RESUMEN
The growth of make-on-demand libraries in recent years has provided completely new possibilities for virtual screening for discovering new hit compounds with specific and favorable properties. However, since these libraries now contain billions of compounds, screening them using traditional methods such as molecular docking has become challenging and requires substantial computational resources. Thus, to take real advantage of the new possibilities introduced by the make-on-demand libraries, different methods have been proposed to accelerate the screening process and prioritize molecules for evaluation. Here, we introduce SpaceGA, a genetic algorithm that leverages the rapid similarity search tool SpaceLight (Bellmann, L.; Penner, P.; Rarey, M. Topological similarity search in large combinatorial fragment spaces. J. Chem. Inf. Model. 2021, 61, 238-251). to constrain the optimization process to accessible compounds within desired combinatorial libraries. As shown herein, SpaceGA is able to efficiently identify molecules with desired properties from trillions of synthesizable compounds by enumerating and evaluating only a small fraction of them. On this basis, SpaceGA represents a promising new tool for accelerating and simplifying virtual screens of ultralarge combinatorial databases.
RESUMEN
Nτ -methylation of His73 in actin by histidine methyltransferase SETD3 plays an important role in stabilising actin filaments in eukaryotes. Mutations in actin and overexpression of SETD3 have been related to human diseases, including cancer. Here, we investigated the importance of Trp79 in ß-actin on productive human SETD3 catalysis. Substitution of Trp79 in ß-actin peptides by its chemically diverse analogues reveals that the hydrophobic Trp79 binding pocket modulates the catalytic activity of SETD3, and that retaining a bulky and hydrophobic amino acid at position 79 is important for efficient His73 methylation by SETD3. Molecular dynamics simulations show that the Trp79 binding pocket of SETD3 is ideally shaped to accommodate large and hydrophobic Trp79, contributing to the favourable release of water molecules upon binding. Our results demonstrate that the distant Trp79 binding site plays an important role in efficient SETD3 catalysis, contributing to the identification of new SETD3 substrates and the development of chemical probes targeting the biomedically important SETD3.
Asunto(s)
Actinas , Metiltransferasas , Humanos , Metiltransferasas/metabolismo , Actinas/química , Histona Metiltransferasas/química , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , Histidina/química , Metilación , CatálisisRESUMEN
Histone lysine methacrylation and crotonylation are epigenetic marks that play important roles in human gene regulation. Here, we explore the molecular recognition of histone H3 peptides possessing methacryllysine and crotonyllysine at positions 18 and 9 (H3K18 and H3K9) by the AF9 YEATS domain. Our binding studies demonstrate that the AF9 YEATS domain displays a higher binding affinity for histones possessing crotonyllysine than the isomeric methacryllysine, indicating that AF9 YEATS distinguishes between the two regioisomers. Molecular dynamics simulations reveal that the crotonyllysine/methacryllysine-mediated desolvation of the AF9 YEATS domain provides an important contribution to the recognition of both epigenetic marks. These results provide important knowledge for the development of AF9 YEATS inhibitors, an area of biomedical interest.
Asunto(s)
Regulación de la Expresión Génica , Histonas , Proteínas Nucleares , Humanos , Histonas/metabolismo , Simulación de Dinámica Molecular , Dominios Proteicos , Proteínas Nucleares/metabolismoRESUMEN
Much effort has been invested in the search for modulators of membrane transport proteins such as P-glycoprotein (P-gp) to improve drug bioavailability and reverse multidrug resistance in cancer. Nonionic surfactants, a class of pharmaceutical excipients, are known to inhibit such proteins, but knowledge about the exact mechanism of this inhibition is scarce. Here, we perform multiscale molecular dynamics simulations of one of these surfactants, polysorbate 20 (PS20), to reveal the behavior of such compounds on the molecular level and thereby discover the molecular mechanism of the P-gp inhibition. We show that the amphiphilic headgroup of PS20 is too hydrophobic to partition in the water phase, which drives the binding of PS20 to the amphiphilic drug-binding domain of P-gp and thereby causes the inhibition of the protein. Based on our findings, we conclude that PS20 primarily inhibits P-gp through direct binding to the drug-binding domain (DBD) from the extracellular leaflet.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Polisorbatos , Subfamilia B de Transportador de Casetes de Unión a ATP , Excipientes/química , Polisorbatos/química , Tensoactivos/químicaRESUMEN
SETD3-catalysed N3-methylation of His73 in ß-actin plays a key role in stabilisation of actin filaments in the metazoan cells. Overexpression and/or dysregulation of SETD3 is associated with several human pathologies, including cancer. Here, we examined the role of the Ile71 residue in ß-actin on human SETD3 catalysis. Substitution of Ile71 in ß-actin peptides by its natural and unnatural mimics reveals that the 'secondary' Ile71 binding pocket modulates the substrate efficiency of ß-actin. Our enzymatic work demonstrates that human SETD3 can accommodate structurally diverse hydrophobic side chains in its Ile71 binding pocket, providing clear limits of the size and shape of Ile analogues. Water thermodynamics calculations reveal that the Ile71 pocket is occupied by high-energy water molecules, that are released upon the Ile71 binding, contributing favourably to the SETD3-ßA complex formation. The work highlights that the hydrophobic Ile71 binding site plays an essential role in SETD3 catalysis, contributing to an ongoing effort in the design and development of chemical probes targeting SETD3.
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Actinas/metabolismo , Histona Metiltransferasas/metabolismo , Isoleucina/metabolismo , Actinas/química , Biocatálisis , Histidina/química , Histidina/metabolismo , Humanos , Isoleucina/química , Modelos Moleculares , Conformación MolecularRESUMEN
Epigenetic readout of the combinatorial posttranslational modification comprised of trimethyllysine and asymmetric dimethylarginine (H3K4me3R8me2a) takes place via biomolecular recognition of tandem Tudor-domain-containing protein Spindlin1. Through comparative thermodynamic data and molecular dynamics simulations, we sought to explore the binding scope of asymmetric dimethylarginine mimics by Spindlin1. Herein, we provide evidence that the biomolecular recognition of H3K4me2R8me2a is not significantly affected when R8me2a is replaced by dimethylarginine analogues, implying that the binding of K4me3 provides the major binding contribution. High-energy water molecules inside both aromatic cages of the ligand binding sites contribute to the reader-histone association upon displacement by histone peptide, with the K4me3 hydration site being lower in free energy due to a flip of Trp151.
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Arginina/análogos & derivados , Proteínas de Ciclo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfoproteínas/metabolismo , Dominio Tudor , Arginina/química , Arginina/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Histonas/metabolismo , Humanos , Proteínas Asociadas a Microtúbulos/química , Simulación de Dinámica Molecular , Fosfoproteínas/química , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of lipid binding to yeast NPC2. We find that NPC2 binds fluorescence- and spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole.
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Proteínas Portadoras/metabolismo , Metabolismo de los Lípidos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Proteínas Portadoras/química , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Intracellular transport of cholesterol and related sterols relies to a large degree on nonvesicular mechanisms, which are only partly understood at the molecular level. Aster proteins belonging to the Lam family of sterol transfer proteins have recently been identified as important catalysts of nonvesicular sterol exchange between the plasma membrane (PM) and endoplasmic reticulum (ER). Here, we used a range of computational tools to study the molecular mechanisms underlying sterol binding as well as multisterol ligand specificity of Aster-A. Our study focused primarily on gaining atomistic insight into the bound ligand-protein complex and was, on this basis, performed in the absence of any membrane. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations provide a rationale for the experimentally found ranking of binding affinities of various sterols to Aster-A. In particular, the polarity of the sterols and the length of their alkyl chain could be identified as being critical determinants of ligand affinity. A Gibbs free energy decomposition identified a charged residue, Glu444, at the base of the binding pose as an important control point for sterol binding. Removing its net charge via protonation was found to cause significant changes to the environment surrounding this residue. In addition, the protonation of Glu444 was found to be paralleled by a large redistribution of molecular flexibility in the Aster domain. This finding was supplemented by multiple branched adaptive steered molecular dynamics (MB-ASMD) simulations by which we defined a possible molecular path for sterol release and demonstrated the importance of Glu444 in this process.
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Membrana Celular , Retículo Endoplásmico , Unión Proteica , Esteroles , Membrana Celular/metabolismo , Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Esteroles/metabolismoRESUMEN
The Nτ-His73 methylation of ß-actin by histidine methyltransferase SETD3 is required for the integrity of the cellular cytoskeleton. Modulation of SETD3 activity in human cells facilitates cancer-like changes to the cell phenotype. SETD3 binds ß-actin in an extended conformation, with a conserved bend-like motif surrounding His73. Here, we report on the catalytic specificity of SETD3 towards i, i + 3 stapled ß-actin peptides possessing a limited conformational freedom surrounding the His73 substrate residue via positions Glu72 and Ile75. Stapled ß-actin peptides were observed to be methylated less efficiently than the linear ß-actin peptide. None of the stapled ß-actin peptides efficiently inhibited the SETD3-catalyzed Nτ-His73 methylation reaction. Molecular dynamics simulations demonstrated that the unbound and SETD3-bound ß-actin peptides display different backbone flexibility and bend-like conformations, highlighting their important role in substrate binding and catalysis. Overall, these findings suggest that reduced backbone flexibility of ß-actin prevents the formation of optimal protein-peptide interactions between the enzyme and substrate, highlighting that the backbone flexibility needs to be considered when designing ß-actin-based probes and inhibitors of biomedically important SETD3.
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Actinas , Simulación de Dinámica Molecular , Péptidos , Actinas/metabolismo , Actinas/química , Especificidad por Sustrato , Humanos , Metilación , Péptidos/química , Péptidos/metabolismo , Histidina/metabolismo , Histidina/química , Unión Proteica , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/química , Histona Metiltransferasas/metabolismo , Histona Metiltransferasas/química , Conformación ProteicaRESUMEN
P-glycoprotein (P-gp), a membrane transport protein overexpressed in certain drug-resistant cancer cells, has been the target of numerous drug discovery projects aimed at overcoming drug resistance in cancer. Most characterized P-gp inhibitors bind at the large hydrophobic drug binding domain (DBD), but none have yet attained regulatory approval. In this study, we explored the potential of designing inhibitors that target the nucleotide binding domains (NBDs), by computationally screening a large library of 2.6 billion synthesizable molecules, using a combination of machine learning-guided molecular docking and molecular dynamics (MD). 14 of the computationally best-scoring molecules were subsequently tested for their ability to inhibit P-gp mediated calcein-AM efflux. In total, five diverse compounds exhibited inhibitory effects in the calcein-AM assay without displaying toxicity. The activity of these compounds was confirmed by their ability to decrease the verapamil-stimulated ATPase activity of P-gp in a subsequent assay. The discovery of these five novel P-gp inhibitors demonstrates the potential of in-silico screening in drug discovery and provides a new stepping point towards future potent P-gp inhibitors.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Nucleótidos , Simulación del Acoplamiento Molecular , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Nucleótidos/metabolismo , Resistencia a AntineoplásicosRESUMEN
Non-haem Fe(ii) and 2-oxoglutarate (2OG) dependent oxygenases catalyse oxidation of multiple proteins in organisms ranging from bacteria to humans. We describe studies on the substrate selectivity and inhibition of the human ribosomal oxygenases (ROX) MINA53 and NO66, members of the JmjC 2OG oxygenase subfamily, which catalyse C-3 hydroxylation of histidine residues in Rpl27a and Rpl8, respectively. Assays with natural and unnatural histidine analogues incorporated into Rpl peptides provide evidence that MINA53 and NO66 have narrow substrate selectivities compared to some other human JmjC hydroxylases, including factor inhibiting HIF and JMJD6. Notably, the results of inhibition assays with Rpl peptides containing histidine analogues with acyclic side chains, including Asn, Gln and homoGln, suggest the activities of MINA53/NO66, and by implication related 2OG dependent protein hydroxylases/demethylases, might be regulated in vivo by competition with non-oxidised proteins/peptides. The inhibition results also provide avenues for development of inhibitors selective for MINA53 and NO66.
RESUMEN
SETD3 was recently identified as the histidine methyltransferase responsible for N3 -methylation of His73 of ß-actin in humans. Overexpression of SETD3 is associated with several diseases, including breast cancer. Here, we report a development of actin-based peptidomimetics as inhibitors of recombinantly expressed human SETD3. Substitution of His73 by simple natural and unnatural amino acids led to selected ß-actin peptides with high potency against SETD3 in MALDI-TOF MS assays. The selenomethionine-containing ß-actin peptide was found to be the most potent SETD3 inhibitor (IC50 =161â nM). Supporting our inhibition assays, a combination of computational docking and molecular dynamics simulations revealed that the His73 binding pocket for ß-actin in SETD3 is rigid and accommodates the inhibitor peptides with similar binding modes. Collectively, our work demonstrates that actin-based peptidomimetics can act as potent SETD3 inhibitors and provide a basis for further development of highly potent and selective inhibitors of SETD3.
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Actinas/farmacología , Inhibidores Enzimáticos/farmacología , Histona Metiltransferasas/antagonistas & inhibidores , Péptidos/farmacología , Actinas/síntesis química , Actinas/química , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Histona Metiltransferasas/aislamiento & purificación , Histona Metiltransferasas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Péptidos/síntesis química , Péptidos/química , Relación Estructura-ActividadRESUMEN
Oxysterols are cholesterol metabolites with multiple functions in controlling cellular homeostasis. In particular, 27-hydroxycholesterol (27-OH-Chol) has been shown to regulate a variety of physiological functions, but little is known about its uptake, intracellular trafficking, and efflux from cells. This is largely due to a lack of suitable analogs of 27-OH-Chol, which mimic this oxysterol closely. Here, we present the intrinsically fluorescent 27-hydroxy-cholestatrienol (27-OH-CTL), which differs from 27-OH-Chol only by having two additional double bonds in the steroid ring system. Based on molecular dynamics (MD) simulations, we show that 27-OH-CTL possesses almost identical membrane properties compared to 27-OH-Chol. By comparative imaging of 27-OH-CTL and of the cholesterol analogue cholestatrienol (CTL) in living cells, we assess the impact of a single hydroxy group on sterol trafficking. We find that human fibroblasts take up more CTL than 27-OH-CTL, but efflux the oxysterol analogue more efficiently. For both sterols, efflux includes shedding of vesicles from the plasma membrane. Intracellular, 27-OH-CTL accumulates primarily in lipid droplets (LDs), while CTL is mostly found in endosomes and lysosomes. Using fluorescence recovery after photobleaching (FRAP), we find for both sterols a rapidly exchanging pool, which moves orders of magnitude faster than sterol containing vesicles and LDs. In summary, by applying a new fluorescent derivative of 27-OH-Chol we demonstrate that human cells can distinguish sterols based on a single hydroxy group in the side chain, resulting in different transport itineraries, dynamics, and efflux kinetics. Both intrinsically fluorescent cholesterol and oxysterol analogues show rapid non-vesicular transport in human fibroblasts.