JAC Advance Access originally published online on April 30, 2007
Journal of Antimicrobial Chemotherapy 2007 59(6):1084-1095; doi:10.1093/jac/dkm101
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Selection of human immunodeficiency virus type 1 resistance against the pyranodipyrimidine V-165 points to a multimodal mechanism of action
1 Laboratory for Molecular Virology and Drug Discovery, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium 2 Laboratory for Molecular Virology and Gene Therapy, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium 3 Laboratory for Biomolecular Dynamics, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium 4 Avexa Limited, Richmond, Victoria, Australia 5 Department of Microbiology, Virology and Immunology, Saint-Petersburg Pavlov State Medical University, Saint-Petersburg, Russia 6 Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, Kortrijk, Flanders, Belgium
* Correspondence address. Laboratory for Molecular Virology and Drug Discovery, Katholieke Universiteit Leuven, Kapucijnenvoer 35, B-3000 Leuven, Belgium. Tel: +32-16-33-21-70; Fax: +32-16-33-63-33; E-mail: myriam.witvrouw{at}med.kuleuven.be
Received 2 January 2007; returned 15 January 2007; revised 12 March 2007; accepted 12 March 2007
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Objectives: 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.
Methods: 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.
Results: 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.
Conclusions: These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.
Keywords: integrase inhibitors , antiviral resistance , HIV-1
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The current therapy for HIV-1 infection is based on a combination of several antiviral agents targeting multiple steps of the HIV-1 life cycle. Drugs that have been formally approved for the treatment of HIV-infected patients belong to four classes: the nucleoside reverse transcriptase inhibitors (NRTIs), the non-NRTIs (NNRTIs), the protease inhibitors (PIs) and the fusion inhibitors.1 Highly active antiretroviral therapy has remarkably reduced the mortality caused by HIV in the developed world. Nevertheless, owing to low-level residual replication and the genetic flexibility of the virus, drug-resistant HIV strains emerge in treated patients. Moreover, transmission of HIV drug-resistant strains has been recognized as a serious threat to the efficacy of current antiretroviral therapy.2,3 In this context, both the understanding and control of antiviral resistance and the continuous development of new drugs targeting alternative steps in the viral replication cycle are warranted.
Integrase (IN), one of the three virally encoded enzymes required for HIV-1 replication, catalyses the insertion of viral DNA into the host cell chromosome.4 The enzyme has three functional domains: the N-terminal domain, a catalytic core domain and the C-terminal domain. Integration requires three distinct steps. The first step occurs in the cytoplasm and involves the assembly of a stable complex at the termini of the viral DNA.5 IN recognizes specific sequences in the long terminal repeats (LTRs) of the viral cDNA, which contain the CAGT sequence. The cleavage of the 3' terminal GT dinucleotides at each DNA end generates new 3'-OH ends and is referred to as 3'-end processing. Next, the pre-integration complex is transported into the nucleus. Strand cleavage of the host DNA and linkage to the viral DNA take place during the strand transfer reaction. Integration is completed by the removal of the two unpaired nucleotides at the 5'-end of the viral DNA and the repair of the single-stranded gaps created between the viral and host DNA by host cell DNA repair enzymes, although retroviral enzymes have been implicated as well.6,7 After integration, the proviral DNA is replicated and genetically transmitted as part of the cellular genome. Therefore, integration defines a point of no return in the establishment of HIV infection. Because no human counterpart of the enzyme is known, there is substantial interest in developing effective inhibitors of HIV IN.8
IN inhibition is typically assayed for in oligonucleotide-based tests that evaluate both the 3' processing and the strand transfer reaction.9 The first decade of research on inhibitors of IN yielded different mechanistic classes of compounds.10–12 However, most of these compounds did not exhibit antiviral activity or were toxic in cell culture. For most of the IN inhibitors with antiviral activity in cell culture, it was not unambiguously shown that integration was the sole target. Both the G-quartets13 and L-chicoric acid derivatives,14 IN inhibitors with proven antiviral activity, were shown to target viral entry as well in cell culture.15,16 The identification of a series of diketo acids (DKAs) as strand transfer inhibitors that prevent integration and HIV-1 replication in cell culture provided the first proof of principle for HIV-1-IN inhibitors as antiviral agents.17 L-731,988 is the prototype of these IN strand transfer inhibitors (INSTIs). The Merck group characterized a series of metabolically stable heterocyclic compounds, represented by L-870,810.18 In HIV-1-infected patients, L-870,810 resulted in a 50-fold reduction in viral load, but clinical studies were halted because of liver and kidney toxicity observed in dogs. However, the latest clinical trial data on the Merck drug MK-0518 and the Gilead IN inhibitor GS-9137 look very promising.19,20 Accordingly, these validated lead compounds are useful in the design and development of second-generation IN inhibitors.21
Next to the DKAs, a series of 5H-pyrano[2,3-d:-6,5-d']dipyrimidines (PDPs) have been identified as a second class of IN inhibitors.22 PDPs interfere with the replication of various HIV-1, HIV-2 and SIV strains in cell culture. The most potent congener V-165 inhibited HIV-1 replication in cell culture and IN enzymatic activity at micromolar concentrations. V-165 retained activity against virus strains resistant to the viral entry antagonists dextran sulphate (DS) or AMD3100 and also proved active against strains resistant to RT inhibitors. Using a series of quantitative PCRs (Q-PCRs) on cells transduced with HIV-1 vectors, V-165 was shown to inhibit integration without marked effect on viral DNA synthesis.23 Mechanism of action studies revealed that V-165 interferes with viral DNA–IN complex formation.22 As such, V-165 is the prototype of the IN binding inhibitors (INBIs).12 Another group of IN inhibitors, namely styrylquinolines that compete with the LTR substrate for IN binding in vitro, have been described.24
Not much is known about development of antiviral resistance against IN inhibitors. Resistance of HIV to INSTIs is typically only observed after 3–6 months of in vitro passaging in the presence of the drug.25–27 HIV-1 strains that were selected in the presence of the DKA L-708,906 carried the mutations T66I, S153Y, M154I17 or T66I, L74M, S230R.26 Mutations that confer resistance to the diketo analogue S-1360 all localized in the catalytic core.27 The mode of action of DKAs is based on the interaction with critical, divalent metal ions in the IN active site, resulting in a subsequent sequestration of the metal cofactor.28 Not surprisingly, mutations that result in resistance to these prototype inhibitors map to the IN active site proximal to residues that coordinate divalent metals.17 HIV-1 strains that were resistant to the naphthyridine carboxamide L-870,810 carried the mutations F121Y, T125K and V72I. Only minor cross-resistance was observed against DKAs.25
As part of the pre-clinical studies with PDP, we have now investigated the development of antiviral resistance against V-165 (Figure 1) in cell culture and we have further characterized the physicochemical properties of the compound.
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Compounds
V-165 was obtained from Ampharm Inc. (Ramsey, NJ, USA).29 AMD3100 was provided by AnorMED (Langley, BC, Canada) and was synthesized as described previously.30 Zidovudine was synthesized according to the method described by Horwitz et al.31 Nevirapine (BI-RG587) was obtained from Boehringer Ingelheim (Ridgefield, CN, USA). Ritonavir (ABT538) was obtained from J. M. Leonard, Abbott Laboratories (Abbott Park, IL, USA). DS (average MW 5000) was purchased from Sigma (Bornem, Belgium) and T-20 from Roche Diagnostics (Vilvoorde, Belgium). All compounds were dissolved in DMSO at 10 mg/mL, DS was dissolved in milli-Q water and T-20 was dissolved in PBS.
MT-432 and 293T cells were grown in a humidified atmosphere with 5% CO2 at 37°C. 293T cells were obtained from O. Danos (Génethon, Evry, France) and grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum (Harlan Sera-Lab Ltd), 2 mM glutamine (Gibco BRL), 100 U/mL penicillin (Gibco BRL) and 100 mg/L streptomycin (Gibco BRL). MT-4 cells were maintained in RPMI 1640 (Gibco BRL) supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 0.1% sodium bicarbonate (Gibco BRL), 100 U/mL penicillin and 100 mg/L streptomycin.
The HIV-1 plasmid pNL4.333 is a molecular clone obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3 from Dr Malcolm Martin (Bethesda, MD, USA). The bacterial expression plasmid pRP1012 (Dr R. H. A. Plasterk, Dutch Cancer Institute, Amsterdam, The Netherlands) encoding HIV-1 IN was used to generate the single IN mutants T206S and S230N and the double IN mutant T206S/S230N. Site-directed mutagenesis was performed using the QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA, USA), as described previously.26
Selection of antiviral resistance
Resistance selection of HIV-1 NL4.3 against V-165 was initiated at a low multiplicity of infection (MOI = 0.01) in MT-4 cells and a drug concentration equal to the 50% effective concentration (IC50), as determined in the MT-4/MTT assay (IC50: 7.2 µM). Every 3 to 4 days, the cell culture was monitored for the appearance of HIV-induced cytopathic effect (CPE). When CPE was observed, the cell-free supernatant was used to infect new MT-4 cells in the presence of an equal or higher compound concentration. When no virus breakthrough was observed, the infected cell culture was subcultivated in the presence of the same compound concentration.
PCR amplification and sequencing of the coding regions for IN, RT and gp160
Proviral DNA extraction of MT-4 cells, infected with different passages of HIV-1 NL4.3 selected in the presence of V-165, was performed using the QIAamp Blood Kit (Qiagen, Hilden, Germany).
PCR amplification and sequencing of IN and gp160 encoding sequences. PCR amplification and sequencing of IN encoding sequences were done as described previously.26 PCR amplification and sequencing of gp160 encoding sequences were done as described elsewhere.34 Mutations present in >25% of the global virus population can be detected as a mixture with the wild-type (WT) amino acid by means of population sequencing.
PCR amplification and sequencing of RT encoding sequences. A 1756 nucleotide base pair fragment was amplified. The PCR reaction was performed using the primers RT1 (5'-GTA GAA TTC TGT TGA CTC AGA TTG G-3', corresponding to positions 2509–2533) and RT2 (5'-GAT AAG CTT GGG CCT TAT CTA TTC CAT-3', corresponding to positions 4238–4265). Primer positions correspond to HIV-1 (HXB2) (GenBank accession no. K03455 [GenBank] ). The cycling conditions were as follows: a denaturation step of 2 min at 95°C was followed by 35 cycles of amplification consisting of 15 s at 95°C and 60 s at 65°C. A final extension was performed at 72°C for 10 min. The primers used to sequence the entire RT gene were AV36 (5'-CAG TAC TGG ATG TGG GTG ATG-3', corresponding to positions 2868–2889), AV44 (5'-TAC TAG GTA TGG TAA ATG CAG T-3', corresponding to positions 2930–2952), AV59 (5'-GGG GCA AGG CCA ATG GAC-3', corresponding to positions 3544–3562), AV181 (5'-TTC ATT TCC TCC AAT TCC TTT GTG-3', corresponding to positions 4164–4187), AV191 (5'-CTT GAT AAA TTT GAT ATG TCC ATT G-3', corresponding to positions 3555–3579) and MW3 (5'-TAT GTA GGA TCT GAC TTA GAA ATA GGG C-3', corresponding to positions 3110–3137).
Construction of HIV-1 clones deleted for IN, RT or gp160
To generate the IN-deleted clone, pNL4.3 was digested with the restriction enzymes AgeI and Van91I. The vector was purified by gel extraction (using ß-agarase I) and subsequent phenol/chloroform extraction. A linker sequence containing the XbaI restriction site was ligated into the vector to recircularize the plasmid, as described previously.26 Competent Escherichia coli (DH5
) (Invitrogen, Merelbeke, Belgium) was transformed with this IN-deleted clone.
For construction of the clone from which gp160 was deleted, pNL4.3 was digested with the restriction enzymes SalI and CelII. A linker sequence containing the SalI and CelII restriction sites was ligated into the vector to recircularize the plasmid. The pNL4.3
RT was kindly provided by B. A. Larder (Virco United Kingdom Limited, Cambridge, UK). It contains a linker sequence with a BstEII restriction site to recircularize the plasmid.
Chimeric virus recombination assay
The chimeric virus recombination assay was performed as described previously.26 For IN-recombination experiments, MT-4 cells were co-transfected with 10 µg of XbaI linearized IN-deleted clone and 2 µg of purified and concentrated HP4149-PCRC PCR product (PCR Purification Kit, Qiagen). For RT-recombination experiments, MT-4 cells were co-transfected with 10 µg of BstEII linearized RT-deleted clone and 2 µg of purified and concentrated RT1–RT2 PCR product. For gp160-recombination experiments, MT-4 cells were co-transfected with 10 µg of SalI linearized gp160-deleted clone and 2 µg of purified and concentrated AV310–AV319 PCR product.
The inhibitory effect of antiviral drugs on the HIV-induced CPE in human MT-4 cell culture was determined by the MTT assay,35 as described previously.26
Determination of replication capacity of HIV-1 strains
Inoculants of various HIV-1 strains containing equal amounts of HIV-1 p24 antigen (10, 5 and 2.5 pg/mL) were added to MT-4 cells (50 000 cells/mL). From 3 days post-infection onwards, aliquots of cell-free supernatants were taken for the determination of viral p24 levels (Alliance HIV-1 P24 antigen ELISA Kit, Perkin Elmer Life Sciences, Milano, Italy).
Production and purification of HIV-1 IN
The purification of WT and mutant HIV-1 IN was done as described elsewhere.36
Oligonucleotide-based integration assay
The enzymatic integration reactions were carried out as described previously,37,38 with minor modifications.39 Overall integration activities of the different enzymes were determined in this assay by measuring the respective amounts of strand transfer products. These data were determined using the OptiQuant Acquisition and Analysis software (Perkin Elmer Corporate, Fremont, CA, USA).
Overall IN assay using an ELISA
To determine the sensitivity of the IN enzymes to different compounds, we optimized an ELISA. This assay uses an oligonucleotide substrate of which one oligo (5'-ACTGCTAGAGATTTTCCACACTGACTAAAAGGGTC-3') is labelled with biotin at the 3' end and the other oligo (5'-GACCCTTTTAGTCAGTGTGGAAAATCTCTAGCATG-3') is labelled with digoxigenin at the 5' end. The IN enzymes were diluted to the same specific activity in 750 mM NaCl, 10 mM Tris pH 7.6, 10% glycerol and 1 mM ß-mercaptoethanol. To perform the reaction, 4 µL of diluted IN (corresponding to a concentration of WT IN of 1.6 µM) and 4 µL of annealed oligos (7 nM) were added to a final reaction volume of 40 µL containing 10 mM MgCl2, 5 mM DTT, 20 mM HEPES pH 7.5, 5% PEG and 15% DMSO. The reaction was carried out for 1 h at 37°C and followed by an ELISA on avidin-coated plates.40
Fluorescence correlation spectroscopy
A commercial fluorescence correlation spectroscopy (FCS) setup (ConfoCor I of Zeiss-EVOTEC) was used as described previously.41,42 The laser beam was focused at 
180 µm above the bottom of the Nunc cuvettes (Nalge Nunc International, Naperville, IL, USA) in a typical volume of 10 µL. The data electronics and software (Borland Delphi) were used as described previously.43
For the DNA binding assay, based on the fluorescence fluctuation analysis, synthetic oligonucleotides resembling the U5 LTR ends of the viral genome were used (INT1 and INT2).44 For DNA substrate preparation, equimolar amounts of complementary oligonucleotides were annealed in 20 mM HEPES, pH 7.5, containing 100 mM NaCl. The samples were incubated at 80°C for 1 min and cooled to 20°C over the course of 90 min. The final DNA concentration of the fluorescent double-stranded DNA was determined using the ConfoCor I. The DNA substrate concentration was kept constant at 30 nM, whereas the IN concentration varied from 0 to 1.6 µM. After an incubation of the samples for 10 min at room temperature, the measurements were performed for 60 s. Each sample was measured 10 times. The data were subsequently analysed as described earlier, using the quantile plot analysis method.44 Briefly, the association constant of IN for DNA (Ka) is calculated by varying the enzyme concentration while measuring the free and bound DNA.37 The Ka value and the enzyme concentration are used for calculating the inhibitor association constant (Ki):
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Quantitative virus adsorption assay
In this assay, the inhibitory effect of antiviral compounds on virus adsorption to MT-4 cells was measured. Therefore, MT-4 cells (5 x 105 cells per tube) were incubated with HIV-1 NL4.3 (corresponding to 100 ng of p24) in the absence or presence of serial dilutions of the test compounds. After 2 h of incubation at 37°C, the cells were washed extensively with PBS to remove the unadsorbed virus particles. Then, the cells were lysed and total RNA was extracted using an RNAqueous®-4PCR Kit (Ambion, Huntingdon, UK). Viral RNA was detected by real-time quantitative RT–PCR, using the primers RT-GAG1 (5'-ATCAAGCAGCCATGCAATGTT-3') and RT-GAG2 (5'-CTGAAGGGTACTAGTAGTTCCTGCTATGTC-3') and a probe (FAM-5'-ACCATCAATGAGGAAGCTGCAGAATGGGA-3'-TAMRA) amplifying a 161 bp nucleotide fragment. The cycling conditions were as follows: reverse transcription step of 30 min at 48°C and an AmpliTaq activation step of 10 min at 95°C were followed by 40 cycles of amplification consisting of 15 s at 95°C and 1 min at 60°C. Reactions were analysed using the ABI Prism 7700 sequence detection system (Applied Biosystems, Lennik, Belgium).
MT-4 cells (1.5 x 106 cells per tube) were incubated with HIV-1 NL4.3 (corresponding to 150 ng of p24) in the absence or presence of the test compounds. Inhibitors were added to the cells 1 h prior to infection. After a 2 h incubation at 37°C, the cells were washed with PBS, replaced with new medium and seeded in a 24-well plate (approximately 300 000 infected cells/well). When the infection medium was replaced with new medium, fresh inhibitors were added. In each 24-well plate, uninfected MT-4 cells were incubated in parallel. Each time a sample was prepared for Q-PCR analysis, an aliquot of uninfected cells was prepared as well.
Lentiviral vector transduction assay
VSV-G pseudotyped HIV-1-derived vector particles were produced by transfecting 293T cells with three plasmids, as described previously.23 The transfer plasmid used was the pCHGFPWS plasmid, as described previously.23 The day prior to transduction, 293T cells were seeded in 24-well plates at approximately 1 x 105 cells per well. Transductions with the lentiviral vectors were carried out at an MOI of 10. Vector was added to the cells in the presence of DMEM/1% fetal calf serum. After 4 h of incubation, the medium was replaced by DMEM containing 10% fetal calf serum. In each 24-well plate, 293T cells that were not transduced were incubated in parallel. Each time a sample was prepared for PCR analysis, an aliquot of untransduced cells was prepared as well. Inhibitors of lentiviral transduction were added to the cells 1 h prior to transduction. When transduction medium was replaced, fresh inhibitors were added.
Quantification of different HIV-1 DNA species by real-time PCR
DNA extractions and quantification of late reverse transcripts, 2-LTR circles and integrants performed done as described previously.23
Evaluation of the physicochemical and pharmacokinetic properties of V-165
The solubility of V-165 was estimated by dissolving the compound in DMSO and spiking into either phosphate buffer (pH 6.5) or 0.01 M HCl (pH 2.0) at a final DMSO concentration of 1% (v/v). Samples were then analysed using nephelometry to determine a solubility range.45 To determine metabolization in and recovery from microsomes, an aqueous solution of V-165 was incubated with human liver microsomes. Metabolic activity was initiated by the addition of an NADPH-regenerating system and quenched at various time points over the incubation period by the addition of acetonitrile. The relative loss of parent compound and the formation of metabolic products were determined by liquid chromatography and mass spectrometry (LC/MS). Plasma protein binding was estimated by spiking V-165 into both 1% BSA and 1% BSA containing 1 mM DTT in pH 7.4 buffer resulting in nominal concentrations of 1 µM. Control samples containing 1 µM compound in buffer alone were also prepared. After incubation at 37°C for 5 min, the spiked solutions were extracted by precipitation of the proteins with 1.7 volumes of acetonitrile, centrifugation for 5 min in a microcentrifuge and the soluble phase was analysed by LC/MS. Compound was quantified from a standard curve constructed using peak areas from LC/MS by running known masses of compounds.
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Selection of HIV-1 strains resistant to the pyranodipyrimidine V-165
V-165-resistant strains were selected by serial passage of HIV-1 NL4.3 in the presence of increasing concentrations of V-165. After 83 passages, the selected strain was able to grow in the presence of 79 µM V-165, a concentration that is 11-fold higher than the concentration of the compound required to inhibit the replication of WT HIV-1 in MT-4 cells infected at an MOI of 0.01 by 50% (IC50) (7.2 µM).
Progressive accumulation of mutations in the IN, gp160 and RT genes of the V-165 selected HIV-1 strains
Figure 2 shows the progressive accumulation of mutations in the IN, gp160 and RT genes of the V-165 selected strains at increasing concentrations of V-165. After 15 passages (#15) in the presence of up to 15 µM V-165, the T206S and the S230N mutations were detected in the IN gene of 60% of the population. In addition, the V212I mutation was present in the gp120 gene of 50% of the selected NL4.3 strains. After 30 passages (#30), the mutations in the IN and gp120 genes were present at a higher frequency. We could also detect the R272T mutation in the gp120 gene of the whole virus population. Selection for 40 passages (#40) in the presence of up to 52 µM V-165 resulted in a virus population with 90% of the strains containing the T206S and the S230N IN mutations. The A29S and the R272T mutation were present in the gp120 gene of the whole virus population after 40 passages (#40), but the original V212I mutation in the gp120 gene was present in only 60% of the virus population. After 63 passages (#63), both IN mutations T206S and S230N were present in the entire virus population. Next to A29S, V212I and R272T, the T168N mutation in the gp120 gene occurred in the virus population. The gp41 gene carried the L33S mutation after 63 passages of the virus. In addition, at high levels of V-165, the K70R mutation emerged in the RT gene. No additional mutations in the IN encoding region of the HIV-1 NL4.3 virus were selected during selection up to 83 passages. The gp120 gene carried the following mutations after 83 passages of the virus in the presence of V-165: A29S, T168N, R272T, N293D and the 364–368 FNSTW deletion. Remarkably, the original V212I mutation in the gp120 gene was lost. The gp41 gene carried the L33S mutation after 83 passages of the virus in the presence of V-165. In addition, the RT gene of the selected HIV-1 NL4.3 virus contained several mutations after 83 passages, namely, T69N, K70R and T215Y/S/F. At low V-165 selective pressure, mutations were thus primarily detected in the IN gene of the selected strain. When V-165 inhibitory pressure was increased, mutations were also detected in the gp160 and RT genes of the passaged virus. No mutations were identified in the gag encoding region or the gag-pol frame shift site of the viruses selected in the presence of V-165. As V-165 interferes with the binding of IN to the viral DNA, we also performed sequencing of the IN attachment sites in the LTR regions, which are the terminal 15 bp adjacent to the conserved CA dinucleotide.46,47 However, no mutations could be detected in these regions (data not shown).
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50% of the virus population.Evaluation of phenotypic (cross)-resistance of the different selected HIV-1 strains using the MT-4/MTT assay
To verify whether the HIV-1 strains selected in the presence of increasing V-165 concentrations were indeed less susceptible to the inhibitory effect of the drug, we determined the antiviral activity of V-165 against the strains HIV-1 NL4.3, NL4.3/V-165(#15), NL4.3/V-165(#30), NL4.3/V-165(#40), NL4.3/V-165(#63) and NL4.3/V-165(#83) using the MT-4/MTT assay (Tables 1 and 2). The susceptibility of the selected strains to the CXCR4 antagonist AMD3100, the fusion inhibitor T-20 and the NRTI zidovudine was determined (Tables 1 and 2). The inhibitory effects of the entry inhibitor DS, the NNRTI nevirapine, the HIV-1 IN inhibitor L-870,810 and the PI ritonavir were measured as well (Table 2).
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NL4.3/V-165(#15) was 2.6-fold less susceptible to V-165 in comparison with WT HIV-1 NL4.3. NL4.3/V-165(#30) was 3.3-fold less susceptible to V-165, and NL4.3/V-165(#40) and NL4.3/V-165(#63) were each 3.4-fold less susceptible to V-165 in comparison with WT NL4.3. NL4.3/V-165(#83) was 6.9-fold less susceptible to the inhibitory effect of V-165 (Table 1). Unexpectedly, the NL4.3/V-165(#83) resistant strain showed reduced susceptibility to AMD3100 (33.3-fold), T-20 (23.3-fold) and zidovudine (8.6-fold). The HIV-1 NL4.3 strains selected for 15, 30, 40 or 63 passages, however, showed the same susceptibility to zidovudine as WT NL4.3 (Table 1). Partial resistance to AMD3100 was observed with the strains selected for 15, 30, 40 and 63 passages (5.0-, 5.0-, 6.6- and 6.6-fold increase in IC50, respectively). Partial resistance to T-20 (14.6-fold) was also observed with NL4.3/V-165(#63) (Table 1). The other inhibitors evaluated retained activity against the resistant strains (Table 2). Cross-resistance with entry inhibitors was thus obtained at later passages of the virus, except for AMD3100.
Evaluation of the IN-, RT- and gp160-recombined HIV-1 strains using the MT-4/MTT assay
To evaluate the importance of the described mutations in the IN, RT and gp160 genes for the observed V-165 resistant phenotype, we constructed recombinant strains carrying either WT or mutated IN, RT or gp160 gene from the selected HIV-1 NL4.3 strains in a WT NL4.3 backbone. Recombination was performed with the following strains: WT HIV-1 NL4.3 and the HIV-1 NL4.3 selected during 83 passages in the presence of V-165 [NL4.3/V-165(#83)]. The recombinant strains are referred to as RINNL4.3, RRTNL4.3, RENVNL4.3, RINNL4.3/V-165(#83), RRTNL4.3/V-165(#83) and RENVNL4.3/V-165(#83). After recombination, the RT, IN and gp160 sequences were identical to those of the parental strains (data not shown).
Antiviral susceptibility of the recombined strains was determined by the MT-4/MTT assay (Table 2). Unexpectedly, IN recombination could not reproduce the phenotypic resistance profile of the corresponding parental selected strains. The IN-recombined strains showed WT susceptibility to most antiviral compounds evaluated, a 1.9-fold decrease in susceptibility to V-165 and a 2.5-fold loss in susceptibility to AMD3100. The loss in susceptibility of the env-recombined strain RENVNL4.3/V-165(#83) with respect to the WT-recombined strain reproduced the decreased susceptibility of the corresponding selected parental strains partially for AMD3100 and completely for T-20. A 2.6-fold loss in susceptibility of RENVNL4.3/V-165(#83) to V-165 was measured as well. Furthermore, RT recombination partially reflected the observed loss in susceptibility of the NL4.3/V-165(#83) strain to the NRTI zidovudine when compared with the respective WT HIV-1 NL4.3 strain. DS, nevirapine, L-870,810 and ritonavir showed WT inhibitory activity against all strains evaluated (Table 2).
Replication kinetics of HIV-1 strains selected in the presence of V-165 and their corresponding HIV-1 IN, RT and gp160 recombinants
To investigate whether the drug-induced mutations affect the viral replication capacity, the HIV-1 strains selected in the presence of V-165 and their corresponding IN, RT and gp160 recombinants were examined for their ability to replicate in MT-4 cells in comparison with their respective parental strains. All recombined strains showed substantially reduced replication fitness in comparison with WT NL4.3 (Figure 3a–c). Replication kinetics of the selected NL4.3/V-165(#83) strain and the recombined strain RENVNL4.3/V-165(#83) were delayed relative to the replication of their corresponding WT strains (Figure 3b). The IN-recombined strain (RINNL4.3/V-165) showed a clear decrease in replication capacity in comparison with the WT recombinant RINNL4.3 (Figure 3a). RRTNL4.3/V-165 showed similar replication kinetics as WT recombinant RRTNL4.3 (Figure 3c).
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Enzymatic activity of mutant INs
Although most mutations observed in env and RT have been described previously as associated with resistance to entry inhibitors34,48 and NRTI,49 respectively, the mutations detected in the IN gene were novel. To study the impact of those IN mutations on enzymatic activity and drug sensitivity, mutant IN was produced by site-directed mutagenesis. The enzymatic activities were determined in the oligonucleotide-based overall integration assay (Figure 4). The S230N IN mutant displayed WT enzymatic activity, whereas the mutants T206S and T206S/S230N showed a 2-fold reduction in the enzymatic activity. To determine the ability of the different enzymes to bind DNA, FCS was used.44 As shown in Table 3, the mutants IN-T206S and IN-T206S/S230N showed, respectively, a 2.0- and 2.1-fold decrease in affinity for DNA in comparison with WT IN.
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Sensitivity of mutant INs to inhibition by V-165 and L-870,810
We determined the sensitivity of mutant IN to the inhibitory effect of V-165 in an ELISA assay. We normalized the different enzyme preparations for equal enzymatic activity. V-165 inhibited the mutants T206S, S230N and T206S/S230N to a 0.9-, 1.3- and 1.6-fold lower extent than WT IN, respectively (Table 4). No cross-resistance towards L-870,810 was observed (Table 4). INBIs were shown to interfere with the interaction of IN with the viral DNA substrate.44 In an FCS-based IN–DNA binding assay, we determined the Ki of WT and mutant IN for inhibition by V-165. The mutant enzymes showed low-level resistance to V-165. T206S and T206S/S230N were 2.1- and 2.3-fold less sensitive to V-165, respectively (Table 3).
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Inhibition of HIV-1 adsorption to MT-4 cells by the pyranodipyrimidine V-165
As the loss in susceptibility to V-165 was also associated with mutations in the envelope of the resistant virus, we investigated the effect of V-165 on virus adsorption to the cells. A known adsorption inhibitor DS, the NNRTI efavirenz and L-870,810 were included as controls (Table 5). DS and V-165 inhibited the adsorption of WT virus to the cells with IC50s of 0.016 and 18.9 µM, respectively. These concentrations are 37.8- and 0.9-fold lower than their respective IC50s in the MT-4/MTT assay. Neither L-870,810 nor efavirenz showed any activity in the virus adsorption assay with inhibitor concentrations up to 1.6 and 0.3 µM (100 x IC50), respectively.
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Effect of V-165 on HIV-1 infection and HIV-1 vector transduction kinetics
Previously, we carried out Q-PCRs on DNA extracted from cells transduced with HIV-1 vector in the absence or presence of V-165.22 In the presence of V-165, no inhibition of early total DNA synthesis was seen, but integration was clearly reduced. In these experiments, however, HIV-1 entry was not assayed for because these lentiviral vectors were pseudotyped with VSV-G glycoprotein. We used this analysis in particular to discriminate between inhibition of reverse transcription and integration. Here, we revisited this Q-PCR analysis in a comparative study of HIV-1 infection and HIV-1 vector transduction in the presence or absence of V-165. The diketo analogue TR-3401 was included as a control INSTI. We infected MT-4 cells with HIV-1 NL4.3 and transduced 293T cells with VSV-G pseudotyped HIV-1 vectors encoding eGFP, either in the absence or in the presence of 12 µM TR-3401 or 72 µM V-165 (15-fold and 5-fold respective IC50 values as determined by the MT-4/MTT assay). Results of DNA quantification are presented in Figure 5. Neither during lentiviral transduction nor during virus infection, was inhibition of DNA synthesis observed in the presence of the diketo analogue (Figure 5a and d, respectively). As expected, no integrated proviral DNA was detected by Q Alu-PCR (Figure 5b and e), whereas the amount of 2-LTR circles clearly increased in the presence of TR-3401 (Figure 5c and f). In the presence of V-165, no clear inhibition of HIV-1 vector DNA synthesis was detected (Figure 5a) and there was also no clear effect on 2-LTR circle formation (Figure 5c) during lentiviral transduction. Still, there was a pronounced defect in the generation of integrated proviral DNA (Figure 5b). These results are highly consistent with our previous report and corroborate integration as a genuine target for PDP. However, during HIV-1 infection of MT-4 cells, inhibition of viral DNA synthesis by PDP, but not by TR-3401, was clearly evidenced from 6 h post-infection (Figure 5d). As reverse transcription during HIV infection and lentiviral vector transduction likely follow the same course, this result indicates that the mechanism of inhibition of PDP is dependent on the viral entry route.
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Evaluation of the physicochemical and pharmacokinetic properties of V-165
As V-165 is the prototype of the INBIs that have not yet entered clinical trials in contrast to INSTIs, we initiated early pre-clinical studies. Solubility experiments using nephelometry indicated that V-165 displays adequate solubility at pH 6.5 (solubility >100), but poor solubility at pH 2 (solubility range 1.6–3.1). Analysis of V-165 stability and protein binding properties revealed 30% recovery from human hepatic microsomal matrix and partial recovery from 1% BSA (49% recovery). This is in contrast to many druggable compounds that show recoveries in excess of 90%. The recovery of the compound from BSA solutions was restored to 100% in the presence of DTT, suggesting disulphide cross-linking to proteins.
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Discussion |
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As part of the pre-clinical studies with PDP, an early INBI prototype, we have now investigated the development of HIV resistance against V-165 (Figure 1) in cell culture and we have characterized the physicochemical properties of the compound. The experiments reported herein point to a multimodal in vivo mechanism of action for PDP.
HIV-1 was grown in the presence of increasing concentrations of V-165 for 45 weeks. After 45 weeks, the selected virus was able to grow as WT virus in the presence of 79 µM V-165, which is 11-fold its IC50. Although selection up to 8 months rendered the selected virus 7-fold less susceptible to V-165 when compared with WT HIV-1, cross-resistance towards the bicyclam AMD3100, the fusion inhibitor T-20 and the NRTI zidovudine was observed as well (Tables 1 and 2). To clarify this multifaceted phenotype, we performed a genotypic analysis of the passaged virus. Numerous mutations were detected in the IN, RT and env genes of the resistant virus. T206S is located in the catalytic core of the viral IN and the S230N mutation is positioned in the DNA binding site of the enzyme. Although both mutations are known polymorphisms,50 this does not exclude a potential relevance in resistance to IN inhibitors. Contrary to mutations in IN, most of the gp160 and RT mutations were only identified in a later phase of the selection process when V-165 selective pressure was elevated (Figure 2). Some of the selected gp120 mutations are known to be associated with resistance towards AMD3100 and entry inhibitors in general (F145L, R272T, Q278H, I288V, N293D, A297T, the 364–368 FNSTW deletion, P390L and S438P).34,48 The L33S mutation that appeared in the gp41 gene has already been described to be associated with resistance to T-20.34 Both the K70R and the T215Y/F mutation in the RT gene are known NRTI resistance mutations.49 These mutations are so-called thymidine-associated mutations (TAMs) and they cause resistance to NRTIs because the mutant RTs more efficiently remove the chain-terminating residue from the growing DNA chain.51 In order to assess the significance of each set of mutations in the IN, RT or gp160 gene for the observed resistance profile, we constructed recombinant strains carrying the WT or resistant IN, RT or gp160 gene in a WT HIV-1 backbone and determined their antiviral susceptibility (Table 2). Surprisingly, IN recombination (i.e. 1.9-fold increase in resistance) could not fully reproduce the observed phenotypic resistance profile of the corresponding parental selected strain (i.e. 7.0-fold increase in resistance). These observations were corroborated on an enzymatic level. Only low-level resistance to V-165 could be detected in the oligonucleotide-based integration assay (Table 4) and in the FCS-based IN/DNA interaction assay (Table 3).
The env-recombined viral strains also exhibited a partial loss in susceptibility to V-165 (2.6-fold) in comparison with the selected strain (6.9-fold) and displayed a nearly similar loss in susceptibility to T-20 and AMD3100 as their original in vitro selected strains, indicating that the mutations in the recombined region are sufficient to reproduce the (cross-)resistant phenotype of the strains selected in the presence of V-165. RT recombination reflected the observed loss in susceptibility of the V-165 selected strains to the NRTI zidovudine when compared with their WT strains, implying a role for these mutations in the observed cross-resistance to zidovudine. They did not seem to be responsible for the observed loss in susceptibility to V-165. These results suggested that the mutations selected in the envelope of the virus strains might play a role in antiviral resistance development at high concentrations of V-165. Although we could not detect a considerable inhibitory effect of V-165 on HIV cDNA synthesis in cell culture, as measured by Q-PCR (Figure 5b), it was shown previously that V-165 does inhibit HIV RT activity in vitro at micromolar concentrations.22 We do not know the reason why specific TAM mutations are selected in the presence of V-165. Perhaps, V-165, by virtue of its dipyrimidine structure, may affect nucleotide incorporation in the growing cDNA chain during reverse transcription, thereby functioning as a chain terminator.
Two explanations for this complex resistance pattern can be put forward. Either the selection experiments were confounded by contamination with entry or RT-inhibitor resistant virus or V-165 exerts a multimodal mechanism of action, targeting HIV replication at different steps in the viral lifecycle. As the selection procedure was carried out twice with a newly synthesized batch of V-165, and this resulted in a similar phenotypic and genotypic resistance profile (data not shown), we can exclude contamination.
The recombination experiment suggested a possible role of the env gene in the observed resistance profile. Therefore, we carried out several experiments to investigate the antiviral effect of V-165 on viral entry. First, we carried out a viral adsorption assay to determine the activity of V-165 on HIV-1 entry (Table 5). The known entry inhibitor DS and the PDP V-165 inhibited the adsorption of WT virus to the cells, with IC50s of 0.016 and 18.9 µM, respectively. Apparently, V-165 affects the adsorption of the virus to the cell, although the compound is much less potent than a typical adsorption inhibitor such as DS, which is 40-fold more active in this assay than in the MT-4/MTT assay. Previously, we excluded viral entry as an antiviral target of V-165 in cell culture.22 Using a series of Q-PCRs on HIV-1 vector-transduced cells in the presence of V-165, it was shown that integration was the major antiviral target. However, as the vectors used were VSV-G glycoprotein pseudotyped, inhibition of HIV-1 entry was not assessed for. Therefore, a multimodal mechanism of action was not excluded. Here, we revisited this Q-PCR analysis in a comparative study of HIV-1 infection and HIV-1 vector transduction in the presence or absence of V-165 (Figure 5). Next to V-165, we tested the experimental diketo analogue TR-4301 as a representative INSTI in parallel. Neither during virus infection nor during lentiviral transduction was inhibition of DNA synthesis observed in the presence of TR-4301. As expected, no integrated proviral DNA was detected by Q Alu-PCR (Figure 5b and e) and there was a clear increase in 2-LTR circles (Figure 5c and f). In the presence of V-165, no clear inhibition of HIV-1 vector DNA synthesis was detected (Figure 5a). Furthermore, there was a pronounced defect in the generation of proviral DNA (Figure 5b), whereas no substantial increase in 2-LTR circles was measured. Possibly, this relates to the inhibitory effect of V-165 on the viral DNA–IN complex formation that takes place in the cytoplasm of the infected cell and may be required for efficient nuclear import. These results are highly consistent with our previous report and corroborate integration as a genuine target for PDP. However, during HIV-1 infection of MT-4 cells, inhibition of viral DNA synthesis was clearly evidenced at 6 h post-infection (Figure 5d). Taking into account the data from the viral adsorption assay, these observations indicate that V-165 also affects HIV-1 entry. However, when HIV-1 entry is not assayed for, the inhibitory activity of V-165 on integration becomes apparent. This comparative study cautions for the use of a pseudotyped lentiviral vector transduction assay to identify the target of an experimental antiviral in cellulo.
In conclusion, this set of experiments illustrates the usefulness of studying antiviral resistance development to pinpoint the viral target(s) of a compound. This has already been illustrated for the IN inhibitors L-chicoric acid16 and Zintevir,15 for which antiviral resistance studies indicated that these compounds also affect viral entry. For V-165, our results also revealed a multimodal mechanism of action. V-165 inhibits viral entry at a concentration of 19 µM and also integration in vivo as indicated by a decrease in the integrated proviruses after VSV-G pseudotyped virus infection. Although the physicochemical properties of V-165 are not optimal, encountering problems with batch variation and stability (data not shown), and V-165 is poorly recovered from human hepatic microsomal matrix and 1% BSA, we still propose IN binding inhibition as a genuine antiviral target and V-165 as the INBI prototype. The quest for more stable and potent derivatives of V-165 that specifically target binding of IN to viral DNA should therefore be pursued.
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None to declare.
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Acknowledgements |
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We gratefully acknowledge expert technical assistance at the K. U. Leuven and K. U. Leuven Campus Kortrijk by Linda Desender, Sofie Janssen, Michela Marongui and An Nijs. The plasmid pNL4.3 was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: pNL4-3 from Dr Malcolm Martin. We thank R. Plasterk for providing the plasmid pRP1012 and B. A. Larder for providing the plasmid pNL4.3
RT. This work was supported by the European Commission (LSHB-CT-2003-503480) (TRIoH project). A. H. is funded by a grant from the Flemish Institute supporting Science-Technological Research in Industry (IWT). |
References |
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