RESUMEN
For most oral small-molecule projects within drug discovery, the extent and duration of the effect are influenced by the total clearance of the compound; hence, designing compounds with low clearance remains a key focus to help enable sufficient protein target engagement. Comprehensive understanding and accurate prediction of animal clearance and pharmacokinetics provides confidence that the same can be observed for human. During a MERTK inhibitor lead optimization project, a series containing a biphenyl ring system with benzylamine meta-substitution on one phenyl and nitrogen inclusion as the meta atom on the other ring demonstrated multiple routes of compound elimination in rats. Here, we describe the identification of a structural pharmacophore involving two key interactions observed for both the MERTK program and an additional internal project. Four strategies to mitigate these clearance liabilities were identified and systematically investigated. We provide evidence that disruption of at least one of the interactions led to a significant reduction in CL that was subsequently predicted from rat hepatocytes using in vitro/in vivo extrapolation and the well-stirred scaling method. These tactics will likely be of general utility to the medicinal chemistry and DMPK community during compound optimization when similar issues are encountered for biphenyl benzylamines.
Asunto(s)
Bencilaminas , Compuestos de Bifenilo , Hepatocitos , Modelos Biológicos , Animales , Bencilaminas/metabolismo , Compuestos de Bifenilo/metabolismo , Hepatocitos/metabolismo , Tasa de Depuración Metabólica , Ratas , Tirosina Quinasa c-Mer/metabolismoRESUMEN
The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.
Asunto(s)
Fagocitosis/efectos de los fármacos , Células Fotorreceptoras de Vertebrados/efectos de los fármacos , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Animales , Línea Celular , Femenino , Humanos , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Imagen Multimodal , Células Fotorreceptoras de Vertebrados/patología , Ratas , Ratas Long-Evans , Ratas Wistar , Degeneración Retiniana/inducido químicamente , Epitelio Pigmentado de la Retina/metabolismo , Distribución Tisular , Tirosina Quinasa c-Mer/genéticaRESUMEN
The activation loop (A-loop) plays a key role in regulating the catalytic activity of protein kinases. Phosphorylation in this region enhances the phosphoryl transfer rate of the kinase domain and increases its affinity for ATP. Furthermore, the A-loop possesses autoinhibitory functions in some kinases, where it collapses onto the protein surface and blocks substrate binding when unphosphorylated. Due to its flexible nature, the A-loop is usually disordered and untraceable in kinase domain crystal structures. The resulting lack of structural information is regrettable as it impedes the design of drug A-loop contacts, which have proven favourable in multiple cases. Here, we characterize the binding with A-loop engagement between type 1.5 kinase inhibitor 'example 172' (EX172) and Mer tyrosine kinase (MerTK). With the help of crystal structures and binding kinetics, we portray how the recruitment of the A-loop elicits a two-step binding mechanism which results in a drug-target complex characterized by high affinity and long residence time. In addition, the type 1.5 compound possesses excellent kinome selectivity and a remarkable preference for the phosphorylated over the dephosphorylated form of MerTK. We discuss these unique characteristics in the context of known type 1 and type 2 inhibitors and highlight opportunities for future kinase inhibitor design.
Asunto(s)
Adenosina Trifosfato/química , Inhibidores de Proteínas Quinasas/química , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Tirosina Quinasa c-Mer/química , Humanos , Estructura Secundaria de ProteínaRESUMEN
When activated by amino acid starvation, the stress sensing protein kinase GCN2 phosphorylates the eukaryotic initiation factor 2 alpha, inhibiting translation to conserve energy and facilitate cell survival. Amino acid starvation, particularly of tryptophan and arginine, affects immune tolerance by suppressing differentiation and proliferation of T-cells via activation of GCN2 kinase. In addition, the GCN2 pathway mediates cancer survival directly within the context of metabolic stress. Here, we report the first crystal structures of the human GCN2 kinase domain (KD) in complex with two inhibitors of different size, shape, and chemical scaffold. Three novel activation loop conformations representative of different activation states of the kinase are described. In addition, a novel dimerization organization for GCN2 is observed. This arrangement is consistent with the hypothesis that the GCN2 KD forms an antiparallel inactive dimer until uncharged tRNA binds to it and triggers conformational changes that shift the equilibrium to the active parallel dimer.
Asunto(s)
Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/química , Cristalografía por Rayos X , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Unión Proteica , Dominios Proteicos , Multimerización de Proteína , ARN de Transferencia/metabolismoRESUMEN
MUS81 is a structure-selective endonuclease that cleaves various branched DNA structures arising from natural physiological processes such as homologous recombination and mitosis. Due to this, MUS81 is able to relieve replication stress, and its function has been reported to be critical to the survival of many cancers, particularly those with dysfunctional DNA-repair machinery. There is therefore interest in MUS81 as a cancer drug target, yet there are currently few small molecule inhibitors of this enzyme reported, and no liganded crystal structures are available to guide hit optimization. Here we report the fragment-based discovery of novel small molecule MUS81 inhibitors with sub-µM biochemical activity. These inhibitors were used to develop a novel crystal system, providing the first structural insight into the inhibition of MUS81 with small molecules.
RESUMEN
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
ATAD2 is an epigenetic bromodomain-containing target which is overexpressed in many cancers and has been suggested as a potential oncology target. While several small molecule inhibitors have been described in the literature, their cellular activity has proved to be underwhelming. In this work, we describe the identification of a novel series of ATAD2 inhibitors by high throughput screening, confirmation of the bromodomain region as the site of action, and the optimization campaign undertaken to improve the potency, selectivity, and permeability of the initial hit. The result is compound 5 (AZ13824374), a highly potent and selective ATAD2 inhibitor which shows cellular target engagement and antiproliferative activity in a range of breast cancer models.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Unión al ADN/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Especificidad por Sustrato , Ensayo de Tumor de Célula MadreRESUMEN
Mer is a member of the TAM (Tyro3, Axl, Mer) kinase family that has been associated with cancer progression, metastasis, and drug resistance. Their essential function in immune homeostasis has prompted an interest in their role as modulators of antitumor immune response in the tumor microenvironment. Here we illustrate the outcomes of an extensive lead-generation campaign for identification of Mer inhibitors, focusing on the results from concurrent, orthogonal high-throughput screening approaches. Data mining, HT (high-throughput), and DECL (DNA-encoded chemical library) screens offered means to evaluate large numbers of compounds. We discuss campaign strategy and screening outcomes, and exemplify series resulting from prioritization of hits that were identified. Concurrent execution of HT and DECL screening successfully yielded a large number of potent, selective, and novel starting points, covering a range of selectivity profiles across the TAM family members and modes of kinase binding, and offered excellent start points for lead development.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Tirosina Quinasa c-Mer/antagonistas & inhibidores , Animales , Cristalografía por Rayos X , Minería de Datos , Descubrimiento de Drogas , Humanos , Modelos Moleculares , Tirosina Quinasa c-Mer/química , Tirosina Quinasa c-Mer/metabolismoRESUMEN
Inhibition of Mer and Axl kinases has been implicated as a potential way to improve the efficacy of current immuno-oncology therapeutics by restoring the innate immune response in the tumor microenvironment. Highly selective dual Mer/Axl kinase inhibitors are required to validate this hypothesis. Starting from hits from a DNA-encoded library screen, we optimized an imidazo[1,2-a]pyridine series using structure-based compound design to improve potency and reduce lipophilicity, resulting in a highly selective in vivo probe compound 32. We demonstrated dose-dependent in vivo efficacy and target engagement in Mer- and Axl-dependent efficacy models using two structurally differentiated and selective dual Mer/Axl inhibitors. Additionally, in vivo efficacy was observed in a preclinical MC38 immuno-oncology model in combination with anti-PD1 antibodies and ionizing radiation.
Asunto(s)
Antineoplásicos/uso terapéutico , Imidazoles/uso terapéutico , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéutico , Piridinas/uso terapéutico , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Imidazoles/síntesis química , Masculino , Ratones Endogámicos C57BL , Ratones Desnudos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Proto-Oncogénicas/metabolismo , Piridinas/síntesis química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Relación Estructura-Actividad , Tirosina Quinasa c-Mer/metabolismo , Tirosina Quinasa del Receptor AxlRESUMEN
JAK1, JAK2, JAK3, and TYK2 belong to the JAK (Janus kinase) family. They play critical roles in cytokine signaling. Constitutive activation of JAK/STAT pathways is associated with a wide variety of diseases. Particularly, pSTAT3 is observed in response to the treatment with inhibitors of oncogenic signaling pathways such as EGFR, MAPK, and AKT and is associated with resistance or poorer response to agents targeting these pathways. Among the JAK family kinases, JAK1 has been shown to be the primary driver of STAT3 phosphorylation and signaling; therefore, selective JAK1 inhibition can be a viable means to overcome such treatment resistances. Herein, an account of the medicinal chemistry optimization from the promiscuous kinase screening hit 3 to the candidate drug 21 (AZD4205), a highly selective JAK1 kinase inhibitor, is reported. Compound 21 has good preclinical pharmacokinetics. Compound 21 displayed an enhanced antitumor activity in combination with an approved EGFR inhibitor, osimertinib, in a preclinical non-small-cell lung cancer (NSCLC) xenograft NCI-H1975 model.
Asunto(s)
Indoles/uso terapéutico , Janus Quinasa 1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Acrilamidas/farmacología , Compuestos de Anilina/farmacología , Animales , Línea Celular Tumoral , Diseño de Fármacos , Descubrimiento de Drogas , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Femenino , Humanos , Indoles/síntesis química , Indoles/farmacocinética , Ratones Desnudos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumors have evolved a variety of methods to reprogram conventional metabolic pathways to favor their own nutritional needs, including glutaminolysis, the first step of which is the hydrolysis of glutamine to glutamate by the amidohydrolase glutaminase 1 (GLS1). A GLS1 inhibitor could potentially target certain cancers by blocking the tumor cell's ability to produce glutamine-derived nutrients. Starting from the known GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide, we describe the medicinal chemistry evolution of a series from lipophilic inhibitors with suboptimal physicochemical and pharmacokinetic properties to cell potent examples with reduced molecular weight and lipophilicity, leading to compounds with greatly improved oral exposure that demonstrate in vivo target engagement accompanied by activity in relevant disease models.
Asunto(s)
Antineoplásicos/farmacología , Glutaminasa/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Piridazinas/farmacología , Tiadiazoles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Disponibilidad Biológica , Línea Celular Tumoral , Descubrimiento de Drogas , Glutaminasa/metabolismo , Humanos , Masculino , Ratones SCID , Simulación del Acoplamiento Molecular , Neoplasias/metabolismo , Neoplasias/patología , Piridazinas/química , Piridazinas/farmacocinética , Piridazinas/uso terapéutico , Tiadiazoles/química , Tiadiazoles/farmacocinética , Tiadiazoles/uso terapéuticoRESUMEN
A high-throughput screen (HTS) of human 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) resulted in several series of compounds with the potential for further optimization. Informatics was used to identify active chemotypes with lead-like profiles and remove compounds that commonly occurred as actives in other HTS screens. The activities were confirmed with IC50 measurements from two orthogonal assay technologies, and further analysis of the Hill slopes and comparison of the ratio of IC50 values at 10 times the enzyme concentration were used to identify artifact compounds. Several series of compounds were rejected as they had both high slopes and poor ratios. A small number of compounds representing the different leading series were assessed using isothermal titration calorimetry, and the X-ray crystal structure of the complex with PFKFB3 was solved. The orthogonal assay technology and isothermal calorimetry were demonstrated to be unreliable in identifying false-positive compounds in this case. Presented here is the discovery of the dihydropyrrolopyrimidinone series of compounds as active and novel inhibitors of PFKFB3, shown by X-ray crystallography to bind to the adenosine triphosphate site. The crystal structures of this series also reveal it is possible to flip the binding mode of the compounds, and the alternative orientation can be driven by a sigma-hole interaction between an aromatic chlorine atom and a backbone carbonyl oxygen. These novel inhibitors will enable studies to explore the role of PFKFB3 in driving the glycolytic phenotype of tumors.
Asunto(s)
Antineoplásicos/farmacología , Ensayos de Selección de Medicamentos Antitumorales/métodos , Inhibidores Enzimáticos/farmacología , Ensayos Analíticos de Alto Rendimiento , Fosfofructoquinasa-2/antagonistas & inhibidores , Antineoplásicos/química , Calorimetría/métodos , Inhibidores Enzimáticos/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Concentración 50 Inhibidora , Ligandos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fosfofructoquinasa-2/química , Fosfofructoquinasa-2/genética , Fosfofructoquinasa-2/metabolismo , Relación Estructura-Actividad Cuantitativa , Bibliotecas de Moléculas Pequeñas , Flujo de TrabajoRESUMEN
MTH1 (NUDT1) is an oncologic target involved in the prevention of DNA damage. We investigate the way MTH1 recognises its substrates and present substrate-bound structures of MTH1 for 8-oxo-dGTP and 8-oxo-rATP as examples of novel strong and weak binding substrate motifs. Investigation of a small set of purine-like fragments using 2D NMR resulted in identification of a fragment with weak potency. The protein-ligand X-Ray structure of this fragment provides insight into the role of water molecules in substrate selectivity. Wider fragment screening by NMR resulted in three new protein structures exhibiting alternative binding configurations to the key Asp-Asp recognition element of the protein. These inhibitor binding modes demonstrate that MTH1 employs an intricate yet promiscuous mechanism of substrate anchoring through its Asp-Asp pharmacophore. The structures suggest that water-mediated interactions convey selectivity towards oxidized substrates over their non-oxidised counterparts, in particular by stabilization of a water molecule in a hydrophobic environment through hydrogen bonding. These findings may be useful in the design of inhibitors of MTH1.