RESUMEN
Multiple viral targets are now available in the clinic to fight HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients due to resistance, with or without treatment-adherence glitches. Accordingly, it is important to better understand how HIV and other retroviruses replicate in order to propose alternative antiviral strategies. Recent studies have shown that multiple cellular factors are implicated during the integration step and, more specifically, that integrase can be regulated through post-translational modifications. We have shown that integrase is phosphorylated by GCN2, a cellular protein kinase of the integrated stress response, leading to a restriction of HIV replication. In addition, we found that this mechanism is conserved among other retroviruses. Accordingly, we developed an in vitro interaction assay, based on the AlphaLISA technology, to monitor the integrase-GCN2 interaction. From an initial library of 133 FDA-approved molecules, we identified nine compounds that either inhibited or stimulated the interaction between GCN2 and HIV integrase. In vitro characterization of these nine hits validated this pilot screen and demonstrated that the GCN2-integrase interaction could be a viable solution for targeting integrase out of its active site.
Asunto(s)
Infecciones por VIH/terapia , Integrasa de VIH/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Replicación Viral/efectos de los fármacos , Dominio Catalítico , Evaluación Preclínica de Medicamentos , VIH , Integrasa de VIH/genética , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Retroviridae , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-Actividad , Replicación Viral/genéticaRESUMEN
Currently, an increasing number of drugs are becoming available to clinics for the treatment of HIV infection. Even if this targeted therapy is highly effective at suppressing viral replication, caregivers are facing growing therapeutic failures in patients, due to resistance with or without treatment adherence concerns. Accordingly, it is important to continue to discover small molecules that have a novel mechanism of inhibition. In this work, HIV integrase inhibitors were selected by high-throughput screening. Chemical structure comparisons enabled the identification of stilbene disulfonic acids as a potential new chemotype. Biochemical characterization of the lead compound stilbenavir (NSC34931) and a few derivatives was performed. Stilbene disulfonic acid derivatives exhibit low to sub-micromolar antiviral activity, and they inhibit integrase through DNA-binding inhibition. They probably bind to the C-terminal domain of integrase, in the cavity normally occupied by the noncleaved strand of the viral DNA substrate. Because of this original mode of action compared to active site strand transfer inhibitors, they do not exhibit cross-resistance to the three main resistance pathways to integrase inhibitors (G140S-Q148H, N155H, and Y143R). Further structure-activity optimization should enable the development of more active and less toxic derivatives with potential clinical relevance.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH/química , Integrasa de VIH/genética , VIH/efectos de los fármacos , Antivirales/química , Antivirales/farmacología , Dominio Catalítico/efectos de los fármacos , Farmacorresistencia Viral , VIH/enzimología , VIH/patogenicidad , Infecciones por VIH/enzimología , Infecciones por VIH/virología , Inhibidores de Integrasa VIH/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Mutación , Replicación Viral/efectos de los fármacosRESUMEN
HIV-1 integrase (IN) inhibitors represent a new class of highly effective anti-AIDS therapeutics. Current FDA-approved IN strand transfer inhibitors (INSTIs) share a common mechanism of action that involves chelation of catalytic divalent metal ions. However, the emergence of IN mutants having reduced sensitivity to these inhibitors underlies efforts to derive agents that antagonize IN function by alternate mechanisms. Integrase along with the 96-residue multifunctional accessory protein, viral protein R (Vpr), are both components of the HIV-1 pre-integration complex (PIC). Coordinated interactions within the PIC are important for viral replication. Herein, we report a 7-mer peptide based on the shortened Vpr (69â»75) sequence containing a biotin group and a photo-reactive benzoylphenylalanyl residue, and which exhibits low micromolar IN inhibitory potency. Photo-crosslinking experiments have indicated that the peptide directly binds IN. The peptide does not interfere with IN-DNA interactions or induce higher-order, aberrant IN multimerization, suggesting a mode of action for the peptide that is distinct from clinically used INSTIs and developmental allosteric IN inhibitors. This compact Vpr-derived peptide may serve as a valuable pharmacological tool to identify a potential new pharmacologic site.
Asunto(s)
Productos del Gen vpr/química , Productos del Gen vpr/metabolismo , Infecciones por VIH/virología , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/fisiología , Péptidos/farmacología , Secuencia de Aminoácidos , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Péptidos/síntesis química , Péptidos/química , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
The synthesis and antiviral evaluation of a series of dihydropyrimidinone and thiopyrimidine derivatives bearing aryl α,γ-diketobutanoic acid moiety are described using the Biginelli multicomponent reaction as key step. The most active among 20 synthesized novel compounds were 4c, 4d and 5b, which possess nanomolar HIV-1 integrase (IN) stand transfer (ST) inhibition activities. In order to understand their mode of interactions within the IN active site, we docked all the compounds into the previously reported X-ray crystal structure of IN. We observed that compounds 4c, 4d and 5b occupied an area close to the two catalytic Mg(2+) ions surrounded by their chelating triad (E221, D128 and D185), DC16, Y212 and the ß-diketo acid moiety of 4c, 4d and 5b chelating Mg(2+). As those compounds lack anti-HIV activities in cell, their prodrugs were synthetized. The prodrug 4c' exhibited an anti-HIV activity of 0.19 µM in primary human lymphocytes with some cytotoxicity. All together, these results indicate that the new analogs potentially interact within the catalytic site with highly conserved residues important for IN catalytic activity.
Asunto(s)
Fármacos Anti-VIH/farmacología , Ácido Butírico/farmacología , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Pirimidinas/farmacología , Animales , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Ácido Butírico/síntesis química , Ácido Butírico/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , Humanos , Modelos Moleculares , Estructura Molecular , Pirimidinas/síntesis química , Pirimidinas/química , Relación Estructura-Actividad , Células VeroRESUMEN
During clinical trials, a number of fully characterized molecules are dropped along the way because they do not provide enough benefit for the patient. Some of them show limited side effects and might be of great use for other applications. AS1411 is a nucleolin-targeting aptamer that underwent phase II clinical trials as anticancer agent. Here, we show that AS1411 exhibits extremely potent antiviral activity and is therefore an attractive new lead as anti-HIV agent.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Oligodesoxirribonucleótidos/farmacología , Antineoplásicos/farmacología , Aptámeros de Nucleótidos , Línea Celular , Proliferación Celular/efectos de los fármacos , HumanosRESUMEN
The development of HIV-1 dual inhibitors is a highly innovative approach aimed at reducing drug toxic side effects as well as therapeutic costs. HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) are both selective targets for HIV-1 chemotherapy, and the identification of dual IN/RNase H inhibitors is an attractive strategy for new drug development. We newly synthesized pyrrolyl derivatives that exhibited good potency against IN and a moderate inhibition of the RNase H function of RT, confirming the possibility of developing dual HIV-1 IN/RNase H inhibitors and obtaining new information for the further development of more effective dual HIV-1 inhibitors.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH/efectos de los fármacos , Pirroles/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , VIH/enzimología , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Estructura Terciaria de Proteína/efectos de los fármacos , Pirroles/síntesis química , Pirroles/química , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Ribonucleasa H/metabolismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
G-rich nucleic acids can form non-canonical G-quadruplex structures (G4s) in which four guanines fold in a planar arrangement through Hoogsteen hydrogen bonds. Although many biochemical and structural studies have focused on DNA sequences containing successive, adjacent guanines that spontaneously fold into G4s, evidence for their in vivo relevance has recently begun to accumulate. Complete sequencing of the human genome highlighted the presence of â¼300,000 sequences that can potentially form G4s. Likewise, the presence of putative G4-sequences has been reported in various viruses genomes [e.g., Human immunodeficiency virus (HIV-1), Epstein-Barr virus (EBV), papillomavirus (HPV)]. Many studies have focused on telomeric G4s and how their dynamics are regulated to enable telomere synthesis. Moreover, a role for G4s has been proposed in cellular and viral replication, recombination and gene expression control. In parallel, DNA aptamers that form G4s have been described as inhibitors and diagnostic tools to detect viruses [e.g., hepatitis A virus (HAV), EBV, cauliflower mosaic virus (CaMV), severe acute respiratory syndrome virus (SARS), simian virus 40 (SV40)]. Here, special emphasis will be given to the possible role of these structures in a virus life cycle as well as the use of G4-forming oligonucleotides as potential antiviral agents and innovative tools.
Asunto(s)
Antivirales/química , ADN Viral/química , G-Cuádruplex , ARN Viral/química , Genoma Humano , HumanosRESUMEN
There are currently three HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) approved by the FDA for the treatment of AIDS. However, the emergence of drug-resistant mutants emphasizes the need to develop additional agents that have improved efficacies against the existent resistant mutants. As reported herein, we modified our recently disclosed 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides IN inhibitors to develop compounds that have improved efficacies against recombinant IN in biochemical assays. These new compounds show single-digit nanomolar antiviral potencies against HIV vectors that carry wild-type (WT) IN in a single round replication assay and have improved potency against vectors harboring the major forms of drug resistant IN mutants. These compounds also have low toxicity for cultured cells, which in several cases, results in selectivity indices (CC50/EC50) of greater than 10000. The compounds have the potential, with additional structural modifications, to yield clinical agents that are effective against the known strains of resistant viruses.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/genética , VIH-1/efectos de los fármacos , Naftiridinas/farmacología , Pirrolidinonas/farmacología , Línea Celular Tumoral , Farmacorresistencia Viral , Células HEK293 , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , VIH-1/enzimología , VIH-1/genética , Humanos , Mutación , Naftiridinas/síntesis química , Naftiridinas/química , Raltegravir Potásico , Proteínas Recombinantes/química , Estereoisomerismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
The increasing efficiency of HAART has helped to transform HIV/AIDS into a chronic disease. Still, resistance and drug-drug interactions warrant the development of new anti-HIV agents. We previously discovered hit 6, active against HIV-1 replication and targeting RNase H in vitro. Because of its diketo-acid moiety, we speculated that this chemotype could serve to develop dual inhibitors of both RNase H and integrase. Here, we describe a new series of 1-benzyl-pyrrolyl diketohexenoic derivatives, 7a-y and 8a-y, synthesized following a parallel solution-phase approach. Those 50 analogues have been tested on recombinant enzymes (RNase H and integrase) and in cell-based assays. Approximately half (22) exibited inhibition of HIV replication. Compounds 7b, 7u, and 8g were the most active against the RNase H activity of reverse-transcriptase, with IC50 values of 3, 3, and 2.5 µM, respectively. Compound 8g was also the most potent integrase inhibitor with an IC50 value of 26 nM.
Asunto(s)
Fármacos Anti-VIH/farmacología , Inhibidores Enzimáticos/farmacología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Cetoácidos/farmacología , Pirroles/farmacología , Ribonucleasa H/antagonistas & inhibidores , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Cetoácidos/síntesis química , Cetoácidos/química , Estructura Molecular , Pirroles/síntesis química , Pirroles/química , Ribonucleasa H/metabolismo , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacosRESUMEN
HIV-1 integrase (IN) is an enzyme which is indispensable for the stable infection of host cells because it catalyzes the insertion of viral DNA into the genome and thus is an attractive target for the development of anti-HIV agents. Earlier, we found Vpr-derived peptides with inhibitory activity against HIV-1 IN. These Vpr-derived peptides are originally located in an α-helical region of the parent Vpr protein. Addition of an octa-arginyl group to the inhibitory peptides caused significant inhibition against HIV replication associated with an increase in cell permeability but also relatively high cytotoxicity. In the current study, stapled peptides, a new class of stabilized α-helical peptidomimetics were adopted to enhance the cell permeability of the above lead peptides. A series of stapled peptides, which have a hydrocarbon link formed by a ruthenium-catalyzed ring-closing metathesis reaction between successive turns of α-helix, were designed, synthesized, and evaluated for biological activity. In cell-based assays some of the stapled peptides showed potent anti-HIV activity comparable with that of the original octa-arginine-containing peptide (2) but with lower cytotoxicity. Fluorescent imaging experiments revealed that these stapled peptides are significantly cell permeable, and CD analysis showed they form α-helical structures, whereas the unstapled congeners form ß-sheet structures. The application of this stapling strategy to Vpr-derived IN inhibitory peptides led to a remarkable increase in their potency in cells and a significant reduction of their cytotoxicity.
Asunto(s)
Inhibidores de Integrasa VIH/química , Integrasa de VIH/metabolismo , VIH-1/genética , Péptidos/química , Secuencia de Aminoácidos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Dicroismo Circular , Sistemas de Liberación de Medicamentos , Ensayo de Inmunoadsorción Enzimática , VIH-1/química , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Péptidos/genética , Péptidos/farmacología , Peptidomiméticos , Unión ProteicaRESUMEN
Since the discovery of HIV as the cause for AIDS 30 years ago, major progress has been made, including the discovery of drugs that now control the disease. Here, we review the integrase (IN) inhibitors from the discovery of the first compounds 20 years ago to the approval of two highly effective IN strand transfer inhibitors (INSTIs), raltegravir (Isentress) and elvitegravir (Stribild), and the promising clinical activity of dolutegravir. After summarizing the molecular mechanism of action of the INSTIs as interfacial inhibitors, we discuss the remaining challenges. Those include: overcoming resistance to clinical INSTIs, long-term safety of INSTIs, cost of therapy, place of the INSTIs in prophylactic treatments, and the development of new classes of inhibitors (the LEDGINs) targeting IN outside its catalytic site. We also discuss the role of chromatin and host DNA repair factor for the completion of integration.
Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH/farmacología , Inhibidores de Integrasa VIH/uso terapéutico , VIH/efectos de los fármacos , Farmacorresistencia Viral , VIH/enzimología , Infecciones por VIH/virología , Inhibidores de Integrasa VIH/efectos adversos , Inhibidores de Integrasa VIH/economía , Humanos , Modelos Biológicos , Replicación Viral/efectos de los fármacosRESUMEN
Although an extensive body of scientific and patent literature exists describing the development of HIV-1 integrase (IN) inhibitors, Merck's raltegravir and Gilead's elvitegravir remain the only IN inhibitors FDA-approved for the treatment of AIDS. The emergence of raltegravir-resistant strains of HIV-1 containing mutated forms of IN underlies the need for continued efforts to enhance the efficacy of IN inhibitors against resistant mutants. We have previously described bicyclic 6,7-dihydroxyoxoisoindolin-1-ones that show good IN inhibitory potency. This report describes the effects of introducing substituents into the 4- and 5-positions of the parent 6,7-dihydroxyoxoisoindolin-1-one platform. We have developed several sulfonamide-containing analogs that enhance potency in cell-based HIV assays by more than two orders-of-magnitude and we describe several compounds that are more potent than raltegravir against the clinically relevant Y143R IN mutant.
Asunto(s)
Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , Indoles/farmacología , Sulfonamidas/farmacología , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/virología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/química , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , Indoles/síntesis química , Indoles/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
HIV-1 integrase (IN) is a validated therapeutic target for the treatment of AIDS. However, the emergence of resistance to raltegravir, the sole marketed FDA-approved IN inhibitor, emphasizes the need to develop second-generation inhibitors that retain efficacy against clinically relevant IN mutants. We report herein bicyclic hydroxy-1H-pyrrolopyridine-triones as a new family of HIV-1 integrase inhibitors that were efficiently prepared using a key 'Pummerer cyclization deprotonation cycloaddition' cascade of imidosulfoxides. In in vitro HIV-1 integrase assays, the analogs showed low micromolar inhibitory potencies with selectivity for strand transfer reactions as compared with 3'-processing inhibition. A representative inhibitor (5e) retained most of its inhibitory potency against the three major raltegravir-resistant IN mutant enzymes, G140S/Q148H, Y143R, and N155H. In antiviral assays employing viral vectors coding these IN mutants, compound 5e was approximately 200- and 20-fold less affected than raltegravir against the G140S/Q148H and Y143R mutations, respectively. Against the N155H mutation, 5e was approximately 10-fold less affected than raltegravir. Thus, our new compounds represent a novel structural class that may be further developed to overcome resistance to raltegravir, particularly in the case of the G140S/Q148H mutations.
Asunto(s)
Compuestos Bicíclicos con Puentes/química , Inhibidores de Integrasa VIH/química , VIH-1 , Imidas/química , Piridinas/química , Piridonas/química , Sustitución de Aminoácidos , Sitios de Unión , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/farmacología , Línea Celular Tumoral , Simulación por Computador , Farmacorresistencia Viral/efectos de los fármacos , Integrasa de VIH/química , Integrasa de VIH/genética , Integrasa de VIH/metabolismo , Inhibidores de Integrasa VIH/síntesis química , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , Imidas/síntesis química , Imidas/farmacología , Mutación , Piridonas/síntesis química , Piridonas/farmacología , Pirrolidinonas/farmacología , Raltegravir PotásicoRESUMEN
Raltegravir (RAL) and related HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) efficiently block viral replication in vitro and suppress viremia in patients. These small molecules bind to the IN active site, causing it to disengage from the deoxyadenosine at the 3' end of viral DNA. The emergence of viral strains that are highly resistant to RAL underscores the pressing need to develop INSTIs with improved resistance profiles. Herein, we show that the candidate second-generation drug dolutegravir (DTG, S/GSK1349572) effectively inhibits a panel of HIV-1 IN variants resistant to first-generation INSTIs. To elucidate the structural basis for the increased potency of DTG against RAL-resistant INs, we determined crystal structures of wild-type and mutant prototype foamy virus intasomes bound to this compound. The overall IN binding mode of DTG is strikingly similar to that of the tricyclic hydroxypyrrole MK-2048. Both second-generation INSTIs occupy almost the same physical space within the IN active site and make contacts with the ß4-α2 loop of the catalytic core domain. The extended linker region connecting the metal chelating core and the halobenzyl group of DTG allows it to enter farther into the pocket vacated by the displaced viral DNA base and to make more intimate contacts with viral DNA, compared with those made by RAL and other INSTIs. In addition, our structures suggest that DTG has the ability to subtly readjust its position and conformation in response to structural changes in the active sites of RAL-resistant INs.
Asunto(s)
Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/metabolismo , Integrasa de VIH/metabolismo , Compuestos Heterocíclicos con 3 Anillos/química , Compuestos Heterocíclicos con 3 Anillos/metabolismo , Sitios de Unión/fisiología , Cristalografía por Rayos X , Variación Genética/genética , Células HEK293 , Integrasa de VIH/química , Inhibidores de Integrasa VIH/farmacología , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Oxazinas , Piperazinas , Piridonas , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Integrase (IN) represents a clinically validated target for the development of antivirals against human immunodeficiency virus (HIV). Inhibitors with a novel structure core are essential for combating resistance associated with known IN inhibitors (INIs). We have previously disclosed a novel dual inhibitor scaffold of HIV IN and reverse transcriptase (RT). Here we report the complete structure-activity relationship (SAR), molecular modeling, and resistance profile of this inhibitor type on IN inhibition. These studies support an antiviral mechanism of dual inhibition against both IN and RT and validate 3-hydroxypyrimidine-2,4-diones as an IN inhibitor scaffold.
Asunto(s)
Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH/enzimología , Pirimidinas/química , Pirimidinas/farmacología , Línea Celular , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Farmacorresistencia Viral/genética , VIH/efectos de los fármacos , Integrasa de VIH/química , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/síntesis química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Mutación , Conformación Proteica , Pirimidinas/síntesis química , Relación Estructura-ActividadRESUMEN
Resistance to raltegravir (RAL), the first HIV-1 integrase (IN) inhibitor approved by the FDA, involves three genetic pathways: IN mutations N155H, Q148H/R/K, and Y143H/R/C. Those mutations are generally associated with secondary point mutations. The resulting mutant viruses show a high degree of resistance against RAL but somehow are affected in their replication capacity. Clinical and virological data indicate the high relevance of the combination G140S + Q148H because of its limited impact on HIV replication and very high resistance to RAL. Here, we report how mutations at the amino acid residues 140, 148, and 155 affect IN enzymatic activity and RAL resistance. We show that single mutations at position 140 have limited impact on 3'-processing (3'-P) but severely inactivate strand transfer (ST). On the other hand, single mutations at position 148 have a more profound effect and inactivate both 3'-P and ST. By examining systematically all of the double mutants at the 140 and 148 positions, we demonstrate that only the combination G140S + Q148H is able to restore the catalytic properties of IN. This rescue only operates in cis when both the 140S and 148H mutations are in the same IN polypeptide flexible loop. Finally, we show that the G140S-Q148H double mutant exhibits the highest resistance to RAL. It also confers cross-resistance to elvitegravir but less to G-quadraduplex inhibitors such as zintevir. Our results demonstrate that IN mutations at positions 140 and 148 in the IN flexible loop can account for the phenotype of RAL-resistant viruses.