Valery Fikkert - Academia.edu (original) (raw)

Papers by Valery Fikkert

Current Medicinal Chemistry -Anti-Infective Agents, 2005

Current strategies for the treatment of human immunodeficiency virus (HIV) infection are based on... more 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.

Antiviral chemistry & chemotherapy, 2005

A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-... more A drug-resistant NL4.3/SPL2923 strain has previously been generated by in vitro selection of HIV-1(NL4.3) in the presence of the polysulfonic dendrimer SPL2923 and mutations were reported in its gp120 gene (Witvrouw et al., 2000). Here, we further analysed the (cross) resistance profile of NL4.3/SPL2923. NL4.3/SPL2923 was found to contain additional mutations in gp41 and showed reduced susceptibility to SPL2923, dextran sulfate (DS) and enfuvirtide. To delineate to what extent the mutations in each env gene were accountable for the phenotypic (cross) resistance of NL4.3/SPL2923, the gp120-, gp41- and gp160-sequences derived from this strain were placed into a wild-type background using env chimeric virus technology (CVT). The cross resistance of NL4.3/SPL2923 towards DS was fully reproduced following gp160-recombination, while it was only partially reproduced following gp120- or gp41-recombination. The mutations in gp41 of NL4.3/SPL2923 were sufficient to reproduce the cross resista...

Antiviral chemistry & chemotherapy, 2003

Prostratin, a non-tumour promoting phorbol ester, exhibit a potent anti-HIV activity against huma... more Prostratin, a non-tumour promoting phorbol ester, exhibit a potent anti-HIV activity against human immunodeficiency virus type 1 (HIV-1). However, the antiviral mechanism of prostratin is not well defined. In the present study, we report that prostratin exhibits potent antiviral activity against different strains of HIV-1 (subtypes B and D), a clinical HIV isolate (L1), HIV-2 (ROD and EHO) and SIV (MAC251) with EC50-values ranging from 0.02-0.09 microg/ml. Prostratin was equally active against HIV strains resistant to the polyanionic binding inhibitor dextran sulphate, the fusion inhibitor T-20 (enfuvirtide), nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs). In contrast, prostratin lost 4.4- and 6.8-fold of its effect against the HIV strains resistant to AMD3100 and the quaternary ammonium salt QAS10+, respectively. As shown by time-of-addition experiments, prostratin needs to be present at the time of viral adsorption to exert its antiviral activity....

Antimicrobial agents and chemotherapy, 2002

A series of diketo derivatives was found to inhibit human immunodeficiency virus type 1 (HIV-1) i... more A series of diketo derivatives was found to inhibit human immunodeficiency virus type 1 (HIV-1) integrase activity. Only L-708,906 inhibited the replication of HIV-1(III(B)) (50% effective concentration, 12 micro M), HIV-1 clinical strains, HIV-1 strains resistant to reverse transcriptase or fusion inhibitors, HIV-2 (ROD strain) and simian immunodeficiency virus (MAC(251)). The combinations of L-708,906 with zidovudine, nevirapine, or nelfinavir proved to be subsynergistic. In cell culture, addition of L-708,906 could be postponed for 7 h after infection, a moment coinciding with HIV integration. Inhibition of integration in cell culture was confirmed by quantitative Alu-PCR.

Proceedings of the National Academy of Sciences, 2006

Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site usin... more Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photocrosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide 128 AACWWAGIK 136 was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling͞MS analysis with site-directed mutagenesis͞molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.

Journal of Virology, 2003

The diketo acid L-708,906 has been reported to be a selective inhibitor of the strand transfer st... more The diketo acid L-708,906 has been reported to be a selective inhibitor of the strand transfer step of the human immunodeficiency virus type 1 (HIV-1) integration process (Science 287:646-650, 2000). We have now studied the development of antiviral resistance to L-708,906 by growing HIV-1 strains in the presence of increasing concentrations of the compound. The mutations T66I, L74M, and S230R emerged successively in the integrase gene. The virus with three mutations (T66I L74M S230R) was 10-fold less susceptible to L-708,906, while displaying the sensitivity of the wild-type virus to inhibitors of the RT or PRO or viral entry process. Chimeric HIV-1 strains containing the mutant integrase genes displayed the same resistance profile as the in vitro-selected strains, corroborating the impact of the reported mutations on the resistance phenotype. Phenotypic cross-resistance to S-1360, a diketo analogue in clinical trials, was observed for all strains. Interestingly, the diketo acid-resistant strain remained fully sensitive to V-165, a novel integrase inhibitor (C. Pannecouque, W. Pluymers, B. Van Maele, V. Tetz, P. Cherepanov, E. De Clercq, M. Witvrouw, and Z. Debyser, Curr. . Antiviral resistance was also studied at the level of recombinant integrase. Single mutations did not appear to impair specific enzymatic activity. However, 3 processing and strand transfer activities of the recombinant integrases with two (T66I L74M) and three (T66I L74M S230R) mutations were notably lower than those of the wild-type integrase. Although the virus with three mutations was resistant to inhibition by diketo acids, the sensitivity of the corresponding enzyme to L-708,906 or S-1360 was reduced only two-to threefold. As to the replication kinetics of the selected strains, the replication fitness for all strains was lower than that of the wild-type HIV-1 strain.

Clinical Infectious Diseases, 1994

Antimicrobial Agents and Chemotherapy, 2003

We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (... more We describe the development of chimeric virus technology (CVT) for human immunodeficiency virus (HIV) type 1 (HIV-1) env genes gp120, gp41, and gp160 for evaluation of the susceptibilities of HIV to entry inhibitors. This env CVT allows the recombination of env sequences derived from different strains into a proviral wild-type HIV-1 clone (clone NL4.3) from which the corresponding env gene has been deleted. An HIV-1 strain (strain NL4.3) resistant to the fusion inhibitor T20 (strain NL4.3/T20) was selected in vitro in the presence of T20. AMD3100-resistant strain NL3.4 (strain NL4.3/AMD3100) was previously selected by De Vreese et al. (K. De Vreese et al., J. Virol. 70:689-696, 1996). NL4.3/AMD3100 contains several mutations in its gp120 gene (De Vreese et al., J. Virol. 70:689-696, 1996), whereas NL4.

AIDS, 2004

Study of HIV-1 resistance development to the diketo analogue S-1360, the first HIV-1 integrase st... more Study of HIV-1 resistance development to the diketo analogue S-1360, the first HIV-1 integrase strand transfer inhibitor that has entered clinical development. HIV-1(IIIB) was passaged in cell culture in the presence of increasing concentrations of S-1360 (IIIB/S-1360(res)). The IIIB/S-1360(res) strains selected for 30, 50 and 70 passages in the presence of S-1360 were evaluated genotypically by sequencing analysis and phenotypically using the MT-4/MTT assay. Multiple mutations, nine in total, emerged progressively in the catalytic domain of integrase as a result of the selection process. They included T66I and L74M that have both been associated with resistance against the diketo acid L-708,906. After 30, 50 and 70 passages in the presence of S-1360, IIIB/S-1360(res) displayed a four-, eight- and more than 62-fold reduced susceptibility for S-1360, respectively. Phenotypic cross-resistance to L-708,906 was modest for the IIIB/S-1360(res) strain selected during 50 passages, but pronounced for the strain selected during 70 passages. Interesting, all IIIB/S-1360(res) strains remained fully susceptible to the pyranodipyrimidine V-165, an integrase DNA binding inhibitor. Recombination of the mutant integrase genes into wild-type background by integrase-chimeric virus technology entirely reproduced the resistance profile of the IIIB/S-1360(res) strains. As for the replication kinetics of the selected and recombined strains, reduced replication fitness was measured for all strains when compared with their respective wild-type strains. The accumulation of integrase mutations coincided with an increasing level of (cross-)resistance of IIIB/S-1360(res). Integrase-chimeric virus technology confirmed that the integrase mutations are indeed fully responsible for the resistance phenotype of IIIB/S-1360.

Molecular pharmacology, 2000

The antiviral activity of L-chicoric acid against HIV-1 has been attributed previously to the inh... more The antiviral activity of L-chicoric acid against HIV-1 has been attributed previously to the inhibition of HIV-1 integration. This conclusion was based on the inhibition of integrase activity in enzymatic assays and the isolation of a resistant HIV strain with a mutation (G140S) in the integrase gene. Here we show that the primary antiviral target of L-CA and its analogs in cell culture is viral entry. L- and D-chicoric acid (L-CA and D-CA) and their respective tetra-acetyl esters inhibit the replication of HIV-1 (III(B) and NL4.3) and HIV-2 (ROD) in MT-4 cells at a 50% effective concentration (EC(50)) ranging from 1.7 to 70.6 microM. In a time-of-addition experiment, L-CA, D-CA, L-CATA, and D-CATA were found to interfere with an early event in the viral replication cycle. Moreover, L-CA, D-CA, and their analogs did not inhibit the replication of virus strains that were resistant toward polyanionic and polycationic compounds at subtoxic concentrations. Furthermore, HIV-1 strains re...