RESUMO
Enhancing virtual screening enrichment has become an urgent problem in computational chemistry, driven by increasingly large databases of commercially available compounds, without a commensurate drop in in vitro screening costs. Docking these large databases is possible with cloud-scale computing. However, rapid docking necessitates compromises in scoring, often leading to poor enrichment and an abundance of false positives in docking results. This work describes a new scoring function composed of two parts - a knowledge-based component that predicts the probability of a particular atom type being in a particular receptor environment, and a tunable weight matrix that converts the probability predictions into a dimensionless score suitable for virtual screening enrichment. This score, the FitScore, represents the compatibility between the ligand and the binding site and is capable of a high degree of enrichment across standardized docking test sets.
Assuntos
Aprendizado de Máquina , Simulação de Acoplamento Molecular , Ligantes , Sítios de Ligação , Humanos , Ligação Proteica , Proteínas/química , Proteínas/metabolismo , Software , Avaliação Pré-Clínica de Medicamentos/métodos , Descoberta de Drogas/métodosRESUMO
Pfizer's Crystal Structure Database (CSDB) is a key enabling technology that allows scientists on structure-based projects rapid access to Pfizer's vast library of in-house crystal structures, as well as a significant number of structures imported from the Protein Data Bank. In addition to capturing basic information such as the asymmetric unit coordinates, reflection data, and the like, CSDB employs a variety of automated methods to first ensure a standard level of annotations and error checking, and then to add significant value for design teams by processing the structures through a sequence of algorithms that prepares the structures for use in modeling. The structures are made available, both as the original asymmetric unit as submitted, as well as the final prepared structures, through REST-based web services that are consumed by several client desktop applications. The structures can be searched by keyword, sequence, submission date, ligand substructure and similarity search, and other common queries.
Assuntos
Algoritmos , Bases de Dados de Proteínas , Humanos , LigantesRESUMO
Several protein engineering approaches were combined to optimize the selectivity and activity of Vibrio fluvialis aminotransferase (Vfat) for the synthesis of (3S,5R)-ethyl 3-amino-5-methyloctanoate; a key intermediate in the synthesis of imagabalin, an advanced candidate for the treatment of generalized anxiety disorder. Starting from wild-type Vfat, which had extremely low activity catalyzing the desired reaction, we engineered an improved enzyme with a 60-fold increase in initial reaction velocity for transamination of (R)-ethyl 5-methyl 3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate. To achieve this, <450 variants were screened, which allowed accurate assessment of enzyme performance using a low-throughput ultra performance liquid chromatography assay. During the course of this work, crystal structures of Vfat wild type and an improved variant (Vfat variant r414) were solved and they are reported here for the first time. This work also provides insight into the critical residues for substrate specificity for the transamination of (R)-ethyl 5-methyl 3-oxooctanoate and structurally related ß-ketoesters.
Assuntos
Aminoácidos/metabolismo , Caprilatos/metabolismo , Engenharia de Proteínas/métodos , Transaminases/genética , Transaminases/metabolismo , Vibrio/enzimologia , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Transaminases/químicaRESUMO
A hierarchical computational approach is used to identify the engineered binding-site cavity at the remodeled intermolecular interface between the mutants of human growth hormone (hGH) and the extracellular domain of its receptor (hGHbp). Multiple docking simulations are conducted with the remodeled hGH-hGHbp complex for a panel of potent benzimidazole-containing inhibitors that can restore the binding affinity of the wild-type complex, and for a set of known nonactive small molecules that contain different heterocyclic motifs. Structural clustering of ligand-bound conformations and binding free-energy calculations, using the AMBER force field and a continuum solvation model, can rapidly locate and screen numerous ligand-binding modes on the protein surface and detect the binding-site hot spot at the intermolecular interface. Structural orientation of the benzimidazole motif in the binding-site cavity closely mimics the position of the hot spot residue W104 in the crystal structure of the wild-type complex, which is recognized as an important structural requirement for restoring binding affinity. Despite numerous pockets on the protein surface of the mutant hGH-hGHbp complex, the binding-site cavity presents the energetically favorable hot spot for the benzimidazole-containing inhibitors, whereas for a set of nonactive molecules, the lowest energy ligand conformations do not necessarily bind in the engineered cavity. The results reveal a dominant role of the intermolecular van der Waals interactions in providing favorable ligand-protein energetics in the redesigned interface, in agreement with the experimental and computational alanine scanning of the hGH-hGHbp complex.
Assuntos
Hormônio do Crescimento Humano/química , Modelos Moleculares , Receptores da Somatotropina/química , Alanina/genética , Benzimidazóis/química , Sítios de Ligação , Biologia Computacional , Simulação por Computador , Hormônio do Crescimento Humano/genética , Hormônio do Crescimento Humano/metabolismo , Humanos , Ligantes , Mutagênese , Mutação , Estrutura Terciária de Proteína , Receptores da Somatotropina/genética , Receptores da Somatotropina/metabolismoRESUMO
A microscopic study of functional disorder-order folding transitions coupled to binding is performed for the p27 protein, which derives a kinetic advantage from the intrinsically disordered unbound form on binding with the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex. Hierarchy of structural loss during p27 coupled unfolding and unbinding is simulated by using high-temperature Monte Carlo simulations initiated from the crystal structure of the tertiary complex. Subsequent determination of the transition-state ensemble and the proposed atomic picture of the folding mechanism coupled to binding provide a microscopic rationale that reconciles the initiation recruitment of p27 at the cyclin A docking site with the kinetic benefit for a disordered alpha-helix in the unbound form of p27. The emerging structural polarization in the ensemble of unfolding/unbinding trajectories and in the computationally determined transition-state ensemble is not determined by the intrinsic folding preferences of p27 but rather is attributed to the topological requirements of the native intermolecular interface to order beta-hairpin and beta-strand of p27 that could be critical for nucleating rapid folding transition coupled to binding. In agreement with the experimental data, the disorder-order folding transition for p27 is largely determined by the functional requirement to form a specific intermolecular interface that ultimately dictates the folding mechanism and overwhelms any local folding preferences for creating a stable alpha-helix in the p27 structure before overcoming the major free energy barrier.
Assuntos
Proteínas/química , Modelos Moleculares , Método de Monte Carlo , Ligação Proteica , Dobramento de Proteína , Proteínas/metabolismoRESUMO
Monte Carlo simulations of molecular recognition at the consensus binding site of the constant fragment (Fc) of human immunoglobulin G (Ig) protein have been performed to analyze structural and thermodynamic aspects of binding for the 13-residue cyclic peptide DCAWHLGELVWCT. The energy landscape analysis of a hot spot at the intermolecular interface using alanine scanning and equilibrium-simulated tempering dynamics with the simplified, knowledge-based energy function has enabled the role of the protein hot spot residues in providing the thermodynamic stability of the native structure to be determined. We have found that hydrophobic interactions between the peptide and the Met-252, Ile-253, His-433, and His-435 protein residues are critical to guarantee the thermodynamic stability of the crystallographic binding mode of the complex. Binding free energy calculations, using a molecular mechanics force field and a solvation energy model, combined with alanine scanning have been conducted to determine the energetic contribution of the protein hot spot residues in binding affinity. The conserved Asn-434, Ser-254, and Tyr-436 protein residues contribute significantly to the binding affinity of the peptide-protein complex, serving as an energetic hot spot at the intermolecular interface. The results suggest that evolutionary conserved hot spot protein residues at the intermolecular interface may be partitioned in fulfilling thermodynamic stability of the native binding mode and contributing to the binding affinity of the complex.
Assuntos
Fragmentos Fc das Imunoglobulinas/química , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Método de Monte Carlo , Peptídeos Cíclicos/metabolismo , Peptídeos/metabolismo , Alanina/química , Sítios de Ligação , Simulação por Computador , Sequência Consenso , Humanos , Regiões Constantes de Imunoglobulina/química , Regiões Constantes de Imunoglobulina/metabolismo , Modelos Moleculares , Peptídeos/química , Peptídeos Cíclicos/química , Ligação Proteica , Conformação Proteica , TermodinâmicaRESUMO
The energy landscape approach has contributed to recent progress in understanding the complexity and simplicity of ligand-macromolecule interactions. Significant advances in computational structure prediction of ligand-protein complexes have been made using approaches that include the effects of protein flexibility and incorporate a hierarchy of energy functions. The results suggest that the complexity of structure prediction in molecular recognition may be determined by low-resolution properties of the underlying binding energy landscapes and by the nature of the energy funnels near the native structures of the complexes.
Assuntos
Ligantes , Substâncias Macromoleculares , Modelos Biológicos , Modelos MolecularesRESUMO
Structure and energetics of the Src Src Homology 2 (SH2) domain binding with the recognition phosphopeptide pYEEI and its mutants are studied by a hierarchical computational approach. The proposed structure prediction strategy includes equilibrium sampling of the peptide conformational space by simulated tempering dynamics with the simplified, knowledge-based energy function, followed by structural clustering of the resulting conformations and binding free energy evaluation of a single representative from each cluster, a cluster center. This protocol is robust in rapid screening of low-energy conformations and recovers the crystal structure of the pYEEI peptide. Thermodynamics of the peptide-SH2 domain binding is analyzed by computing the average energy contributions over conformations from the clusters, structurally similar to the predicted peptide bound structure. Using this approach, the binding thermodynamics for a panel of studied peptides is predicted in a better agreement with the experiment than previously suggested models. However, the overall correlation between computed and experimental binding affinity remains rather modest. The results of this study show that small differences in binding free energies between the Ala and Gly mutants of the pYEEI peptide are considerably more difficult to predict than the structure of the bound peptides, indicating that accurate computational prediction of binding affinities still remains a major methodological and technical challenge.