Myriam Witvrouw

To gain further insight into the understanding of the antiviral resistance patterns and mechanisms of the integrase strand transfer inhibitor L-870,810, the prototypical naphthyridine analogue, we passaged the human immunodeficiency virus type 1 strain HIV-1(III(B)) in cell culture in the presence of increasing concentrations of L-870,810 (III(B)/L-870,810). The mutations L74M, E92Q, and S230N were successively selected in the integrase. The L74M and E92Q mutations have both been associated in the past with resistance against the diketo acid (DKA) analogues L-708,906 and S-1360 and the clinical trial drugs MK-0518 and GS-9137. After 20, 40, and 60 passages in the presence of L-870,810, III(B)/L-870,810 displayed 22-, 34-, and 110-fold reduced susceptibility to L-870,810, respectively. Phenotypic cross-resistance against the DKA analogue CHI-1043 and MK-0518 was modest but that against GS-9137 was pronounced. Recombination of the mutant integrase genes into the wild-type background reproduced the resistance profile of the resistant III(B)/L-870,810 strains. In addition, resistance against L-870,810 was accompanied by reduced viral replication kinetics and reduced enzymatic activity of integrase. In conclusion, the accumulation of L74M, E92Q, and S230N mutations in the integrase causes resistance to the naphthyridine L-870,810 and cross-resistance to GS-9137. These data may have implications for cross-resistance of different integrase inhibitors in the clinic.

We have previously identified the pyranodipyrimidines (PDPs) as a new class of integrase (IN) inhibitors. The most potent congener V-165 inhibits HIV-1 integration at low micromolar concentrations by inhibiting the binding of IN to the DNA. As part of pre-clinical studies with PDP, we wanted to investigate HIV resistance development against V-165 and to further characterize the physicochemical properties of the compound. We selected PDP-resistant HIV-1 strains by growing the virus in the presence of increasing concentrations of V-165. The selected strains were analysed genotypically and phenotypically. Mutant IN enzymes were generated and evaluated in an enzymatic oligonucleotide-based assay for their activity and sensitivity to the different IN inhibitors. In addition, the antiviral effect of the compound on viral entry and integration was measured using quantitative PCR. Numerous mutations were detected in the RT, IN and env genes of the virus selected in the presence of V-165. Although V-165 inhibited integration in vivo as indicated by a decrease in the number of integrated proviruses, the compound also inhibited viral entry at a concentration of 19 µM. V-165 was poorly recovered from human hepatic microsomal matrix and 1% BSA. These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.

Current strategies for the treatment of human immunodeficiency virus (HIV) infection are based on cocktails of drugs that target the viral entry step and the enzymes reverse transcriptase or protease. At present, the clinical benefit of this combination therapy for HIV-infected patients is considerable, although it is not clear how long this effect will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting additional steps of the viral replication cycle may increase the potency of inhibition and prevent significant resistance development. During HIV replication, integration of the viral genome into the cellular chromosome is an essential step catalyzed by the viral integrase. Although HIV integrase is an attractive target for antiviral therapy and the focus of intensive research, to date only two classes of compounds that selectively inhibit HIV integration have been identified, namely the diketo acids and the pyranodipyrimidines. In this review we address the question why it has proven so difficult to find potent and selective integrase inhibitors; we point to potential pitfalls in defining an inhibitor as an authentic integrase inhibitor and we propose new strategies and new technologies for the discovery of genuine HIV integration inhibitors. For the diketo acids and the pyranodipyrimidines we will discuss in detail the antiviral activity, the molecular mechanism of anti-HIV action, the in vitro HIV resistance development and the clinical perspectives.

Integration of the proviral DNA is a crucial step in the life cycle of the human immunodeficiency virus (HIV). Basic understanding of the cell biology of HIV integration will help the development of new antiviral drugs to complement the currently used cocktails of reverse transcriptase and protease inhibitors. Our research team has made major advancement in the field of antiviral research in recent years. The scientific-technological achievements were possible by the collaboration of a group of researchers with complementary research expertise. The integrase team is headed by Myriam Witvrouw and Zeger Debyser. After a general overview we summarized our most relevant publications. In the perspectives we referred to the ongoing work.