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HIV-1 integrase (IN) is a validated therapeutic target for antiviral agents. However, the emergence of viral strains resistant to clinically studied IN inhibitors demands new structure and new mechanism IN inhibitors. Herein, we describe the design and discovery of novel IN inhibitors targeting the catalytic domain as well as its interaction with LEDGF/p75, which is essential for the HIV-1 integration as an IN cofactor. By merging the pharmacophores of salicylate and catechol, the 2,3-dihydroxybenzamide (5a) was identified as a new scaffold to inhibit the strand transfer reaction efficiently. Further structural modifications on the 2,3-dihydroxybenzamide scaffold revealed that the heteroaromatic functionality attached on the carboxamide portion and the piperidin-1-ylsulfonyl substituted at the phenyl ring are beneficial for the activity, resulting in a low micromolar IN inhibitor (5p, IC50 = 5 μM) with more than 40-fold selectivity for the strand transfer over the 3′-processing reaction. More significantly, this active scaffold remarkably inhibited the interaction between IN and LEDGF/p75 cofactor. The prototype example, N-(cyclohexylmethyl)-2,3-dihydroxy-5-(piperidin-1-ylsulfonyl) benzamide (5u) inhibited the IN-LEDGF/p75 interaction with an IC50 value of 8 μM. Based on the molecular modeling, the mechanism of action was hypothesized to involve the chelation of the divalent metal ions inside the IN active site. And the inhibitor of IN-LEDGF/p75 interaction was properly bound to the LEDGF/p75 binding site in IN protein. This work provided a new and efficient approach to evolve novel HIV-1 IN inhibitors from rational integration and optimization of previously reported inhibitors.

The diketo acid (DKA) class of HIV-1 integrase (IN) inhibitors is thought to function by chelating divalent metal ions on the enzyme catalytic site. However, differences in mutations conferring resistance to various DKA inhibitors suggest that multiple binding orientations may exist. In order to facilitate identification of DKA binding sites, a series of photoactivable analogues of two potent DKAs was prepared as novel photoaffinity probes. In cross-linking assays designed to measure disruption of substrate DNA binding, the photoprobes behaved similarly to a reference DKA inhibitor. Molecular modeling studies suggest that such photoprobes interact within the IN active site in a manner similar to that of the parent DKAs. Analogues Ia-c are novel photoaffinity ligands useful in clarifying the HIV-1 binding interactions of DKA inhibitors.

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