Antileishmanial activity of antiretroviral drugs combined with miltefosine.

Co-infection of Leishmaniasis, a neglected tropical disease, with human immunodeficiency virus (HIV) has hindered treatment efficacy. In this study, we aim to evaluate the antileishmanial activity of two protease inhibitors (darunavir and atazanavir) and four reverse transcriptase inhibitors (tenofovir, efavirenz, neviraprine, and delavirdine mesylate) on Leishmania infantum. The activity of different antiretrovirals combinations and of antiretroviral with miltefosine, a drug used on leishmaniasis treatment, was also evaluated. Only two non-nucleoside reverse transcriptase inhibitors (NNRTIs) were active on L. infantum. Efavirenz showed the best antileishmanial activity on promastigotes cells with IC50 value of 26.1 µM followed by delavirdine mesylate with an IC50 value of 136.2 µM. Neviraprine, tenofovir, atazanavir, and darunavir were not active at the concentrations tested (IC50 > 200 µM). The efavirenz also showed high antileishmanial activity on intramacrophage amastigotes with IC50 of 12.59 µM. The interaction of efavirenz with miltefosine improved antileishmanial activity on promastigotes and intracellular amastigotes (IC50 values of 11. 8 µM and 8.89 µM, respectively). These results suggest that combined-therapy including efavirenz and miltefosine could be alternative options for treating Leishmaniasis and Leishmania/HIV co-infections.

Role of the K101E substitution in HIV-1 reverse transcriptase in resistance to rilpivirine and other nonnucleoside reverse transcriptase inhibitors.

Resistance to the recently approved nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) commonly involves substitutions at positions E138K and K101E in HIV-1 reverse transcriptase (RT), together with an M184I substitution that is associated with resistance to coutilized emtricitabine (FTC). Previous biochemical and virological studies have shown that compensatory interactions between substitutions E138K and M184I can restore enzyme processivity and the viral replication capacity. Structural modeling studies have also shown that disruption of the salt bridge between K101 and E138 can affect RPV binding. The current study was designed to investigate the impact of K101E, alone or in combination with E138K and/or M184I, on drug susceptibility, viral replication capacity, and enzyme function. We show here that K101E can be selected in cell culture by the NNRTIs etravirine (ETR), efavirenz (EFV), and dapivirine (DPV) as well as by RPV. Recombinant RT enzymes and viruses containing K101E, but not E138K, were highly resistant to nevirapine (NVP) and delavirdine (DLV) as well as ETR and RPV, but not EFV. The addition of K101E to E138K slightly enhanced ETR and RPV resistance compared to that obtained with E138K alone but restored susceptibility to NVP and DLV. The K101E substitution can compensate for deficits in viral replication capacity and enzyme processivity associated with M184I, while M184I can compensate for the diminished efficiency of DNA polymerization associated with K101E. The coexistence of K101E and E138K does not impair either viral replication or enzyme fitness. We conclude that K101E can play a significant role in resistance to RPV.

Synthesis and biological evaluation of pyridazine derivatives as novel HIV-1 NNRTIs.

In continuation of our efforts toward identification and optimization of novel non-nucleoside reverse transcriptase inhibitors (NNRTIs), we have employed a structure-based bioisosterism strategy, with which a new series of diarylpyridazine (DAPD) derivatives were synthesized and evaluated for their anti-HIV-1 (human immunodeficiency virus type 1) activity. Most of the title compounds displayed excellent anti-HIV-1 activity at submicromolar concentrations ranging from 34 nM to 5.08 µM. The most promising compound 8g inhibited HIV-1 IIIB in MT-4 cells at a low EC50 value (0.034 µM), which was lower than the reference drug nevirapine and delavirdine. The structure activity relationships (SARs) were discussed and rationalized by docking simulations.

Hybrids of delavirdine and piperdin-4-yl-aminopyrimidines (DPAPYs) as potent HIV-1 NNRTIs: Design, synthesis and biological activities.

The hybrids of delavirdine and piperdin-4-yl-aminopyrimidine (DPAPYs) were designed from two excellent HIV-1 NNRTIs delavirdine and piperidin-4-yl-aminopyrimidine via molecular hybridization. The target compounds 4a-r were prepared and evaluated for their cellular anti-HIV activities and cytotoxicities as well as the inhibitory activities against HIV-1 reverse transcriptase (RT). All the newly synthesized compounds demonstrated moderate to excellent potency against wild-type (WT) HIV-1 with EC50 values in a range of 5.7 to 0.0086 µM and against RT with IC50 values ranging from 12.0 to 0.11 µM, indicating that the DPAPYs were specific RT inhibitors. Among all, 4d displayed the most potent activity against WT HIV-1 (EC50 = 8.6 nM, SI = 2151). Gratifyingly, it exhibited good to excellent potency against the single HIV-1 mutants L100I, K103N, Y181C, Y188L, E138K, as well as the double mutant F227L + V106A. Furthermore, the preliminary structure-activity relationships were summarized, molecular modeling was conducted to explore the binding mode of DPAPYs and HIV-1 RT, and their physicochemical properties were also predicted.

In silico design, synthesis and anti-HIV activity of quinoline derivatives as non-nucleoside reverse transcriptase inhibitors (NNRTIs).

A series of quinoline derivatives has been designed, synthesized and screened for their anti-HIV properties. The drug-like properties of compounds were evaluated first and then molecular docking using DS v20.1.0.19295 software showed that the compounds behaved as non-nucleoside reverse transcriptase inhibitors (NNRTIs) while interacting at the allosteric site of target HIV-RT protein (PDB:3MEC). The docking results revealed that all compounds formed hydrogen bonds with Lys101, Lys103, Val179, Tyr188, Gln190, Gly190, Pro225, Phe227, and Tyr318, and showed π-interaction with Tyr188 and Tyr318. TOPKAT (Toxicity Prediction by Komputer Assisted Technology) results confirmed that the compounds were found to be less toxic than the reference drugs. Density functional theory (DFT) analysis was performed to assess the binding affinity of all compounds. Further, molecular dynamics (MD) simulations were performed on compound 6 and delavirdine with HIV-RT enzyme. Comprehensive MD analyses showed a similar pattern of conformational stability and flexibility in both the complexes suggesting alike inhibitory action. The hydrogen-bonding interactions and the binding energy of active-site residues for the compound 6 complex revealed strong inhibitory activity than the reference (delavirdine) complex. Thus, the compound 6 might act as a potential inhibitor against HIV-RT. Overall, this study revealed that compound 6 (5-hydroxy-N-(4-methyl-2-oxo-1,2-dihydroquinolin-8-yl) thiophene-2-sulfonamide) has prudent anti-HIV activity against both HIV-1 (SI = 2.65) and HIV-2 (SI = 2.32) that can further be utilised in drug discovery against HIV virus.

Human immunodeficiency virus type 1 resistance or cross-resistance to nonnucleoside reverse transcriptase inhibitors currently under development as microbicides.

Microbicides based on nonnucleoside reverse transcriptase inhibitors (NNRTIs) are currently being developed to protect women from HIV acquisition through sexual contact. However, the large-scale introduction of these products raises two major concerns. First, when these microbicides are used by undiagnosed HIV-positive women, they could potentially select for viral resistance, which may compromise subsequent therapeutic options. Second, NNRTI-based microbicides that are inactive against NNRTI-resistant strains might promote the selective transmission of these viruses. In order to address these concerns, drug resistance was selected in vitro by the serial passage of three viral isolates from subtypes B and C and CRF02_AG (a circulating recombinant form) in activated peripheral blood mononuclear cells (PBMCs) under conditions of increasing concentrations of three NNRTIs (i.e., TMC120, UC781, and MIV-160) that are currently being developed as candidate microbicides. TMC120 and MIV-160 displayed a high genetic barrier to resistance development, whereas resistance to UC781 emerged rapidly, similarly to efavirenz and nevirapine. Phenotypically, the selected viruses appeared to be highly cross-resistant to current first-line therapeutic NNRTIs (i.e., delavirdine, nevirapine, and efavirenz), although they retained some susceptibility to the more recently developed NNRTIs lersivirine and etravirine. The ability of UC781, TMC120, and MIV-160 to inhibit the in vitro-selected NNRTI-resistant viruses was also limited, although residual activity could be observed for the candidate microbicide NNRTI MIV-170. Interestingly, only four p2/p7/p1/p6/PR/RT/INT recombinant NNRTI-resistant viruses (i.e., TMC120-resistant VI829, EFV-resistant VI829, MIV-160-resistant VI829, and EFV-resistant MP568) showed impairments in replicative fitness. Overall, these in vitro analyses demonstrate that due to potential cross-resistance, the large-scale introduction of single-NNRTI-based microbicides should be considered with caution.

The human immunodeficiency virus type 1 nonnucleoside reverse transcriptase inhibitor resistance mutation I132M confers hypersensitivity to nucleoside analogs.

We previously identified a rare mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), I132M, which confers high-level resistance to the nonnucleoside RT inhibitors (NNRTIs) nevirapine and delavirdine. In this study, we have further characterized the role of this mutation in viral replication capacity and in resistance to other RT inhibitors. Surprisingly, our data show that I132M confers marked hypersusceptibility to the nucleoside analogs lamivudine (3TC) and tenofovir at both the virus and enzyme levels. Subunit-selective mutagenesis studies revealed that the mutation in the p51 subunit of RT was responsible for the increased sensitivity to the drugs, and transient kinetic analyses showed that this hypersusceptibility was due to I132M decreasing the enzyme's affinity for the natural dCTP substrate but increasing its affinity for 3TC-triphosphate. Furthermore, the replication capacity of HIV-1 containing I132M is severely impaired. This decrease in viral replication capacity could be partially or completely compensated for by the A62V or L214I mutation, respectively. Taken together, these results help to explain the infrequent selection of I132M in patients for whom NNRTI regimens are failing and furthermore demonstrate that a single mutation outside of the polymerase active site and inside of the p51 subunit of RT can significantly influence nucleotide selectivity.

Modifying the diffusion layer of soluble salts of poorly soluble basic drugs to improve dissolution performance.

The dissolution mechanism of soluble salts of poorly soluble bases can be complex because both the dissolution of the salt and precipitation of the free base can occur depending on the experimental conditions and properties of the molecule. The dissolution of three soluble salts of poorly soluble bases is described in this paper. Two of these compounds precipitate as free base under normal stomach pH conditions (pH from 2-4) during dissolution. This free base precipitation is a result of formation of free base on the surface of the dissolving salt. Diffusion Layer modulated (DLM) solids are defined and presented that can effectively counteract this precipitation mechanism. These DLM materials employ excipients in order to modify the pH or solubility conditions at the surface of the dissolving salt to minimize precipitation of the free base that can occur. Rotating disk dissolution data is presented which shows how these formulated solids can act to improve the dissolution profile for these materials.

A study of 3-substituted benzylidene-1,3-dihydro-indoline derivatives as antimicrobial and antiviral agents.

3-Substituted benzylidene-1,3-dihydro-indoline derivatives were tested for their in vitro antibacterial activity against the Gram-negative bacteria Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and the Gram-positive bacteria Bacillus subtilis, Staphylococcus aureus, and for their their in vitro antifungal activity against Candida krusei and Candida albicans. The minimum inhibitory concentration (MIC) values were determined by the 2-fold serial dilution technique in Mueller Hinton broth and Sabouraud dextrose agar using antibacterial and antifungal assays, respectively. For comparison of the antimicrobial activity, rifampicin, ampicillin trihydrate, gentamicin sulfate, and ofloxacin were used as reference antibacterial agents, and fluconazole and amphotericin B were employed as reference antifungal agents. The most active compound 10 showed notable inhibition against Bacillus subtilis, Staphylococcus aureus, and Candida krusei. Compounds 1 and 6 were found slightly effective against Klebsiella pneumoniae and Escherichia coli. In addition, compounds 13 and 14 showed inhibition against Bacillus subtilis and Staphylococcus aureus. Indole derivatives were also tested in vitro for replication of the HepAD38 cell line and compared with lamivudine (3TC, L-2',3'-dideoxy-3'-thiacytidine). The IC50 values of the compounds were found to be >1000 microM against HBV except for compound 13 which exhibited activity with an IC50 value of 500 microM.

[Establishment of pharmacological evaluation system for non-nucleoside reverse-transcriptase inhibitors resistant HIV-1].

Consistent non-nucleoside reverse-transcriptase inhibitors (NNRTIs) resistant HIV-1 strains occurred due to the clinical use for more than ten years of efavirenz (EFV), nevirapine (NVP), and delavirdine (DLV). In this study, we established nine cell-based pharmacological models according to most NNRTIs-resistant clinical tested strains, Resistant mutations were introduced into vector, pNL4-3.Luc.R-E-, by overlapping PCR. Then, pseudovirions were produced by co-transfection of VSV-G plasmid and pNL4-3.Luc.R-E- -mut. All nine recombinant VSVG/HIV-mut pseudovirions (VSVG/HIV-wt, VSVG/HIV(-K103N), VSVG/HIV(-Y181C), VSVG/HIV(-L100I,K103N), VSVG/HIV(-Y188L), VSVG/HIV(-K103N,Y181C), VSVG/HIV(-K103N,P225H), VSVG/HIV(-K103N,Y188L), VSVG/HIV(-K103N,G109A) and VSVG/HIV(-K103N,V108I)) had high efficient infectivity. Furthermore, they all showed resistant characteristics to EFV and NVP with IC50 changes consisting with clinical reports, not to nucleoside reverse-transcriptase inhibitors (AZT and d4T). This series safe cell-based model, which could be carried out in BSL-2 laboratory, can be used for evaluating NNRTIs candidates.

Prevalence of predicted resistance to doravirine in HIV-1-positive patients after exposure to non-nucleoside reverse transcriptase inhibitors.

This study investigated the prevalence of doravirine (DOR) resistance mutations in non-nucleoside reverse transcriptase inhibitor (NNRTI)-experienced patients. DOR resistance was assessed in samples from NNRTI-experienced patients who underwent genotypic testing for virological failure from the Antiretroviral Response Cohort Analysis (ARCA) database. Intermediate DOR resistance was defined as detection of any of V106A/M, Y188C/H, V108I, and K103N+P225H. High-level DOR resistance was defined as detection of any of Y188L, M230L, G190E, V106A/M+F227L, and V106A/M+L234I. Overall, 6893 patients were included in the study: 64.2% had experienced efavirenz (EFV), 54.4% nevirapine (NVP), 6.8% etravirine (ETR), 7.7% rilpivirine (RPV) and 0.7% delavirdine. Among NNRTI-experienced patients, 12.7% and 6.1% of subjects had intermediate and high-level DOR resistance, respectively. The most common DOR resistance mutation was Y188L. In multivariable analysis, previous EFV use (OR = 1.52, 95% CI 1.15-2.02) and ETR use (OR = 1.91, 95% CI 1.34-2.73) were associated with detection of high-level DOR resistance, whilst RPV use was associated with a lower probability of high-level DOR resistance (OR = 0.39, 95% CI 0.22-0.71). Moreover, EFV use (OR = 1.76, 95% CI 1.19-2.58) and ETR use (OR = 1.72, 95% CI 1.10-2.68) were associated with detection of the Y188L mutation, whereas RPV use was not (OR = 0.16, 95% CI 0.05-0.50). In Italy, DOR resistance is uncommon among NNRTI-experienced patients, confirming a distinguishing resistance pattern within NNRTIs. However, previous EFV and ETR experience poses a higher risk of DOR resistance. These results support the use of DOR in NNRTI-experienced patients.

Addressing ceiling effects in health status measures: a comparison of techniques applied to measures for people with HIV disease.

OBJECTIVES: To compare different approaches to address ceiling effects when predicting EQ-5D index scores from the 10 subscales of the MOS-HIV Health Survey. STUDY DESIGN: Data were collected from an HIV treatment trial. Statistical methods included ordinary least squares (OLS) regression, the censored least absolute deviations (CLAD) approach, a standard two-part model (TPM), a TPM with a log-transformed EQ-5D index, and a latent class model (LCM). Predictive accuracy was evaluated using percentage of absolute error (R(1)) and squared error (R(2)) predicted by statistical methods. FINDINGS: A TPM with a log-transformed EQ-5D index performed best on R(1); a LCM performed best on R(2). In contrast, the CLAD was worst. Performance of the OLS and a standard TPM were intermediate. Values for R(1) ranged from 0.33 (CLAD) to 0.42 (TPM-L); R(2) ranged from 0.37 (CLAD) to 0.53 (LCM). CONCLUSIONS: The LCM and TPM with a log-transformed dependent variable are superior to other approaches in handling data with ceiling effects.

Design and synthesis of dual inhibitors of HIV reverse transcriptase and integrase: introducing a diketoacid functionality into delavirdine.

Cost and toxicity problems associated with highly active antiretroviral therapy (HAART) in HIV/AIDS treatment could be alleviated by using designed multiple ligands (DMLs). Dual inhibitors of HIV reverse transcriptase (RT) and integrase (IN) were rationally designed by introducing a diketoacid (DKA) functionality into the tolerant C-5 site of RT inhibitor delavirdine. The resulting compounds all demonstrate good activity against both RT and IN in enzymatic assays and HIV in cell-based assay, whereas their C-7 regioisomers are all inactive in these assays. Balanced activities were observed with C-3 halogenated inhibitors.

Probing nonnucleoside inhibitor-induced active-site distortion in HIV-1 reverse transcriptase by transient kinetic analyses.

Nonnucleoside reverse transcriptase inhibitors (NNRTI) are a group of structurally diverse compounds that bind to a single site in HIV-1 reverse transcriptase (RT), termed the NNRTI-binding pocket (NNRTI-BP). NNRTI binding to RT induces conformational changes in the enzyme that affect key elements of the polymerase active site and also the association between the two protein subunits. To determine which conformational changes contribute to the mechanism of inhibition of HIV-1 reverse transcription, we used transient kinetic analyses to probe the catalytic events that occur directly at the enzyme's polymerase active site when the NNRTI-BP was occupied by nevirapine, efavirenz, or delavirdine. Our results demonstrate that all NNRTI-RT-template/primer (NNRTI-RT-T/P) complexes displayed a metal-dependent increase in dNTP binding affinity (K(d) ) and a metal-independent decrease in the maximum rate of dNTP incorporation (k (pol)). The magnitude of the decrease in k (pol) was dependent on the NNRTI used in the assay: Efavirenz caused the largest decrease followed by delavirdine and then nevirapine. Analyses that were designed to probe direct effects on phosphodiester bond formation suggested that the NNRTI mediate their effects on the chemistry step of the DNA polymerization reaction via an indirect manner. Because each of the NNRTI analyzed in this study exerted largely similar phenotypic effects on single nucleotide addition reactions, whereas each of them are known to exert differential effects on RT dimerization, we conclude that the NNRTI effects on subunit association do not directly contribute to the kinetic mechanism of inhibition of DNA polymerization.

Emergence of NNRTI drug resistance mutations after single-dose nevirapine exposure in HIV type 1 subtype C-infected infants in India.

A feasibility study for providing single-dose nevirapine (SD-NVP) prophylaxis for prevention of mother-to-child transmission (PMTCT) of HIV infection provided an opportunity to study the emergence of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations as a result of single-dose administration. The study aimed at the detection of NNRTI drug resistance mutations arising as a result of SD-NVP. A total of 19 and 13 samples collected at 48 h and 2 months postpartum, respectively, from infants that were given SD-NVP were studied for the presence of NNRTI drug resistance mutations by PCR amplification and sequencing of the HIV-1 pol gene using HIV proviral DNA. The drug resistance mutational analysis of final sequences was carried out using the Stanford University HIV Drug Resistance database (http://hivdb.stanford.edu/hiv). Mutations associated with NNRTI drug resistance were observed in two (10.5%) and six (46.15%) samples at 48 h and at 2 months, respectively. K103N, one of the most common mutations, was not observed in any of the samples. The emergence of NVP resistance must be weighed against the simplicity, efficacy, and cost effectiveness of SD-NVP prophylaxis in PMTCT settings in developing countries.

Non-nucleoside phenotypic hypersusceptibility cut-point determination from ACTG 359.

PURPOSE: Non-nucleoside reverse transcriptase inhibitor (NNRTI) hypersusceptibility (HS) improves virologic response to those agents, but phenotypic susceptibility cut-points, and methods to determine the cut-points, have not been completely defined. METHOD: Phenotypic drug susceptibility (fold change in IC50 [FC]) was determined for 96 randomly selected antiretroviral-experienced, NNRTI-naive patients who received a delavirdine (DLV)-containing regimen in ACTG 359. A weighted FC score was used to account for other regimen agents. Regression models were used to define baseline DLV HS cut-points using week 4 or week 16 responses. RESULTS: At study entry, DLV HS was present in 36% (35/96) of patients. Models explored HS cut-points from 0.2-1.0 using the week 4 virologic response. Using either a binary or continuous endpoint, DLV HS cut-points between 0.3 and 0.4 were identified. The classification and regression tree (CART) analysis identified baseline DLV FC <0.44 as a predictor of week 4 response. CONCLUSIONS: In relating drug HS to virologic response, several different analytic methods identified a DLV HS FC cut-point of 0.3-0.4. In refining phenotypic cut-points, early virologic responses (not confounded by rebound) may be better metrics than later responses, especially for drugs with low genetic barriers, such as DLV.

Transport of stavudine, delavirdine, and saquinavir across the blood-brain barrier by polybutylcyanoacrylate, methylmethacrylate-sulfopropylmethacrylate, and solid lipid nanoparticles.

Permeability of the anti-human immunodeficiency virus (HIV) agents, including stavudine (D4T), delavirdine (DLV), and saquinavir (SQV), across the in vitro blood-brain barrier (BBB) was studied. Here, the anti-HIV agents were incorporated with polybutylcyanoacrylate (PBCA) nanoparticles (NPs), methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) NPs, and solid lipid nanoparticles (SLNs). Transport of the anti-HIV agents across BBB is a key factor in their applications to the therapy of the acquired immunodeficiency syndrome (AIDS). Experimental results revealed that the drug order of the loading efficiency (LE) on PBCA and MMA-SPM was D4T>DLV>SQV. For the entrapment efficiency (EE) in SLNs, this order was reversed. Also, LE of D4T on MMA-SPM was larger than that on PBCA; however, the reverse was true for DLV and SQV. As the particle size increased, LE decreased and EE increased. For a fixed drug carrier, an increase in the particle size yielded a decrease in the BBB permeability coefficient of the anti-HIV agents. Moreover, enhancement in the BBB permeability was on the carrier order of PBCA>MMA-SPM>SLNs for D4T, and for DLV and SQV, the order became PBCA>SLNs>MMA-SPM. PBCA, MMA-SPM, and SLNs were efficacious carriers of D4T, DLV, and SQV to meliorate BBB permeability by 3-16 folds, indicating the clinical potential of the present NP formulations for the AIDS treatment.

[HIV-1 genotypic resistance profiles in children failing highly active antiretroviral therapy].

OBJECTIVE: To study the genotypic resistance profiles of HIV-1 children failing highly active antiretroviral therapy (HAART) so as to provide helpful information for the treatment regime of Chinese children infected with HIV-1. METHODS: Peripheral venous blood samples were collected from 20 HIV-1 infected children of Henan province, aged 9 (3 - 14). Nested RT-PCR was used to amplify part of the RT (40 -250 aa) gene. The PCR products of RT gene underwent nucleotide sequencing, the resulting nucleotide sequences were analyzed by the HIVdb data offered by the Stanford University web site to find the drug resistance mutations. RESULTS: (1) Phylogenetic analysis revealed that 20 of the RT sequences were classified as subtype B. (2) According to the genotypic analysis, 20 , 15, and 13 children showed high level resistance to the nevirapine. (NVP), delavirdine (DLV), and efavirenz (EFV) respectively; 7 and 5 children showed high and intermediate level resistance to azidothymidine (AZT) respectively. Five children showed potential low-level and intermediate level resistance to lamivudine (3TC), and 11 showed high level resistance to 3TC; 11 showed intermediate and high level resistance to stavudine (d4T) and didanoside (ddI) respectively; and 19 and 12 children showed resistance to abacavir (ABC) and tenofovir (TDF) which had never been taken by these children. CONCLUSION: The emergence of HIV resistant strains during antiretroviral therapy is one of the main reasons for treatment failure in HIV-infected children.

Reverse transcriptase mutations 118I, 208Y, and 215Y cause HIV-1 hypersusceptibility to non-nucleoside reverse transcriptase inhibitors.

BACKGROUND: HIV-1 hypersusceptibility to non-nucleoside reverse transcriptase inhibitors (NNRTI) improves the response to NNRTI-containing regimens. The genetic basis for NNRTI hypersusceptibility was partly defined in our earlier analyses of a paired genotype-phenotype dataset of viral isolates from treatment-experienced patients, in which we identified reverse transcriptase mutations V118I, H208Y, and T215Y as being strongly associated with NNRTI hypersusceptibility. OBJECTIVES: We evaluated the role of these mutations in NNRTI hypersusceptibility by site-directed mutagenesis and phenotypic analysis of HIV-1 recombinants. METHODS: Drug susceptibility and replication capacity were determined in single cycle assays. Hypersusceptibility was defined by a statistically significant (P < 0.01; Student's t-test) mean fold-change in 50% inhibitory concentration (IC50) of less than 0.4. RESULTS: The single mutations V118I, H208Y, and T215Y did not show hypersusceptibility to efavirenz with mean fold-change of 0.58, 0.55, and 0.70, respectively (P < 0.01 and P = 0.12). The H208Y/T215Y and V118I/H208Y/T215Y mutants showed marked hypersusceptibility to efavirenz, having mean fold-change values of 0.27 and 0.20, respectively (P < 0.001). In addition, H208Y/T215Y, V118I/T215Y, and V118I/H208Y/T215Y were hypersusceptible to delavirdine and nevirapine. The V118I/T215Y mutant was not replication impaired; whereas H208Y/T215Y and V118I/H208Y/T215Y had significantly (P < 0.01) reduced replication capacities of 40 and 35% of wild-type, respectively. CONCLUSION: Different combinations of V118I, H208Y, and T215Y produce NNRTI hypersusceptibility. The V118I/T215Y mutant is hypersusceptible to delavirdine and nevirapine without reduced replication capacity, whereas the H208Y/T215Y and V118I/H208Y/T215Y mutants are hypersusceptible to all NNRTI and show impaired replication. These findings suggest that more than one mechanism is involved in NNRTI hypersusceptibility.