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1.
ACS Med Chem Lett ; 15(7): 1151-1158, 2024 Jul 11.
Artículo en Inglés | MEDLINE | ID: mdl-39015284

RESUMEN

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.

2.
J Med Chem ; 66(4): 2918-2945, 2023 02 23.
Artículo en Inglés | MEDLINE | ID: mdl-36727211

RESUMEN

Herein, we report the optimization of a meta-substituted series of selective estrogen receptor degrader (SERD) antagonists for the treatment of ER+ breast cancer. Structure-based design together with the use of modeling and NMR to favor the bioactive conformation led to a highly potent series of basic SERDs with promising physicochemical properties. Issues with hERG activity resulted in a strategy of zwitterion formation and ultimately in the identification of 38. This compound was shown to be a highly potent SERD capable of effectively degrading ERα in both MCF-7 and CAMA-1 cell lines. The low lipophilicity and zwitterionic nature led to a SERD with a clean secondary pharmacology profile and no hERG activity. Favorable physicochemical properties resulted in good oral bioavailability in preclinical species and potent in vivo activity in a mouse xenograft model.


Asunto(s)
Neoplasias de la Mama , Receptores de Estrógenos , Ratones , Humanos , Animales , Femenino , Receptores de Estrógenos/metabolismo , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Antagonistas de Estrógenos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Receptor alfa de Estrógeno/metabolismo , Línea Celular
3.
J Med Chem ; 64(6): 3165-3184, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33683117

RESUMEN

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/metabolismo
4.
Biochem J ; 477(22): 4443-4452, 2020 11 27.
Artículo en Inglés | MEDLINE | ID: mdl-33119085

RESUMEN

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 Proteína
5.
Prog Biophys Mol Biol ; 147: 4-16, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31255703

RESUMEN

ATM, ATR and DNA-PKCs are key effectors of DNA Damage response and have been extensively linked to tumourigenesis and survival of cancer cells after radio/chemotherapy. Despite numerous efforts, the structures of these proteins remained elusive until very recently. The resolution revolution in Cryo-EM allowed for molecular details of these proteins to be seen for the first time. Here we provide a comprehensive review of the structures of ATM, ATR and DNA-PKcs and their complexes and expand with observations springing from our own cryo-EM studies. These observations include a novel conformation of ATR and novel dimeric arrangements of DNA-PKcs.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Daño del ADN , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Humanos
6.
Sci Adv ; 3(5): e1700933, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28508083

RESUMEN

ATM (ataxia-telangiectasia mutated) is a phosphatidylinositol 3-kinase-related protein kinase (PIKK) best known for its role in DNA damage response. ATM also functions in oxidative stress response, insulin signaling, and neurogenesis. Our electron cryomicroscopy (cryo-EM) suggests that human ATM is in a dynamic equilibrium between closed and open dimers. In the closed state, the PIKK regulatory domain blocks the peptide substrate-binding site, suggesting that this conformation may represent an inactive or basally active enzyme. The active site is held in this closed conformation by interaction with a long helical hairpin in the TRD3 (tetratricopeptide repeats domain 3) domain of the symmetry-related molecule. The open dimer has two protomers with only a limited contact interface, and it lacks the intermolecular interactions that block the peptide-binding site in the closed dimer. This suggests that the open conformation may be more active. The ATM structure shows the detailed topology of the regulator-interacting N-terminal helical solenoid. The ATM conformational dynamics shown by the structures represent an important step in understanding the enzyme regulation.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/química , Multimerización de Proteína , Microscopía por Crioelectrón , Humanos , Dominios Proteicos , Estructura Cuaternaria de Proteína
7.
Anal Biochem ; 503: 58-64, 2016 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-27036617

RESUMEN

Poly(ADP-ribose) (PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed and, from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition.


Asunto(s)
Fluorescencia , Glicósido Hidrolasas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Mediciones Luminiscentes/métodos , Línea Celular , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/análisis , Glicósido Hidrolasas/antagonistas & inhibidores , Humanos , Relación Estructura-Actividad , Factores de Tiempo
8.
Protein Sci ; 23(5): 627-38, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24677421

RESUMEN

The EphB receptors have key roles in cell morphology, adhesion, migration and invasion, and their aberrant action has been linked with the development and progression of many different tumor types. Their conflicting expression patterns in cancer tissues, combined with their high sequence and structural identity, present interesting challenges to those seeking to develop selective therapeutic molecules targeting this large receptor family. Here, we present the first structure of the EphB1 tyrosine kinase domain determined by X-ray crystallography to 2.5Å. Our comparative crystalisation analysis of the human EphB family kinases has also yielded new crystal forms of the human EphB2 and EphB4 catalytic domains. Unable to crystallize the wild-type EphB3 kinase domain, we used rational engineering (based on our new structures of EphB1, EphB2, and EphB4) to identify a single point mutation which facilitated its crystallization and structure determination to 2.2 Å. This mutation also improved the soluble recombinant yield of this kinase within Escherichia coli, and increased both its intrinsic stability and catalytic turnover, without affecting its ligand-binding profile. The partial ordering of the activation loop in the EphB3 structure alludes to a potential cis-phosphorylation mechanism for the EphB kinases. With the kinase domain structures of all four catalytically competent human EphB receptors now determined, a picture begins to emerge of possible opportunities to produce EphB isozyme-selective kinase inhibitors for mechanistic studies and therapeutic applications.


Asunto(s)
Receptor EphB1/química , Receptor EphB2/química , Receptor EphB4/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutagénesis , Conformación Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Receptor EphB3/química , Receptor EphB3/genética
9.
Biosci Rep ; 33(3)2013 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-23627399

RESUMEN

The Eph (erythropoietin-producing hepatocellular carcinoma) B receptors are important in a variety of cellular processes through their roles in cell-to-cell contact and signalling; their up-regulation and down-regulation has been shown to have implications in a variety of cancers. A greater understanding of the similarities and differences within this small, highly conserved family of tyrosine kinases will be essential to the identification of effective therapeutic opportunities for disease intervention. In this study, we have developed a route to production of multi-milligram quantities of highly purified, homogeneous, recombinant protein for the kinase domain of these human receptors in Escherichia coli. Analyses of these isolated catalytic fragments have revealed stark contrasts in their amenability to recombinant expression and their physical properties: e.g., a >16°C variance in thermal stability, a 3-fold difference in catalytic activity and disparities in their inhibitor binding profiles. We find EphB3 to be an outlier in terms of both its intrinsic stability, and more importantly its ligand-binding properties. Our findings have led us to speculate about both their biological significance and potential routes for generating EphB isozyme-selective small-molecule inhibitors. Our comprehensive methodologies provide a template for similar in-depth studies of other kinase superfamily members.


Asunto(s)
Receptores de la Familia Eph/química , Receptores de la Familia Eph/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica
10.
PLoS One ; 7(12): e50889, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-23251397

RESUMEN

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5'-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.


Asunto(s)
Dominio Catalítico , Glicósido Hidrolasas/química , Biología Computacional , Humanos , Conformación Proteica , Relación Estructura-Actividad
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