RESUMEN
BACKGROUND: Coexpression of CD160 and PD-1 on HIV-specific CD8+ T-cells defines a highly exhausted T-cell subset. CD160 binds to Herpes Virus Entry Mediator (HVEM) and blocking this interaction with HVEM antibodies reverses T-cell exhaustion. As HVEM binds both inhibitory and activatory receptors, our aim in the current study was to assess the impact of CD160-specific antibodies on the enhancement of T-cell activation. METHODS: Expression of the two CD160 isoforms; glycosylphosphatidylinositol-anchored (CD160-GPI) and the transmembrane isoforms (CD160-TM) was assessed in CD4 and CD8 primary T-cells by quantitative RT-PCR and Flow-cytometry. Binding of these isoforms to HVEM ligand and the differential capacities of CD160 and HVEM specific antibodies to inhibit this binding were further evaluated using a Time-Resolved Fluorescence assay (TRF). The impact of both CD160 and HVEM specific antibodies on enhancing T-cell functionality upon antigenic stimulation was performed in comparative ex vivo studies using primary cells from HIV-infected subjects stimulated with HIV antigens in the presence or absence of blocking antibodies to the key inhibitory receptor PD-1. RESULTS: We first show that both CD160 isoforms, CD160-GPI and CD160-TM, were expressed in human primary CD4+ and CD8+ T-cells. The two isoforms were also recognized by the HVEM ligand, although this binding was less pronounced with the CD160-TM isoform. Mechanistic studies revealed that although HVEM specific antibodies blocked its binding to CD160-GPI, surprisingly, these antibodies enhanced HVEM binding to CD160-TM, suggesting that potential antibody-mediated HVEM multimerization and/or induced conformational changes may be required for optimal CD160-TM binding. Triggering of CD160-GPI over-expressed on Jurkat cells with either bead-bound HVEM-Fc or anti-CD160 monoclonal antibodies enhanced cell activation, consistent with a positive co-stimulatory role for CD160-GPI. However, CD160-TM did not respond to this stimulation, likely due to the lack of optimal HVEM binding. Finally, ex vivo assays using PBMCs from HIV viremic subjects showed that the use of CD160-GPI-specific antibodies combined with blockade of PD-1 synergistically enhanced the proliferation of HIV-1 specific CD8+ T-cells upon antigenic stimulation. CONCLUSIONS: Antibodies targeting CD160-GPI complement the blockade of PD-1 to enhance HIV-specific T-cell responses and warrant further investigation in the development of novel immunotherapeutic approaches.
Asunto(s)
Antígenos CD/genética , Antígenos CD/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Activación de Linfocitos/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Adulto , Animales , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células CHO , Cricetinae , Cricetulus , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Humanos , Células Jurkat , Masculino , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Receptores Inmunológicos/inmunología , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismoRESUMEN
ATR is a key kinase in the DNA-damage response (DDR) that is synthetic lethal with several other DDR proteins, making it an attractive target for the treatment of genetically selected solid tumors. Herein we describe the discovery of a novel ATR inhibitor guided by a pharmacophore model to position a key hydrogen bond. Optimization was driven by potency and selectivity over the related kinase mTOR, resulting in the identification of camonsertib (RP-3500) with high potency and excellent ADME properties. Preclinical evaluation focused on the impact of camonsertib on myelosuppression, and an exploration of intermittent dosing schedules to allow recovery of the erythroid compartment and mitigate anemia. Camonsertib is currently undergoing clinical evaluation both as a single agent and in combination with talazoparib, olaparib, niraparib, lunresertib, or gemcitabine (NCT04497116, NCT04972110, NCT04855656). A preliminary recommended phase 2 dose for monotherapy was identified as 160 mg QD given 3 days/week.
Asunto(s)
Neoplasias , Humanos , Proteínas de la Ataxia Telangiectasia Mutada , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , GemcitabinaRESUMEN
Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.
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Ataxia Telangiectasia , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Proteína Quinasa Activada por ADN/metabolismo , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of RP-6685: a potent, selective, and orally bioavailable Polθ inhibitor that showed in vivo efficacy in an HCT116 BRCA2-/- mouse tumor xenograft model.
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ADN Polimerasa Dirigida por ADN , Neoplasias Ováricas , Animales , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Diseño de Fármacos , Descubrimiento de Drogas , Femenino , Humanos , RatonesRESUMEN
The interaction of the hepatitis C virus (HCV) RNA-dependent RNA polymerase with RNA substrate is incompletely defined. We have characterized the activities of the HCV NS5B polymerase, modified by different deletions and affinity tags, with a routinely used homopolymeric substrate, and established apparent affinities of the various NS5B constructs both for the NTP and the template/primer substrates. We identified a uniquely tagged HCV NS5B RNA polymerase construct with a lower affinity (higher K(m)) than mature HCV NS5B for template/ primer substrate and highlighted the use of such a polymerase for the identification of inhibitors of NS5B activity, particularly inhibitors of productive RNA binding. The characterization of specific benzimidazole-5-carboxamide-based inhibitors, identified in a screening campaign, revealed that this class of compounds was non-competitive with regard to NTP incorporation and had no effect on processive elongation, but inhibited an initiation phase of the HCV polymerase activity. The potency of these compounds versus a panel of different NS5B polymerase constructs was inversely proportional to the enzymes' affinities for template/primer substrate. The benzimidazole-5-carboxamide compounds also inhibited the full-length, untagged NS5B de novo initiation reaction using HCV 3'-UTR substrate RNA and expand the diversifying pool of potential HCV replication inhibitors.
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Inhibidores Enzimáticos/farmacología , Hepacivirus/enzimología , ARN Polimerasa Dependiente del ARN/antagonistas & inhibidores , ARN Polimerasa Dependiente del ARN/metabolismo , ARN/metabolismo , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Amidas/química , Amidas/farmacología , Animales , Bencimidazoles/química , Bencimidazoles/farmacología , Bovinos , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Hepacivirus/genética , Hepacivirus/fisiología , Concentración 50 Inhibidora , Cinética , Poliovirus/enzimología , ARN/genética , ARN Polimerasa II/metabolismo , ARN Viral/biosíntesis , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Especificidad por Sustrato , Moldes Genéticos , Proteínas no Estructurales Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Biobanking biological samples involve multiple handling, processing, and labeling steps. Each step may be a source of error, which if unnoticed or uncorrected may have consequences for research. We aimed to develop a simple and inexpensive genotyping method that would be valuable to detect such errors and confirm sample identity. For this purpose, seven variable number of tandem repeat (VNTR) loci were selected, analyzed by polymerase chain reaction (PCR) amplification, and organized in a PCR-based DNA profiling algorithm that proved useful to minimize the number of steps required for the procedure. Match probability calculations suggest that this method/algorithm has the potential to discriminate every participant of a biobank. As a proof of concept, the algorithm was applied on samples taken from the PROCURE Prostate Cancer Biobank. It was applied on 403 DNA samples from 101 randomly chosen patients who provided prostate tissues at surgery and blood at two to three different time points over a period of up to 7 years. A unique DNA profile requiring the analysis of no more than four VNTR loci (D16S83, D17S5, D1S80, D19S20) was successfully obtained for each of the 101 cases studied and led to the identification of two mismatches among the 403 samples evaluated (0.5% error rate). Further investigations using the same genotyping method revealed that one of the errors was due to tissue mishandling and that the other was due to tissue mislabeling. These errors, typical to the complex biobanking process, highlight the importance to implement a routine genotyping method as part of quality assurance in biobanking.
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Dermatoglifia del ADN/métodos , Repeticiones de Minisatélite , Manejo de Especímenes/normas , Algoritmos , Bancos de Muestras Biológicas/normas , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patologíaRESUMEN
A previously disclosed series of non-nucleoside allosteric inhibitors of the NS5B polymerase of the hepatitis C virus (HCV) was optimized to yield novel compounds with improved physicochemical properties and activity in cell-based assays. Replacement of ionizable carboxylic acids with neutral substituents in lead compounds produced inhibitors with cellular permeability and antiviral activity in a cell-based assay of subgenomic HCV RNA replication (replicon EC(50) as low as 1.7 microM). The improvement in potency in this ex vivo model of HCV RNA replication validates, in part, the mechanism by which this class of allosteric benzimidazole derivatives inhibits the polymerase and represents a significant step forward in the discovery of novel HCV therapeutics.
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Antivirales/síntesis química , Bencimidazoles/síntesis química , Inhibidores Enzimáticos/síntesis química , Hepacivirus/efectos de los fármacos , Replicón/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Regulación Alostérica , Antivirales/farmacología , Bencimidazoles/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/genética , Humanos , Relación Estructura-ActividadRESUMEN
Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with (33)P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.
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Genoma Viral , Hepacivirus/genética , Riñón/virología , Replicón , Replicación Viral/fisiología , Fusión Celular , Línea Celular , Hepacivirus/fisiología , Humanos , Riñón/citología , Riñón/embriología , ARN Viral/biosíntesis , Células Tumorales CultivadasRESUMEN
A variety of 3'-untranslated regions (UTRs) were cloned from infectious hepatitis C virus human samples and examined in NS5B polymerase de novo initiation reactions. We isolated and characterized four distinct 3'-UTRs that harbor the conserved terminal 98 nucleotides, but have poly(U/UC) tracts of 25, 93, 98, and 101 nucleotides, respectively. Reconstitution of de novo initiation by the mature NS5B with the different 3'-UTR RNA substrates revealed distinctively sized products that are consistent with internal initiation at specific sites within the polypyrimidine tract. These sites were further mapped by demonstrating that nucleotide substitutions of the cytidylate stretches within the poly(U/UC) template eliminate specific products of de novo synthesis and by showing that these products could be radiolabeled by [gamma-32P]GTP. We also examine analogs that can substitute for GTP in this reaction.
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Regiones no Traducidas 3' , Hepacivirus/genética , ARN/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas no Estructurales Virales/genética , Secuencia de Bases , Clonación Molecular , Relación Dosis-Respuesta a Droga , Guanosina Trifosfato/metabolismo , Modelos Genéticos , Datos de Secuencia Molecular , Plásmidos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The hepatitis C virus (HCV) NS3 protease is essential for polyprotein maturation and viral propagation, and it has been proposed as a suitable target for antiviral drug discovery. An N-terminal hexapeptide cleavage product of a dodecapeptide substrate identified as a weak competitive inhibitor of the NS3 protease activity was optimized to a potent and highly specific inhibitor of the enzyme. The effect of this potent NS3 protease inhibitor was evaluated on replication of subgenomic HCV RNA and compared with interferon-alpha (IFN-alpha), which is currently used in the treatment of HCV-infected patients. Treatment of replicon-containing cells with the NS3 protease inhibitor or IFN-alpha showed a dose-dependent decrease in subgenomic HCV RNA that reached undetectable levels following a 14-day treatment. Kinetic studies in the presence of either NS3 protease inhibitor or IFN-alpha also revealed similar profiles in HCV RNA decay with half-lives of 11 and 14 h, respectively. The finding that an antiviral specifically targeting the NS3 protease activity inhibits HCV RNA replication further validates the NS3 enzyme as a prime target for drug discovery and supports the development of NS3 protease inhibitors as a novel therapeutic approach for HCV infection.
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Hepacivirus/efectos de los fármacos , ARN Viral/efectos de los fármacos , Inhibidores de Serina Proteinasa/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Antivirales/farmacología , Antivirales/uso terapéutico , Hepacivirus/enzimología , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Humanos , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , ARN Viral/biosíntesis , Inhibidores de Serina Proteinasa/uso terapéuticoRESUMEN
Benzimidazole 5-carboxamide derivatives from a combinatorial screening library were discovered as specific inhibitors of the NS5B polymerase of the hepatitis C virus (HCV). Initial hit-to-lead activities taking advantage of high-throughput parallel synthetic techniques, identified a 1,2-disubstituted benzimidazole 5-carboxylic acid scaffold as the minimum core for biological activity. Potent analogues in this series inhibit the polymerase at low micromolar concentrations and provide an attractive "drug-like" lead structure for further optimization and the development of potential HCV therapeutics.