RESUMEN
The HIV integrase (IN) strand transfer inhibitor (INSTI) bictegravir (BIC) has a long dissociation half-life (t1/2) from wild-type IN-DNA complexes: BIC 163 hr > dolutegravir (DTG) 96 hr > raltegravir (RAL) 10 hr > elvitegravir (EVG) 3.3 hr. In cells, BIC had more durable antiviral activity against wild-type HIV after drug washout than RAL or EVG. BIC also had a longer t1/2 and maintained longer antiviral activity after drug washout than DTG with the clinically relevant resistance IN mutant G140S+Q148H. Structural analyses indicate that BIC makes more contacts with the IN-DNA complex than DTG mainly via its bicyclic ring system which may contribute to more prolonged residence time and resilience against many resistance mutations.
RESUMEN
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
During HIV-1 reverse transcription, there are increasing opportunities for nucleos(t)ide (NRTI) or nonnucleoside (NNRTI) reverse transcriptase (RT) inhibitors to stop elongation of the nascent viral DNA (vDNA). In addition, RT inhibitors appear to influence the kinetics of vDNA synthesis differently. While cell-free kinetic inhibition constants have provided detailed mechanistic insight, these assays are dependent on experimental conditions that may not mimic the cellular milieu. Here we describe a novel cell-based strategy to provide a measure of the intrinsic inhibition efficiencies of clinically relevant RT inhibitors on a per-stop-site basis. To better compare inhibition efficiencies among HIV-1 RT inhibitors that can stop reverse transcription at any number of different stop sites, their basic probability, p, of getting stopped at any potential stop site was determined. A relationship between qPCR-derived 50% effective inhibitory concentrations (EC50s) and this basic probability enabled determination of p by successive approximation. On a per-stop-site basis, tenofovir (TFV) exhibited 1.4-fold-greater inhibition efficiency than emtricitabine (FTC), and as a class, both NRTIs exhibited an 8- to 11-fold greater efficiency than efavirenz (EFV). However, as more potential stops sites were considered, the probability of reverse transcription failing to reach the end of the template approached equivalence between both classes of RT inhibitors. Overall, this novel strategy provides a quantitative measure of the intrinsic inhibition efficiencies of RT inhibitors in the natural cellular milieu and thus may further understanding of drug efficacy. This approach also has applicability for understanding the impact of viral polymerase-based inhibitors (alone or in combination) in other virus systems.
Asunto(s)
Desoxicitidina/análogos & derivados , Transcriptasa Inversa del VIH/efectos de los fármacos , Inhibidores de la Transcriptasa Inversa/farmacología , Línea Celular , Desoxicitidina/farmacología , Emtricitabina , HumanosRESUMEN
An extract of Humicola fuscoatra (UCSC strain no. 108111A) was shown to reactivate latent HIV-1 expression in an in vitro model of central memory CD4+ T cells. We report the bioassay-guided isolation and structure determination of several resorcyclic acid lactones, including four known compounds, radicicol (1, aka. monorden) and pochonins B (2), C (3), and N (4), and three new analogues, radicicols B-D (5-7). Compounds 1-3 and 5 showed moderate activities in the memory T cell model of HIV-1 latency. Radicicol (1) displayed lower potency in reactivating latent HIV-1 (EC50 = 9.1 µM) relative to the HDAC inhibitors apicidin (EC50 = 0.3 µM), romidepsin (EC50 = 0.003 µM), and SAHA (EC50 = 0.6 µM); however, it achieved equivalent maximum efficacy relative to the positive control compounds (98% of SAHA and romidepsin).
Asunto(s)
Ascomicetos/química , Productos Biológicos/farmacología , Linfocitos T CD4-Positivos/virología , VIH-1/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Lactonas/química , Macrólidos/farmacología , Productos Biológicos/química , Infecciones por VIH/virología , Inhibidores de Histona Desacetilasas/química , Humanos , Lactonas/farmacología , Macrólidos/química , Biología Marina , Modelos Biológicos , Estructura Molecular , Latencia del Virus/efectos de los fármacosRESUMEN
tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction.
Asunto(s)
Acetatos/farmacología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Cromatina/metabolismo , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/enzimología , Quinolinas/farmacología , Factores de Transcripción/metabolismo , Integración Viral/efectos de los fármacos , Acetatos/química , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Cromatina/genética , ADN Viral/genética , ADN Viral/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Integrasa de VIH/química , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/química , VIH-1/genética , Humanos , Quinolinas/química , Factores de Transcripción/genética , Integración Viral/fisiologíaRESUMEN
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.
Asunto(s)
Proteínas de la Cápside/química , Cápside/química , Infecciones por VIH/virología , VIH-1/química , Multimerización de Proteína , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Simulación por Computador , VIH-1/genética , VIH-1/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Mutación , PolimerizacionRESUMEN
HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors.
Asunto(s)
ADN Viral/metabolismo , Inhibidores de Integrasa VIH/farmacocinética , Integrasa de VIH/metabolismo , Catálisis , Disulfuros , Integrasa de VIH/genética , VIH-1/genética , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Multimerización de ProteínaRESUMEN
We have developed a homogeneous time-resolved fluorescence resonance energy transfer (FRET)-based assay that detects the formation of HIV-1 integrase (IN) dimers. The assay utilizes IN monomers that express two different epitope tags that are recognized by their respective antibodies, coupled to distinct fluorophores. Surprisingly, we found that dithiothreitol (DTT), a reducing agent essential for in vitro enzymatic activity of IN, weakened the interaction between IN monomers. This effect of DTT on IN is dependent on its thiol groups, since the related chemical threitol, which contains hydroxyls in place of thiols, had no effect on IN dimer formation. By studying mutants of IN, we determined that cysteines in IN appear to be dispensable for the dimer dissociation effect of DTT. Peptides derived from the IN binding domain (IBD) of lens epithelium derived growth factor/transcriptional coactivator p75 (LEDGF), a cellular cofactor that interacts with the IN dimer interface, were tested in this IN dimerization assay. These peptides, which compete with LEDGF for binding to IN, displayed an intriguing equilibrium binding dose-response curve characterized by a plateau rising to a peak, then descending to a second plateau. Mathematical modeling of this binding system revealed that these LEDGF-derived peptides promote IN dimerization and block subunit exchange between IN dimers. This dose-response behavior was also observed with a small molecule that interacts with the IN dimer interface and inhibits LEDGF binding to IN. In conclusion, this novel IN dimerization assay revealed that peptide and small molecule inhibitors of the IN-LEDGF interaction also stabilize IN dimers and promote their formation.
Asunto(s)
Ditiotreitol/farmacología , Integrasa de VIH/química , VIH-1/efectos de los fármacos , VIH-1/enzimología , Multimerización de Proteína/efectos de los fármacos , Integrasa de VIH/metabolismo , Cinética , Unión ProteicaRESUMEN
The interaction between lens epithelium-derived growth factor/transcriptional co-activator p75 (LEDGF) and human immunodeficiency virus type 1 (HIV-1) integrase (IN) is essential for HIV-1 replication. Homogeneous time-resolved fluorescence resonance energy transfer assays were developed to characterize HIV-1 integrase dimerization and the interaction between LEDGF and IN dimers. Using these assays in an equilibrium end point dose-response format with mathematical modeling, we determined the dissociation constants of IN dimers (K(dimer) = 67.8 pm) and of LEDGF from IN dimers (K(d) = 10.9 nm). When used in a kinetic format, the assays allowed the determination of the on- and off-rate constants for these same interactions. Integrase dimerization had a k(on) of 0.1247 nm(-1) x min(-1) and a k(off) of 0.0080 min(-1) resulting in a K(dimer) of 64.5 pm. LEDGF binding to IN dimers had a k(on) of 0.0285 nm(-1).min(-1) and a k(off) of 0.2340 min(-1) resulting in a K(d) of 8.2 nm. These binding assays can also be used in an equilibrium end point competition format. In this format, the IN catalytic core domain produced a K(i) of 15.2 nm while competing for integrase dimerization, confirming the very tight interaction of IN with itself. In the same format, LEDGF produced a K(i) value of 35 nm when competing for LEDGF binding to IN dimers. In summary, this study describes a methodology combining homogeneous time-resolved fluorescence resonance energy transfer and mathematical modeling to derive the affinities between IN monomers and between LEDGF and IN dimers. This study revealed the significantly tighter nature of the IN-IN dimer compared with the IN-LEDGF interaction.
Asunto(s)
Integrasa de VIH/química , Integrasa de VIH/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Algoritmos , Secuencia de Aminoácidos , Unión Competitiva , Transferencia Resonante de Energía de Fluorescencia , Integrasa de VIH/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Cinética , Modelos Biológicos , Modelos Químicos , Datos de Secuencia Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
GS-9160 is a novel and potent inhibitor of human immunodeficiency virus type 1 (HIV-1) integrase (IN) that specifically targets the process of strand transfer. It is an authentic inhibitor of HIV-1 integration, since treatment of infected cells results in an elevation of two-long terminal repeat circles and a decrease of integration junctions. GS-9160 has potent and selective antiviral activity in primary human T lymphocytes producing a 50% effective concentration (EC(50)) of approximately 2 nM, with a selectivity index (50% cytotoxic concentration/EC(50)) of approximately 2,000. The antiviral potency of GS-9160 decreased by 6- to 10-fold in the presence of human serum. The antiviral activity of GS-9160 is synergistic in combination with representatives from three different classes of antiviral drugs, namely HIV-1 protease inhibitors, nonnucleoside reverse transcriptase inhibitors, and nucleotide reverse transcriptase inhibitors. Viral resistance selections performed with GS-9160 yielded a novel pattern of mutations within the catalytic core domain of IN; E92V emerged initially, followed by L74M. While E92V as a single mutant conferred 12-fold resistance against GS-9160, L74M had no effect as a single mutant. Together, these mutations conferred 67-fold resistance to GS-9160, indicating that L74M may potentiate the resistance caused by E92V. The pharmacokinetic profile of GS-9160 in healthy human volunteers revealed that once-daily dosing was not likely to achieve antiviral efficacy; hence, the clinical development of this compound was discontinued.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Linfocitos T/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/virología , Dominio Catalítico/genética , Línea Celular , Células Cultivadas , Evaluación Preclínica de Medicamentos , Farmacorresistencia Viral , Sinergismo Farmacológico , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacocinética , Inhibidores de la Proteasa del VIH/farmacología , VIH-1/enzimología , VIH-1/genética , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Sensibilidad y EspecificidadRESUMEN
A series of C3 halobenzyl-substituted tricyclic HIV integrase inhibitors was prepared. Improvement in cell-based inhibitor potency was observed in comparison to previously disclosed tricyclic pyrroloquinolines carrying the 'halobenzyl tail' at the lactam nitrogen. Animal PK for several of the C3-substituted inhibitors was examined, with a dihaloaryl analog achieving good balance in protein-shifted EC(50) and t(1/2) in animal PK studies.
Asunto(s)
Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/farmacología , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/farmacología , Pirroles/síntesis química , Pirroles/farmacología , Quinolinas/síntesis química , Quinolinas/farmacología , Administración Oral , Animales , Fármacos Anti-VIH/química , Perros , Diseño de Fármacos , Inhibidores de Integrasa VIH/química , Humanos , Estructura Molecular , Pirroles/química , Quinolinas/química , Ratas , Relación Estructura-ActividadRESUMEN
SAR studies on the para-fluorobenzyl moiety of tricyclic HIV integrase inhibitors are discussed and lead compounds with potency and PK properties comparable to raltegravir were identified.
Asunto(s)
Fluorobencenos/química , Inhibidores de Integrasa VIH/síntesis química , Animales , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Fármacos Anti-VIH/farmacología , Unión Competitiva/fisiología , Disponibilidad Biológica , Línea Celular , Perros , Fluorobencenos/metabolismo , Fluorobencenos/farmacología , Inhibidores de Integrasa VIH/administración & dosificación , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/metabolismo , Humanos , Ratas , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
A series of C5-aza tricyclic HIV integrase inhibitors was prepared. A highly potent and orally bioavailable compound (compound 9) was identified and selected for development.
Asunto(s)
Azacitidina/análogos & derivados , Inhibidores de Integrasa VIH/farmacología , Administración Oral , Animales , Azacitidina/química , Azacitidina/farmacología , Disponibilidad Biológica , Cristalografía por Rayos X , Perros , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacocinética , Humanos , Compuestos Orgánicos/química , Compuestos Orgánicos/farmacocinética , Compuestos Orgánicos/farmacología , Pirrolidinonas , Raltegravir Potásico , RatasRESUMEN
Screening of a marine natural products library afforded three new analogues of the tetronic acid containing polyketide abyssomicin family and identified abyssomicin 2 as a selective reactivator of latent HIV virus. Examination of the mode of action of this new latent HIV reactivating agent demonstrated that it functions via a distinct mechanism compared to that of existing reactivating agents and is effective at reactivating latent virus in a subset of primary patient cell lines.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Furanos/química , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Policétidos/química , Proteína Quinasa C/química , Latencia del Virus/efectos de los fármacos , Línea Celular , Inhibidores de Histona Desacetilasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Estructura Molecular , Proteína Quinasa C/metabolismo , Proteína Quinasa C/farmacologíaRESUMEN
Human rhinoviruses (HRV) represent the single most important causative agent of the common cold. The HRV genome encodes an RNA-dependent RNA polymerase (RdRp) designated 3D polymerase that is required for replication of the HRV RNA genome. We have expressed and purified recombinant HRV-16 3D polymerase to near homogeneity from Escherichia coli transformed with an expression plasmid containing the full-length 460 amino acid HRV-16 3D sequence with a methionine at the N-terminus and a glycine-serine linker followed by a 6-histidine affinity tag at the C-terminus. The purified recombinant protein has rifampicin-resistant activity in a poly(A)-dependent poly(U) polymerase assay while corresponding fractions similarly purified from E. coli transformed with an expression plasmid without the HRV-16 3D sequence showed no activity. The optimal conditions for temperature, pH, divalent cations Mg(2+) and Mn(2+), and KCl were determined. The recombinant protein has RNA polymerase activity on homopolymeric templates poly(A) and poly(C) and heteropolymeric RNA templates primed with either RNA or DNA oligonucleotide primers or self-primed by a copy-back mechanism. A unique, secondary structureless heteropolymeric RNA template that is an efficient substrate was developed to facilitate kinetic characterizations of the enzyme. In the presence of Mg(2+), the enzyme displayed strong base and sugar specificity. However, when Mg(2+) was replaced by Mn(2+) specificity for ribonucleotides was lost, utilization of deoxynucleotides became possible and primer-independent activity was observed on the poly(C) template. Zn(2+) was found to inhibit HRV-16 3D polymerase with an IC(50) as low as 0.6 microM by a mechanism distinct from the magnesium ion stimulation. The activity of this 6His-tagged HRV-16 3D polymerase was compared with that of a recombinant HRV-16 3D polymerase expressed without the 6His-tag and was found to be identical. The availability of recombinant rhinovirus RdRp in a purified form will facilitate the structure-function analysis of this enzyme as well as the identification of specific inhibitors to the rhinovirus 3D polymerase that have therapeutic value in the treatment of the common cold.
Asunto(s)
ARN Polimerasa Dependiente del ARN/metabolismo , Rhinovirus/enzimología , Cationes Bivalentes , Escherichia coli , Expresión Génica , Células HeLa , Histidina , Humanos , Cinética , ARN/biosíntesis , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/genética , Especificidad por Sustrato , Moldes Genéticos , ZincRESUMEN
Elvitegravir is a strand transfer inhibitor of HIV-1 integrase that is currently undergoing phase 3 clinical testing. The two predominant metabolites of elvitegravir, M1 and M4 (elvitegravir hydroxide and elvitegravir glucuronide), have been shown to inhibit HIV-1 integrase in vitro. While they are markedly less potent than elvitegravir and present only at low levels in plasma clinically, we investigated their potential to select for elvitegravir resistance in vitro. Resistance selection experiments using metabolites M1 and M4 led to the development of the previously reported elvitegravir integrase resistance mutations H51Y, T66A, E92G, and S147G, as well as a novel S153F substitution. Additional resistance selection experiments using elvitegravir led to the development of previously reported integrase inhibitor resistance mutations (T66I, F121Y, and S153Y) as well as a novel R263K integrase mutation. Phenotypic analyses of site-directed mutants with these mutations demonstrated broad cross-resistance between elvitegravir and its M1 and M4 metabolites with more limited cross-resistance to the integrase inhibitor raltegravir. Overall, our in vitro studies demonstrate that the resistance profile of the M1 and M4 metabolites of elvitegravir overlaps with that of the parent molecule elvitegravir; as such, their presence at low levels is not considered clinically relevant.
Asunto(s)
Farmacorresistencia Viral , Infecciones por VIH/virología , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , Pirrolidinonas/farmacología , Quinolonas/farmacología , Línea Celular , Infecciones por VIH/tratamiento farmacológico , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , VIH-1/enzimología , VIH-1/genética , Humanos , Mutación , Quinolonas/metabolismo , Raltegravir PotásicoRESUMEN
LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.
Asunto(s)
ADN/metabolismo , Integrasa de VIH/metabolismo , VIH-1/enzimología , Péptidos y Proteínas de Señalización Intercelular/farmacología , ADN/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Sustancias Macromoleculares/metabolismoRESUMEN
This communication details both the syntheses and biological evaluation of a novel class of HIV-1 integrase inhibitors. When the quinoline moiety is replaced with the quinoxoline moiety, the antiviral activity is significantly compromised. Similarly, introduction of imidazole to replace the pyridine ring is deleterious to the potency of the compound against the enzyme. Substitution at the 3-position of the pyridine has been investigated. The presence of the pyridine ring in the tricyclic core is preferred for antiviral activity against HIV integrase.
Asunto(s)
Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/efectos de los fármacos , Piridinas/química , Diseño de Fármacos , Inhibidores de Integrasa VIH/química , VIH-1/efectos de los fármacosRESUMEN
A series of novel tricyclic inhibitors of HIV-1 integrase enzyme was prepared. The effect of substitution at C-6 of the 9-hydroxy-6,7-dihydropyrrolo[3,4-g]quinolin-8-one compounds was studied in vitro. Inhibitors with small side chains at C-6 were generally well tolerated by the enzyme, and the physicochemical properties of the inhibitors were improved by substitution of a small alkyl group at this position. A second series of analogs bearing a sulfamate at the C-5 position with various C-6 substituents were prepared to explore the interplay between the two groups. The SAR of the two classes are not parallel; modification at C-5 impacts the effect of substitutions at C-6.