RESUMEN

CONTEXT: Melanoma is one of the cancers with the highest mortality rate for its ability to metastasize. Several targets have undergone investigation for the development of drugs against this pathology. One of the main targets is the kinase BRAF (RAF, rapidly accelerated fibrosarcoma). The most common mutation in melanoma is BRAFV600E and has been reported in 50-90% of patients with melanoma. Due to the relevance of the BRAFV600E mutation, inhibitors to this kinase have been developed, vemurafenib-OMe and dabrafenib. Ursolic acid (UA) is a pentacyclic triterpene with a privileged structure, the pentacycle scaffold, which allows to have a broad variety of biological activity; the most studied is its anticancer capacity. In this work, we reported the interaction profile of vemurafenib-OMe, dabrafenib, and UA, to define whether UA has binding capacity to BRAFWT, BRAFV600E, and BRAFV600K. Homology modeling of BRAFWT, V600E, and V600K; molecular docking; and molecular dynamics simulations were carried out and interactions and residues relevant to the binding of the inhibitors were obtained. We found that UA, like the inhibitors, presents hydrogen bond interactions, and hydrophobic interactions of van der Waals, and π-stacking with I463, Q530, C532, and F583. The ΔG of ursolic acid in complex with BRAFV600K (- 63.31 kcal/mol) is comparable to the ΔG of the selective inhibitor dabrafenib (- 63.32 kcal/mol) in complex to BRAFV600K and presents a ΔG like vemurafenib-OMe with BRAFWT and V600E. With this information, ursolic acid could be considered as a lead compound for design cycles and to optimize the binding profile and the selectivity towards mutations for the development of new selective inhibitors for BRAFV600E and V600K to new potential melanoma treatments. METHODS: The homology modeling calculations were executed on the public servers I-TASSER and ROBETTA, followed by molecular docking calculations using AutoGrid 4.2.6, AutoDockGPU 1.5.3, and AutoDockTools 1.5.6. Molecular dynamics and metadynamics simulations were performed in the Desmond module of the academic version of the Schrödinger-Maestro 2020-4 program, utilizing the OPLS-2005 force field. Ligand-protein interactions were evaluated using Schrödinger-Maestro program, LigPlot + , and PLIP (protein-ligand interaction profiler). Finally, all of the protein figures presented in this article were made in the PyMOL program.

Asunto(s)

Melanoma , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas Proto-Oncogénicas B-raf , Triterpenos , Ácido Ursólico , Triterpenos/química , Triterpenos/farmacología , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Proteínas Proto-Oncogénicas B-raf/genética , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genética , Imidazoles/química , Imidazoles/farmacología , Unión Proteica , Vemurafenib/farmacología , Vemurafenib/química , Oximas/química , Oximas/farmacología , Mutación , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Sitios de Unión

RESUMEN

Inhibition of HIV-1 protease is a cornerstone of antiretroviral therapy. However, the notorious ability of HIV-1 to develop resistance to protease inhibitors (PIs), particularly darunavir (DRV), poses a major challenge. Using quantum chemistry and computer simulations, this study aims to investigate the interactions between two novel PIs, GRL-004 and GRL-063, as well as a wild-type (WT) HIV strain and a DRV-resistant mutant strain. To do this, we used molecular docking, molecular dynamics simulations, and quantum mechanical calculations to check how well GRL-004 and GRL-063 bound to both WT and DRV-resistant proteases. The results show that GRL-004 and GRL-063 bind very well to ASP29 in the WT strain. ASP29 is an important amino acid in the HIV protease dimer. Remarkably, amino acids such as ILE50 in the WT strains showed substantial binding energies to both drugs. Quantum energy calculations showed a slight reduction in the energy affinity of the interaction between the MUT strain and the ligand GRL-063, compared to the WT strain. GRL-004 showed similar interaction energy for both strains, suggesting that it has greater plasticity than GRL-063 despite its lower interaction affinity. Furthermore, GLY49B demonstrated strong binding energies regardless of mutations. Other relevant residues with strong binding energies include GLY49B, PHE82A, PRO81A, ASP29A, ASP25A and ALA28B. This study improves our understanding of receptor-ligand dynamics and the adaptability of new protease inhibitors (PIs), which has profound implications for the innovation of future antiretroviral drugs.

Asunto(s)

Inhibidores de la Proteasa del VIH , Proteasa del VIH , VIH-1 , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Teoría Cuántica , Inhibidores de la Proteasa del VIH/química , Inhibidores de la Proteasa del VIH/farmacología , Inhibidores de la Proteasa del VIH/metabolismo , Proteasa del VIH/metabolismo , Proteasa del VIH/química , Proteasa del VIH/genética , VIH-1/enzimología , VIH-1/efectos de los fármacos , Darunavir/farmacología , Darunavir/química , Darunavir/metabolismo , Farmacorresistencia Viral , Unión Proteica , Sitios de Unión , Humanos

RESUMEN

Molecular dynamics (MD) simulations produce a substantial volume of high-dimensional data, and traditional methods for analyzing these data pose significant computational demands. Advances in MD simulation analysis combined with deep learning-based approaches have led to the understanding of specific structural changes observed in MD trajectories, including those induced by mutations. In this study, we model the trajectories resulting from MD simulations of the SARS-CoV-2 spike protein-ACE2, specifically the receptor-binding domain (RBD), as interresidue distance maps, and use deep convolutional neural networks to predict the functional impact of point mutations, related to the virus's infectivity and immunogenicity. Our model was successful in predicting mutant types that increase the affinity of the S protein for human receptors and reduce its immunogenicity, both based on MD trajectories (precision = 0.718; recall = 0.800; [Formula: see text] = 0.757; MCC = 0.488; AUC = 0.800) and their centroids. In an additional analysis, we also obtained a strong positive Pearson's correlation coefficient equal to 0.776, indicating a significant relationship between the average sigmoid probability for the MD trajectories and binding free energy (BFE) changes. Furthermore, we obtained a coefficient of determination of 0.602. Our 2D-RMSD analysis also corroborated predictions for more infectious and immune-evading mutants and revealed fluctuating regions within the receptor-binding motif (RBM), especially in the [Formula: see text] loop. This region presented a significant standard deviation for mutations that enable SARS-CoV-2 to evade the immune response, with RMSD values of 5Å in the simulation. This methodology offers an efficient alternative to identify potential strains of SARS-CoV-2, which may be potentially linked to more infectious and immune-evading mutations. Using clustering and deep learning techniques, our approach leverages information from the ensemble of MD trajectories to recognize a broad spectrum of multiple conformational patterns characteristic of mutant types. This represents a strategic advantage in identifying emerging variants, bypassing the need for long MD simulations. Furthermore, the present work tends to contribute substantially to the field of computational biology and virology, particularly to accelerate the design and optimization of new therapeutic agents and vaccines, offering a proactive stance against the constantly evolving threat of COVID-19 and potential future pandemics.

Asunto(s)

Enzima Convertidora de Angiotensina 2 , Aprendizaje Profundo , Simulación de Dinámica Molecular , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Humanos , SARS-CoV-2/genética , SARS-CoV-2/química , SARS-CoV-2/metabolismo , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , COVID-19/virología , Unión Proteica , Conformación Proteica , Mutación , Sitios de Unión , Dominios Proteicos

RESUMEN

Selective recognition and sensing of catecholamine-based neurotransmitters by fluorescent synthetic receptors capable of operating in pure water is a central topic of modern supramolecular chemistry that impacts biological and analytical chemistry. Despite advances achieved in the recognition of some neurotransmitters such as dopamine, little effort has been invested in the optical recognition of other neurotransmitters of paramount importance in biochemistry and medicinal chemistry such as the drug L-dihydroxy-phenylalanine (levodopa). Herein, a cationic Cu(II)-terpyridine complex bearing an intramolecular fluorescent quinolinium ring covalently linked to phenylboronic acid (CuL1) was synthesized, structurally described by single-crystal X-ray diffraction and studied in-depth as a fluorescent receptor for neurotransmitters in water. The complex CuL1 was designed to act as a receptor for levodopa through two Lewis acids of different natures (Cu(II) and B atoms) as cooperative binding points. The receptor CuL1 was found to have a strongly acidified -B(OH)2 group (pKa = 6.2) and exceptionally high affinity for levodopa (K = 4.8 × 106 M-1) with selectivity over other related neurotransmitters such as dopamine, epinephrine, norepinephrine and nucleosides in the micromolar concentration range at physiological pH. Such levodopa affinity/selectivity for a boronic acid-based receptor in water is still rare. On the basis of spectroscopic tools (11B NMR, UV-vis, EPR, and fluorescence), high-resolution ESI-MS, crystal structure, and DFT calculations, the interaction mode of CuL1 with levodopa is proposed in a 1 : 1 model using two-point recognition involving a boronate-catechol esterification and a coordination bond Cu(II)-carboxylate. Furthermore, a visual sensing ensemble was constructed using CuL1 and the commercial fluorescent dye eosin Y. Levodopa is efficiently detected by the displacement of the eosin Y bound to the Cu(II)-receptor, monitoring its green emission. The use of Cu(II)-boronate complexes for fast and selective neurotransmitter sensing was unexplored until now.

Asunto(s)

Ácidos Borónicos , Complejos de Coordinación , Cobre , Agua , Ácidos Borónicos/química , Agua/química , Cobre/química , Complejos de Coordinación/química , Complejos de Coordinación/síntesis química , Modelos Moleculares , Levodopa/química , Estructura Molecular , Sitios de Unión

RESUMEN

Carbohydrate binding modules (CBMs) are protein domains that typically reside near catalytic domains, increasing substrate-protein proximity by constraining the conformational space of carbohydrates. Due to the flexibility and variability of glycans, the molecular details of how these protein regions recognize their target molecules are not always fully understood. Computational methods, including molecular docking and molecular dynamics simulations, have been employed to investigate lectin-carbohydrate interactions. In this study, we introduce a novel approach that integrates multiple computational techniques to identify the critical amino acids involved in the interaction between a CBM located at the tip of bacteriophage J-1's tail and its carbohydrate counterparts. Our results highlight three amino acids that play a significant role in binding, a finding we confirmed through in vitro experiments. By presenting this approach, we offer an intriguing alternative for pinpointing amino acids that contribute to protein-sugar interactions, leading to a more thorough comprehension of the molecular determinants of protein-carbohydrate interactions.

Asunto(s)

Aminoácidos , Biología Computacional , Aminoácidos/química , Aminoácidos/metabolismo , Simulación de Dinámica Molecular , Carbohidratos/química , Simulación del Acoplamiento Molecular , Unión Proteica , Sitios de Unión , Proteínas Virales/química , Proteínas Virales/metabolismo , Proteínas Virales/genética

RESUMEN

Drug repositioning is an important therapeutic strategy for treating breast cancer. Hsp90ß chaperone is an attractive target for inhibiting cell progression. Its structure has a disordered and flexible linker region between the N-terminal and central domains. Geldanamycin was the first Hsp90ß inhibitor to interact specifically at the N-terminal site. Owing to the toxicity of geldanamycin, we investigated the repositioning of ritonavir as an Hsp90ß inhibitor, taking advantage of its proven efficacy against cancer. In this study, we used molecular modeling techniques to analyze the contribution of the Hsp90ß linker region to the flexibility and interaction between the ligands geldanamycin, ritonavir, and Hsp90ß. Our findings indicate that the linker region is responsible for the fluctuation and overall protein motion without disturbing the interaction between the inhibitors and the N-terminus. We also found that ritonavir established similar interactions with the substrate ATP triphosphate, filling the same pharmacophore zone.

Asunto(s)

Benzoquinonas , Proteínas HSP90 de Choque Térmico , Lactamas Macrocíclicas , Ritonavir , Lactamas Macrocíclicas/farmacología , Lactamas Macrocíclicas/química , Ritonavir/química , Ritonavir/farmacología , Benzoquinonas/química , Benzoquinonas/farmacología , Benzoquinonas/metabolismo , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Humanos , Unión Proteica , Simulación de Dinámica Molecular , Simulación del Acoplamiento Molecular , Modelos Moleculares , Sitios de Unión , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química

RESUMEN

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

Asunto(s)

Calcio , Receptores Nicotínicos , Animales , Ratas , Acetilcolina/metabolismo , Acetilcolina/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Simulación de Dinámica Molecular , Técnicas de Placa-Clamp , Subunidades de Proteína/metabolismo , Subunidades de Proteína/genética , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Xenopus laevis

RESUMEN

The Glycine Transporter Type 1 (GlyT1) significantly impacts central nervous system functions, influencing glycinergic and glutamatergic neurotransmission. Bitopertin, the first GlyT1 inhibitor in clinical trials, was developed for schizophrenia treatment but showed limited efficacy. Despite this, bitopertin's repositioning could advance treating various pathologies. This study aims to understand bitopertin's mechanism of action using computational methods, exploring off-target effects, and providing a comprehensive pharmacological profile. Similarity Ensemble Approach (SEA) and SwissTargetPrediction initially predicted targets, followed by molecular modeling on SWISS-MODEL and GalaxyWeb servers. Binding sites were identified using PrankWeb, and molecular docking was performed with DockThor and GOLD software. Molecular dynamics analyses were conducted on the Visual Dynamics platform. Reverse screening on SEA and SwissTargetPrediction identified GlyT1 (SLC6A9), GlyT2 (SLC6A5), PROT (SLC6A7), and DAT (SLC6A3) as potential bitopertin targets. Homology modeling on SwissModel generated high-resolution models, optimized further on GalaxyWeb. PrankWeb identified similar binding sites in GlyT1, GlyT2, PROT, and DAT, indicating potential interaction. Docking studies suggested bitopertin's interaction with GlyT1 and proximity to GlyT2 and PROT. Molecular dynamics confirmed docking results, highlighting bitopertin's target stability beyond GlyT1. The study concludes that bitopertin potentially interacts with multiple SLC6 family targets, indicating a broader pharmacological property.

Asunto(s)

Proteínas de Transporte de Glicina en la Membrana Plasmática , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de Transporte de Glicina en la Membrana Plasmática/metabolismo , Proteínas de Transporte de Glicina en la Membrana Plasmática/antagonistas & inhibidores , Humanos , Sitios de Unión , Piperazinas/farmacología , Piperazinas/química , Simulación por Computador , Reposicionamiento de Medicamentos , Sulfonas

RESUMEN

The initial adoption of penicillin as an antibiotic marked the start of exploring other compounds essential for pharmaceuticals, yet resistance to penicillins and their side effects has compromised their efficacy. The N-terminal nucleophile (Ntn) amide-hydrolases S45 family plays a key role in catalyzing amide bond hydrolysis in various compounds, including antibiotics like penicillin and cephalosporin. This study comprehensively analyzes the structural and functional traits of the bacterial N-terminal nucleophile (Ntn) amide-hydrolases S45 family, covering penicillin G acylases, cephalosporin acylases, and D-succinylase. Utilizing structural bioinformatics tools and sequence analysis, the investigation delineates structurally conserved regions (SCRs) and substrate binding site variations among these enzymes. Notably, sixteen SCRs crucial for substrate interaction are identified solely through sequence analysis, emphasizing the significance of sequence data in characterizing functionally relevant regions. These findings introduce a novel approach for identifying targets to enhance the biocatalytic properties of N-terminal nucleophile (Ntn) amide-hydrolases, while facilitating the development of more accurate three-dimensional models, particularly for enzymes lacking structural data. Overall, this research advances our understanding of structure-function relationships in bacterial N-terminal nucleophile (Ntn) amide-hydrolases, providing insights into strategies for optimizing their enzymatic capabilities.

Asunto(s)

Amidohidrolasas , Amidohidrolasas/química , Amidohidrolasas/metabolismo , Amidohidrolasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Relación Estructura-Actividad , Secuencia Conservada , Bacterias/enzimología , Secuencia de Aminoácidos , Modelos Moleculares , Especificidad por Sustrato

RESUMEN

Exploring therapeutic options is crucial in the ongoing COVID-19 pandemic caused by SARS-CoV-2. Nirmatrelvir, which is a potent inhibitor that targets the SARS-CoV-2 Mpro, shows promise as an antiviral treatment. Additionally, Ivermectin, which is a broad-spectrum antiparasitic drug, has demonstrated effectiveness against the virus in laboratory settings. However, its clinical implications are still debated. Using computational methods, such as molecular docking and 100 ns molecular dynamics simulations, we investigated how Nirmatrelvir and Ivermectin interacted with SARS-CoV-2 Mpro(A). Calculations using density functional theory were instrumental in elucidating the behavior of isolated molecules, primarily by analyzing the frontier molecular orbitals. Our analysis revealed distinct binding patterns: Nirmatrelvir formed strong interactions with amino acids, like MET49, MET165, HIS41, HIS163, HIS164, PHE140, CYS145, GLU166, and ASN142, showing stable binding, with a root-mean-square deviation (RMSD) of around 2.0 Å. On the other hand, Ivermectin interacted with THR237, THR239, LEU271, LEU272, and LEU287, displaying an RMSD of 1.87 Å, indicating enduring interactions. Both ligands stabilized Mpro(A), with Ivermectin showing stability and persistent interactions despite forming fewer hydrogen bonds. These findings offer detailed insights into how Nirmatrelvir and Ivermectin bind to the SARS-CoV-2 main protease, providing valuable information for potential therapeutic strategies against COVID-19.

Asunto(s)

Antivirales , Tratamiento Farmacológico de COVID-19 , Proteasas 3C de Coronavirus , Ivermectina , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , SARS-CoV-2 , Ivermectina/química , Ivermectina/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/enzimología , Proteasas 3C de Coronavirus/química , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Proteasas 3C de Coronavirus/metabolismo , Humanos , Antivirales/química , Antivirales/farmacología , Unión Proteica , Sulfonamidas/química , Sulfonamidas/farmacología , Sitios de Unión , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Lactamas , Leucina , Nitrilos , Prolina

RESUMEN

BACKGROUND: Klebsiella pneumoniae is a Gram-negative pathogen that has become a threat to public health worldwide due to the emergence of hypervirulent and multidrug-resistant strains. Cell-surface components, such as polysaccharide capsules, fimbriae, and lipopolysaccharides (LPS), are among the major virulence factors for K. pneumoniae. One of the genes involved in LPS biosynthesis is the uge gene, which encodes the uridine diphosphate galacturonate 4-epimerase enzyme. Although essential for the LPS formation in K. pneumoniae, little is known about the mechanisms that regulate the expression of uge. Ferric uptake regulator (Fur) is an iron-responsive transcription factor that modulates the expression of capsular and fimbrial genes, but its role in LPS expression has not yet been identified. This work aimed to investigate the role of the Fur regulator in the expression of the K. pneumoniae uge gene and to determine whether the production of LPS by K. pneumoniae is modulated by the iron levels available to the bacterium. RESULTS: Using bioinformatic analyses, a Fur-binding site was identified on the promoter region of the uge gene; this binding site was validated experimentally through Fur Titration Assay (FURTA) and DNA Electrophoretic Mobility Shift Assay (EMSA) techniques. RT-qPCR analyses were used to evaluate the expression of uge according to the iron levels available to the bacterium. The iron-rich condition led to a down-regulation of uge, while the iron-restricted condition resulted in up-regulation. In addition, LPS was extracted and quantified on K. pneumoniae cells subjected to iron-replete and iron-limited conditions. The iron-limited condition increased the amount of LPS produced by K. pneumoniae. Finally, the expression levels of uge and the amount of the LPS were evaluated on a K. pneumoniae strain mutant for the fur gene. Compared to the wild-type, the strain with the fur gene knocked out presented a lower LPS amount and an unchanged expression of uge, regardless of the iron levels. CONCLUSIONS: Here, we show that iron deprivation led the K. pneumoniae cells to produce higher amount of LPS and that the Fur regulator modulates the expression of uge, a gene essential for LPS biosynthesis. Thus, our results indicate that iron availability modulates the LPS biosynthesis in K. pneumoniae through a Fur-dependent mechanism.

Asunto(s)

Proteínas Bacterianas , Regulación Bacteriana de la Expresión Génica , Hierro , Klebsiella pneumoniae , Lipopolisacáridos , Regiones Promotoras Genéticas , Proteínas Represoras , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/efectos de los fármacos , Lipopolisacáridos/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Hierro/metabolismo , Sitios de Unión , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo

RESUMEN

AIMS: Validating the docking procedure and maintaining the structural water molecules at HDAC8 catalytic site. BACKGROUND: Molecular docking simulations play a significant role in Computer-Aided Drug Design, contributing to the development of new molecules. To ensure the reliability of these simulations, a validation process called "self-docking or re-docking" is employed, focusing on the binding mode of a ligand co-crystallized with the protein of interest. OBJECTIVE: In this study, several molecular docking studies were conducted using five X-ray structures of HDAC8-ligand complexes from the PDB. METHODS: Ligands initially complexed with HDAC8 were removed and re-docked onto the free protein, revealing a poor reproduction of the expected binding mode. In response to this, we observed that most HDAC8-ligand complexes contained one to two water molecules in the catalytic site, which were crucial for maintaining the cocrystallized ligand. RESULTS: These water molecules enhance the binding mode of the co-crystallized ligand by stabilizing the proteinligand complex through hydrogen bond interactions between ligand and water molecules. Notably, these interactions are lost if water molecules are removed, as is often done in classical docking methodologies. Considering this, molecular docking simulations were repeated, both with and without one or two conserved water molecules near Zn+2 in the catalytic cavity. Simulations indicated that replicating the native binding pose of co-crystallized ligands on free HDAC8 without these water molecules was challenging, showing greater coordinate displacements (RMSD) compared to those including conserved water molecules from crystals. CONCLUSION: The study highlighted the importance of conserved water molecules within the active site, as their presence significantly influenced the successful reproduction of the ligands' native binding modes. The results suggest an optimal molecular docking procedure for validating methods suitable for filtering new HDAC8 inhibitors for future experimental assays.

Asunto(s)

Antineoplásicos , Diseño de Fármacos , Inhibidores de Histona Desacetilasas , Histona Desacetilasas , Simulación del Acoplamiento Molecular , Proteínas Represoras , Agua , Histona Desacetilasas/metabolismo , Histona Desacetilasas/química , Agua/química , Humanos , Antineoplásicos/química , Antineoplásicos/farmacología , Ligandos , Proteínas Represoras/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/química , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Estructura Molecular , Relación Estructura-Actividad , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X

RESUMEN

The MC1R protein is a receptor found in melanocytes that plays a role in melanin synthesis. Mutations in this protein can impact hair color, skin tone, tanning ability, and increase the risk of skin cancer. The MC1R protein is activated by the alpha-melanocyte-stimulating hormone (α-MSH). Previous studies have shown that mutations affect the interaction between MC1R and α-MSH; however, the mechanism behind this process is poorly understood. Our study aims to shed light on this mechanism using molecular dynamics (MD) simulations to analyze the Asp84Glu and Asp294His variants. We simulated both the wild-type (WT) protein and the mutants with and without ligand. Our results reveal that mutations induce unique conformations during state transitions, hindering the switch between active and inactive states and decreasing cellular levels of cAMP. Interestingly, Asp294His showed increased ligand affinity but decreased protein activity, highlighting that tighter binding does not always lead to increased activation. Our study provides insights into the molecular mechanisms underlying the impact of MC1R mutations on protein activity.

Asunto(s)

AMP Cíclico , Mutación , Receptor de Melanocortina Tipo 1 , alfa-MSH , Humanos , alfa-MSH/química , alfa-MSH/metabolismo , alfa-MSH/genética , Sitios de Unión , AMP Cíclico/metabolismo , AMP Cíclico/química , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Receptor de Melanocortina Tipo 1/genética , Receptor de Melanocortina Tipo 1/química , Receptor de Melanocortina Tipo 1/metabolismo

RESUMEN

LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists, and the phorbol ester-induced LPA3 internalization. Pitstop 2 (clathrin heavy chain inhibitor) markedly reduced LPA-induced receptor internalization; in contrast, phorbol ester-induced internalization was only delayed. LPA induced rapid ß-arrestin-LPA3 receptor association. The agonist and the phorbol ester-induced marked LPA3 receptor phosphorylation, and phosphorylation sites were detected using mass spectrometry. Phosphorylated residues were detected in the intracellular loop 3 (S221, T224, S225, and S229) and in the carboxyl terminus (S321, S325, S331, T333, S335, Y337, and S343). Interestingly, phosphorylation sites are within sequences predicted to constitute ß-arrestin binding sites. These data provide insight into LPA3 receptor signaling and regulation.

Asunto(s)

Lisofosfolípidos , Receptores del Ácido Lisofosfatídico , Transducción de Señal , Humanos , beta-Arrestinas/metabolismo , Sitios de Unión , Señalización del Calcio , Células HEK293 , Lisofosfolípidos/metabolismo , Fosforilación , Receptores del Ácido Lisofosfatídico/metabolismo

RESUMEN

CONTEXT: SARS-CoV-2, responsible for COVID-19, has led to over 500 million infections and more than 6 million deaths globally. There have been limited effective treatments available. The study aims to find a drug that can prevent the virus from entering host cells by targeting specific sites on the virus's spike protein. METHOD: We examined 13,397 compounds from the Malaria Box library against two specific sites on the spike protein: the receptor-binding domain (RBD) and a predicted cryptic pocket. Using virtual screening, molecular docking, molecular dynamics, and MMPBSA techniques, they evaluated the stability of two compounds. TCMDC-124223 showed high stability and binding energy in the RBD, while TCMDC-133766 had better binding energy in the cryptic pocket. The study also identified that the interacting residues are conserved, which is crucial for addressing various virus variants. The findings provide insights into the potential of small molecules as drugs against the spike protein.

Asunto(s)

Antivirales , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , SARS-CoV-2/efectos de los fármacos , Humanos , Sitios de Unión , Antivirales/química , Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , Unión Proteica , Dominios Proteicos , COVID-19/virología , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología

RESUMEN

The neural plate border (NPB) of vertebrate embryos is segregated from the neural plate (NP) and epidermal regions, and comprises an intermingled group of progenitors with multiple fate potential. Recent studies have shown that, during the gastrula stage, TFAP2A acts as a pioneer factor in remodeling the epigenetic landscape required to activate components of the NPB induction program. Here, we show that chick Tfap2a has two highly conserved binding sites for miR-137, and both display a reciprocal expression pattern at the NPB and NP, respectively. In addition, ectopic miR-137 expression reduced TFAP2A, whereas its functional inhibition expanded their territorial distribution overlapping with PAX7. Furthermore, we demonstrate that loss of the de novo DNA methyltransferase DNMT3A expanded miR-137 expression to the NPB. Bisulfite sequencing revealed a markedly elevated presence of non-canonical CpH methylation within the miR-137 promoter region when comparing NPB and NP samples. Our findings show that miR-137 contributes to the robustness of NPB territorial restriction in vertebrate development.

Asunto(s)

Metilación de ADN , Regulación del Desarrollo de la Expresión Génica , MicroARNs , Placa Neural , Factor de Transcripción AP-2 , Animales , MicroARNs/genética , MicroARNs/metabolismo , Embrión de Pollo , Metilación de ADN/genética , Placa Neural/metabolismo , Placa Neural/embriología , Factor de Transcripción AP-2/metabolismo , Factor de Transcripción AP-2/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A/metabolismo , Regiones Promotoras Genéticas/genética , Sitios de Unión

RESUMEN

Light chain amyloidosis is a conformational disease caused by the abnormal proliferation and deposition of antibody light chains as amyloid fibers in organs and tissues. The effect of Cu(II) binding to the model recombinant protein 6aJL2-R24G was previously characterized in our group, and we found an acceleration of the aggregation kinetics of the protein. In this study, in order to confirm the Cu(II) binding sites, histidine variants of 6aJL2-R24G were prepared and the effects of their interaction with Cu(II) were analyzed by circular dichroism, fluorescence spectroscopy, isothermal calorimetry titrations, and molecular dynamics simulations. Confirming our earlier work, we found that His8 and His99 are the highest affinity Cu(II) binding sites, and that Cu(II) binding to both sites is a cooperative event.

Asunto(s)

Cobre , Histidina , Unión Proteica , Cobre/metabolismo , Cobre/química , Histidina/química , Histidina/metabolismo , Humanos , Sitios de Unión , Simulación de Dinámica Molecular , Cadenas Ligeras de Inmunoglobulina/metabolismo , Cadenas Ligeras de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/química , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/metabolismo , Amiloidosis de Cadenas Ligeras de las Inmunoglobulinas/genética , Amiloidosis/metabolismo , Amiloidosis/genética , Cinética

RESUMEN

PhoX is a high-affinity phosphate binding protein, present in Xanthomonas citri, a phytopathogen responsible for the citrus canker disease. Performing molecular dynamics simulations and different types of computational analyses, we study the molecular mechanisms at play in relation to phosphate binding, revealing the global functioning of the protein: PhoX naturally oscillates along its global normal modes, which allow it to explore both bound and unbound conformations, eventually attracting a nearby negative phosphate ion to the highly positive electrostatic potential on its surface, particularly close to the binding pocket. There, several hydrogen bonds are formed with the two main domains of the structure. Phosphate creates, in this way, a strong bridge that connects the domains, keeping itself between them, in a tight closed conformation, explaining its high binding affinity.

Asunto(s)

Proteínas Bacterianas , Simulación de Dinámica Molecular , Fosfatos , Xanthomonas , Fosfatos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Unión Proteica , Proteínas de Unión a Fosfato/metabolismo , Enlace de Hidrógeno , Sitios de Unión , Electricidad Estática

RESUMEN

Melatonin receptors MT1 and MT2 are G protein-coupled receptors that mediate the effects of melatonin, a hormone involved in circadian rhythms and other physiological functions. Understanding the molecular interactions between these receptors and their ligands is crucial for developing novel therapeutic agents. In this study, we used molecular docking, molecular dynamics simulations, and quantum mechanics calculation to investigate the binding modes and affinities of three ligands: melatonin (MLT), ramelteon (RMT), and 2-phenylmelatonin (2-PMT) with both receptors. Based on the results, we identified key amino acids that contributed to the receptor-ligand interactions, such as Gln181/194, Phe179/192, and Asn162/175, which are conserved in both receptors. Additionally, we described new meaningful interactions with Gly108/Gly121, Val111/Val124, and Val191/Val204. Our results provide insights into receptor-ligand recognition's structural and energetic determinants and suggest potential strategies for designing more optimized molecules. This study enhances our understanding of receptor-ligand interactions and offers implications for future drug development.

Asunto(s)

Melatonina , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Receptor de Melatonina MT1 , Receptor de Melatonina MT2 , Melatonina/metabolismo , Melatonina/química , Receptor de Melatonina MT2/metabolismo , Receptor de Melatonina MT2/química , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT1/química , Humanos , Ligandos , Teoría Cuántica , Sitios de Unión , Indenos/química , Indenos/metabolismo

RESUMEN

The spike protein determines the host-range specificity of coronaviruses. In particular, the Receptor-Binding Motif in the spike protein from SARS-CoV-2 contains the amino acids involved in molecular recognition of the host Angiotensin Converting Enzyme 2. Therefore, to understand how SARS-CoV-2 acquired its capacity to infect humans it is necessary to reconstruct the evolution of this important motif. Early during the pandemic, it was proposed that the SARS-CoV-2 Receptor-Binding Domain was acquired via recombination with a pangolin infecting coronavirus. This proposal was challenged by an alternative explanation that suggested that the Receptor-Binding Domain from SARS-CoV-2 did not originated via recombination with a coronavirus from a pangolin. Instead, this alternative hypothesis proposed that the Receptor-Binding Motif from the bat coronavirus RaTG13, was acquired via recombination with an unidentified coronavirus. And as a consequence of this event, the Receptor-Binding Domain from the pangolin coronavirus appeared as phylogenetically closer to SARS-CoV-2. Recently, the genomes from coronaviruses from Cambodia (bat_RShST182/200) and Laos (BANAL-20-52/103/247) which are closely related to SARS-CoV-2 were reported. However, no detailed analysis of the evolution of the Receptor-Binding Motif from these coronaviruses was reported. Here we revisit the evolution of the Receptor-Binding Domain and Motif in the light of the novel coronavirus genome sequences. Specifically, we wanted to test whether the above coronaviruses from Cambodia and Laos were the source of the Receptor-Binding Domain from RaTG13. We found that the Receptor-Binding Motif from these coronaviruses is phylogenetically closer to SARS-CoV-2 than to RaTG13. Therefore, the source of the Receptor-Binding Domain from RaTG13 is still unidentified. In accordance with previous studies, our results are consistent with the hypothesis that the Receptor-Binding Motif from SARS-CoV-2 evolved by vertical inheritance from a bat-infecting population of coronaviruses.

Asunto(s)

Evolución Molecular , Filogenia , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/química , Humanos , Animales , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/química , Secuencias de Aminoácidos , COVID-19/virología , Unión Proteica , Betacoronavirus/genética , Quirópteros/virología , Pangolines/virología , Sitios de Unión , Genoma Viral , Receptores Virales/metabolismo , Receptores Virales/genética , Receptores Virales/química