RESUMEN
Protease inhibitors (PIs) remain an important component of antiretroviral therapy for the treatment of HIV-1 infection due to their high genetic barrier to resistance development. Nevertheless, the two most commonly prescribed HIV PIs, atazanavir and darunavir, still require co-administration with a pharmacokinetic boosting agent to maintain sufficient drug plasma levels which can lead to undesirable drug-drug interactions. Herein, we describe GS-9770, a novel investigational non-peptidomimetic HIV PI with unboosted once-daily oral dosing potential due to improvements in its metabolic stability and its pharmacokinetic properties in preclinical animal species. This compound demonstrates potent inhibitory activity and high on-target selectivity for recombinant HIV-1 protease versus other aspartic proteases tested. In cell culture, GS-9770 inhibits Gag polyprotein cleavage and shows nanomolar anti-HIV-1 potency in primary human cells permissive to HIV-1 infection and against a broad range of HIV subtypes. GS-9770 demonstrates an improved resistance profile against a panel of patient-derived HIV-1 isolates with resistance to atazanavir and darunavir. In resistance selection experiments, GS-9770 prevented the emergence of breakthrough HIV-1 variants at all fixed drug concentrations tested and required multiple protease substitutions to enable outgrowth of virus exposed to escalating concentrations of GS-9770. This compound also remained fully active against viruses resistant to drugs from other antiviral classes and showed no in vitro antagonism when combined pairwise with drugs from other antiretroviral classes. Collectively, these preclinical data identify GS-9770 as a potent, non-peptidomimetic once-daily oral HIV PI with potential to overcome the persistent requirement for pharmacological boosting with this class of antiretroviral agents.
Asunto(s)
Infecciones por VIH , Inhibidores de la Proteasa del VIH , VIH-1 , Humanos , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Proteasa del VIH/uso terapéutico , Darunavir/farmacología , Darunavir/uso terapéutico , Sulfato de Atazanavir/farmacología , Sulfato de Atazanavir/uso terapéutico , Farmacorresistencia Viral , VIH-1/genética , Antirretrovirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Proteasa del VIH/genética , Proteasa del VIH/metabolismoRESUMEN
Lenacapavir (LEN) is a picomolar first-in-class capsid inhibitor of human immunodeficiency virus type 1 (HIV-1) with a multistage mechanism of action and no known cross resistance to other existing antiretroviral (ARV) drug classes. LEN exhibits a low aqueous solubility and exceptionally low systemic clearance following intravenous (IV) administration in nonclinical species and humans. LEN formulated in an aqueous suspension or a PEG/water solution formulation showed sustained plasma exposure levels with no unintended rapid drug release following subcutaneous (SC) administration to rats and dogs. A high total fraction dose release was observed with both formulations. The long-acting pharmacokinetics (PK) were recapitulated in humans following SC administration of both formulations. The SC PK profiles displayed two-phase absorption kinetics in both animals and humans with an initial fast-release absorption phase, followed by a slow-release absorption phase. Noncompartmental and compartmental analyses informed the LEN systemic input rate from the SC depot and exit rate from the body. Modeling-enabled deconvolution of the input rates from two processes: absorption of the soluble fraction (minor) from a direct fast-release process leading to the early PK phase and absorption of the precipitated fraction (major) from an indirect slow-release process leading to the later PK phase. LEN SC PK showed flip-flop kinetics due to the input rate being substantially slower than the systemic exit rate. LEN input rates via the slow-release process in humans were slower than those in both rats and dogs. Overall, the combination of high potency, exceptional stability, and optimal release rate from the injection depot make LEN well suited for a parenteral long-acting formulation that can be administered once up to every 6 months in humans for the prevention and treatment of HIV-1.
Asunto(s)
Fármacos Anti-VIH , VIH-1 , Humanos , Ratas , Animales , Perros , Antirretrovirales , Cápside , Fármacos Anti-VIH/farmacología , Proteínas de la CápsideRESUMEN
BACKGROUND: Long-acting subcutaneous lenacapavir (LEN), a first-in-class HIV capsid inhibitor approved by the US FDA for the treatment of multidrug-resistant HIV-1 with twice yearly dosing, is under investigation for HIV-1 pre-exposure prophylaxis (PrEP). We previously derived a simian-tropic HIV-1 clone (stHIV-A19) that encodes an HIV-1 capsid and replicates to high titres in pigtail macaques (PTM), resulting in a nonhuman primate model well-suited for evaluating LEN PrEP in vivo. METHODS: Lenacapavir potency against stHIV-A19 in PTM peripheral blood mononuclear cells in vitro was determined and subcutaneous LEN pharmacokinetics were evaluated in naïve PTMs in vivo. To evaluate the protective efficacy of LEN PrEP, naïve PTMs received either a single subcutaneous injection of LEN (25 mg/kg, N = 3) or vehicle (N = 4) 30 days before a high-dose intravenous challenge with stHIV-A19, or 7 daily subcutaneous injections of a 3-drug control PrEP regimen starting 3 days before stHIV-A19 challenge (N = 3). FINDINGS: In vitro, LEN showed potent antiviral activity against stHIV-A19, comparable to its potency against HIV-1. In vivo, subcutaneous LEN displayed sustained plasma drug exposures in PTMs. Following stHIV-A19 challenge, while all vehicle control animals became productively infected, all LEN and 3-drug control PrEP animals were protected from infection. INTERPRETATION: These findings highlight the utility of the stHIV-A19/PTM model and support the clinical development of long-acting LEN for PrEP in humans. FUNDING: Gilead Sciences as part of a Cooperative Research and Development Agreement between Gilead Sciences and Frederick National Lab; federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024/HHSN261201500003I; NIH grant R01AI078788.
Asunto(s)
Fármacos Anti-VIH , Seropositividad para VIH , VIH-1 , Estados Unidos , Animales , Humanos , Macaca , Leucocitos Mononucleares , Administración Intravenosa , Proteínas de la CápsideRESUMEN
Long-acting antiretroviral agents for preexposure prophylaxis (PrEP) represent a promising new alternative to daily oral regimens for HIV prevention. Lenacapavir (LEN) is a first-in-class long-acting capsid inhibitor approved for the treatment of HIV-1 infection. Here, we assessed the efficacy of LEN for PrEP using a single high-dose simian-human immunodeficiency virus (SHIV) rectal challenge macaque model. In vitro, LEN showed potent antiviral activity against SHIV, as it did for HIV-1. In macaques, a single subcutaneous administration of LEN demonstrated dose proportional increases in and durability of drug plasma levels. A high-dose SHIV inoculum for the PrEP efficacy evaluation was identified via virus titration in untreated macaques. LEN-treated macaques were challenged with high-dose SHIV 7 weeks after drug administration, and the majority remained protected from infection, as confirmed by plasma PCR, cell-associated proviral DNA, and serology testing. Complete protection and superiority to the untreated group was observed among animals whose LEN plasma exposure exceeded its model-adjusted clinical efficacy target at the time of challenge. All infected animals had subprotective LEN concentrations and showed no emergent resistance. These data demonstrate effective SHIV prophylaxis in a stringent macaque model at clinically relevant LEN exposures and support the clinical evaluation of LEN for HIV PrEP in humans.
Asunto(s)
Fármacos Anti-VIH , Infecciones por VIH , VIH-1 , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Humanos , Macaca , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/prevención & control , Infecciones por VIH/tratamiento farmacológico , VIH-1/genéticaRESUMEN
The integrase strand transfer inhibitor (INSTI)-based regimens bictegravir/emtricitabine/tenofovir alafenamide (BIC/FTC/TAF), dolutegravir (DTG)+FTC/TAF, DTG/lamivudine (3TC), and DTG/rilpivirine (RPV) are all approved for treatment of HIV-infected patients, with various limitations. Here, time to in vitro viral breakthrough (VB) and resistance barrier using simulated human drug exposures at either full or suboptimal treatment adherence to each regimen were compared. At drug concentrations corresponding to full adherence and 1 missed dose (Cmin and Cmin-1), no VB occurred with any regimen. At Cmin-2, VB occurred only with DTG+3TC, with emergent resistance to both drugs. At Cmin-3, VB occurred with all regimens: 100% of DTG+3TC cultures had VB by day 12, and <15% of BIC+FTC+TAF, DTG+FTC+TAF, and DTG+RPV cultures had VB. Emergent reverse transcriptase (RT) or integrase (IN) resistance was seen with DTG+RPV and DTG+3TC but not with BIC+FTC+TAF or DTG+FTC+TAF. At Cmin-4, 100% VB occurred with DTG+3TC and DTG+FTC+TAF by day 12, while 94% VB occurred with DTG+RPV by day 25 and only 50% VB occurred with BIC+FTC+TAF by day 35. Emergent Cmin-4 drug resistance was seen with all regimens but at differing frequencies; DTG+RPV had the most cultures with resistance. Emergent resistance was consistent with clinical observations. Overall, under high adherence conditions, no in vitro VB or resistance development occurred with these INSTI-based regimens. However, when multiple missed doses were simulated in vitro, BIC+FTC+TAF had the highest forgiveness and barrier to resistance of all tested regimens. Compared to DTG+3TC and DTG+FTC+TAF, DTG+RPV had higher forgiveness but lower resistance barrier after several simulated missed doses.
Asunto(s)
Fármacos Anti-VIH , Perdón , Infecciones por VIH , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Emtricitabina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Humanos , Integrasas , Lamivudine/uso terapéutico , Piridonas/uso terapéuticoRESUMEN
Because no currently available vaccine can prevent HIV infection, pre-exposure prophylaxis (PrEP) with antiretrovirals (ARVs) is an important tool for combating the HIV pandemic1,2. Long-acting ARVs promise to build on the success of current PrEP strategies, which must be taken daily, by reducing the frequency of administration3. GS-CA1 is a small-molecule HIV capsid inhibitor with picomolar antiviral potency against a broad array of HIV strains, including variants resistant to existing ARVs, and has shown long-acting therapeutic potential in a mouse model of HIV infection4. Here we show that a single subcutaneous administration of GS-CA1 provides long-term protection against repeated rectal simian-human immunodeficiency virus (SHIV) challenges in rhesus macaques. Whereas all control animals became infected after 15 weekly challenges, a single 300 mg kg-1 dose of GS-CA1 provided per-exposure infection risk reduction of 97% for 24 weeks. Pharmacokinetic analysis showed a correlation between GS-CA1 plasma concentration and protection from SHIV challenges. GS-CA1 levels greater than twice the rhesus plasma protein-adjusted 95% effective concentration conferred 100% protection in this model. These proof-of-concept data support the development of capsid inhibitors as a novel long-acting PrEP strategy in humans.
Asunto(s)
Antirretrovirales , Proteínas de la Cápside , Cápside , Síndrome de Inmunodeficiencia Adquirida del Simio , Virus de la Inmunodeficiencia de los Simios , Animales , Antirretrovirales/farmacología , Cápside/efectos de los fármacos , Proteínas de la Cápside/antagonistas & inhibidores , Proteínas de la Cápside/metabolismo , Macaca mulatta , Síndrome de Inmunodeficiencia Adquirida del Simio/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida del Simio/prevención & control , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacosRESUMEN
BACKGROUND: Barriers to lifelong HIV-1 suppression by antiretrovirals include poor adherence and drug resistance; regimens with higher tolerance to missed doses (forgiveness) would be beneficial to patients. To model short-term nonadherence, in vitro experiments monitoring viral breakthrough (VB) and resistance development were conducted. METHODS: HIV breakthrough experiments simulated drug exposures at full adherence or suboptimal adherence to bictegravir+emtricitabine+tenofovir alafenamide (BIC+FTC+TAF) or dolutegravir + lamivudine (DTG+3TC). MT-2 cells were infected with wild-type or low frequency M184V HIV-1, exposed to drug combinations, monitored for VB, and rebound virus was deep sequenced. Drug concentrations were determined using human plasma-free adjusted clinical trough concentrations (Cmin), at simulated Cmin after missing 1 to 3 consecutive doses (Cmin - 1 or Cmin - 2, and Cmin - 3) based on drug or active metabolite half-lives. RESULTS: Cultures infected with wild-type or low frequency M184V HIV-1 showed no VB with BIC+FTC+TAF at drug concentrations corresponding to Cmin, Cmin - 1, or Cmin - 2 but breakthrough did occur in 26 of 36 cultures at Cmin - 3, where the M184V variant emerged in one culture. Experiments using DTG + 3TC prevented most breakthrough at Cmin concentrations (9/60 had breakthrough) but showed more breakthroughs as drug concentrations decreased (up to 36/36) and variants associated with resistance to both drugs emerged in some cases. CONCLUSIONS: These in vitro VB results suggest that the high potency, long half-lives, and antiviral synergy provided by the BIC/FTC/TAF triple therapy regimen may protect from viral rebound and resistance development after short-term lapses in drug adherence.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Cumplimiento de la Medicación , Línea Celular , Humanos , Linfocitos T/efectos de los fármacos , Linfocitos T/virologíaRESUMEN
People living with HIV (PLWH) have expressed concern about the life-long burden and stigma associated with taking pills daily and can experience medication fatigue that might lead to suboptimal treatment adherence and the emergence of drug-resistant viral variants, thereby limiting future treatment options1-3. As such, there is strong interest in long-acting antiretroviral (ARV) agents that can be administered less frequently4. Herein, we report GS-CA1, a new archetypal small-molecule HIV capsid inhibitor with exceptional potency against HIV-2 and all major HIV-1 types, including viral variants resistant to the ARVs currently in clinical use. Mechanism-of-action studies indicate that GS-CA1 binds directly to the HIV-1 capsid and interferes with capsid-mediated nuclear import of viral DNA, HIV particle production and ordered capsid assembly. GS-CA1 selects in vitro for unfit GS-CA1-resistant capsid variants that remain fully susceptible to other classes of ARVs. Its high metabolic stability and low solubility enabled sustained drug release in mice following a single subcutaneous dosing. GS-CA1 showed high antiviral efficacy as a long-acting injectable monotherapy in a humanized mouse model of HIV-1 infection, outperforming long-acting rilpivirine. Collectively, these results demonstrate the potential of ultrapotent capsid inhibitors as new long-acting agents for the treatment of HIV-1 infection.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteínas de la Cápside/antagonistas & inhibidores , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Indazoles/farmacología , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Fármacos Anti-VIH/uso terapéutico , Cápside/efectos de los fármacos , Cápside/metabolismo , Proteínas de la Cápside/genética , ADN Viral/efectos de los fármacos , Preparaciones de Acción Retardada , Farmacorresistencia Viral/efectos de los fármacos , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/genética , VIH-1/patogenicidad , VIH-2/efectos de los fármacos , VIH-2/patogenicidad , Humanos , Indazoles/uso terapéutico , Cumplimiento de la Medicación , Ratones , Piridinas/uso terapéuticoRESUMEN
GS-9620 is a potent and selective oral Toll-like receptor 7 (TLR7) agonist that directly activates plasmacytoid dendritic cells (pDCs). GS-9620 suppressed hepatitis B virus (HBV) in animal models of chronic infection and transiently activated HIV expression ex vivo in latently infected peripheral blood mononuclear cells (PBMCs) from virally suppressed patients. Currently, GS-9620 is under clinical evaluation for treating chronic HBV infection and for reducing latent reservoirs in virally suppressed HIV-infected patients. Here, we investigated the in vitro anti-HIV-1 activity of GS-9620. GS-9620 potently inhibited viral replication in PBMCs, particularly when it was added 24 to 48 h prior to HIV infection (50% effective concentration = 27 nM). Depletion of pDCs but not other immune cell subsets from PBMC cultures suppressed GS-9620 antiviral activity. Although GS-9620 was inactive against HIV in purified CD4+ T cells and macrophages, HIV replication was potently inhibited by conditioned medium derived from GS-9620-treated pDC cultures when added to CD4+ T cells prior to infection. This suggests that GS-9620-mediated stimulation of PBMCs induced the production of a soluble factor(s) inhibiting HIV replication in trans GS-9620-treated PBMCs primarily showed increased production of interferon alpha (IFN-α), and cotreatment with IFN-α-blocking antibodies reversed the HIV-1-inhibitory effect of GS-9620. Additional studies demonstrated that GS-9620 inhibited a postentry event in HIV replication at a step coincident with or prior to reverse transcription. The simultaneous activation of HIV-1 expression and inhibition of HIV-1 replication are important considerations for the clinical evaluation of GS-9620 since these antiviral effects may help restrict potential local HIV spread upon in vivo latency reversal.
Asunto(s)
Antivirales/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Leucocitos Mononucleares/virología , Pteridinas/uso terapéutico , Receptor Toll-Like 7/antagonistas & inhibidores , Anticuerpos/uso terapéutico , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , Humanos , Interferón-alfa/antagonistas & inhibidores , Interferones/metabolismo , Interleucina-6/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Bictegravir (BIC; GS-9883), a novel, potent, once-daily, unboosted inhibitor of HIV-1 integrase (IN), specifically targets IN strand transfer activity (50% inhibitory concentration [IC50] of 7.5 ± 0.3 nM) and HIV-1 integration in cells. BIC exhibits potent and selective in vitro antiretroviral activity in both T-cell lines and primary human T lymphocytes, with 50% effective concentrations ranging from 1.5 to 2.4 nM and selectivity indices up to 8,700 relative to cytotoxicity. BIC exhibits synergistic in vitro antiviral effects in pairwise combinations with tenofovir alafenamide, emtricitabine, or darunavir and maintains potent antiviral activity against HIV-1 variants resistant to other classes of antiretrovirals. BIC displayed an in vitro resistance profile that was markedly improved compared to the integrase strand transfer inhibitors (INSTIs) raltegravir (RAL) and elvitegravir (EVG), and comparable to that of dolutegravir (DTG), against nine INSTI-resistant site-directed HIV-1 mutants. BIC displayed statistically improved antiviral activity relative to EVG, RAL, and DTG against a panel of 47 patient-derived HIV-1 isolates with high-level INSTI resistance; 13 of 47 tested isolates exhibited >2-fold lower resistance to BIC than DTG. In dose-escalation experiments conducted in vitro, BIC and DTG exhibited higher barriers to resistance than EVG, selecting for HIV-1 variants with reduced phenotypic susceptibility at days 71, 87, and 20, respectively. A recombinant virus with the BIC-selected M50I/R263K dual mutations in IN exhibited only 2.8-fold reduced susceptibility to BIC compared to wild-type virus. All BIC-selected variants exhibited low to intermediate levels of cross-resistance to RAL, DTG, and EVG (<8-fold) but remained susceptible to other classes of antiretrovirals. A high barrier to in vitro resistance emergence for both BIC and DTG was also observed in viral breakthrough studies in the presence of constant clinically relevant drug concentrations. The overall virologic profile of BIC supports its ongoing clinical investigation in combination with other antiretroviral agents for both treatment-naive and -experienced HIV-infected patients.
Asunto(s)
Farmacorresistencia Viral/efectos de los fármacos , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Amidas , Fármacos Anti-VIH/farmacología , Línea Celular , Sinergismo Farmacológico , Integrasa de VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Mutación , Oxazinas , Piperazinas , Piridonas , Raltegravir Potásico/farmacologíaRESUMEN
Transposases are important tools in genome engineering, and there is considerable interest in engineering more efficient ones. Here, we seek to understand the factors determining their activity using the Sleeping Beauty transposase. Recent work suggests that protein coevolutionary information can be used to classify groups of physically connected, coevolving residues into elements called "sectors", which have proven useful for understanding the folding, allosteric interactions, and enzymatic activity of proteins. Using extensive mutagenesis data, protein modeling and analysis of folding energies, we show that (i) The Sleeping Beauty transposase contains two sectors, which span across conserved domains, and are enriched in DNA-binding residues, indicating that the DNA binding and endonuclease functions of the transposase coevolve; (ii) Sector residues are highly sensitive to mutations, and most mutations of these residues strongly reduce transposition rate; (iii) Mutations with a strong effect on free energy of folding in the DDE domain of the transposase significantly reduce transposition rate. (iv) Mutations that influence DNA and protein-protein interactions generally reduce transposition rate, although most hyperactive mutants are also located on the protein surface, including residues with protein-protein interactions. This suggests that hyperactivity results from the modification of protein interactions, rather than the stabilization of protein fold.
Asunto(s)
Elementos Transponibles de ADN , Transposasas/genética , Transposasas/metabolismo , Proteínas Portadoras/metabolismo , Secuencia Conservada , Vectores Genéticos/genética , Recombinación Homóloga , Modelos Moleculares , Mutagénesis , Mutación , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Relación Estructura-Actividad , Transposasas/químicaRESUMEN
Failure of combination antiretroviral (ARV) therapy in HIV-infected patients is often associated with the emergence of drug resistance-associated mutations (RAMs). To facilitate analysis of the barrier to resistance at therapeutically relevant ARV concentrations, we performed fixed-dose in vitro HIV-1 drug resistance selection assays using the immortalized MT-2 T-cell line and primary human CD4+ T cells with a panel of FDA-approved ARVs, each at their respective cell culture equivalent clinical trough concentration (CCE Cmin). At high multiples of its CCE Cmin, emtricitabine (FTC) selected for the rapid emergence of M184I/V, a result consistent with resistance emergence in vivo. While the rate of viral breakthrough in the presence of rilpivirine or efavirenz was delayed relative to FTC, both inhibitors selected for virus with known clinically relevant RAMs. No viral breakthrough was observed for the protease inhibitor atazanavir even at subtherapeutic drug concentrations, which is consistent with its previously characterized high in vivo barrier to resistance. Depending on assay conditions, treatment with integrase inhibitors elvitegravir and raltegravir resulted in breakthrough of both resistant and wild-type virus. The RAMs observed in drug selections were not detected above a 2% threshold by deep sequencing in the in vitro virus inoculum, and only rarely in isolates from treatment-naive HIV+ patients. These new viral breakthrough assays facilitate the analysis of multiple experimental replicates and conditions in parallel and provide a rapid quantitative means to evaluate drug resistance emergence at therapeutically relevant drug concentrations, which should facilitate the identification of new ARVs with a high barrier to resistance.
Asunto(s)
Antirretrovirales/farmacología , Farmacorresistencia Viral , VIH-1/efectos de los fármacos , Pruebas de Sensibilidad Microbiana/métodos , Selección Genética , Linfocitos T CD4-Positivos/virología , Células Cultivadas , HumanosRESUMEN
Tenofovir alafenamide fumarate (TAF) is an oral phosphonoamidate prodrug of the HIV reverse transcriptase nucleotide inhibitor tenofovir (TFV). Previous studies suggested a principal role for the lysosomal serine protease cathepsin A (CatA) in the intracellular activation of TAF. Here we further investigated the role of CatA and other human hydrolases in the metabolism of TAF. Overexpression of CatA or liver carboxylesterase 1 (Ces1) in HEK293T cells increased intracellular TAF hydrolysis 2- and 5-fold, respectively. Knockdown of CatA expression with RNA interference (RNAi) in HeLa cells reduced intracellular TAF metabolism 5-fold. Additionally, the anti-HIV activity and the rate of CatA hydrolysis showed good correlation within a large set of TFV phosphonoamidate prodrugs. The covalent hepatitis C virus (HCV) protease inhibitors (PIs) telaprevir and boceprevir potently inhibited CatA-mediated TAF activation (50% inhibitory concentration [IC50] = 0.27 and 0.16 µM, respectively) in vitro and also reduced its anti-HIV activity in primary human CD4(+) T lymphocytes (21- and 3-fold, respectively) at pharmacologically relevant concentrations. In contrast, there was no inhibition of CatA or any significant effect on anti-HIV activity of TAF observed with cobicistat, noncovalent HIV and HCV PIs, or various prescribed inhibitors of host serine proteases. Collectively, these studies confirm that CatA plays a pivotal role in the intracellular metabolism of TAF, whereas the liver esterase Ces1 likely contributes to the hepatic activation of TAF. Moreover, this work demonstrates that a wide range of viral and host PIs, with the exception of telaprevir and boceprevir, do not interfere with the antiretroviral activity of TAF.
Asunto(s)
Adenina/análogos & derivados , Fármacos Anti-VIH/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Profármacos/metabolismo , Tenofovir/metabolismo , Adenina/metabolismo , Adenina/farmacología , Alanina , Fármacos Anti-VIH/farmacología , Biotransformación , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/virología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Catepsina A/antagonistas & inhibidores , Catepsina A/genética , Catepsina A/metabolismo , Cobicistat/farmacología , Interacciones Farmacológicas , Expresión Génica , Células HEK293 , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Oligopéptidos/farmacología , Cultivo Primario de Células , Profármacos/farmacología , Prolina/análogos & derivados , Prolina/farmacología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Tenofovir/farmacologíaRESUMEN
HIV capsid protein is an important target for antiviral drug design. High-throughput screening campaigns have identified two classes of compounds (PF74 and BI64) that directly target HIV capsid, resulting in antiviral activity against HIV-1 and HIV-2 laboratory strains. Using recombinant proteins, we developed a suite of label-free assays to mechanistically understand how these compounds modulate capsid activity. PF74 preferentially binds to the preassembled hexameric capsid form and prevents disruption of higher-order capsid structures by stabilizing capsid intersubunit interactions. BI64 binds only the monomeric capsid and locks the protein in the assembly incompetent monomeric form by disrupting capsid intersubunit interactions. We also used these assays to characterize the interaction between capsid and the host protein cleavage and polyadenylation specific factor 6 (CPSF6). Consistent with recently published results, our assays revealed CPSF6 activates capsid polymerization and preferentially binds to the preassembled hexameric capsid form similar to the small molecule compound, PF74. Furthermore, these label-free assays provide a robust method for facilitating the identification of a different class of small molecule modulators of capsid function.
Asunto(s)
Fármacos Anti-VIH/farmacología , Técnicas Biosensibles/métodos , Cápside/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Secuencia de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Bencimidazoles/farmacología , Cápside/química , VIH-1 , Interacciones Huésped-Patógeno/efectos de los fármacos , Indoles/química , Indoles/metabolismo , Indoles/farmacología , Datos de Secuencia Molecular , Fenilalanina/análogos & derivados , Fenilalanina/química , Fenilalanina/metabolismo , Fenilalanina/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Proteínas de la Cápside/metabolismo , ADN Viral/biosíntesis , VIH-1/genética , Replicación Viral/genética , Proteínas de la Cápside/antagonistas & inhibidores , Proteínas de la Cápside/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Núcleo Celular/virología , Citosol/metabolismo , Citosol/virología , Regulación de la Expresión Génica , Células HEK293 , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , VIH-1/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Indoles/farmacología , Macrófagos/metabolismo , Macrófagos/virología , Microscopía Confocal/métodos , Fenilalanina/análogos & derivados , Fenilalanina/farmacología , Cultivo Primario de Células , Transcripción Reversa/efectos de los fármacos , Transducción de Señal , Linfocitos T/metabolismo , Linfocitos T/virología , Integración Viral/efectos de los fármacos , Integración Viral/genética , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , beta Carioferinas/genética , beta Carioferinas/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
BACKGROUND: Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir that shows enhanced antiretroviral effect and reduced plasma tenofovir exposures at approximately one-tenth the clinically approved dose of tenofovir disoproxil fumarate (TDF). Tenofovir released from TDF undergoes active renal secretion via organic anion transporters (OAT1 and OAT3), leading to higher exposure of renal proximal tubules to tenofovir and a potential for renal adverse effects in a small subset of TDF-treated patients. Here, we evaluate the interaction of TAF with OAT1 and OAT3 to assess the potential for its active accumulation in proximal tubules. METHODS: OAT-mediated transport and cytotoxicity (CC50) of TAF and tenofovir were assessed in cells expressing OATs and compared with matched transporter-null cells. RESULTS: While OAT1 and OAT3 expression increased tenofovir cellular uptake by >70-fold and 8.2-fold, respectively, the expression of either OAT did not significantly change TAF intracellular accumulation under identical conditions. In addition, although tenofovir was significantly more cytotoxic in OAT1- and OAT3-expressing cells (>21 and >3.6 fold change in CC50 values, respectively), TAF in vitro cytotoxicity showed little to no change upon overexpression of either renal transporter (0.5-3.5 fold change in CC50). CONCLUSIONS: Unlike tenofovir, TAF does not interact with renal transporters OAT1 or OAT3 and exhibits no OAT-dependent cytotoxicity. TAF is thus unlikely to actively accumulate in renal proximal tubules in an OAT-dependent manner, supporting the potential for an improved renal safety profile.
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Adenina/análogos & derivados , Túbulos Renales Proximales/metabolismo , Transportadores de Anión Orgánico/metabolismo , Adenina/química , Adenina/metabolismo , Adenina/farmacología , Alanina , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Transporte Biológico , Línea Celular , Células Cultivadas , Expresión Génica , Humanos , Proteína 1 de Transporte de Anión Orgánico/genética , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico Sodio-Independiente/genética , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Tenofovir/análogos & derivadosRESUMEN
BACKGROUND: Tenofovir alafenamide (TAF) is a novel investigational prodrug of tenofovir (TFV) that permits enhanced delivery of TFV into peripheral blood mononuclear cells (PBMCs) and lymphatic tissues. A critical step in the intracellular metabolic activation of TAF is mediated by the lysosomal protease cathepsin A (CatA). Here, we investigated CatA levels together with intracellular metabolism and antiretroviral activity of TAF in primary CD4+ T-lymphocytes (CD4s) and monocyte-derived macrophages (MDMs) isolated from a demographically diverse group of blood donors. METHODS: CD4s and MDMs were prepared from fresh PBMCs. CatA levels were quantified in cell extracts by monitoring TAF hydrolysis using HPLC. Intracellular TAF metabolites were quantified by HPLC combined with mass spectrometry. Antiviral activities in activated CD4s and MDMs were determined using HIV-1 single-cycle reporter and p24 antigen production assays, respectively. RESULTS: The levels of CatA and intracellular TAF metabolites differed minimally in CD4s and MDMs among 13 tested donors. TAF was >600-fold and 80-fold more potent than parent TFV in CD4s and MDMs, respectively, and its relative range of antiviral activity across all tested donors was comparable to that of other HIV-1 reverse transcriptase inhibitors, with mean ±sd (range) EC50 values of 11.0 ±3.4 (6.6-19.9) nM and 9.7 ±4.6 (2.5-15.7) nM in CD4s and MDMs, respectively. CONCLUSIONS: These results indicate consistent intracellular metabolism and antiretroviral potency of TAF in relevant target cells of HIV-1 infection across multiple donors of variable gender, age and ethnicity, supporting further clinical investigation of TAF.
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Adenina/análogos & derivados , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Adenina/química , Adenina/metabolismo , Adenina/farmacología , Alanina , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Catepsina A/metabolismo , Células Cultivadas , Activación Enzimática/efectos de los fármacos , VIH-1/efectos de los fármacos , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Tenofovir/análogos & derivados , Donantes de TejidosRESUMEN
HIV-1 integrase (IN) is the target for two classes of antiretrovirals: i) the integrase strand-transfer inhibitors (INSTIs) and ii) the non-catalytic site integrase inhibitors (NCINIs). NCINIs bind at the IN dimer interface and are thought to interfere primarily with viral DNA (vDNA) integration in the target cell by blocking IN-vDNA assembly as well as the IN-LEDGF/p75 interaction. Herein we show that treatment of virus-producing cells, but not of mature virions or target cells, drives NCINI antiviral potency. NCINIs target an essential late-stage event in HIV replication that is insensitive to LEDGF levels in the producer cells. Virus particles produced in the presence of NCINIs displayed normal Gag-Pol processing and endogenous reverse transcriptase activity, but were defective at initiating vDNA synthesis following entry into the target cell. NCINI-resistant virus carrying a T174I mutation in the IN dimer interface was less sensitive to the compound-induced late-stage effects, including the reverse transcription block. Wild-type, but not T174I virus, produced in the presence of NCINIs exhibited striking defects in core morphology and an increased level of IN oligomers that was not observed upon treatment of mature cell-free particles. Collectively, these results reveal that NCINIs act through a novel mechanism that is unrelated to the previously observed inhibition of IN activity or IN-LEDGF interaction, and instead involves the disruption of an IN function during HIV-1 core maturation and assembly.
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ADN Viral/antagonistas & inhibidores , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/genética , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular Tumoral , ADN Viral/genética , Farmacorresistencia Viral , Expresión Génica , Genes Reporteros , Vectores Genéticos , Integrasa de VIH/metabolismo , Inhibidores de Integrasa VIH/química , Transcriptasa Inversa del VIH/genética , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , VIH-1/crecimiento & desarrollo , VIH-1/metabolismo , Interacciones Huésped-Patógeno , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Mutación , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , Linfocitos T/virología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Virión/efectos de los fármacos , Virión/genética , Ensamble de Virus/efectos de los fármacos , Integración Viral/efectos de los fármacosRESUMEN
A once-daily single-tablet antiretroviral regimen containing tenofovir (TFV) disoproxil fumarate, emtricitabine (FTC), elvitegravir (EVG), and cobicistat (COBI) is an approved combination for the treatment of patients infected with HIV. COBI and TFV have been reported to interact with distinct transporters in renal proximal tubules; while TFV is renally eliminated by a combination of glomerular filtration and tubular secretion via anion transporters OAT1, OAT3, and MRP4, COBI inhibits renal cation transporters, particularly MATE1, resulting in a measurable decrease in the tubular secretion of creatinine. To investigate the potential for a renal drug-drug interaction between TFV and COBI in vitro, the uptake of TFV in the presence and absence of COBI was determined in fresh human renal cortex tissue and in cells expressing the relevant renal transporters. At concentrations exceeding clinical protein-unbound plasma levels, COBI did not significantly inhibit the transport of TFV by the anion transporters OAT1, OAT3, and MRP4 (50% inhibitory concentrations [IC50s] of >15, 6.6, and 8.5 µM, respectively). Conversely, TFV had little or no effect on the cation transporters OCT2 and MATE1 (IC50 > 100 µM). Consistent with studies using individual transporters, no increase in the accumulation of TFV in freshly isolated human renal cortex tissue or renal proximal tubule cells (RPTECs) was observed in the presence of COBI. Finally, COBI alone or in combination with FTC and EVG did not affect the sensitivity to TFV of cultured primary RPTECs or cells coexpressing OAT1 and MRP4. These results illustrate that COBI and TFV interact primarily with distinct renal transporters and indicate a low potential for pharmacokinetic renal drug-drug interaction.
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Adenina/análogos & derivados , Fármacos Anti-VIH/farmacología , Carbamatos/farmacología , Organofosfonatos/farmacología , Tiazoles/farmacología , Adenina/farmacología , Adulto , Línea Celular , Cobicistat , Interacciones Farmacológicas , Humanos , Técnicas In Vitro , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 2 de Cátion Orgánico , TenofovirRESUMEN
Human immunodeficiency virus-1 (HIV-1) capsid protein (CA) has become a target of antiviral drug design in recent years. The recognition that binding of small molecules to the CA protein can result in the perturbation of capsid assembly or disassembly has led to mathematical modeling of the process. Although a number of capsid assembly models have been developed using biophysical parameters of the CA protein obtained experimentally, there is currently no model of CA polymerization that can be practically used to analyze in vitro CA polymerization data to facilitate drug discovery. Herein, we describe an equilibrium model of CA polymerization for the kinetic analysis of in vitro assembly of CA into polymer tubes. This new mathematical model has been used to assess whether a triangular trimer of dimers rather than a hexagonal hexamer can be the basic capsomere building block of CA polymer. The model allowed us to quantify for the first time the affinity for each of the four crucial interfaces involved in the polymerization process and indicated that the trimerization of CA dimers is a relatively slow step in CA polymerization in vitro. For wild-type CA, these four interfaces include the interface between two monomers of a CA dimer (K(D) = 6.6 µM), the interface between any two dimers within a CA trimer of dimers (K(D) = 32 nM), and two types of interfaces between neighboring trimers of dimers, either within the same ring around the perimeter of the polymer tube (K(D) = 438 nM) or from two adjacent rings (K(D) = 147 nM). A comparative analysis of the interface dissociation constants between wild-type and two mutant CA proteins, cross-linked hexamer (A14C/E45C/W184A/M185A) and A14C/E45C, yielded results that are consistent with the trimer of dimers with a triangular geometry being the capsomere building block involved in CA polymer growth. This work provides additional insights into the mechanism of HIV-1 CA assembly and may prove useful in elucidating how small molecule CA binding agents may disturb this essential step in the HIV-1 life cycle.