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1.
J Med Chem ; 66(1): 804-821, 2023 01 12.
Artículo en Inglés | MEDLINE | ID: mdl-36516442

RESUMEN

Owing to their central role in regulating cell signaling pathways, the phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are attractive therapeutic targets in diseases such as cancer, neurodegeneration, and immunological disorders. Until now, tool molecules for these kinases have been either limited in potency or isoform selectivity, which has hampered further investigation of biology and drug development. Herein we describe the virtual screening workflow which identified a series of thienylpyrimidines as PI5P4Kγ-selective inhibitors, as well as the medicinal chemistry optimization of this chemotype, to provide potent and selective tool molecules for further use. In vivo pharmacokinetics data are presented for exemplar tool molecules, along with an X-ray structure for ARUK2001607 (15) in complex with PI5P4Kγ, along with its selectivity data against >150 kinases and a Cerep safety panel.


Asunto(s)
Neoplasias , Transducción de Señal , Humanos , Isoformas de Proteínas , Encéfalo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química
2.
J Med Chem ; 65(4): 3359-3370, 2022 02 24.
Artículo en Inglés | MEDLINE | ID: mdl-35148092

RESUMEN

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4Ks) are emerging as attractive therapeutic targets in diseases, such as cancer, immunological disorders, and neurodegeneration, owing to their central role in regulating cell signaling pathways that are either dysfunctional or can be modulated to promote cell survival. Different modes of binding may enhance inhibitor selectivity and reduce off-target effects in cells. Here, we describe efforts to improve the physicochemical properties of the selective PI5P4Kγ inhibitor, NIH-12848 (1). These improvements enabled the demonstration that this chemotype engages PI5P4Kγ in intact cells and that compounds from this series do not inhibit PI5P4Kα or PI5P4Kß. Furthermore, the first X-ray structure of PI5P4Kγ bound to an inhibitor has been determined with this chemotype, confirming an allosteric binding mode. An exemplar from this chemical series adopted two distinct modes of inhibition, including through binding to a putative lipid interaction site which is 18 Å from the ATP pocket.


Asunto(s)
Adenosina Trifosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/síntesis química , Quinazolinas/farmacología , Tiofenos/síntesis química , Tiofenos/farmacología , Regulación Alostérica/efectos de los fármacos , Unión Competitiva , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Especificidad por Sustrato
3.
Cell Chem Biol ; 28(6): 835-847.e5, 2021 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-33662256

RESUMEN

BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.


Asunto(s)
Proteína BRCA2/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Muerte Celular/efectos de los fármacos , Cristalografía por Rayos X , Daño del ADN , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica/efectos de los fármacos , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Células Tumorales Cultivadas
4.
ACS Med Chem Lett ; 11(8): 1539-1547, 2020 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-32832021

RESUMEN

Bifunctional molecules known as PROTACs simultaneously bind an E3 ligase and a protein of interest to direct ubiquitination and clearance of that protein, and they have emerged in the past decade as an exciting new paradigm in drug discovery. In order to investigate the permeability and properties of these large molecules, we synthesized two panels of PROTAC molecules, constructed from a range of protein-target ligands, linkers, and E3 ligase ligands. The androgen receptor, which is a well-studied protein in the PROTAC field was used as a model system. The physicochemical properties and permeability of PROTACs are discussed.

5.
J Mol Biol ; 428(23): 4589-4607, 2016 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-27725183

RESUMEN

Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.


Asunto(s)
Proteína BRCA2/metabolismo , Inhibidores Enzimáticos/aislamiento & purificación , Unión Proteica/efectos de los fármacos , Ingeniería de Proteínas/métodos , Recombinasa Rad51/metabolismo , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas/métodos , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Pyrococcus/enzimología , Recombinasa Rad51/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
FEBS Open Bio ; 6(5): 372-85, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-27419043

RESUMEN

Homologous recombination is essential for repair of DNA double-strand breaks. Central to this process is a family of recombinases, including archeal RadA and human RAD51, which form nucleoprotein filaments on damaged single-stranded DNA ends and facilitate their ATP-dependent repair. ATP binding and hydrolysis are dependent on the formation of a nucleoprotein filament comprising RadA/RAD51 and single-stranded DNA, with ATP bound between adjacent protomers. We demonstrate that truncated, monomeric Pyrococcus furiosus RadA and monomerised human RAD51 retain the ability to bind ATP and other nucleotides with high affinity. We present crystal structures of both apo and nucleotide-bound forms of monomeric RadA. These structures reveal that while phosphate groups are tightly bound, RadA presents a shallow, poorly defined binding surface for the nitrogenous bases of nucleotides. We suggest that RadA monomers would be constitutively bound to nucleotides in the cell and that the bound nucleotide might play a structural role in filament assembly.

7.
Nat Rev Drug Discov ; 15(8): 533-50, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27050677

RESUMEN

Protein-protein interactions (PPIs) are of pivotal importance in the regulation of biological systems and are consequently implicated in the development of disease states. Recent work has begun to show that, with the right tools, certain classes of PPI can yield to the efforts of medicinal chemists to develop inhibitors, and the first PPI inhibitors have reached clinical development. In this Review, we describe the research leading to these breakthroughs and highlight the existence of groups of structurally related PPIs within the PPI target class. For each of these groups, we use examples of successful discovery efforts to illustrate the research strategies that have proved most useful.


Asunto(s)
Descubrimiento de Drogas/tendencias , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteínas/efectos de los fármacos , Animales , Biología Computacional , Humanos , Modelos Moleculares , Estructura Terciaria de Proteína , Proteínas/química , Bibliotecas de Moléculas Pequeñas
8.
FEBS Lett ; 590(8): 1094-102, 2016 04.
Artículo en Inglés | MEDLINE | ID: mdl-26992456

RESUMEN

RAD51 is a recombinase involved in the homologous recombination of double-strand breaks in DNA. RAD51 forms oligomers by binding to another molecule of RAD51 via an 'FxxA' motif, and the same recognition sequence is similarly utilised to bind BRCA2. We have tabulated the effects of mutation of this sequence, across a variety of experimental methods and from relevant mutations observed in the clinic. We use mutants of a tetrapeptide sequence to probe the binding interaction, using both isothermal titration calorimetry and X-ray crystallography. Where possible, comparison between our tetrapeptide mutational study and the previously reported mutations is made, discrepancies are discussed and the importance of secondary structure in interpreting alanine scanning and mutational data of this nature is considered.


Asunto(s)
Proteína BRCA2/metabolismo , Péptidos/metabolismo , Recombinasa Rad51/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia Conservada , Humanos , Modelos Moleculares , Mutación/genética , Unión Proteica , Estructura Secundaria de Proteína , Relación Estructura-Actividad
9.
ChemMedChem ; 10(2): 296-303, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25470112

RESUMEN

The development of small molecules that inhibit protein-protein interactions continues to be a challenge in chemical biology and drug discovery. Herein we report the development of indole-based fragments that bind in a shallow surface pocket of a humanised surrogate of RAD51. RAD51 is an ATP-dependent recombinase that plays a key role in the repair of double-strand DNA breaks. It both self-associates, forming filament structures with DNA, and interacts with the BRCA2 protein through a common "FxxA" tetrapeptide motif. We elaborated previously identified fragment hits that target the FxxA motif site and developed small-molecule inhibitors that are approximately 500-fold more potent than the initial fragments. The lead compounds were shown to compete with the BRCA2-derived Ac-FHTA-NH2 peptide and the self-association peptide of RAD51, but they had no effect on ATP binding. This study is the first reported elaboration of small-molecular-weight fragments against this challenging target.


Asunto(s)
Proteína BRCA2/metabolismo , Recombinasa Rad51/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Secuencias de Aminoácidos , Proteína BRCA2/antagonistas & inhibidores , Sitios de Unión , Diseño de Fármacos , Humanos , Simulación de Dinámica Molecular , Unión Proteica , Estructura Terciaria de Proteína , Pyrococcus furiosus/enzimología , Recombinasa Rad51/antagonistas & inhibidores , Recombinasa Rad51/genética , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-Actividad , Termodinámica
10.
Chembiochem ; 14(3): 332-42, 2013 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-23344974

RESUMEN

The ability to identify inhibitors of protein-protein interactions represents a major challenge in modern drug discovery and in the development of tools for chemical biology. In recent years, fragment-based approaches have emerged as a new methodology in drug discovery; however, few examples of small molecules that are active against chemotherapeutic targets have been published. Herein, we describe the fragment-based approach of targeting the interaction between the tumour suppressor BRCA2 and the recombination enzyme RAD51; it makes use of a screening pipeline of biophysical techniques that we expect to be more generally applicable to similar targets. Disruption of this interaction in vivo is hypothesised to give rise to cellular hypersensitivity to radiation and genotoxic drugs. We have used protein engineering to create a monomeric form of RAD51 by humanising a thermostable archaeal orthologue, RadA, and used this protein for fragment screening. The initial fragment hits were thoroughly validated biophysically by isothermal titration calorimetry (ITC) and NMR techniques and observed by X-ray crystallography to bind in a shallow surface pocket that is occupied in the native complex by the side chain of a phenylalanine from the conserved FxxA interaction motif found in BRCA2. This represents the first report of fragments or any small molecule binding at this protein-protein interaction site.


Asunto(s)
Proteína BRCA2/metabolismo , Mapas de Interacción de Proteínas , Recombinasa Rad51/metabolismo , Archaea/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Proteína BRCA2/química , Sitios de Unión , Calorimetría , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Recombinasa Rad51/química , Recombinasa Rad51/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo
11.
Biochemistry ; 51(25): 4990-5003, 2012 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-22697260

RESUMEN

Fragment-based approaches to finding novel small molecules that bind to proteins are now firmly established in drug discovery and chemical biology. Initially developed primarily in a few centers in the biotech and pharma industry, this methodology has now been adopted widely in both the pharmaceutical industry and academia. After the initial success with kinase targets, the versatility of this approach has now expanded to a broad range of different protein classes. Herein we describe recent fragment-based approaches to a wide range of target types, including Hsp90, ß-secretase, and allosteric sites in human immunodeficiency virus protease and fanesyl pyrophosphate synthase. The role of fragment-based approaches in an academic research environment is also examined with an emphasis on neglected diseases such as tuberculosis. The development of a fragment library, the fragment screening process, and the subsequent fragment hit elaboration will be discussed using examples from the literature.


Asunto(s)
Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Fragmentos de Péptidos/síntesis química , Cristalografía por Rayos X , Descubrimiento de Drogas/tendencias , Ensayos Analíticos de Alto Rendimiento/tendencias , Humanos , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/fisiología , Unión Proteica/fisiología , Bibliotecas de Moléculas Pequeñas/síntesis química
12.
Curr Opin Chem Biol ; 14(3): 299-307, 2010 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-20223699

RESUMEN

Fragment-based approaches have now become firmly established in the drug discovery armoury. After notable early successes against protein kinases, the versatility and power of fragment-based approaches are increasingly being demonstrated on more diverse and difficult protein targets. This review highlights seven examples including targeting protein-protein interactions, a RNA polymerase and a DNA-binding protein. It shows how fragment-based approaches using small libraries have been successful when large HTS screens have failed. It also highlights the range of biophysical approaches being used and the interplay between experimental and in silico screens. The examples all show the iterative way in which potency is built up by synthetic elaboration of the initial fragment hits.


Asunto(s)
Descubrimiento de Drogas/métodos , Mapeo de Interacción de Proteínas/métodos , Simulación por Computador , Proteínas de Unión al ADN , ARN Polimerasas Dirigidas por ADN , Humanos , Bibliotecas de Moléculas Pequeñas
13.
Chembiochem ; 10(17): 2772-9, 2009 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-19827080

RESUMEN

A new strategy that combines the concepts of fragment-based drug design and dynamic combinatorial chemistry (DCC) for targeting adenosine recognition sites on enzymes is reported. We demonstrate the use of 5'-deoxy-5'-thioadenosine as a noncovalent anchor fragment in dynamic combinatorial libraries templated by Mycobacterium tuberculosis pantothenate synthetase. A benzyl disulfide derivative was identified upon library analysis by HPLC. Structural and binding studies of protein-ligand complexes by X-ray crystallography and isothermal titration calorimetry informed the subsequent optimisation of the DCC hit into a disulfide containing the novel meta-nitrobenzyl fragment that targets the pantoate binding site of pantothenate synthetase. Given the prevalence of adenosine-recognition motifs in enzymes, our results provide a proof-of-concept for using this strategy to probe adjacent pockets for a range of adenosine binding enzymes, including other related adenylate-forming ligases, kinases, and ATPases, as well as NAD(P)(H), CoA and FAD(H2) binding proteins.


Asunto(s)
Adenosina/análogos & derivados , Técnicas Químicas Combinatorias/métodos , Diseño de Fármacos , Tionucleósidos/química , Adenosina/síntesis química , Adenosina/química , Cristalografía por Rayos X , Disulfuros/química , Datos de Secuencia Molecular , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Péptido Sintasas/metabolismo , Conformación Proteica , Tionucleósidos/síntesis química
15.
Nat Prod Rep ; 24(5): 1009-26, 2007 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-17898895

RESUMEN

This review highlights five key reactions in vitamin biosynthesis and in particular focuses on their mechanisms and inhibition and insights from structural studies. Each of the enzymes has the potential to be a target for novel antimicrobial agents.


Asunto(s)
Antiinfecciosos , Coenzimas , Enzimas , Vitaminas , Coenzimas/biosíntesis , Coenzimas/química , Coenzimas/metabolismo , Enzimas/efectos de los fármacos , Enzimas/metabolismo , Estructura Molecular , Vitaminas/biosíntesis , Vitaminas/química , Vitaminas/metabolismo
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