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1.
Mol Cell ; 83(5): 698-714.e4, 2023 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-36724784

RESUMO

Non-homologous end joining is the major double-strand break repair (DSBR) pathway in mammals. DNA-PK is the hub and organizer of multiple steps in non-homologous end joining (NHEJ). Recent high-resolution structures show how two distinct NHEJ complexes "synapse" two DNA ends. One complex includes a DNA-PK dimer mediated by XLF, whereas a distinct DNA-PK dimer forms via a domain-swap mechanism where the C terminus of Ku80 from one DNA-PK protomer interacts with another DNA-PK protomer in trans. Remarkably, the distance between the two synapsed DNA ends in both dimers is the same (∼115 Å), which matches the distance observed in the initial description of an NHEJ long-range synaptic complex. Here, a mutational strategy is used to demonstrate distinct cellular function(s) of the two dimers: one promoting fill-in end processing, while the other promotes DNA end resection. Thus, the specific DNA-PK dimer formed (which may be impacted by DNA end structure) dictates the mechanism by which ends will be made ligatable.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA , Animais , Proteínas de Ligação a DNA/genética , Subunidades Proteicas/metabolismo , Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA/genética , Proteína Quinase Ativada por DNA/genética , Autoantígeno Ku/genética , Mamíferos/metabolismo
2.
Mol Cell ; 81(16): 3400-3409.e3, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352203

RESUMO

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.


Assuntos
DNA Ligase Dependente de ATP/ultraestrutura , Enzimas Reparadoras do DNA/ultraestrutura , Proteína Quinase Ativada por DNA/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Apoptose/genética , Microscopia Crioeletrônica , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/ultraestrutura , Complexos Multiproteicos/genética , Fosforilação/genética
3.
Nature ; 601(7894): 643-648, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34987222

RESUMO

The DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has a central role in non-homologous end joining, one of the two main pathways that detect and repair DNA double-strand breaks (DSBs) in humans1,2. DNA-PKcs is of great importance in repairing pathological DSBs, making DNA-PKcs inhibitors attractive therapeutic agents for cancer in combination with DSB-inducing radiotherapy and chemotherapy3. Many of the selective inhibitors of DNA-PKcs that have been developed exhibit potential as treatment for various cancers4. Here we report cryo-electron microscopy (cryo-EM) structures of human DNA-PKcs natively purified from HeLa cell nuclear extracts, in complex with adenosine-5'-(γ-thio)-triphosphate (ATPγS) and four inhibitors (wortmannin, NU7441, AZD7648 and M3814), including drug candidates undergoing clinical trials. The structures reveal molecular details of ATP binding at the active site before catalysis and provide insights into the modes of action and specificities of the competitive inhibitors. Of note, binding of the ligands causes movement of the PIKK regulatory domain (PRD), revealing a connection between the p-loop and PRD conformations. Electrophoretic mobility shift assay and cryo-EM studies on the DNA-dependent protein kinase holoenzyme further show that ligand binding does not have a negative allosteric or inhibitory effect on assembly of the holoenzyme complex and that inhibitors function through direct competition with ATP. Overall, the structures described in this study should greatly assist future efforts in rational drug design targeting DNA-PKcs, demonstrating the potential of cryo-EM in structure-guided drug development for large and challenging targets.


Assuntos
Reparo do DNA por Junção de Extremidades , Proteína Quinase Ativada por DNA , Trifosfato de Adenosina , Domínio Catalítico , Microscopia Crioeletrônica , DNA/metabolismo , Reparo do DNA , Proteína Quinase Ativada por DNA/metabolismo , Células HeLa , Holoenzimas/metabolismo , Humanos , Autoantígeno Ku/metabolismo , Piridazinas , Quinazolinas
4.
Nat Methods ; 21(7): 1340-1348, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38918604

RESUMO

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein-nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.


Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Microscopia Crioeletrônica/métodos , Ligantes , SARS-CoV-2 , COVID-19/virologia , Escherichia coli , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Conformação Proteica , Reprodutibilidade dos Testes
5.
Proc Natl Acad Sci U S A ; 121(17): e2403206121, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38630725

RESUMO

Mycobacterium abscessus is increasingly recognized as the causative agent of chronic pulmonary infections in humans. One of the genes found to be under strong evolutionary pressure during adaptation of M. abscessus to the human lung is embC which encodes an arabinosyltransferase required for the biosynthesis of the cell envelope lipoglycan, lipoarabinomannan (LAM). To assess the impact of patient-derived embC mutations on the physiology and virulence of M. abscessus, mutations were introduced in the isogenic background of M. abscessus ATCC 19977 and the resulting strains probed for phenotypic changes in a variety of in vitro and host cell-based assays relevant to infection. We show that patient-derived mutational variations in EmbC result in an unexpectedly large number of changes in the physiology of M. abscessus, and its interactions with innate immune cells. Not only did the mutants produce previously unknown forms of LAM with a truncated arabinan domain and 3-linked oligomannoside chains, they also displayed significantly altered cording, sliding motility, and biofilm-forming capacities. The mutants further differed from wild-type M. abscessus in their ability to replicate and induce inflammatory responses in human monocyte-derived macrophages and epithelial cells. The fact that different embC mutations were associated with distinct physiologic and pathogenic outcomes indicates that structural alterations in LAM caused by nonsynonymous nucleotide polymorphisms in embC may be a rapid, one-step, way for M. abscessus to generate broad-spectrum diversity beneficial to survival within the heterogeneous and constantly evolving environment of the infected human airway.


Assuntos
Mycobacterium abscessus , Humanos , Proteínas de Bactérias/genética , Lipopolissacarídeos/química , Mutação
6.
Bioinformatics ; 2024 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-39037932

RESUMO

SUMMARY: Analysing protein structure similarities is an important step in protein engineering and drug discovery. Methodologies that are more advanced than simple RMSD are available but often require extensive mathematical or computational knowledge for implementation. Grouping and optimising such tools in an efficient open-source library increases accessibility and encourages the adoption of more advanced metrics. Melodia is a Python library with a complete set of components devised for describing, comparing and analysing the shape of protein structures using differential geometry of three-dimensional curves and knot theory. It can generate robust geometric descriptors for thousands of shapes in just a few minutes. Those descriptors are more sensitive to structural feature variation than RMSD deviation. Melodia also incorporates sequence structural annotation and three-dimensional visualisations. AVAILABILITY AND IMPLEMENTATION: Melodia is an open-source Python library freely available on https://github.com/rwmontalvao/Melodia_py, along with interactive Jupyter Notebook tutorials. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

7.
Nucleic Acids Res ; 51(21): 11732-11747, 2023 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-37870477

RESUMO

The classical Non-Homologous End Joining (c-NHEJ) pathway is the predominant process in mammals for repairing endogenous, accidental or programmed DNA Double-Strand Breaks. c-NHEJ is regulated by several accessory factors, post-translational modifications, endogenous chemical agents and metabolites. The metabolite inositol-hexaphosphate (IP6) stimulates c-NHEJ by interacting with the Ku70-Ku80 heterodimer (Ku). We report cryo-EM structures of apo- and DNA-bound Ku in complex with IP6, at 3.5 Å and 2.74 Å resolutions respectively, and an X-ray crystallography structure of a Ku in complex with DNA and IP6 at 3.7 Å. The Ku-IP6 interaction is mediated predominantly via salt bridges at the interface of the Ku70 and Ku80 subunits. This interaction is distant from the DNA, DNA-PKcs, APLF and PAXX binding sites and in close proximity to XLF binding site. Biophysical experiments show that IP6 binding increases the thermal stability of Ku by 2°C in a DNA-dependent manner, stabilizes Ku on DNA and enhances XLF affinity for Ku. In cells, selected mutagenesis of the IP6 binding pocket reduces both Ku accrual at damaged sites and XLF enrolment in the NHEJ complex, which translate into a lower end-joining efficiency. Thus, this study defines the molecular bases of the IP6 metabolite stimulatory effect on the c-NHEJ repair activity.


Assuntos
Proteínas de Ligação a DNA , Ácido Fítico , Animais , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA/genética , Autoantígeno Ku/metabolismo , Mamíferos/genética , Humanos
8.
Proteins ; 2024 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-38483037

RESUMO

The number of antibiotic resistant pathogens is increasing rapidly, and with this comes a substantial socioeconomic cost that threatens much of the world. To alleviate this problem, we must use antibiotics in a more responsible and informed way, further our understanding of the molecular basis of drug resistance, and design new antibiotics. Here, we focus on a key drug-resistant pathogen, Mycobacterium tuberculosis, and computationally analyze trends in drug-resistant mutations in genes of the proteins embA, embB, embC, and katG, which play essential roles in the action of the first-line drugs ethambutol and isoniazid. We use docking to predict binding modes of isoniazid to katG that agree with suggested binding sites found in our laboratory using cryo-EM. Using mutant stability predictions, we recapitulate the idea that resistance occurs when katG's heme cofactor is destabilized rather than due to a decrease in affinity to isoniazid. Conversely, we have identified resistance mutations that affect the affinity of ethambutol more drastically than the affinity of the natural substrate of embB. With this, we illustrate that we can distinguish between the two types of drug resistance-cofactor destabilization and drug affinity reduction-suggesting potential uses in the prediction of novel drug-resistant mutations.

9.
Brief Bioinform ; 23(4)2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35656714

RESUMO

Proteins are capable of highly specific interactions and are responsible for a wide range of functions, making them attractive in the pursuit of new therapeutic options. Previous studies focusing on overall geometry of protein-protein interfaces, however, concluded that PPI interfaces were generally flat. More recently, this idea has been challenged by their structural and thermodynamic characterisation, suggesting the existence of concave binding sites that are closer in character to traditional small-molecule binding sites, rather than exhibiting complete flatness. Here, we present a large-scale analysis of binding geometry and physicochemical properties of all protein-protein interfaces available in the Protein Data Bank. In this review, we provide a comprehensive overview of the protein-protein interface landscape, including evidence that even for overall larger, more flat interfaces that utilize discontinuous interacting regions, small and potentially druggable pockets are utilized at binding sites.


Assuntos
Proteínas , Sítios de Ligação , Bases de Dados de Proteínas , Ligação Proteica , Proteínas/química
10.
Brief Bioinform ; 23(3)2022 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-35229157

RESUMO

SARS-CoV-2 is a novel positive-sense single-stranded RNA virus from the Coronaviridae family (genus Betacoronavirus), which has been established as causing the COVID-19 pandemic. The genome of SARS-CoV-2 is one of the largest among known RNA viruses, comprising of at least 26 known protein-coding loci. Studies thus far have outlined the coding capacity of the positive-sense strand of the SARS-CoV-2 genome, which can be used directly for protein translation. However, it has been recently shown that transcribed negative-sense viral RNA intermediates that arise during viral genome replication from positive-sense viruses can also code for proteins. No studies have yet explored the potential for negative-sense SARS-CoV-2 RNA intermediates to contain protein-coding loci. Thus, using sequence and structure-based bioinformatics methodologies, we have investigated the presence and validity of putative negative-sense ORFs (nsORFs) in the SARS-CoV-2 genome. Nine nsORFs were discovered to contain strong eukaryotic translation initiation signals and high codon adaptability scores, and several of the nsORFs were predicted to interact with RNA-binding proteins. Evolutionary conservation analyses indicated that some of the nsORFs are deeply conserved among related coronaviruses. Three-dimensional protein modeling revealed the presence of higher order folding among all putative SARS-CoV-2 nsORFs, and subsequent structural mimicry analyses suggest similarity of the nsORFs to DNA/RNA-binding proteins and proteins involved in immune signaling pathways. Altogether, these results suggest the potential existence of still undescribed SARS-CoV-2 proteins, which may play an important role in the viral lifecycle and COVID-19 pathogenesis.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/genética , Genoma Viral , Humanos , Pandemias , RNA Viral/química , RNA Viral/genética , Proteínas de Ligação a RNA/genética , SARS-CoV-2/genética
11.
Mol Cell ; 61(3): 434-448, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26778126

RESUMO

BRCA1 accumulation at DNA damage sites is an important step for its function in the DNA damage response and in DNA repair. BRCA1-BRCT domains bind to proteins containing the phosphorylated serine-proline-x-phenylalanine (pSPxF) motif including Abraxas, Bach1/FancJ, and CtIP. In this study, we demonstrate that ionizing radiation (IR)-induces ATM-dependent phosphorylation of serine 404 (S404) next to the pSPxF motif. Crystal structures of BRCT/Abraxas show that phosphorylation of S404 is important for extensive interactions through the N-terminal sequence outside the pSPxF motif and leads to formation of a stable dimer. Mutation of S404 leads to deficiency in BRCA1 accumulation at DNA damage sites and cellular sensitivity to IR. In addition, two germline mutations of BRCA1 are found to disrupt the dimer interface and dimer formation. Thus, we demonstrate a mechanism involving IR-induced phosphorylation and dimerization of the BRCT/Abraxas complex for regulating Abraxas-mediated recruitment of BRCA1 in response to IR.


Assuntos
Proteína BRCA1/metabolismo , Neoplasias Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Núcleo Celular/metabolismo , Dano ao DNA , Osteossarcoma/metabolismo , Sequência de Aminoácidos , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/química , Proteína BRCA1/genética , Neoplasias Ósseas/genética , Neoplasias Ósseas/patologia , Proteínas de Transporte/química , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Núcleo Celular/patologia , Núcleo Celular/efeitos da radiação , Cristalografia por Raios X , Mutação em Linhagem Germinativa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Osteossarcoma/genética , Osteossarcoma/patologia , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Interferência de RNA , Serina , Transfecção
12.
Nucleic Acids Res ; 50(19): 11058-11071, 2022 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-36263813

RESUMO

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that 'maximizes the fidelity of DNA repair'. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH - thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).


Assuntos
Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA/genética , Quebras de DNA de Cadeia Dupla , DNA Ligase Dependente de ATP/genética , DNA Ligases/genética , DNA Ligases/metabolismo , Biocatálise
13.
Angew Chem Int Ed Engl ; 63(37): e202406846, 2024 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-38896426

RESUMO

Ligand binding hotspots are regions of protein surfaces that form particularly favourable interactions with small molecule pharmacophores. Targeting interactions with these hotspots maximises the efficiency of ligand binding. Existing methods are capable of identifying hotspots but often lack assays to quantify ligand binding and direct elaboration at these sites. Herein, we describe a fragment-based competitive 19F Ligand Based NMR (LB-NMR) screening platform that enables routine, quantitative ligand profiling focused at ligand-binding hotspots. As a proof of concept, the method was applied to 4'-phosphopantetheine adenylyltransferase (PPAT) from Mycobacterium abscessus (Mabs). X-ray crystallographic characterisation of the hits from a 960-member fragment screen identified three ligand-binding hotspots across the PPAT active site. From the fragment hits a collection of 19F reporter candidates were designed and synthesised. By rigorous prioritisation and use of optimisation workflows, a single 19F reporter molecule was generated for each hotspot. Profiling the binding of a set of structurally characterised ligands by competitive 19F LB-NMR with this suite of 19F reporters recapitulated the binding affinity and site ID assignments made by ITC and X-ray crystallography. This quantitative mapping of ligand binding events at hotspot level resolution establishes the utility of the fragment-based competitive 19F LB-NMR screening platform for hotspot-directed ligand profiling.


Assuntos
Bibliotecas de Moléculas Pequenas , Ligantes , Cristalografia por Raios X , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Molecular , Flúor/química , Espectroscopia de Ressonância Magnética/métodos
14.
Biochem Biophys Res Commun ; 666: 61-67, 2023 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-37178506

RESUMO

The RGD motif on the SARS-CoV-2 spike protein has been suggested to interact with RGD-binding integrins αVß3 and α5ß1 to enhance viral cell entry and alter downstream signaling cascades. The D405N mutation on the Omicron subvariant spike proteins, resulting in an RGN motif, has recently been shown to inhibit binding to integrin αVß3. Deamidation of asparagines in protein ligand RGN motifs has been demonstrated to generate RGD and RGisoD motifs that permit binding to RGD-binding integrins. Two asparagines, N481 and N501, on the Wild-type spike receptor-binding domain have been previously shown to have deamidation half-lives of 16.5 and 123 days, respectively, which may occur during the viral life cycle. Deamidation of Omicron subvariant N405 may recover the ability to interact with RGD-binding integrins. Thus, herein, all-atom molecular dynamics simulations of the Wild-type and Omicron subvariant spike protein receptor-binding domains were conducted to investigate the potential for asparagines, the Omicron subvariant N405 in particular, to assume the optimized geometry for deamidation to occur. In summary, the Omicron subvariant N405 was primarily found to be stabilized in a state unfavourable for deamidation after hydrogen bonding with downstream E406. Nevertheless, a small number of RGD or RGisoD motifs on the Omicron subvariant spike proteins may restore the ability to interact with RGD-binding integrins. The simulations also provided structural clarification regarding the deamidation rates of Wild-type N481 and N501 and highlighted the utility of tertiary structure dynamics information in predicting asparagine deamidation. Further work is needed to characterize the effects of deamidation on spike-integrin interactions.


Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Asparagina , Integrina alfaVbeta3
15.
Brief Bioinform ; 22(6)2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34015821

RESUMO

The rapid developments in gene sequencing technologies achieved in the recent decades, along with the expansion of knowledge on the three-dimensional structures of proteins, have enabled the construction of proteome-scale databases of protein models such as the Genome3D and ModBase. Nevertheless, although gene products are usually expressed as individual polypeptide chains, most biological processes are associated with either transient or stable oligomerisation. In the PDB databank, for example, ~40% of the deposited structures contain at least one homo-oligomeric interface. Unfortunately, databases of protein models are generally devoid of multimeric structures. To tackle this particular issue, we have developed ProtCHOIR, a tool that is able to generate homo-oligomeric structures in an automated fashion, providing detailed information for the input protein and output complex. ProtCHOIR requires input of either a sequence or a protomeric structure that is queried against a pre-constructed local database of homo-oligomeric structures, then extensively analyzed using well-established tools such as PSI-Blast, MAFFT, PISA and Molprobity. Finally, MODELLER is employed to achieve the construction of the homo-oligomers. The output complex is thoroughly analyzed taking into account its stereochemical quality, interfacial stabilities, hydrophobicity and conservation profile. All these data are then summarized in a user-friendly HTML report that can be saved or printed as a PDF file. The software is easily parallelizable and also outputs a comma-separated file with summary statistics that can straightforwardly be concatenated as a spreadsheet-like document for large-scale data analyses. As a proof-of-concept, we built oligomeric models for the Mabellini Mycobacterium abscessus structural proteome database. ProtCHOIR can be run as a web-service and the code can be obtained free-of-charge at http://lmdm.biof.ufrj.br/protchoir.


Assuntos
Biologia Computacional/métodos , Modelos Moleculares , Multimerização Proteica , Proteoma , Proteômica/métodos , Software , Algoritmos , Bases de Dados de Proteínas , Regiões Promotoras Genéticas , Ligação Proteica , Conformação Proteica , Reprodutibilidade dos Testes , Interface Usuário-Computador
16.
Brief Bioinform ; 22(6)2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34137435

RESUMO

Mutations in hallmark genes are believed to be the main drivers of cancer progression. These mutations are reported in the Catalogue of Somatic Mutations in Cancer (COSMIC). Structural appreciation of where these mutations appear, in protein-protein interfaces, active sites or deoxyribonucleic acid (DNA) interfaces, and predicting the impacts of these mutations using a variety of computational tools are crucial for successful drug discovery and development. Currently, there are 723 genes presented in the COSMIC Cancer Gene Census. Due to the complexity of the gene products, structures of only 87 genes have been solved experimentally with structural coverage between 90% and 100%. Here, we present a comprehensive, user-friendly, web interface (https://cancer-3d.com/) of 714 modelled cancer-related genes, including homo-oligomers, hetero-oligomers, transmembrane proteins and complexes with DNA, ribonucleic acid, ligands and co-factors. Using SDM and mCSM software, we have predicted the impacts of reported mutations on protein stability, protein-protein interfaces affinity and protein-nucleic acid complexes affinity. Furthermore, we also predicted intrinsically disordered regions using DISOPRED3.


Assuntos
Biomarcadores Tumorais , Biologia Computacional/métodos , Bases de Dados Genéticas , Mutação , Neoplasias/genética , Oncogenes , Software , Análise de Dados , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , Interface Usuário-Computador , Fluxo de Trabalho
17.
Brief Bioinform ; 22(6)2021 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-34013350

RESUMO

Graph machine learning (GML) is receiving growing interest within the pharmaceutical and biotechnology industries for its ability to model biomolecular structures, the functional relationships between them, and integrate multi-omic datasets - amongst other data types. Herein, we present a multidisciplinary academic-industrial review of the topic within the context of drug discovery and development. After introducing key terms and modelling approaches, we move chronologically through the drug development pipeline to identify and summarize work incorporating: target identification, design of small molecules and biologics, and drug repurposing. Whilst the field is still emerging, key milestones including repurposed drugs entering in vivo studies, suggest GML will become a modelling framework of choice within biomedical machine learning.


Assuntos
Gráficos por Computador , Desenvolvimento de Medicamentos/métodos , Descoberta de Drogas/métodos , Aprendizado de Máquina , Modelos Moleculares , Estrutura Molecular , Algoritmos , Reposicionamento de Medicamentos , Redes Neurais de Computação
18.
Brief Bioinform ; 22(2): 769-780, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33416848

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a rapidly growing infectious disease, widely spread with high mortality rates. Since the release of the SARS-CoV-2 genome sequence in March 2020, there has been an international focus on developing target-based drug discovery, which also requires knowledge of the 3D structure of the proteome. Where there are no experimentally solved structures, our group has created 3D models with coverage of 97.5% and characterized them using state-of-the-art computational approaches. Models of protomers and oligomers, together with predictions of substrate and allosteric binding sites, protein-ligand docking, SARS-CoV-2 protein interactions with human proteins, impacts of mutations, and mapped solved experimental structures are freely available for download. These are implemented in SARS CoV-2 3D, a comprehensive and user-friendly database, available at https://sars3d.com/. This provides essential information for drug discovery, both to evaluate targets and design new potential therapeutics.


Assuntos
Antivirais/farmacologia , COVID-19/virologia , Bases de Dados de Proteínas , Sistemas de Liberação de Medicamentos , Proteoma , SARS-CoV-2/efeitos dos fármacos , Humanos , SARS-CoV-2/isolamento & purificação
19.
Angew Chem Int Ed Engl ; 62(17): e202300221, 2023 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-36757665

RESUMO

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20 µM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.


Assuntos
Mycobacterium tuberculosis , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Nucleotidiltransferases/metabolismo , Antituberculosos/farmacologia
20.
J Med Virol ; 94(9): 4181-4192, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35575289

RESUMO

Cleavage of the severe respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein has been demonstrated to contribute to viral-cell fusion and syncytia formation. Studies have shown that variants of concern (VOC) and variants of interest (VOI) show differing membrane fusion capacity. Mutations near cleavage motifs, such as the S1/S2 and S2' sites, may alter interactions with host proteases and, thus, the potential for fusion. The biochemical basis for the differences in interactions with host proteases for the VOC/VOI spike proteins has not yet been explored. Using sequence and structure-based bioinformatics, mutations near the VOC/VOI spike protein cleavage sites were inspected for their structural effects. All mutations found at the S1/S2 sites were predicted to increase affinity to the furin protease but not TMPRSS2. Mutations at the spike residue P681 in several strains, such P681R in the Delta strain, resulted in the disruption of a proline-directed kinase phosphorylation motif at the S1/S2 site, which may lessen the impact of phosphorylation for these variants. However, the unique N679K mutation in the Omicron strain was found to increase the propensity for O-linked glycosylation at the S1/S2 cleavage site, which may prevent recognition by proteases. Such glycosylation in the Omicron strain may hinder entry at the cell surface and, thus, decrease syncytia formation and induce cell entry through the endocytic pathway as has been shown in previous studies. Further experimental work is needed to confirm the effect of mutations and posttranslational modifications on SARS-CoV-2 spike protein cleavage sites.


Assuntos
SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Glicosilação , Mutação , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genética
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