities to UNG2 and dUTPase. [Copyright &y& Elsevier]