The human immunodeficiency virus (HIV) integrase (IN) must covalently join the viral cDNA into a host chromosome for productive HIV infection. l-Chicoric acid (l-CA) enters cells poorly but is a potent inhibitor of IN in vitro. Using quantitative real-time polymerase chain reaction (PCR), l-CA inhibits integration at concentrations from 500 nM to 10 microM but also inhibits entry at concentrations above 1 microM. Using recombinant HIV IN, steady-state kinetic analyses with l-CA were consistent with a noncompetitive or irreversible mechanism of inhibition. IN, in the presence or absence of l-CA, was successively washed. Inhibition of IN diminished, demonstrating that l-CA was reversibly bound to the protein. These data demonstrate that l-CA is a noncompetitive but reversible inhibitor of IN in vitro and of HIV integration in vivo. Thus, l-CA likely interacts with amino acids other than those which bind substrate.

L-chicoric acid (L-CA) is a potent inhibitor of HIV integrase (IN) in vitro. In this report, the effects of a glycine to serine mutation at position 140 (G140S) on HIV IN and its effects on IN inhibitor resistance are described. HIV containing the G140S mutation showed a delay in replication. Using real-time polymerase chain reaction, the delay was secondary to a failure in integration. The mutant protein (IN(G140S)) was attenuated approximately four-fold for catalysis under equilibrium conditions compared to wild-type IN (IN(WT)) and attenuated five-fold in steady-state kinetic analysis of disintegration. Fifty percent inhibitory concentration assays were performed with IN inhibitors against both IN proteins in disintegration and strand transfer reactions. IN(G140S) was resistant to both L-CA and L-731,988, a diketoacid. HIV containing the mutation was resistant to both inhibitors as well. The G140S mutation attenuates IN activity and confers resistance to IN inhibitors, suggesting that diketoacids and L-CA interact with a similar binding site on HIV IN.

The human immunodeficiency virus type 1 (HIV-1) is a major health problem worldwide. In this study, 17 analogues of L-chicoric acid, a potent inhibitor of HIV integrase, were studied. Of these analogues, five submicromolar inhibitors of integrase were discovered and 13 compounds with activity against integrase at less than 10 microM were identified. Six demonstrated greater than 10-fold selectivity for HIV replication over cellular toxicity. Ten analogues inhibited HIV replication at nontoxic concentrations. Alteration of the linkages between the two bis-catechol rings, including the use of amides, mixed amide esters, cholate, and alkyl bridges, was explored. Amides were as active as esters but were more toxic in tissue culture. Alkyl and cholate bridges were significantly less potent against HIV-1 integrase in vitro and were inactive against HIV-1 replication. Two amino acid derivates and one digalloylderivative of L-chicoric acid (L-CA) showed improved selectivity over L-CA against integration in cell culture. These data suggest that in addition to the bis-catechols and free carboxylic acid groups reported previously, polar linkages are important constituents for optimal activity against HIV-1 integrase and that new derivatives can be developed with increased specificity for integration over HIV entry in vivo.

These results demonstrate that integrase genes derived from HIV-infected individuals can differ from reference sequences but these mutations do not result in loss of function, including susceptibility to an integrase inhibitor; therefore, integrase remains an attractive target for antiviral drug design, as mutability appears to be restricted by function.