RESUMO
Recent literature has both suggested and questioned MTH1 as a novel cancer target. BAY-707 was just published as a target validation small molecule probe for assessing the effects of pharmacological inhibition of MTH1 on tumor cell survival, both in vitro and in vivo. (1) In this report, we describe the medicinal chemistry program creating BAY-707, where fragment-based methods were used to develop a series of highly potent and selective MTH1 inhibitors. Using structure-based drug design and rational medicinal chemistry approaches, the potency was increased over 10,000 times from the fragment starting point while maintaining high ligand efficiency and drug-like properties.
Assuntos
Antineoplásicos/farmacologia , Enzimas Reparadoras do DNA/antagonistas & inibidores , Morfolinas/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Animais , Antineoplásicos/química , Antineoplásicos/farmacocinética , Células CACO-2 , Permeabilidade da Membrana Celular , Desenho de Fármacos , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Hepatócitos/metabolismo , Humanos , Camundongos , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Morfolinas/química , Morfolinas/farmacocinética , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Nucleotide methylations are the most common type of rRNA modification in bacteria, and are introduced post-transcriptionally by a wide variety of site-specific enzymes. Three 5-methylcytidine (m(5)C) bases are found in the rRNAs of Escherichia coli and one of these, at nucleotide 1407 in 16 S rRNA, is the modification product of the methyltransferase (MTase) YebU (also called RsmF). YebU requires S-adenosyl-l-methionine (SAM) and methylates C1407 within assembled 30 S subunits, but not in naked 16 S rRNA or within tight-couple 70 S ribosomes. Here, we describe the three-dimensional structure of YebU determined by X-ray crystallography, and we present a molecular model for how YebU specifically recognizes, binds and methylates its ribosomal substrate. The YebU protein has an N-terminal SAM-binding catalytic domain with structural similarity to the equivalent domains in several other m(5)C RNA MTases including RsmB and PH1374. The C-terminal one-third of YebU contains a domain similar to that in pseudouridine synthases and archaeosine-specific transglycosylases (PUA-domain), which was not predicted by sequence alignments. Furthermore, YebU is predicted to contain extended regions of positive electrostatic potential that differ from other RNA-MTase structures, suggesting that YebU interacts with its RNA target in a different manner. Docking of YebU onto the 30 S subunit indicates that the PUA and MTase domains make several contacts with 16 S rRNA as well as with the ribosomal protein S12. The ribosomal protein interactions would explain why the assembled 30 S subunit, and not naked 16 S rRNA, is the preferred substrate for YebU.
Assuntos
Escherichia coli/enzimologia , Metiltransferases/química , RNA Ribossômico/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Propriedades de SuperfícieRESUMO
MTH1 is a hydrolase responsible for sanitization of oxidized purine nucleoside triphosphates to prevent their incorporation into replicating DNA. Early tool compounds published in the literature inhibited the enzymatic activity of MTH1 and subsequently induced cancer cell death; however recent studies have questioned the reported link between these two events. Therefore, it is important to validate MTH1 as a cancer dependency with high quality chemical probes. Here, we present BAY-707, a substrate-competitive, highly potent and selective inhibitor of MTH1, chemically distinct compared to those previously published. Despite superior cellular target engagement and pharmacokinetic properties, inhibition of MTH1 with BAY-707 resulted in a clear lack of in vitro or in vivo anticancer efficacy either in mono- or in combination therapies. Therefore, we conclude that MTH1 is dispensable for cancer cell survival.
Assuntos
Enzimas Reparadoras do DNA/metabolismo , Sistemas de Liberação de Medicamentos , Morfolinas/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Células CACO-2 , Células Cultivadas , Enzimas Reparadoras do DNA/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Células HeLa , Hepatócitos/efeitos dos fármacos , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Morfolinas/química , Neoplasias/fisiopatologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , RatosRESUMO
Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA. The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life. We report here the crystal structure of Escherichia coli TruD at 2.0 A resolution. The structure reveals an overall V-shaped molecule with an RNA-binding cleft formed between two domains: a catalytic domain and an insertion domain. The catalytic domain has a fold similar to that of the catalytic domains of previously characterised pseudouridine synthases, whereas the insertion domain displays a novel fold.
Assuntos
Proteínas de Escherichia coli/química , Transferases Intramoleculares/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Elétrons , Escherichia coli/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA de Transferência/química , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de AminoácidosRESUMO
Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.
Assuntos
Proteínas/química , Soluções Tampão , Cristalização , Modelos Moleculares , Conformação Proteica , Temperatura , TermodinâmicaRESUMO
Proteins that bind transition metals make up a substantial portion of the proteome, and the identification of a metal cofactor in a protein can greatly facilitate its functional assignment and help place it in the context of known cellular pathways. Existing methods for the detection of metalloproteins generally consume large amounts of protein, require expensive equipment, or are very labor intensive, rendering them unsuitable for use in high throughput proteomic initiatives. Here we present a method for the identification of metalloproteins that contain iron, copper, manganese, cobalt, nickel, and/or zinc that is sensitive, quick, robust, inexpensive, and can be performed with standard laboratory equipment. The assay is based on a combination of chemiluminescence and colorimetric detection methods, it typically consumes only 10 microg of protein, and most common chemical components of protein solutions do not interfere with metal detection. Analysis of 52 protein samples was compared with the results from inductively coupled plasma-atomic emission spectrometry to verify the accuracy and sensitivity of the method. The assay is conducted in a 384-well format and requires about 3 h for completion, including a 2-h wait; so whole proteomes can be assayed for metal content in a matter of days.
Assuntos
Metaloproteínas/análise , Metais/análise , Proteoma , Colorimetria , Medições Luminescentes , Espectrofotometria AtômicaRESUMO
Pseudouridine, the 5-ribosyl isomer of uridine, is the most common modification of structural RNA. The recently identified pseudouridine synthase TruD belongs to a widespread class of pseudouridine synthases without significant sequence homology to previously known families. TruD from Escherichia coli was overexpressed, purified and crystallized. The crystals diffract to a minimum Bragg spacing of 2.4 A and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 63.4, b = 108.6, c = 111.7 A.