RESUMEN
Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.
RESUMEN
KRAS, which is mutated in â¼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit.
Asunto(s)
Carcinogénesis/metabolismo , Dimerización , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Línea Celular Tumoral , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación/fisiología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Proteínas ras/genéticaRESUMEN
Structure-based design was utilized to optimize 6,6-diaryl substituted dihydropyrone and hydroxylactam to obtain inhibitors of lactate dehydrogenase (LDH) with low nanomolar biochemical and single-digit micromolar cellular potencies. Surprisingly the replacement of a phenyl with a pyridyl moiety in the chemical structure revealed a new binding mode for the inhibitors with subtle conformational change of the LDHA active site. This led to the identification of a potent, cell-active hydroxylactam inhibitor exhibiting an in vivo pharmacokinetic profile suitable for mouse tumor xenograft study.
Asunto(s)
Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Lactamas/farmacología , Animales , Línea Celular , Perros , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , L-Lactato Deshidrogenasa/metabolismo , Lactamas/química , Ratones , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Although RAF monomer inhibitors (type I.5, BRAF(V600)) are clinically approved for the treatment of BRAFV600-mutant melanoma, they are ineffective in non-BRAFV600 mutant cells1-3. Belvarafenib is a potent and selective RAF dimer (type II) inhibitor that exhibits clinical activity in patients with BRAFV600E- and NRAS-mutant melanomas. Here we report the first-in-human phase I study investigating the maximum tolerated dose, and assessing the safety and preliminary efficacy of belvarafenib in BRAFV600E- and RAS-mutated advanced solid tumours (NCT02405065, NCT03118817). By generating belvarafenib-resistant NRAS-mutant melanoma cells and analysing circulating tumour DNA from patients treated with belvarafenib, we identified new recurrent mutations in ARAF within the kinase domain. ARAF mutants conferred resistance to belvarafenib in both a dimer- and a kinase activity-dependent manner. Belvarafenib induced ARAF mutant dimers, and dimers containing mutant ARAF were active in the presence of inhibitor. ARAF mutations may serve as a general resistance mechanism for RAF dimer inhibitors as the mutants exhibit reduced sensitivity to a panel of type II RAF inhibitors. The combination of RAF plus MEK inhibition may be used to delay ARAF-driven resistance and suggests a rational combination for clinical use. Together, our findings reveal specific and compensatory functions for the ARAF isoform and implicate ARAF mutations as a driver of resistance to RAF dimer inhibitors.
Asunto(s)
Resistencia a Antineoplásicos/genética , Melanoma/tratamiento farmacológico , Melanoma/genética , Mutación , Proteínas Proto-Oncogénicas A-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas A-raf/genética , Quinasas raf/antagonistas & inhibidores , Animales , Línea Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Humanos , Melanoma/patología , Ratones , Multimerización de Proteína/efectos de los fármacos , Proteínas Proto-Oncogénicas A-raf/química , Quinasas raf/químicaRESUMEN
Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.
RESUMEN
Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.
Asunto(s)
Neoplasias/tratamiento farmacológico , Compuestos de Fenilurea/uso terapéutico , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinazolinonas/uso terapéutico , Quinasas raf/antagonistas & inhibidores , Animales , Azetidinas/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Combinación de Medicamentos , Sinergismo Farmacológico , Femenino , Humanos , Células de Riñón Canino Madin Darby , Ratones Desnudos , Estructura Molecular , Mutación , Compuestos de Fenilurea/química , Compuestos de Fenilurea/metabolismo , Piperidinas/uso terapéutico , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Quinazolinonas/química , Quinazolinonas/metabolismo , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de Xenoinjerto , Quinasas raf/genética , Quinasas raf/metabolismoRESUMEN
Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.
Asunto(s)
Fosfatidilinositol 3-Quinasas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas p21(ras)/efectos de los fármacos , Línea Celular Tumoral , Guanosina Trifosfato/metabolismo , Humanos , Mutación/efectos de los fármacos , Mutación/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas B-raf/efectos de los fármacos , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas ras/efectos de los fármacos , Proteínas ras/genéticaRESUMEN
A series of trisubstituted hydroxylactams was identified as potent enzymatic and cellular inhibitors of human lactate dehydrogenase A. Utilizing structure-based design and physical property optimization, multiple inhibitors were discovered with <10 µM lactate IC50 in a MiaPaca2 cell line. Optimization of the series led to 29, a potent cell active molecule (MiaPaca2 IC50 = 0.67 µM) that also possessed good exposure when dosed orally to mice.
RESUMEN
The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Pirazoles/farmacología , Piridonas/farmacología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Perros , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Células de Riñón Canino Madin Darby , Ratones , Ratones Desnudos , Modelos Moleculares , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Piridonas/síntesis química , Piridonas/química , Relación Estructura-ActividadRESUMEN
Metabolic reprogramming in tumors represents a potential therapeutic target. Herein we used shRNA depletion and a novel lactate dehydrogenase (LDHA) inhibitor, GNE-140, to probe the role of LDHA in tumor growth in vitro and in vivo. In MIA PaCa-2 human pancreatic cells, LDHA inhibition rapidly affected global metabolism, although cell death only occurred after 2 d of continuous LDHA inhibition. Pancreatic cell lines that utilize oxidative phosphorylation (OXPHOS) rather than glycolysis were inherently resistant to GNE-140, but could be resensitized to GNE-140 with the OXPHOS inhibitor phenformin. Acquired resistance to GNE-140 was driven by activation of the AMPK-mTOR-S6K signaling pathway, which led to increased OXPHOS, and inhibitors targeting this pathway could prevent resistance. Thus, combining an LDHA inhibitor with compounds targeting the mitochondrial or AMPK-S6K signaling axis may not only broaden the clinical utility of LDHA inhibitors beyond glycolytically dependent tumors but also reduce the emergence of resistance to LDHA inhibition.
Asunto(s)
Plasticidad de la Célula/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Piridonas/farmacología , Tiofenos/farmacología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Piridonas/química , Relación Estructura-Actividad , Tiofenos/químicaRESUMEN
Inhibitors targeting the activating mutants of the epidermal growth factor receptor (EGFR) have found success in the treatment of EGFR mutant positive non-small-cell lung cancer. A secondary point mutation (T790M) in the inhibitor binding site has been linked to the acquired resistance against those first generation therapeutics. Herein, we describe the lead optimization of a series of reversible, pan-mutant (L858R, del746-750, T790M/L858R, and T790M/del746-750) EGFR inhibitors. By use of a noncovalent double mutant (T790M/L858R and T790M/del746-750) selective EGFR inhibitor (2) as a starting point, activities against the single mutants (L858R and del746-750) were introduced through a series of structure-guided modifications. The in vitro ADME-PK properties of the lead molecules were further optimized through a number of rational structural changes. The resulting inhibitor (21) exhibited excellent cellular activity against both the single and double mutants of EGFR, demonstrating target engagement in vivo and ADME-PK properties that are suitable for further evaluation. The reversible, noncovalent inhibitors described complement the covalent pan-mutant EGFR inhibitors that have shown encouraging results in recent clinical trials.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Resistencia a Antineoplásicos , Receptores ErbB/genética , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Modelos Moleculares , Mutación , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Activating mutations in protein kinases drive many cancers. While how recurring point mutations affect kinase activity has been described, the effect of in-frame deletions is not well understood. We show that oncogenic deletions within the ß3-αC loop of HER2 and BRAF are analogous to the recurrent EGFR exon 19 deletions. We identify pancreatic carcinomas with BRAF deletions mutually exclusive with KRAS mutations. Crystal structures of BRAF deletions reveal the truncated loop restrains αC in an active "in" conformation, imparting resistance to inhibitors like vemurafenib that bind the αC "out" conformation. Characterization of loop length explains the prevalence of five amino acid deletions in BRAF, EGFR, and HER2 and highlights the importance of this region for kinase activity and inhibitor efficacy.
Asunto(s)
Genes erbB-1 , Genes erbB-2 , Mutación , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Proto-Oncogénicas B-raf/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antineoplásicos/farmacología , Emparejamiento Base/genética , Secuencia Conservada , Dimerización , Resistencia a Antineoplásicos/genética , Activación Enzimática/genética , Receptores ErbB/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Conformación Proteica , Mapeo de Interacción de Proteínas , Inhibidores de Proteínas Quinasas/farmacología , Estructura Secundaria de Proteína , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The rapid advancement of a series of noncovalent inhibitors of T790M mutants of EGFR is discussed. The optimization of pyridone 1, a nonselective high-throughput screening hit, to potent molecules with high levels of selectivity over wtEGFR and the broader kinome is described herein.
RESUMEN
The treatment of epidermal growth factor receptor (EGFR)-driven non-small cell lung cancers with the T790M resistance mutation remains a significant unmet medical need. We report the identification of 4-aminoindazolyl-dihydrofuro[3,4-d]pyrimidines as non-covalent inhibitors of EGFR, with excellent activity against the T790M resistance double mutants and initial single activating mutants. Using an optimization strategy focused on structure-based design and improving PK properties through metabolite identification, we obtained advanced leads with high oral exposure.
Asunto(s)
Receptores ErbB/antagonistas & inhibidores , Furanos/farmacología , Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Acrilamidas/farmacología , Compuestos de Anilina/farmacología , Animales , Sitios de Unión , Cristalografía por Rayos X , Perros , Receptores ErbB/química , Clorhidrato de Erlotinib/farmacología , Furanos/síntesis química , Furanos/química , Furanos/farmacocinética , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Enlace de Hidrógeno , Indazoles/síntesis química , Indazoles/química , Indazoles/farmacocinética , Ratones , Microsomas Hepáticos/metabolismo , Mutación Puntual , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/química , Pirimidinas/farmacocinética , RatasRESUMEN
Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Genes erbB-1/efectos de los fármacos , Animales , Antineoplásicos/farmacocinética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Perros , Diseño de Fármacos , Técnicas de Silenciamiento del Gen , Humanos , Técnicas In Vitro , Lípidos/química , Neoplasias Pulmonares/tratamiento farmacológico , Macaca fascicularis , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Mutación , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Especificidad por Sustrato , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.
Asunto(s)
Acetilcolinesterasa/metabolismo , Carbazoles/química , Carbazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Acetilcolinesterasa/química , Acetilcolinesterasa/farmacocinética , Acetilcolinesterasa/uso terapéutico , Animales , Compuestos Aza/química , Compuestos Aza/farmacocinética , Compuestos Aza/farmacología , Compuestos Aza/uso terapéutico , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacocinética , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/uso terapéutico , Cristalografía por Rayos X , Perros , Humanos , Ratones , Ratones Desnudos , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas/química , RatasRESUMEN
Optimization of 5-(2,6-dichlorophenyl)-3-hydroxy-2-mercaptocyclohex-2-enone using structure-based design strategies resulted in inhibitors with considerable improvement in biochemical potency against human lactate dehydrogenase A (LDHA). These potent inhibitors were typically selective for LDHA over LDHB isoform (410 fold) and other structurally related malate dehydrogenases, MDH1 and MDH2 (>500 fold). An X-ray crystal structure of enzymatically most potent molecule bound to LDHA revealed two additional interactions associated with enhanced biochemical potency.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Perros , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , L-Lactato Deshidrogenasa/metabolismo , Células de Riñón Canino Madin DarbyRESUMEN
A series of 3,6-disubstituted dihydropyrones were identified as inhibitors of human lactate dehydrogenase (LDH)-A. Structure activity relationships were explored and a series of 6,6-spiro analogs led to improvements in LDHA potency (IC50 <350 nM). An X-ray crystal structure of an improved compound bound to human LDHA was obtained and it illustrated additional opportunities to enhance the potency of these compounds, resulting in the identification of 51 (IC50=30 nM).
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Pironas/síntesis química , Pironas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Humanos , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Checkpoint kinase 1 (ChK1) is activated in response to DNA damage, acting to temporarily block cell cycle progression and allow for DNA repair. It is envisaged that inhibition of ChK1 will sensitize tumor cells to treatment with DNA-damaging therapies, and may enhance the therapeutic window. High throughput screening identified carboxylate-containing diarylpyrazines as a prominent hit series, but with limited biochemical potency and no cellular activity. Through a series of SAR investigations and X-ray crystallographic analysis the critical role of polar contacts with conserved waters in the kinase back pocket was established. Structure-based design, guided by in silico modeling, transformed the series to better satisfy these contacts and the novel 1,7-diazacarbazole class of inhibitors was discovered. Here we present the genesis of this novel series and the identification of GNE-783, a potent, selective and orally bioavailable inhibitor of ChK1.