| One of the greatest achievements is the development and application of anti-HIV-1 drugs during fighting against HIV/AIDS for more than 30 years. Combinations of antiretroviral drugs were introduced in 1996 — so-called highly active antiretroviraltherapy(HAART). It is the best option for longlasting viral suppression and, subsequently, for reduction of mortality and for improvement of quality of life. Based on the huge success of antiretroviral therapy and the control of HIV-1 spread, the expand treatment and prophylactic treatment, which advocate the early treatment and prophylaxis before/after exposure for whom under the risk of infection, have been taken as new strategies for AIDS prevention and control in the recent years. As of June 2015, 16 million people living with HIV-1 were accessing antiretroviral. Unlike 20 years ago, the transmission and evolution of HIV-1 are under the widespread drug stress now. Because of its high mutation and replication rate, HIV-1 is prone to resistance in a matter of weeks. At present, a number of reports have described HIV-1 strains with resistance to each type of antiretroviral drugs, which will lead to the failure of drug and treatment. Therefore, the development of novel anti-HIV-1 drugs against drug-resistant strains is still urgent for AIDS prevention and control.Nonclinical studies are the foundation of the antiretroviral drug development, and the assessment of drug’s activit ies against resistant strains is an important part of nonclinical studies. FDA’s guidance for industry HIV-1 infection: developing antiretroviral drugs for treatment recommends that the resistance testing is essential for developing the anti-HIV-1 drugs. Unlike the assessment of activities against wild type isolates, there are many special problems against resistant isolates:(1) In our country, the research on drug resistance is mainly based on the results of genotypic resistance, and there is no phenotypic resistance method and, subsequently, we do not understand the phenotypic resistance characteristics of epidemic strains.(2) Due to many kinds of anti-HIV-1 drugs available, patients often bear multiple amino-acid substitutions, which have complicated effects on the treatment efficacy.(3) The tranditional pharmacodynamic parameters such as the IC50 s and the fold changes do not evaluate the activity changes of protease inhibitors(PIs) and non-nucleoside reverse transcriptase inhibitors(NNRTIs).In view of these problems, this research has established an in-house HIV-1 phenotypic resistance assay based on the recombinant virus, and understood the drug resistance of the main epidemic strains in our country. Then the activities of commonly used antiretroviral drugs against strains bearing multiple resistance mutation and mutant were evaluated with this method, and a new parameter was proposed. According to the understanding of HIV-1 replication and anti-HIV-1 drug, we conducted the nonclinical studies on two anti-HIV-1 candidates(a novel protease inhibitor and polypeptides argeting Vpu-tetherin interaction) by the means of in-house recombinant virus phenotypic resistance assay and other assays based on the established parameters, and explored the possible mechanisms.Chapter Ⅰ The establishment of HIV-1 phenotypic resistance assay based on the recombinant virus and the study of phenotypic resistance of epidemic strainsBoth genotypic and phenotypic methods for HIV-1 drug resistance evaluation are now available. Because of its rapid and low-cost, genotypic assay is widely carried out. Though the high requirements for laboratory and technology limit the application of phenotypic assay, it is essential for identifying the resistance of mutations and studying the activities of novel anti-HIV-1 agents. The epidemic of HIV-1 in China is diversity and there are many researches on HIV-1 genotypic resistance. However, there is no standard phenotypic resistance method and, therefore, we have not any knowledge on the phenotypic resistance of HIV-1 epidemic strains. We are to establish a phenotypic resistance method based on the recombinant virus and to examine HIV-1 variants susceptibilities to antiretroviral drugs, so as to supplement to genotypic resistance assay and provide evidence for the treatment strategies.Methods and results:(1) Twenty eight plasma specimens were collected from 10 provinces and metropolitan cities across China. All patients provided a written informed consent and their basic personal and clinical information was acquired by face to face interview. HIV-1 RNA was extracted and nested RT-PCR was employed to amplify HIV-1 gag-pol gene which comprised the entire protease(codons 1–99) and the former reverse transcriptase(codons 1–312). The patient-derived PCR products were directly sequenced, and the sequences were edited and aligned. Genetic resistance was determined by submitting the sequences to the Stanford University Network HIV database. Subtype was determined by phylogenetic tree analysis of the RT fragments. The identified viral genetic subtypes included B, CRF01_AE and CRF07_BC. Major drug resistance mutations were identified in HN2009001 and GD2005004, and no primary or secondary drug resistance mutations were found in the other 17 patients.(2) Recombinant viruses were generated with a p NL4.3 vector. The patient-derived target segments were subcloned into a p NL4.3-Δ(Sph I-Age I) vector and recombinant infectious clones were obtained. Then 293 T cells were transfected with the constructed plasmids and 19 homologous recombinant viruses which were replication-competent were harvested. Each recombinant viral activities in the presence of 6 antiretroviral drugs(AZT/3TC/d4T/dd I/NVP/EFV) widely used in China were measured using the luciferase reporter from MT2/TZM-bl cells in biosafety level-3 laboratory. Three replicate determinations were performed in duplicate plates for each concentration of antiretroviral drug. And 114 drug-resistance phenotypic results were obtained.(3) The relationship between phenotypic drug resistance and genetic polymorphisms was determined. The agreement rate was 96.5% between the genotypic results and phenotypic results. For a treatment-experienced sample from Henan(HN2009001) with major drug resistance mutations including M41 L, L210 W, T215 Y, K103 N, and K238 T, both phenotypic and genotypic assays suggested that it was resistant to all drugs tested, with the fold changes in IC50 were 3.92-126.75. There were no any recognizable genotypic resistance-associated mutations noted for 14 samples, which were susceptible to 6 drugs tested according to the phenotypic assay. The other 4 samples had no any recognizable genotypic resistance-associated mutations, with large reductions in susceptibilities to NVP and EFV(3.41-7.84).Conclusions: This study provides a new useful tool based on the recombinant virus for assessing drug phenotypic resistance to antiretroviral drugs. The phenotypic results agree with the genotypic results for the most part, permitting the straightforward quantitation of the level of resistance, and serving as a basis for studying HIV-1 resistance and activities of novel anti-HIV-1 agents. The disparity in HIV-1 susceptibility poses a need to invest the clinical effectiveness of antiretroviral therapy of special individuals.Chapter Ⅱ The effects of multiple resistance mutations and mutants on the pharmacodynamic properties of anti-HIV-1 drugs and the proposal of a new parameterThe currently pharmacodynamic properties to determine the inhibitory activities of drugs against resistant strains are the concentration of drug required for 50% inhibition(IC50s) and fold changes relative to the wild type strains. It is assumed that resistance mutations shift dose-response curves to the right alone without affecting their slopes. It is noted that a resistance mutation may not only increase IC50, but decrease the slope of the dose-response curve, and even have influence on the slope alone. At present, the effects of multiple resistance mutations and mutants on the pharmacodynamic properties of anti-HIV-1 drugs are unclear, and there is not a parameter applying for accurately determining the phenotypic resistance of various types of drugs. We are to elucidate the effects of multiple resistance mutations and mutants on the pharmacodynamic properties of anti-HIV-1 drugs, and to put forward a new parameter under the consideration of the IC50 and the slope.Methods and results:(1) Site-directed infectious clone plasmids were constructed with a wild type p NL4.3 vector. Then 293 T cells were transfected with the constructed plasmids and 8 replication-competent recombinant virus strains bearing different resistant mutations and mutants were constructed.(2) Phenotypic susceptibilities of the recombinant viruses to 10 antiretroviral drugs were measured in biosafety level-3 laboratory and dose response curves were drawn. Three replicate determinations were performed in duplicate plates for each concentration of antiretroviral drug. Combining with the former phenotypic resistance results, we assessed the activities of 10 widely used drugs against 22 common HIV-1 resistance mutation complexes in China.(3) The pharmacodynamic characteristics of various drugs against each mutant strain were summarized. There were intrinsic differences in the method by which resistance mutations affect pharmacodynamic parameters, including three cases:(1) The mutations influenced the IC50, without the slope. All of nucleoside reverse transcriptase inhibitor(NRTIs) resistant mutations resulted in large increases in the IC50 s for NRTIs, without affecting the slopes of the curves.(2)The mutations affected both the IC50 and the slope. The K103N\H221Y\Y181C\T215Y mutation led to an intermediate shift in the IC50(13.36-fold) but a marked reduction in the slope for EFV.(3) The mutations had no effect on the IC50, but reduced the slope of the dose-response curve. This was the most apparent when examining the effects of the mutations on PIs and NNRTIs activities. For example, the mutation G48V\I54V had no effect on the IC50, but reduced the slope of the dose-response curve for RTV. And the mutation V82 A reduced the slope by 0.41 but not the IC50 for SQV.(4) We proposed a new index, termed IIP atoxic, which incorporated the IC50, the slope, and the maximum nontoxic concentration of each drug. The IIP atoxic values of 10 approved drugs were calculated, and there were strong correlations(r > 0.95, P < 0.001 each) between the fractional changes in IIP max(from literature, peak plasma concentrations of the drugs were used) and those in IIP atoxic for all drugs tested.Conclusions: The study assesses the pharmacodynamic characteristics of 10 widely used drugs against common HIV-1 resistance mutations and mutants strains. The effect of resistance mutations on the slope of the dose-response curve is more evident for PIs and NNRTIs than for NRTIs. We propose a new comprehensive index IIP atoxic, applying for nonclinical study, thereby enabling predict the efficacy of pre-clinical drugs for which human pharmacokinetic is not available against resistant viruses.Chapter Ⅲ The study on the anti-HIV-1 activity of the novel protease inhibitor DG35There are not any anti-HIV-1 drugs available with independent intellectual property rights in our country. DG35 is a small molecular compound targeting HIV-1 protease, which is developed in our country, while the activity is unknown. We are to evaluate the anti-HIV-1 activity of DG35, providing nonclinical pharmacodynamic data for the subsequent development of the drug.Methods and results: According to the FDA’s guidance for developing the anti-HIV-1 drugs, the antiretroviral activity of DG35 was evaluated using high-throughput screening methods. Nonclinical studies, such as cytotoxicity, inhibition against viruses with various genetic background, combination antiviral activity, and selection of drug-resistant HIV-1 variants were completed using the established phenotypic resistance methods and the constructed recombinant viruses, especially the study of drug resistance. The results shown that:(1) On the basis of the methodologies used, it was certain that DG35 had a high anti-HIV-1 activity. The IC50 of DG35 against the wild type reference strain(HIV-1NL4.3)(3.58±0.10 n M) below that of the approved drug IDV(7.19±1.01 n M) and the slope(3.42±0.58) higher than that of IDV(2.49±0.23).(2) The inhibitory activities of DG35 against the mutant strains bearing M46 I, I54 V, V82 A, M46I\N88S, and M46I\V82T\I84V agreeed with that of DG35 against the wild type reference strains(1.02-12.46 n M). Compared with HIV-1NL4.3, not only the IC50 of DG35 against the strain carrying the PI resistant mutation(G48V\I54V) increased by 65.57, but the slope and the IIP atoxic value decreased. And there were high inhibitory activities of DG35 against 3 NNRTIs resistant strains.(3) Combinations of DG35 with the AZT(NRTI), EFV(NNRTI) and RTV(PI) were studied. And all combinations were scored as highly synergism, with synergy indexes 592.28 n M2%, 661.33 n M2% and 528.72 n M2%, respectively.(4) After 6 cell passages, no viral replications was detected in the presence of DG35.Conclusions: There are high inhibitory activities of DG35 against both laboratory reference strains and PI resistant strains. But resistant strains bearing G48V\I54V and G48V\I54V\V82A have influence on the activities of DG35, and there is no cross resistance with NNRTIs. All combinations with 3 approved anti-HIV-1 drugs are scored as highly synergism. And it has a high resistance gene barrier. These results laid the foundation for the following study and development of the novel drug.Chapter Ⅳ Polypeptides inhibit HIV-1 replication by interfering Vpu-mediated tetherin degradationDue to at present a number of reports describing HIV-1 strains with resistance to each type of antiretroviral drugs, there is a need to discover novel anti-HIV-1 drugs with different mode of action. Tetherin(THN, also known as BST-2) is a protein expressing at the surface of host cell, and can block the release of virus through tethering virions to infected cells, which is the innate antiviral mechanism of human body cell. Viral protein U(Vpu) is gained in the process of HIV-1 evolution, and the degradation of tetherin is mediated by intracellular protein degradation, thereby blocking the natural antiviral function. It may be a promising new target for anti-HIV-1 drugs to inhibit the degradation of tetherin mediated by Vpu. Up to now, however, neither polypeptide nor small molecule has been reported to block the interaction between Vpu and tetherin. We are to design the polypeptides inhibiting HIV-1 replication by interfering Vpu-mediated tetherin degradation, to verify the activities, and to explore the possible active mechanisms, providing a new insight for the study and development of new anti-HIV-1 drugs.Methods and results:(1) We designed Vpu derived polypeptides that covered the amino acid sequence on the interface of Vpu-tetherin complex. Seven 20-mer polypeptides from wild type Vpu amino acid sequence based on HIV-1NL4.3 reference strain were designed, with each polypeptide shifted 10 amino acids. Poly-arginine was introduced in all designed polypeptides as trans-membranepeptide, and also to increase the solubility of the polypeptides. Two scramble 20-mer polypeptides were also designed as controls.(2) The inhibitory activity was determined using the established phenotypic efficacy methods. Three polypeptides with significant anti-HIV-1 activity were identified, termed as CJ128, CJ130 and CJ131, with IC50 4.29±0.05μM, 3.75±0.13μM and 3.50±0.21μM, respectively, while the two scramble polypeptides controls showed no activity against HIV NL4.3 replication.(3) Vpu and tetherin plasmids were co-transfected into HEK293 T cells to construct a model of Vpu-tetherin interaction, and the expression level of tetherin in the presence of polypeptides were monitored by western blot. The results indicated that CJ128 and CJ130 restored tetherin expression level, indicating that the polypeptides inhibit HIV-1 infection through blocking Vpu-mediated tetherin degradation.(4) We prepared fluorescently labeled(5-FAM) polypeptides and studied their in situ binding. After 6h incubation, laser confocal scanning were used to image the polypeptides binding at the cell surface. All active polypeptides displayed discrete spots on the cell surface, typical for specific cell surface receptor binding, in contrast to inactive control which was eventually distributed on the cell surface, typical for non-specific binding.Conclusions: We identified 3 Vpu-derived polypeptides that could efficiently inhibit HIV-1 replication at μM co ncentration. A pilot mechanism study showed that the active polypeptide could counteract Vpu mediated tetherin down regulation. Laser confocal image scanning study showed that the polypeptides bound on the cell surface with a receptor specific binding manner, which may target tetherin that expressed on cell surface. These results suggested that the polypeptides inhibited HIV-1 infection by blocking Vpu-mediated tetherin degradation. Our work provided the first evidence that Vpu-tetherin interaction could be a target for novel anti-HIV-1 drug design. It provides a promising strategy for the further research and development of novel anti-HIV-1 drugs. |
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