The 4-aryl-2-hydroxy-4-oxo-2-butenoic acids and their isosteric tetrazoles are among an emerging class of aryl beta-diketo (ADK)-based agents which exhibit potent inhibition of HIV-1 integrase (IN)-catalyzed strand transfer (ST) processes, while having much reduced potencies against 3'-processing (3'-P) reactions. In the current study, L-708,906 (10e) and 5CITEP (13b), which are two examples of ADK inhibitors that have been reported by Merck and Shionogi pharmaceutical companies, served as model ADK leads. Structural variations to both the "left" and "right" sides of these molecules were made in order to examine effects on HIV-1 integrase inhibitory potencies. It was found that a variety of groups could be introduced onto the left side aryl ring with maintenance of good ST inhibitory potency. However, introduction of carboxylic acid-containing substituents onto the left side aryl ring enhanced 3'-P inhibitory potency and reduced selectivity toward ST reactions. Although both L-708,906 and 5CITEP show potent inhibition of IN in biochemical assays, there is a disparity of antiviral activity in cellular assays using HIV-1-infected cells. Neither 5CITEP nor any other of the indolyl-containing inhibitors exhibit significant antiviral effects in cellular systems. Alternatively, consistent with literature reports, L-708,906 does provide antiviral protection at low micromolar concentrations. Interestingly, several analogues of L-708,906 with varied substituents on the left side aryl ring, while having good inhibitory potencies against IN in extracellular assays, are not antiviral in whole-cell systems.

Among all the HIV-1 integrase inhibitors, the ␤-diketo acids (DKAs) represent a major lead in anti-HIV-1 integrase drug design. These derivatives inhibit the integration reaction in vitro with a strong specificity for the 3-end joining step. They are also antiviral and inhibit integration in vivo. The aim of the present study has been to investigate the molecular interactions between DKAs and HIV-1 integrase. We have compared 5CITEP with one of the most potent DKAs reported by the Merck group (L-708,906) and found that 5CITEP inhibits 3-processing at concentrations where L-708,906 is only active on strand transfer. We also report a novel bifunctional DKA derivative that inhibits 3-processing even more effectively than 5CITEP. The interactions of these inhibitors with the viral DNA donor ends have been studied by performing experiments with oligonucleotides containing defined modifications. We propose that the bifunctional DKA derivative binds to both the acceptor and donor sites of HIV-1 integrase, whereas the monofunctional L-708,906 derivative binds selectively to the acceptor site.

The ␤-diketo acids (DKAs) represent a major advance for anti-HIV-1 integrase drug development. We compared the inhibition of HIV-1 integrase by six DKA derivatives using the wild-type enzyme or the double-mutant F185K/C280S, which has been previously used for crystal structure determinations. With the wild-type enzyme, we found that DKAs could be classified into two groups: those similarly potent in the presence of magnesium and manganese and those potent in manganese and relatively ineffective in the presence of magnesium. Both the aromatic and the carboxylic or tetrazole functions of DKAs determined their metal selectivity. The F185K/C280S enzyme was markedly more active in the presence of manganese than magnesium. The F185K/C280S integrase was also relatively resistant to the same group of DKAs that were potent in the presence of magnesium with the wild-type enzyme. Resistance was caused by a synergistic effect from both the F185K and C280S mutations. Molecular modeling and docking suggested metal-dependent differences for binding of DKAs. Molecular modeling also indicated that the tetrazole or the azido groups of some derivatives could directly chelate magnesium or manganese in the integrase catalytic site. Together, these experiments suggest that DKAs recognize conformational differences between wild-type and the double-mutant HIV-1 integrase, because they chelate the magnesium or manganese in the enzyme active site and compete for DNA binding.