Myriam Witvrouw

Sulfated polysaccharides extracted from sea algae as potential antiviral drugs

The inhibitory effects of polyanionic substances on the replication of herpes simplex virus (HSV) and other viruses were reported almost four decades ago. However, these observations did not generate much interest, because the antiviral action of the compounds was considered to be largely nonspecific. Shortly after the identification of human immunodeficiency virus (HIV) as the causative agent of the acquired immune deficiency syndrome (AIDS) in 1984, heparin and other sulfated polysaccharides were found to be potent and selective inhibitors of HIV-1 replication in cell culture. Since 1988, the activity spectrum of the sulfated polysaccharides has been shown to extend to various enveloped viruses, including viruses that emerge as opportunistic pathogens (e.g., herpes simplex virus [HSV] and cytomegalovirus [CMV]) in immunosuppressed (e.g., AIDS) patients. As potential anti-HIV drug candidates, sulfated polysaccharides offer a number of promising features. They are able to block HIV replication in cell culture at concentrations as low as 0.1 to 0.01 microgram ml-1 without toxicity to the host cells at concentrations up to 2.5 mg ml-1. We noted that some polysulfates show a differential inhibitory activity against different HIV strains, suggesting that marked differences exist in the target molecules with which polysulfates interact. They not only inhibit the cytopathic effect of HIV, but also prevent HIV-induced syncytium (giant cell) formation. Furthermore, experiments carried out with dextran sulfate samples of increasing molecular weight and with sulfated cyclodextrins of different degrees of sulfation have shown that antiviral activity increases with increasing molecular weight and degree of sulfation. A sugar backbone is not strictly needed for the anti-HIV activity of polysulfates because sulfated polymers composed of a carbon-carbon backbone have also proved to be highly efficient anti-HIV agents in vitro. Other, yet to be defined, structural features may also play an important role. Sulfated polysaccharides may act synergistically with other anti-HIV drugs (e.g., azidothymidine [AZT]). They are known to lead very slowly to virus-drug resistance development and they show activity against HIV mutants that have become resistant to reverse transcriptase inhibitors, such as AZT, tetrahydro-imidazo [4,5,l-jk] [1,4]-benzodiazepin-2(1H)-thione (TIBO) and others. From studies on their mechanism of action we concluded that polysulfates exert their anti-HIV activity by shielding off the positively charged sites in the V3 loop of the viral envelope glycoprotein (gp120). The V3 loop is necessary for virus attachment to cell surface heparan sulfate, a primary binding site, before more specific binding occurs to the CD4 receptor of CD4+ cells. This general mechanism also explains the broad antiviral activity of polysulfates against enveloped viruses. Variations in the viral envelope glycoprotein region may result in differences in the susceptibility of different enveloped viruses to compounds that interact with their envelope glycoproteins. The efficacy of polysulfates in the therapy and/or prophylaxis of retroviral infections and opportunistic infections remains to be demonstrated both in animal models and humans. It is important to consider not only treatment of patients who are already infected with HIV, but also prophylaxis and protection from HIV and/or other virus infections. Because (i) sexual transmission is responsible for the large majority of HIV infections worldwide; (ii) this transmission is mostly mediated via mononuclear cells that infect epithelial cells of the genital tract; and because (iii) polysulfates effectively inhibit cell-cell adhesion, polysulfates may be considered as potentially effective in a vaginal formulation to protect against HIV infection.

Transient and Stable Knockdown of the Integrase Cofactor LEDGF/p75 Reveals Its Role in the Replication Cycle of Human Immunodeficiency Virus

ABSTRACT After identifying the interaction between the transcriptional coactivator lens epithelium-derived growth factor (LEDGF/p75) and the human immunodeficiency virus type 1 (HIV-1) integrase (IN), we have now investigated the role of LEDGF/p75 during HIV replication. Transient small interfering RNA-mediated knockdown of LEDGF/p75 in HeLaP4 cells resulted in a three- to fivefold inhibition of HIV-1 (strain NL4.3) replication. Quantitative PCR was used to pinpoint the replication block to the integration step. Next, polyclonal and monoclonal HeLaP4-derived cell lines were selected with a stable knockdown of LEDGF/p75 mediated by a lentiviral vector (lentivector) encoding a short hairpin RNA (shRNA) targeting this protein. Cell lines stably transduced with a lentivector encoding an unrelated hairpin or a double-mismatch hairpin served as controls. Again, a two- to fourfold reduction of HIV-1 replication was observed. The extent of LEDGF/p75 knockdown closely correlated with the reduction of HIV-1 replication. After the back-complementation of LEDGF/p75 in the poly- and monoclonal knockdown cell lines using an shRNA-resistant expression plasmid, viral replication was restored to nearly wild-type levels. The Q168A mutation in integrase has been shown to interfere with the interaction with LEDGF/p75 without reducing the enzymatic activity. Transduction by HIV-1-derived lentivectors carrying the Q168A IN mutant was severely hampered, pointing again to a requirement for LEDGF/p75. Altogether, our data validate LEDGF/p75 as an important cellular cofactor for HIV integration and as a potential target for antiviral drug development.

Discovery of 2,3-diaryl-1,3-thiazolidin-4-ones as potent anti-HIV-1 agents

Design, synthesis and anti-HIV activity of a series of 2,3-diaryl-1,3-thiazolidin-4-ones are reported. Some derivatives proved to be highly effective in inhibiting HIV-1 replication at nanomolar concentrations thereby acting as non-nucleoside HIV-1 RT inhibitors (NNRTIs). SAR studies evidenced that the nature of the substituents at the 2 and 3 positions of the thiazolidinone nucleus largely influenced the in vitro anti-HIV activity of this new class of potent antiviral agents.

Synthesis and antiviral activity evaluation of some new 6-substituted 3-(1-adamantyl)-1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles

A number of novel 3,6-disubstituted 1,2,4-triazolo[3,4-b][1,3,4]thiadiazole derivatives, containing the adamantyl moiety, were synthesized and examined in various viral test systems. No antiviral effects were noted with any of the compounds at subtoxic concentrations in cell culture.

Virus Evolution Reveals an Exclusive Role for LEDGF/p75 in Chromosomal Tethering of HIV

Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75–integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase–LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites.

Antiviral activity against human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) of ethnobotanically selected ethiopian medicinal plants

Ethiopian medicinal plants used for the treatment of a variety of ailments including infectious diseases were screened for activity against human immunodeficiency virus type 1 (HIV‐1) and type 2 (HIV‐2). Seventy‐one polar and nonpolar extracts derived from 21 plants belonging to 14 families were tested for inhibition of viral replication using HIV‐1 (IIIB) and HIV‐2 (ROD) strains. Selective inhibition of viral growth was assessed by the simultaneous determination of the in vitro cytotoxicity of each of the extracts against MT‐4 cells. Six extracts made from the root bark of Bersama abyssinica Fresen, the leaves of Combretum paniculatum Vent., and Dodonaea angustifolia L.f., and the stem bark of Ximenia americana L. displayed antiviral activity at concentrations that were nontoxic to MT‐4 cells. The highest selective inhibition of HIV‐1 replication was observed with the acetone fraction of C. paniculatum and the methanol fraction of D. angustifolia which showed selectivity indices (ratio of 50% cytotoxic concentration to 50% effective antiviral concentration) of 6.4 and 4.9, and afforded cell protection of viral induced cytopathic effect of 100% and 99%, respectively, when compared with control samples. The greatest degree of antiviral activity against HIV‐2 was achieved with the acetone extract of C. paniculatum (EC50: 3 µg/mL), which also showed the highest selectivity index (32). The 50% cytotoxic concentration ranged from 0.5 µg/mL for the hexane extract of D. angustifolia L.f., the most cytotoxic of the extracts tested, to >250 µg/mL for some extracts such as the methanol fraction of Alcea rosea L., the least toxic tested. Only the polar extracts that were obtained by extraction with hydroalcohol, methanol or acetone exhibited inhibition of viral growth at subtoxic concentrations. The results obtained in this study enable the selection of extracts which show some specificity of action and support the further investigation of these extracts for their potential as new lead antiretroviral compounds. Copyright

Investigations on the 4-quinolone-3-carboxylic acid motif. 1. Synthesis and structure-activity relationship of a class of human immunodeficiency virus type 1 integrase inhibitors.

A set of 4-quinolone-3-carboxylic acids bearing different substituents on the condensed benzene ring was designed and synthesized as potential HIV-1 integrase inhibitors structurally related to elvitegravir. Some of the new compounds proved to be able to inhibit the strand transfer step of the virus integration process in the micromolar range. Docking studies and quantum mechanics calculations were used to rationalize these data.

Antiviral Portrait Series: 4. Polysuifates as Inhibitors of HIV and Other Enveloped Viruses

Polysulfates are highly potent and selective inhibitors of the in vitro replication of HIV and other enveloped viruses. They not only inhibit the cytopathic effect of HIV, but also prevent HIV-induced syncytium (giant cell) formation. They also act synergistically with other anti-HIV drugs. The anti-HIV activity of polysulfates is a result of their shielding of the positively charged sites in the V3 loop of the viral envelope glycoprotein gp120. When polysulfates were administered intravenously to rabbits, their half-life was approximately 2h. Although they are very poorly absorbed following oral administration, they can be made orally bioavailable with the appropriate chemical modifications. Also, polysulfates may lose (much of) their anticoagulant activity upon chemical modification without giving up their anti-HIV activity. Their efficacy in the therapy and/or prophylaxis of retroviral infections remains to be demonstrated both in animal models and in humans.

Prevalence and characteristics of multinucleoside-resistant human immunodeficiency virus type 1 among European patients receiving combinations of nucleoside analogues.

ABSTRACT The prevalence and the genotypic and phenotypic characteristics of multinucleoside-resistant (MNR) human immunodeficiency virus type 1 (HIV-1) variants in Europe were investigated in a multicenter study that involved centers in nine European countries. Study samples (n = 363) collected between 1991 and 1997 from patients exposed to two or more nucleoside analogue reverse transcriptase inhibitors (NRTIs) and 274 control samples from patients exposed to no or one NRTI were screened for two marker mutations of multinucleoside resistance (the Q151M mutation and a mutation with a 2-amino-acid insertion at codon 69, T69S-XX). Q151M was identified in six of the study samples (1.6%), and T69S-XX was identified in two of the study samples (0.5%; both of them T69S-SS), but both patterns were absent among control samples. Non-NRTI (NNRTI)-related changes were observed in viral strains from two patients, which displayed the Q151M resistance pattern, although the patients were NNRTI naive. The patients whose isolates displayed multinucleoside resistance had received treatment with zidovudine and either didanosine, zalcitabine, or stavudine. Both resistance patterns conferred broad cross-resistance to NRTIs in vitro and a poor response to treatment in vivo. MNR HIV-1 is found only among multinucleoside-experienced patients. Its prevalence is low in Europe, but it should be closely monitored since it seriously limits treatment options.

Development of Resistance against Diketo Derivatives of Human Immunodeficiency Virus Type 1 by Progressive Accumulation of Integrase Mutations

ABSTRACT 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 (D. Hazuda, P. Felock, M. Witmer, A. Wolfe, K. Stillmock, J. A. Grobler, A. Espeseth, L. Gabryelski, W. Schleif, C. Blau, and M. D. Miller, 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. Biol. 12:1169-1177, 2002). 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.