RESUMO
Trypanosome parasites control their virulence and spread by using quorum sensing (QS) to generate transmissible "stumpy forms" in their host bloodstream. However, the QS signal "stumpy induction factor" (SIF) and its reception mechanism are unknown. Although trypanosomes lack G protein-coupled receptor signaling, we have identified a surface GPR89-family protein that regulates stumpy formation. TbGPR89 is expressed on bloodstream "slender form" trypanosomes, which receive the SIF signal, and when ectopically expressed, TbGPR89 drives stumpy formation in a SIF-pathway-dependent process. Structural modeling of TbGPR89 predicts unexpected similarity to oligopeptide transporters (POT), and when expressed in bacteria, TbGPR89 transports oligopeptides. Conversely, expression of an E. coli POT in trypanosomes drives parasite differentiation, and oligopeptides promote stumpy formation in vitro. Furthermore, the expression of secreted trypanosome oligopeptidases generates a paracrine signal that accelerates stumpy formation in vivo. Peptidase-generated oligopeptide QS signals being received through TbGPR89 provides a mechanism for both trypanosome SIF production and reception.
Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Percepção de Quorum/fisiologia , Trypanosoma/metabolismo , Diferenciação Celular , Sequência Conservada/genética , Proteínas de Ligação ao GTP/metabolismo , Proteínas de Membrana Transportadoras/genética , Oligopeptídeos/genética , Oligopeptídeos/fisiologia , Filogenia , Proteínas de Protozoários/metabolismo , Percepção de Quorum/genética , Transdução de Sinais , Trypanosoma/fisiologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologia , Virulência/fisiologiaRESUMO
Within drug discovery, the goal of AI scientists and cheminformaticians is to help identify molecular starting points that will develop into safe and efficacious drugs while reducing costs, time and failure rates. To achieve this goal, it is crucial to represent molecules in a digital format that makes them machine-readable and facilitates the accurate prediction of properties that drive decision-making. Over the years, molecular representations have evolved from intuitive and human-readable formats to bespoke numerical descriptors and fingerprints, and now to learned representations that capture patterns and salient features across vast chemical spaces. Among these, sequence-based and graph-based representations of small molecules have become highly popular. However, each approach has strengths and weaknesses across dimensions such as generality, computational cost, inversibility for generative applications and interpretability, which can be critical in informing practitioners' decisions. As the drug discovery landscape evolves, opportunities for innovation continue to emerge. These include the creation of molecular representations for high-value, low-data regimes, the distillation of broader biological and chemical knowledge into novel learned representations and the modeling of up-and-coming therapeutic modalities.
Assuntos
Descoberta de Drogas , Intuição , Humanos , AprendizagemRESUMO
Ligand-based virtual screening (LBVS) can be pivotal for identifying potential drug leads, especially when the target protein's structure is unknown. However, current LBVS methods are limited in their ability to consider the ligand conformational flexibility. This study presents AutoDock-SS (Similarity Searching), which adapts protein-ligand docking for use in LBVS. AutoDock-SS integrates novel ligand-based grid maps and AutoDock-GPU into a novel three-dimensional LBVS workflow. Unlike other approaches based on pregenerated conformer libraries, AutoDock-SS's built-in conformational search optimizes conformations dynamically based on the reference ligand, thus providing a more accurate representation of relevant ligand conformations. AutoDock-SS supports two modes: single and multiple ligand queries, allowing for the seamless consideration of multiple reference ligands. When tested on the Directory of Useful DecoysâEnhanced (DUD-E) data set, AutoDock-SS surpassed alternative 3D LBVS methods, achieving a mean AUROC of 0.775 and an EF1% of 25.72 in single-reference mode. The multireference mode, evaluated on the augmented DUD-E+ data set, demonstrated superior accuracy with a mean AUROC of 0.843 and an EF1% of 34.59. This enhanced performance underscores AutoDock-SS's ability to treat compounds as conformationally flexible while considering the ligand's shape, pharmacophore, and electrostatic potential, expanding the potential of LBVS methods.
Assuntos
Simulação de Acoplamento Molecular , Ligantes , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas/química , Proteínas/metabolismo , Interface Usuário-Computador , Conformação Proteica , Conformação MolecularRESUMO
Membrane targeting of autophagy-related complexes is an important step that regulates their activities and prevents their aberrant engagement on non-autophagic membranes. ATG16L1 is a core autophagy protein implicated at distinct phases of autophagosome biogenesis. In this study, we dissected the recruitment of ATG16L1 to the pre-autophagosomal structure (PAS) and showed that it requires sequences within its coiled-coil domain (CCD) dispensable for homodimerisation. Structural and mutational analyses identified conserved residues within the CCD of ATG16L1 that mediate direct binding to phosphoinositides, including phosphatidylinositol 3-phosphate (PI3P). Mutating putative lipid binding residues abrogated the localisation of ATG16L1 to the PAS and inhibited LC3 lipidation. On the other hand, enhancing lipid binding of ATG16L1 by mutating negatively charged residues adjacent to the lipid binding motif also resulted in autophagy inhibition, suggesting that regulated recruitment of ATG16L1 to the PAS is required for its autophagic activity. Overall, our findings indicate that ATG16L1 harbours an intrinsic ability to bind lipids that plays an essential role during LC3 lipidation and autophagosome maturation.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membrana Celular/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animais , Proteínas Relacionadas à Autofagia/fisiologia , Células Cultivadas , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Endossomos/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/fisiologia , Enzimas de Conjugação de Ubiquitina/fisiologia , Proteínas rab de Ligação ao GTP/fisiologiaRESUMO
The ATP binding sites of many enzymes are structurally related, which complicates their development as therapeutic targets. In this work, we explore a diverse set of ATPases and compare their ATP binding pockets using different strategies, including direct and indirect structural methods, in search of pockets attractive for drug discovery. We pursue different direct and indirect structural strategies, as well as ligandability assessments to help guide target selection. The analyses indicate human RAD51, an enzyme crucial in homologous recombination, as a promising, tractable target. Inhibition of RAD51 has shown promise in the treatment of certain cancers but more potent inhibitors are needed. Thus, we design compounds computationally against the ATP binding pocket of RAD51 with consideration of multiple criteria, including predicted specificity, drug-likeness, and toxicity. The molecules designed are evaluated experimentally using molecular and cell-based assays. Our results provide two novel hit compounds against RAD51 and illustrate a computational pipeline to design new inhibitors against ATPases.
Assuntos
Descoberta de Drogas , Recombinação Homóloga , Adenosina Trifosfatases , Trifosfato de Adenosina/química , Sítios de Ligação , Humanos , Ligação ProteicaRESUMO
Modified peptides, such as stapled peptides, which replicate the structure of α-helical protein segments, represent a potential therapeutic advance. However, the 3D solution structure of these stapled peptides is rarely explored beyond the acquisition of circular dichroism (CD) data to quantify bulk peptide helicity; the detailed backbone structure, which underlies this, is typically undefined. Diastereomeric stapled peptides based on helical sections of three proteins (αSyn, Cks1 and CK1α) were generated; their overall helicity was quantified by CD; and the most helical peptide from each series was selected for structural analysis. Solution-phase models for the optimised peptides were generated using NMR-derived restraints and a modified CHARMM22 force field. Comparing these models with PDB structures allowed deviation between the stapled peptides and critical helical regions to be evaluated. These studies demonstrate that CD alone is not sufficient to assess the structural fidelity of a stapled peptide.
RESUMO
The inverted repeat (IR) sequences delimiting the left and right ends of many naturally active mariner DNA transposons are non-identical and have different affinities for their transposase. We have compared the preferences of two active mariner transposases, Mos1 and Mboumar-9, for their imperfect transposon IRs in each step of transposition: DNA binding, DNA cleavage, and DNA strand transfer. A 3.1 Å resolution crystal structure of the Mos1 paired-end complex containing the pre-cleaved left IR sequences reveals the molecular basis for the reduced affinity of the Mos1 transposase DNA-binding domain for the left IR as compared with the right IR. For both Mos1 and Mboumar-9, in vitro DNA transposition is most efficient when the preferred IR sequence is present at both transposon ends. We find that this is due to the higher efficiency of cleavage and strand transfer of the preferred transposon end. We show that the efficiency of Mboumar-9 transposition is improved almost 4-fold by changing the 3' base of the preferred Mboumar-9 IR from guanine to adenine. This preference for adenine at the reactive 3' end for both Mos1 and Mboumar-9 may be a general feature of mariner transposition.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Drosophila/enzimologia , Sequências Repetidas Invertidas/genética , Plasmídeos/genética , Transposases/química , Transposases/metabolismo , Adenina/química , Animais , Sequência de Bases , Cristalografia por Raios X , DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Enzimológica da Expressão Gênica , Guanina/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Transposases/genéticaRESUMO
Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that 'cut and paste' certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell-wall polysaccharides (xyloglucan, mannans, mixed-linkage ß-glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero-trans-ß-glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early-diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero-transglycosylation: its preferred donor substrates (cellulose or mixed-linkage ß-glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose-xyloglucan and mixed-linkage ß-glucan-xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter-linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp â Pro, Gly â Ser and Arg â Leu) are responsible for the evolution of HTG's unique specificity from the better-known xyloglucan-acting homo-transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable 'green' technology for industrially manipulating biomass.
Assuntos
Celulose/metabolismo , Equisetum/metabolismo , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Proteínas Recombinantes/metabolismo , Substituição de Aminoácidos , Clonagem Molecular , Equisetum/genética , Evolução Molecular , Glicosídeo Hidrolases/genética , Glicosiltransferases/metabolismo , Pichia/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Conformação Proteica , Proteínas Recombinantes/genética , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Newly designed triazolothiadiazines incorporating with structural motifs of nonsteroidal analgesic anti-inflammatory drugs were synthesized and screened for their bioactivity against epithelial cancer cells. Compounds with bioactivities less then â¼5µM (IC50) were further analyzed and showed to induce apoptotic cell death and SubG1 cell cycle arrest in liver cancer cells. Among this group, two compounds (1g and 1h) were then studied to identify the mechanism of action. These molecules triggered oxidative stress induced apoptosis through ASK-1 protein activation and Akt protein inhibition as demonstrated by downstream targets such as GSK3ß, ß-catenin and cyclin D1. QSAR and molecular docking models provide insight into the mechanism of inhibition and indicate the optimal direction of future synthetic efforts. Furthermore, molecular docking results were confirmed with in vitro COX bioactivity studies. This study demonstrates that the novel triazolothiadiazine derivatives are promising drug candidates for epithelial cancers, especially liver cancer.
Assuntos
Antineoplásicos/síntese química , Regulação Neoplásica da Expressão Gênica , Tiadiazinas/síntese química , Triazóis/síntese química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Células HCT116 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Concentração Inibidora 50 , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Células MCF-7 , Simulação de Acoplamento Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estrutura Secundária de Proteína , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Relação Quantitativa Estrutura-Atividade , Transdução de Sinais , Tiadiazinas/farmacologia , Triazóis/farmacologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Infection by parasitic nematodes is widespread in the developing world causing extensive morbidity and mortality. Furthermore, infection of animals is a global problem, with a substantial impact on food production. Here we identify small molecule inhibitors of a nematode-specific metalloprotease, DPY-31, using both known metalloprotease inhibitors and virtual screening. This strategy successfully identified several µM inhibitors of DPY-31 from both the human filarial nematode Brugia malayi, and the parasitic gastrointestinal nematode of sheep Teladorsagia circumcincta. Further studies using both free living and parasitic nematodes show that these inhibitors elicit the severe body morphology defect 'Dumpy' (Dpy; shorter and fatter), a predominantly non-viable phenotype consistent with mutants lacking the DPY-31 gene. Taken together, these results represent a start point in developing DPY-31 inhibition as a totally novel mechanism for treating infection by parasitic nematodes in humans and animals.
Assuntos
Proteínas de Helminto/antagonistas & inibidores , Nematoides/enzimologia , Inibidores de Proteases/química , Animais , Sítios de Ligação , Brugia Malayi/enzimologia , Caenorhabditis elegans/enzimologia , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Helminto/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Concentração Inibidora 50 , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Metaloproteases/antagonistas & inibidores , Metaloproteases/metabolismo , Simulação de Acoplamento Molecular , Inibidores de Proteases/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , OvinosRESUMO
Non-coding transcription can trigger histone post-translational modifications forming specialized chromatin. In fission yeast, heterochromatin formation requires RNAi and the histone H3K9 methyltransferase complex CLRC, composed of Clr4, Raf1, Raf2, Cul4, and Rik1. CLRC mediates H3K9 methylation and siRNA production; it also displays E3-ubiquitin ligase activity in vitro. DCAFs act as substrate receptors for E3 ligases and may couple ubiquitination with histone methylation. Here, structural alignment and mutation of signature WDxR motifs in Raf1 indicate that it is a DCAF for CLRC. We demonstrate that Raf1 promotes H3K9 methylation and siRNA amplification via two distinct, separable functions. The association of the DCAF Raf1 with Cul4-Rik1 is critical for H3K9 methylation, but dispensable for processing of centromeric transcripts into siRNAs. Thus the association of a DCAF, Raf1, with its adaptor, Rik1, is required for histone methylation and to allow RNAi to signal to chromatin.
Assuntos
Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Histonas/genética , Proteínas Proto-Oncogênicas c-raf/genética , RNA Interferente Pequeno/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Metiltransferases/genética , Complexos Multiproteicos/genética , Mutação , Processamento de Proteína Pós-Traducional , Schizosaccharomyces/metabolismo , Homologia Estrutural de Proteína , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
BACKGROUND: The control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. METHODS: Here, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared. RESULTS: An increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the µ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene. CONCLUSION: An increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum.
Assuntos
Substituição de Aminoácidos , Antimaláricos/farmacologia , Artemisininas/farmacologia , Resistência a Medicamentos , Plasmodium chabaudi/efeitos dos fármacos , Plasmodium chabaudi/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Análise Mutacional de DNA , Marcadores Genéticos , Humanos , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Plasmodium chabaudi/isolamento & purificação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Conformação Proteica , Proteínas de Protozoários/químicaRESUMO
Structure-based virtual screening relies on scoring the predicted binding modes of compounds docked into the target. Because the accuracy of this scoring relies on the accuracy of the docking, methods that increase docking accuracy are valuable. Here, we present a relatively straightforward method for improving the probability of identifying accurately docked poses. The method is similar in concept to consensus scoring schemes, which have been shown to increase ranking power and thus hit rates, but combines information about predicted binding modes rather than predicted binding affinities. The pose prediction success rate of each docking program alone was found in this trial to be 55% for Autodock, 58% for DOCK, and 64% for Vina. By using more than one docking program to predict the binding pose, correct poses were identified in 82% or more of cases, a significant improvement. In a virtual screen, these more reliably posed compounds can be preferentially advanced to subsequent scoring stages to improve hit rates. Consensus docking can be easily introduced into established structure-based virtual screening methodologies.
Assuntos
Bacteroides/enzimologia , Hepacivirus/enzimologia , Simulação de Acoplamento Molecular/métodos , RNA Polimerase Dependente de RNA/antagonistas & inibidores , beta-N-Acetil-Hexosaminidases/antagonistas & inibidores , Bases de Dados de Proteínas , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ligantes , Probabilidade , Ligação Proteica , RNA Polimerase Dependente de RNA/metabolismo , Software , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
INTRODUCTION: The discovery of a new drug is a costly and lengthy endeavour. The computational prediction of which small molecules can bind to a protein target can accelerate this process if the predictions are fast and accurate enough. Recent machine-learning scoring functions re-evaluate the output of molecular docking to achieve more accurate predictions. However, previous scoring functions were trained on crystalised protein-ligand complexes and datasets of decoys. The limited availability of crystal structures and biases in the decoy datasets can lower the performance of scoring functions. OBJECTIVES: To address key limitations of previous scoring functions and thus improve the predictive performance of structure-based virtual screening. METHODS: A novel machine-learning scoring function was created, named SCORCH (Scoring COnsensus for RMSD-based Classification of Hits). To develop SCORCH, training data is augmented by considering multiple ligand poses and labelling poses based on their RMSD from the native pose. Decoy bias is addressed by generating property-matched decoys for each ligand and using the same methodology for preparing and docking decoys and ligands. A consensus of 3 different machine learning approaches is also used to improve performance. RESULTS: We find that multi-pose augmentation in SCORCH improves its docking power and screening power on independent benchmark datasets. SCORCH outperforms an equivalent scoring function trained on single poses, with a 1 % enrichment factor (EF) of 13.78 vs. 10.86 on 18 DEKOIS 2.0 targets and a mean native pose rank of 5.9 vs 30.4 on CSAR 2014. Additionally, SCORCH outperforms widely used scoring functions in virtual screening and pose prediction on independent benchmark datasets. CONCLUSION: By rationally addressing key limitations of previous scoring functions, SCORCH improves the performance of virtual screening. SCORCH also provides an estimate of its uncertainty, which can help reduce the cost and time required for drug discovery.
Assuntos
Aprendizado de Máquina , Proteínas , Simulação de Acoplamento Molecular , Proteínas/química , Proteínas/metabolismo , Ligação Proteica , Ligantes , IncertezaRESUMO
Hepatitis E Virus (HEV) infection is an emergent zoonotic disease, where chronic hepatitis E associated to solid organ transplant (SOT) recipients, related to genotype 3, is the clinical manifestation of major concern. In this setting, ribavirin (RBV) treatment is the only available therapy, though drug-resistant variants could emerge leading to a therapeutic failure. Crystallographic structures have not been reported for most of the HEV proteins, including the RNA-polymerase (RdRp). Therefore, the mechanism of action of RBV against HEV and the molecular interactions between this drug and RdRp are largely unknown. In this work, we aimed to model in silico the 3 D structure of a novel HEV3 RdRp (HEV_C1_Uy) from a chronically HEV infected-SOT recipient treated with RBV and to perform a molecular docking simulation between RBV triphosphate (RBVT), 7-methyl-guanosine-5'-triphosphate and the modelled protein. The models were generated using I-TASSER server and validated with multiple bioinformatics tools. The docking analysis were carried out with AutoDock Vina and LeDock software. We obtained a suitable model for HEV_C1_Uy (C-Score=-1.33, RMSD = 10.4 ± 4.6 Å). RBVT displayed a binding affinity of -7.6 ± 0.2 Kcal/mol by molecular docking, mediated by 6 hydrogen-bonds (Q195-O14, S198-O11, E257-O13, S260-O2, O3, S311-O11) between the finger's-palm-domains and a free binding energy of 31.26 ± 16.81 kcal/mol by molecular dynamics simulations. We identified the possible HEV RdRp interacting region for incoming nucleotides or analogs and provide novel insights that will contribute to better understand the molecular interactions of RBV and the enzyme and the mechanism of action of this antiviral drug.Communicated by Ramaswamy H. Sarma.
Assuntos
Vírus da Hepatite E , Hepatite E , Humanos , Ribavirina/farmacologia , Ribavirina/uso terapêutico , Vírus da Hepatite E/genética , Simulação de Acoplamento Molecular , RNA Polimerase Dependente de RNA/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , Hepatite E/tratamento farmacológico , GenótipoRESUMO
Ligand-activated nuclear receptors (NRs) orchestrate development, growth, and reproduction across all animal lifeforms - the Metazoa - but how NRs evolved remains mysterious. Given the NR ligands including steroids and retinoids are predominantly terpenoids, we asked whether NRs might have evolved from enzymes that catalyze terpene synthesis and metabolism. We provide evidence suggesting that NRs may be related to the terpene synthase (TS) enzyme superfamily. Based on over 10,000 3D structural comparisons, we report that the NR ligand-binding domain and TS enzymes share a conserved core of seven α-helical segments. In addition, the 3D locations of the major ligand-contacting residues are also conserved between the two protein classes. Primary sequence comparisons reveal suggestive similarities specifically between NRs and the subfamily of cis-isoprene transferases, notably with dehydrodolichyl pyrophosphate synthase and its obligate partner, NUS1/NOGOB receptor. Pharmacological overlaps between NRs and TS enzymes add weight to the contention that they share a distant evolutionary origin, and the combined data raise the possibility that a ligand-gated receptor may have arisen from an enzyme antecedent. However, our findings do not formally exclude other interpretations such as convergent evolution, and further analysis will be necessary to confirm the inferred relationship between the two protein classes.
Assuntos
Evolução Molecular , Receptores Citoplasmáticos e Nucleares , Alquil e Aril Transferases , Animais , Filogenia , TerpenosRESUMO
5-Fluorouracil (5-FU) is an antineoplastic antimetabolite that is widely administered to cancer patients by bolus injection, especially to those suffering from colorectal and pancreatic cancer. Because of its suboptimal route of administration and dose-limiting toxicities, diverse 5-FU prodrugs have been developed to confer oral bioavailability and increase the safety profile of 5-FU chemotherapy regimens. Our contribution to this goal is presented herein with the development of a novel palladium-activated prodrug designed to evade the metabolic machinery responsible for 5-FU anabolic activation and catabolic processing. The new prodrug is completely innocuous to cells and highly resistant to metabolization by primary hepatocytes and liver S9 fractions (the main metabolic route for 5-FU degradation), whereas it is rapidly converted into 5-FU in the presence of a palladium (Pd) source. In vivo pharmokinetic analysis shows the prodrug is rapidly and completely absorbed after oral administration and exhibits a longer half-life than 5-FU. In vivo efficacy studies in a xenograft colon cancer model served to prove, for the first time, that orally administered prodrugs can be locally converted to active drugs by intratumorally inserted Pd implants.
Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Fluoruracila/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Paládio/química , Pró-Fármacos/metabolismo , Animais , Antimetabólitos Antineoplásicos/toxicidade , Biotransformação , Fluoruracila/análogos & derivados , Fluoruracila/toxicidade , Células HCT116 , Meia-Vida , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Pró-Fármacos/toxicidade , Ligação Proteica , Ratos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Aminoglycoside 2''-phosphotransferase IVa [APH(2'')-IVa] is a member of a family of bacterial enzymes responsible for medically relevant resistance to antibiotics. APH(2'')-IVa confers high-level resistance against several clinically used aminoglycoside antibiotics in various pathogenic Enterococcus species by phosphorylating the drug, thereby preventing it from binding to its ribosomal target and producing a bactericidal effect. We describe here three crystal structures of APH(2'')-IVa, one in its apo form and two in complex with a bound antibiotic, tobramycin and kanamycin A. The apo structure was refined to a resolution of 2.05 Å, and the APH(2'')-IVa structures with tobramycin and kanamycin A bound were refined to resolutions of 1.80 and 2.15 Å, respectively. Comparison among the structures provides insight concerning the substrate selectivity of this enzyme. In particular, conformational changes upon substrate binding, involving rotational shifts of two distinct segments of the enzyme, are observed. These substrate-induced shifts may also rationalize the altered substrate preference of APH(2'')-IVa in comparison to those of other members of the APH(2'') subfamily, which are structurally closely related. Finally, analysis of the interactions between the enzyme and aminoglycoside reveals a distinct binding mode as compared to the intended ribosomal target. The differences in the pattern of interactions can be utilized as a structural basis for the development of improved aminoglycosides that are not susceptible to these resistance factors.
Assuntos
Farmacorresistência Bacteriana , Enterococcus/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Apoenzimas/química , Apoenzimas/metabolismo , Cristalografia por Raios X , Enterococcus/efeitos dos fármacos , Canamicina/química , Canamicina/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Tobramicina/química , Tobramicina/metabolismoRESUMO
Cyclodipeptide synthases (CDPSs) produce a variety of cyclic dipeptide products by utilising two aminoacylated tRNA substrates. We sought to investigate the minimal requirements for substrate usage in this class of enzymes as the relationship between CDPSs and their substrates remains elusive. Here, we investigated the Bacillus thermoamylovorans enzyme, BtCDPS, which synthesises cyclo(l-Leu-l-Leu). We systematically tested where specificity arises and, in the process, uncovered small molecules (activated amino esters) that will suffice as substrates, although catalytically poor. We solved the structure of BtCDPS to 1.7 Å and combining crystallography, enzymatic assays and substrate docking experiments propose a model for how the minimal substrates interact with the enzyme. This work is the first report of a CDPS enzyme utilizing a molecule other than aa-tRNA as a substrate; providing insights into substrate requirements and setting the stage for the design of improved simpler substrates.
RESUMO
[This corrects the article DOI: 10.1039/D0CB00142B.].