RESUMEN
Casitas B-lymphoma proto-oncogene-b (Cbl-b) is a RING finger E3 ligase that has an important role in effector T cell function, acting as a negative regulator of T cell, natural killer (NK) cell, and B cell activation. A discovery effort toward Cbl-b inhibitors was pursued in which a generative AI design engine, REINVENT, was combined with a medicinal chemistry structure-based design to discover novel inhibitors of Cbl-b. Key to the success of this effort was the evolution of the "Design" phase of the Design-Make-Test-Analyze cycle to involve iterative rounds of an in silico structure-based drug design, strongly guided by physics-based affinity prediction and machine learning DMPK predictive models, prior to selection for synthesis. This led to the accelerated discovery of a potent series of carbamate Cbl-b inhibitors.
Asunto(s)
Carbamatos , Diseño de Fármacos , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-cbl , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Carbamatos/química , Carbamatos/farmacología , Carbamatos/síntesis química , Humanos , Relación Estructura-Actividad , Modelos Moleculares , Inteligencia Artificial , Descubrimiento de Drogas , Proteínas Adaptadoras Transductoras de SeñalesRESUMEN
PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. With the aim of developing MTA-cooperative PRMT5 inhibitors suitable for MTAP-deficient cancers, herein we report our efforts to develop novel "MTA-cooperative" compounds identified through a high-throughput biochemical screening approach. Optimization of hits was achieved through structure-based design with a focus on improvement of oral drug-like properties. Bioisosteric replacement of the original thiazole guanidine headgroup, spirocyclization of the isoindolinone amide scaffold to both configurationally and conformationally lock the bioactive form, and fine-tuning of the potency, MTA cooperativity, and DMPK properties through specific substitutions of the azaindole headgroup were conducted. We have identified an orally available in vivo lead compound, 28 ("AZ-PRMT5i-1"), which shows sub-10 nM PRMT5 cell potency, >50-fold MTA cooperativity, suitable DMPK properties for oral dosing, and significant PRMT5-driven in vivo efficacy in several MTAP-deficient preclinical cancer models.
Asunto(s)
Inhibidores Enzimáticos , Proteína-Arginina N-Metiltransferasas , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Humanos , Animales , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Relación Estructura-Actividad , Ratones , Descubrimiento de Drogas , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis químicaRESUMEN
The optimization of an allosteric fragment, discovered by differential scanning fluorimetry, to an in vivo MAT2a tool inhibitor is discussed. The structure-based drug discovery approach, aided by relative binding free energy calculations, resulted in AZ'9567 (21), a potent inhibitor in vitro with excellent preclinical pharmacokinetic properties. This tool showed a selective antiproliferative effect on methylthioadenosine phosphorylase (MTAP) KO cells, both in vitro and in vivo, providing further evidence to support the utility of MAT2a inhibitors as potential anticancer therapies for MTAP-deficient tumors.
Asunto(s)
Neoplasias , Humanos , Entropía , Metionina Adenosiltransferasa/metabolismoRESUMEN
The inhibition of ataxia-telangiectasia mutated (ATM) has been shown to chemo- and radio-sensitize human glioma cells in vitro and therefore might provide an exciting new paradigm in the treatment of glioblastoma multiforme (GBM). The effective treatment of GBM will likely require a compound with the potential to efficiently cross the blood-brain barrier (BBB). Starting from clinical candidate AZD0156, 4, we investigated the imidazoquinolin-2-one scaffold with the goal of improving likely CNS exposure in humans. Strategies aimed at reducing hydrogen bonding, basicity, and flexibility of the molecule were explored alongside modulating lipophilicity. These studies identified compound 24 (AZD1390) as an exceptionally potent and selective inhibitor of ATM with a good preclinical pharmacokinetic profile. 24 showed an absence of human transporter efflux in MDCKII-MDR1-BCRP studies (efflux ratio <2), significant BBB penetrance in nonhuman primate PET studies (Kp,uu 0.33) and was deemed suitable for development as a clinical candidate to explore the radiosensitizing effects of ATM in intracranial malignancies.
Asunto(s)
Ataxia Telangiectasia , Glioblastoma , Piridinas , Quinolonas , Animales , Humanos , Barrera Hematoencefálica/metabolismo , Ataxia Telangiectasia/tratamiento farmacológico , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Neoplasias , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Glioblastoma/tratamiento farmacológicoRESUMEN
Casitas B-lymphoma proto-oncogene-b (Cbl-b), a member of the Cbl family of RING finger E3 ubiquitin ligases, has been demonstrated to play a central role in regulating effector T-cell function. Multiple studies using gene-targeting approaches have provided direct evidence that Cbl-b negatively regulates T, B, and NK cell activation via a ubiquitin-mediated protein modulation. Thus, inhibition of Cbl-b ligase activity can lead to immune activation and has therapeutic potential in immuno-oncology. Herein, we describe the discovery and optimization of an arylpyridone series as Cbl-b inhibitors by structure-based drug discovery to afford compound 31. This compound binds to Cbl-b with an IC50 value of 30 nM and induces IL-2 production in T-cells with an EC50 value of 230 nM. Compound 31 also shows robust intracellular target engagement demonstrated through inhibition of Cbl-b autoubiquitination, inhibition of ubiquitin transfer to ZAP70, and the cellular modulation of phosphorylation of a downstream signal within the TCR axis.
Asunto(s)
Proteínas Proto-Oncogénicas c-cbl , Ubiquitina-Proteína Ligasas , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Linfocitos T/metabolismo , Fosforilación , Ubiquitina/metabolismoRESUMEN
The glycine to cysteine mutation at codon 12 of Kirsten rat sarcoma (KRAS) represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 14, AZD4747, a clinical development candidate for the treatment of KRASG12C-positive tumors, including the treatment of central nervous system (CNS) metastases. Building on our earlier discovery of C5-tethered quinazoline AZD4625, excision of a usually critical pyrimidine ring yielded a weak but brain-penetrant start point which was optimized for potency and DMPK. Key design principles and measured parameters that give high confidence in CNS exposure are discussed. During optimization, divergence between rodent and non-rodent species was observed in CNS exposure, with primate PET studies ultimately giving high confidence in the expected translation to patients. AZD4747 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.
Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Neoplasias , Animales , Humanos , Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas p21(ras)/genética , Neoplasias/tratamiento farmacológico , Diseño de Fármacos , Glicina/uso terapéutico , Mutación , Neoplasias Pulmonares/tratamiento farmacológicoRESUMEN
Alchemical absolute binding free energy calculations are of increasing interest in drug discovery. These calculations require restraints between the receptor and ligand to restrict their relative positions and, optionally, orientations. Boresch restraints are commonly used, but they must be carefully selected in order to sufficiently restrain the ligand and to avoid inherent instabilities. Applying multiple distance restraints between anchor points in the receptor and ligand provides an alternative framework without inherent instabilities which may provide convergence benefits by more strongly restricting the relative movements of the receptor and ligand. However, there is no simple method to calculate the free energy of releasing these restraints due to the coupling of the internal and external degrees of freedom of the receptor and ligand. Here, a method to rigorously calculate free energies of binding with multiple distance restraints by imposing intramolecular restraints on the anchor points is proposed. Absolute binding free energies for the human macrophage migration inhibitory factor/MIF180, system obtained using a variety of Boresch restraints and rigorous and nonrigorous implementations of multiple distance restraints are compared. It is shown that several multiple distance restraint schemes produce estimates in good agreement with Boresch restraints. In contrast, calculations without orientational restraints produce erroneously favorable free energies of binding by up to approximately 4 kcal mol-1. These approaches offer new options for the deployment of alchemical absolute binding free energy calculations.
Asunto(s)
Simulación de Dinámica Molecular , Humanos , Termodinámica , Ligandos , Entropía , Unión ProteicaRESUMEN
KRAS is an archetypal high-value intractable oncology drug target. The glycine to cysteine mutation at codon 12 represents an Achilles heel that has now rendered this important GTPase druggable. Herein, we report our structure-based drug design approach that led to the identification of 21, AZD4625, a clinical development candidate for the treatment of KRASG12C positive tumors. Highlights include a quinazoline tethering strategy to lock out a bio-relevant binding conformation and an optimization strategy focused on the reduction of extrahepatic clearance mechanisms seen in preclinical species. Crystallographic analysis was also key in helping to rationalize unusual structure-activity relationship in terms of ring size and enantio-preference. AZD4625 is a highly potent and selective inhibitor of KRASG12C with an anticipated low clearance and high oral bioavailability profile in humans.
Asunto(s)
Antineoplásicos , Neoplasias Pulmonares , Antineoplásicos/farmacología , Diseño de Fármacos , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Mutación , Proteínas Proto-Oncogénicas p21(ras)/genética , Quinazolinas/farmacología , Relación Estructura-ActividadRESUMEN
In this article, we report the discovery of a series of pyrimidopyridones as inhibitors of IRAK4 kinase. From a previously disclosed 5-azaquinazoline series, we found that switching the pyridine ring for an N-substituted pyridone gave a novel hinge binding scaffold which retained potency against IRAK4. Importantly, introduction of the carbonyl established an internal hydrogen bond with the 4-NH, establishing a conformational lock and allowing truncation of the large basic substituent to a 1-methylcyclopyl group. Subsequent optimisation, facilitated by X-ray crystal structures, allowed identification of preferred substituents at both the pyridone core and pyrazole. Subsequent combinations of optimal groups allowed control of lipophilicity and identification of potent and selective inhibitors of IRAK4 with better in vitro permeability and lower clearance.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1 , Piridonas , Conformación Molecular , Piridonas/farmacología , Relación Estructura-ActividadRESUMEN
In recent years, the emergence of targeted covalent inhibitors which bind to the G12C mutant of KRAS have offered a solution to this previously intractable target. Inhibitors of KRASG12C tend to be structurally complex, displaying features such as atropisomerism, chiral centres and a reactive covalent warhead. Such molecules result in lengthy and challenging syntheses, and as a consequence critical decisions need to be made at the design level to maximise the chances of success. Here we take a retrospective look into how computational chemistry can help guide and prioritise medicinal chemistry efforts in the context of a series of conformationally restricted tetracyclic quinolines.
RESUMEN
MAT2a is a methionine adenosyltransferase that synthesizes the essential metabolite S-adenosylmethionine (SAM) from methionine and ATP. Tumors bearing the co-deletion of p16 and MTAP genes have been shown to be sensitive to MAT2a inhibition, making it an attractive target for treatment of MTAP-deleted cancers. A fragment-based lead generation campaign identified weak but efficient hits binding in a known allosteric site. By use of structure-guided design and systematic SAR exploration, the hits were elaborated through a merging and growing strategy into an arylquinazolinone series of potent MAT2a inhibitors. The selected in vivo tool compound 28 reduced SAM-dependent methylation events in cells and inhibited proliferation of MTAP-null cells in vitro. In vivo studies showed that 28 was able to induce antitumor response in an MTAP knockout HCT116 xenograft model.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Metionina Adenosiltransferasa/antagonistas & inhibidores , Sitio Alostérico , Animales , Proliferación Celular , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Técnicas de Inactivación de Genes , Células HCT116 , Semivida , Humanos , Metionina Adenosiltransferasa/genética , Metionina Adenosiltransferasa/metabolismo , Ratones , Simulación de Dinámica Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , Ratas , S-Adenosilmetionina/metabolismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
In this article, we report our efforts towards improving in vitro human clearance in a series of 5-azaquinazolines through a series of C4 truncations and C2 expansions. Extensive DMPK studies enabled us to tackle high Aldehyde Oxidase (AO) metabolism and unexpected discrepancies in human hepatocyte and liver microsomal intrinsic clearance. Our efforts culminated with the discovery of 5-azaquinazoline 35, which also displayed exquisite selectivity for IRAK4, and showed synergistic in vitro activity against MyD88/CD79 double mutant ABC-DLBCL in combination with the covalent BTK inhibitor acalabrutinib.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/metabolismo , Quinazolinas/química , Aldehído Oxidasa/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Perros , Estabilidad de Medicamentos , Semivida , Hepatocitos/metabolismo , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Ratones , Microsomas Hepáticos/metabolismo , Simulación de Dinámica Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/metabolismo , Quinazolinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Attempts to directly drug the important oncogene KRAS have met with limited success despite numerous efforts across industry and academia. The KRASG12C mutant represents an "Achilles heel" and has recently yielded to covalent targeting with small molecules that bind the mutant cysteine and create an allosteric pocket on GDP-bound RAS, locking it in an inactive state. A weak inhibitor at this site was optimized through conformational locking of a piperazine-quinazoline motif and linker modification. Subsequent introduction of a key methyl group to the piperazine resulted in enhancements in potency, permeability, clearance, and reactivity, leading to identification of a potent KRASG12C inhibitor with high selectivity and excellent cross-species pharmacokinetic parameters and in vivo efficacy.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Quinazolinas/uso terapéutico , Quinolonas/uso terapéutico , Regulación Alostérica , Animales , Antineoplásicos/síntesis química , Antineoplásicos/farmacocinética , Células CACO-2 , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Masculino , Ratones Desnudos , Conformación Molecular , Mutación , Piperazinas/síntesis química , Piperazinas/farmacocinética , Proteínas Proto-Oncogénicas p21(ras)/genética , Quinazolinas/síntesis química , Quinazolinas/farmacocinética , Quinolonas/síntesis química , Quinolonas/farmacocinética , Ratas Wistar , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this article, we report the discovery of a series of 5-azaquinazolines as selective IRAK4 inhibitors. From modestly potent quinazoline 4, we introduced a 5-aza substitution to mask the 4-NH hydrogen bond donor (HBD). This allowed us to substitute the core with a 2-aminopyrazole, which showed large gains in cellular potency despite the additional formal HBD. Further optimization led to 6-cyanomethyl-5-azaquinazoline 13, a selective IRAK4 inhibitor, which proved efficacious in combination with ibrutinib, while showing very little activity as a single agent up to 100 mg/kg. This contrasted to previously reported IRAK4 inhibitors that exhibited efficacy in the same model as single agents and was attributed to the enhanced specificity of 13 toward IRAK4.
Asunto(s)
Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Terapia Molecular Dirigida , Factor 88 de Diferenciación Mieloide/genética , Quinazolinas/química , Quinazolinas/farmacología , Administración Oral , Animales , Línea Celular Tumoral , Diseño de Fármacos , Femenino , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Modelos Moleculares , Mutación , Conformación Proteica , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/administración & dosificación , Quinazolinas/farmacocinética , Ratas , Ratas Wistar , Relación Estructura-Actividad , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The three-dimensional conformations adopted by a free ligand in solution impact bioactivity and physicochemical properties. Solution 1D NMR spectra inherently contain information on ligand conformational flexibility and three-dimensional shape, as well as the propensity of the free ligand to fully preorganize into the bioactive conformation. Herein we discuss some key learnings, distilled from our experience developing potent and selective synthetic macrocyclic inhibitors, including Mcl-1 clinical candidate AZD5991. Case studies have been selected from recent oncology research projects, demonstrating how 1D NMR conformational signatures can complement X-ray protein-ligand structural information to guide medicinal chemistry optimization. Learning to extract free ligand conformational information from routinely available 1D NMR signatures has proven to be fast enough to guide medicinal chemistry decisions within design cycles for compound optimization.
Asunto(s)
Diseño de Fármacos , Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/antagonistas & inhibidores , Cinética , Ligandos , Compuestos Macrocíclicos/síntesis química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
B-cell lymphoma 6 (BCL6) inhibition is a promising mechanism for treating hematological cancers but high quality chemical probes are necessary to evaluate its therapeutic potential. Here we report potent BCL6 inhibitors that demonstrate cellular target engagement and exhibit exquisite selectivity for BCL6 based on mass spectrometry analyses following chemical proteomic pull down. Importantly, a proteolysis-targeting chimera (PROTAC) was also developed and shown to significantly degrade BCL6 in a number of diffuse large B-cell lymphoma (DLBCL) cell lines, but neither BCL6 inhibition nor degradation selectively induced marked phenotypic response. To investigate, we monitored PROTAC directed BCL6 degradation in DLBCL OCI-Ly1 cells by immunofluorescence and discovered a residual BCL6 population. Analysis of subcellular fractions also showed incomplete BCL6 degradation in all fractions despite having measurable PROTAC concentrations, together providing a rationale for the weak antiproliferative response seen with both BCL6 inhibitor and degrader. In summary, we have developed potent and selective BCL6 inhibitors and a BCL6 PROTAC that effectively degraded BCL6, but both modalities failed to induce a significant phenotypic response in DLBCL despite achieving cellular concentrations.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Proto-Oncogénicas c-bcl-6/antagonistas & inhibidores , Quinolonas/farmacología , Talidomida/análogos & derivados , Talidomida/farmacología , Proteínas Adaptadoras Transductoras de Señales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Ligandos , Linfoma de Células B Grandes Difuso/tratamiento farmacológico , Péptido Hidrolasas/metabolismo , Unión Proteica , Proteolisis , Proteínas Proto-Oncogénicas c-bcl-6/química , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Quinolonas/síntesis química , Quinolonas/metabolismo , Talidomida/síntesis química , Talidomida/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
We report the discovery of a novel series of 3-cinnoline carboxamides as highly potent and selective ataxia telangiectasia mutated (ATM) kinase inhibitors. Optimization of this series focusing on potency and physicochemical properties (especially permeability) led to the identification of compound 21, a highly potent ATM inhibitor (ATM cell IC50 0.0028 µM) with excellent kinase selectivity and favorable physicochemical and pharmacokinetics properties. In vivo, 21 in combination with irinotecan showed tumor regression in the SW620 colorectal tumor xenograft model, superior inhibition to irinotecan alone. Compound 21 was selected for preclinical evaluation alongside AZD0156.
RESUMEN
ATM inhibitors, such as 7, have demonstrated the antitumor potential of ATM inhibition when combined with DNA double-strand break-inducing agents in mouse xenograft models. However, the properties of 7 result in a relatively high predicted clinically efficacious dose. In an attempt to minimize attrition during clinical development, we sought to identify ATM inhibitors with a low predicted clinical dose (<50 mg) and focused on strategies to increase both ATM potency and predicted human pharmacokinetic half-life (predominantly through the increase of volume of distribution). These efforts resulted in the discovery of 64 (AZD0156), an exceptionally potent and selective inhibitor of ATM based on an imidazo[4,5- c]quinolin-2-one core. 64 has good preclinical phamacokinetics, a low predicted clinical dose, and a high maximum absorbable dose. 64 has been shown to potentiate the efficacy of the approved drugs irinotecan and olaparib in disease relevant mouse models and is currently undergoing clinical evaluation with these agents.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/antagonistas & inhibidores , Diseño de Fármacos , Piridinas/farmacocinética , Quinolinas/farmacocinética , Quinolonas/farmacología , Quinolonas/farmacocinética , Administración Oral , Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Disponibilidad Biológica , Humanos , Concentración 50 Inhibidora , Modelos Moleculares , Conformación Proteica , Inhibidores de Proteínas Quinasas , Piridinas/administración & dosificación , Piridinas/química , Quinolinas/administración & dosificación , Quinolinas/química , Quinolonas/administración & dosificación , Quinolonas/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
We have developed a series of orally efficacious IRAK4 inhibitors, based on a scaffold hopping strategy and using rational structure based design. Efforts to tackle low permeability and high efflux in our previously reported pyrrolopyrimidine series (Scott et al., 2017) led to the identification of pyrrolotriazines which contained one less formal hydrogen bond donor and were intrinsically more lipophilic. Further optimisation of substituents on this pyrrolotriazine core culminated with the discovery of 30 as a promising in vivo probe to assess the potential of IRAK4 inhibition for the treatment of MyD88 mutant DLBCL in combination with a BTK inhibitor. When tested in an ABC-DLBCL model with a dual MyD88/CD79 mutation (OCI-LY10), 30 demonstrated tumour regressions in combination with ibrutinib.