Dicaffeoyltartaric Acid Analogues Inhibit Human Immunodeficiency Virus Type 1 (HIV-1) Integrase and HIV-1 Replication at Nontoxic Concentrations

Abstract

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.

Human Immunodeficiency Virus Type 1 (HIV-1) Integrase: Resistance to Diketo Acid Integrase Inhibitors Impairs HIV-1 Replication and Integration and Confers Cross-Resistance to l -Chicoric Acid

“…In triplicate infections, equal amounts of each virus as determined by RT activity were inoculated onto H9 cells. At g of proteinase K) as described previously (34,49,61). The samples were incubated at 60°C for 1 h and heat inactivated at 95°C for 15 min.…”

“…The samples were incubated at 60°C for 1 h and heat inactivated at 95°C for 15 min. DNA from each sample was amplified by SYBR Green-I PCR as previously described (35,49,61). Primers used were AA55 and M667, which amplify minus-strand strongstop DNA (69); M661 and M667, which amplify products of complete cDNA synthesis (69); and MH535 and MH536, which amplify two-LTR-circle DNA, the products of failed integration (8).…”

“…DNA from each standard and each cell lysate sample was amplified first with Alu (5Ј-GCCTCCCAAAGTGC TGGGATTACAG-3Ј) (8) and M661 (69) primers. PCR conditions were as follows: initial denaturation at 94°C for 2 min, followed by 20 cycles of 94°C for 30 s, 63.8°C for 30 s, and 72°C for 1 min 15 s. The resulting PCR product was diluted in an equal volume of H9 cell lysate (10 6 cells/ml), and 2 l of the diluted PCR product was amplified in the presence of SYBR Green-I with the AA55 and M667 primers as previously described (35,49,61).…”

“…The susceptibility of each virus to IN or RT inhibitors was determined in MT-2 cells as described previously (42,49). Triplicate samples of L-CA, L-731,988, ZDV, or NVP were serially diluted 128-fold in 96-well plates.…”

“…The susceptibility of each mutant IN to L-CA or L-731,988 was determined essentially as previously described (36,37,49). Briefly, in triplicate, IN was incubated with V1-V2 as described above in the presence of 0.3 to 10.0 M inhibitor.…”