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1.
J Colloid Interface Sci ; 581(Pt A): 218-225, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771733

RESUMO

We used the Surface Forces Apparatus to elucidate the interaction mechanism between grafted 5 heptad-long peptides engineered to spontaneously form a heterodimeric coiled-coil complex. The results demonstrated that when intimate contact between peptides is reached, binding occurs first via weakly interacting but more mobile distal heptads, suggesting an induced-fit association process. Precise control of the distance between peptide-coated surfaces allowed to quantitatively monitor the evolution of their biding energy. The binding energy of the coiled-coil complex increased in a stepwise fashion rather than monotonically with the overlapping distance, each step corresponding to the interaction between a quantized number of heptads. Surface forces data were corroborated to surface plasmon resonance measurements and molecular dynamics simulations and allowed the calculation of the energetic contribution of each heptad within the coiled-coil complex.

2.
MAbs ; 12(1): 1682866, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31777319

RESUMO

Recent development of monoclonal antibodies as mainstream anticancer agents demands further optimization of their safety for use in humans. Potent targeting and/or effector activities on normal tissues is an obvious toxicity concern. Optimization of specific tumor targeting could be achieved by taking advantage of the extracellular acidity of solid tumors relative to normal tissues. Here, we applied a structure-based computational approach to engineer anti-human epidermal growth factor receptor 2 (Her2) antibodies with selective binding in the acidic tumor microenvironment. We used an affinity maturation platform in which dual-pH histidine-scanning mutagenesis was implemented for pH selectivity optimization. Testing of a small set of designs for binding to the recombinant Her2 ectodomain led to the identification of antigen-binding fragment (Fab) variants with the desired pH-dependent binding behavior. Binding selectivity toward acidic pH was improved by as much as 25-fold relative to the parental bH1-Fab. In vitro experiments on cells expressing intact Her2 confirmed that designed variants formatted as IgG1/k full-size antibodies have high affinity and inhibit the growth of tumor spheroids at a level comparable to that of the benchmark anti-Her2 antibody trastuzumab (Herceptin®) at acidic pH, whereas these effects were significantly reduced at physiological pH. In contrast, both Herceptin and the parental bH1 antibody exhibited strong cell binding and growth inhibition irrespective of pH. This work demonstrates the feasibility of computational optimization of antibodies for selective targeting of the acidic environment such as that found in many solid tumors.

3.
MAbs ; 11(7): 1300-1318, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31318308

RESUMO

Solution stability is an important factor in the optimization of engineered biotherapeutic candidates such as monoclonal antibodies because of its possible effects on manufacturability, pharmacology, efficacy and safety. A detailed atomic understanding of the mechanisms governing self-association of natively folded protein monomers is required to devise predictive tools to guide screening and re-engineering along the drug development pipeline. We investigated pairs of affinity-matured full-size antibodies and observed drastically different propensities to aggregate from variants differing by a single amino-acid. Biophysical testing showed that antigen-binding fragments (Fabs) from the aggregating antibodies also reversibly associated with equilibrium dissociation constants in the low-micromolar range. Crystal structures (PDB accession codes 6MXR, 6MXS, 6MY4, 6MY5) and bottom-up hydrogen-exchange mass spectrometry revealed that Fab self-association occurs in a symmetric mode that involves the antigen complementarity-determining regions. Subtle local conformational changes incurred upon point mutation of monomeric variants foster formation of complementary polar interactions and hydrophobic contacts to generate a dimeric Fab interface. Testing of popular in silico tools generally indicated low reliabilities for predicting the aggregation propensities observed. A structure-aggregation data set is provided here in order to stimulate further improvements of in silico tools for prediction of native aggregation. Incorporation of intermolecular docking, conformational flexibility, and short-range packing interactions may all be necessary features of the ideal algorithm.

4.
Horm Cancer ; 10(1): 24-35, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30565014

RESUMO

The androgen-directed treatment of prostate cancer (PCa) is fraught with the recurrent profile of failed treatment due to drug resistance and must be addressed if we are to provide an effective therapeutic option. The most singular difficulty in the treatment of PCa is the failure to respond to classical androgen withdrawal or androgen blockade therapy, which often develops as the malignancy incurs genetic alterations and gain-of-function somatic mutations in the androgen receptor (AR). Physical cellular damaging therapeutic agents, such as radiation or activatable heat-generating transducers would circumvent classical "anti-functional" biological resistance, but to become ultimately effective would require directed application modalities. To this end, we have developed a novel AR-directed therapeutic agent by creating bivalent androgen hormone-AF-2 compounds that bind with high affinity to AR within cells. Here, we used molecular modeling and synthetic chemistry to create a number of compounds by conjugating 5α-dihydrotestosterone (DHT) to various AF-2 motif sequence peptides, through the use of a glycine and other spacer linkers. Our data indicates these compounds will bind to the AR in vitro and that altering the AF-2 peptide composition of the compound does indeed improve affinity for the AR. We also show that many of these bivalent compounds can readily pass through the plasma membrane and effectively compete against androgens alone.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Motivos de Aminoácidos , Antagonistas de Receptores de Andrógenos/farmacologia , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Cristalografia por Raios X , Di-Hidrotestosterona/farmacologia , Resistencia a Medicamentos Antineoplásicos , Glicina/metabolismo , Humanos , Concentração Inibidora 50 , Masculino , Simulação de Dinâmica Molecular , Mutação , Peptídeos/química , Próstata/metabolismo , Neoplasias da Próstata/terapia , Ligação Proteica
5.
Sci Rep ; 8(1): 17680, 2018 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-30518942

RESUMO

Conjugation of small molecules to proteins through N-hydroxysuccinimide (NHS) esters results in a random distribution of small molecules on lysine residues and the protein N-terminus. While mass spectrometry methods have improved characterization of these protein conjugates, it remains a challenge to quantify the occupancy at individual sites of conjugation. Here, we present a method using Tandem Mass Tags (TMT) that enabled the accurate and sensitive quantification of occupancy at individual conjugation sites in the NIST monoclonal antibody. At conjugation levels relevant to antibody drug conjugates in the clinic, site occupancy data was obtained for 37 individual sites, with average site occupancy data across 2 adjacent lysines obtained for an additional 12 sites. Thus, altogether, a measure of site occupancy was obtained for 98% of the available primary amines. We further showed that removal of the Fc-glycan on the NIST mAb increased conjugation at two specific sites in the heavy chain, demonstrating the utility of this method to identify changes in the susceptibility of individual sites to conjugation. This improved site occupancy data allowed calibration of a bi-parametric linear model for predicting the susceptibility of individual lysines to conjugation from 3D-structure based on their solvent exposures and ionization constants. Trained against the experimental data for lysines from the Fab fragment, the model provided accurate predictions of occupancies at lysine sites from the Fc region and the protein N-terminus (R2 = 0.76). This predictive model will enable improved engineering of antibodies for optimal labeling with fluorophores, toxins, or crosslinkers.


Assuntos
Anticorpos Monoclonais/química , Imunoconjugados/química , Lisina/análise , Succinimidas/química , Sequência de Aminoácidos , Esterificação , Modelos Moleculares , Espectrometria de Massas em Tandem
6.
J Chem Theory Comput ; 14(9): 4938-4947, 2018 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-30107730

RESUMO

Despite decades of development, protein-protein docking remains a largely unsolved problem. The main difficulties are the immense space spanned by the translational and rotational degrees of freedom and the prediction of the conformational changes of proteins upon binding. FFT is generally the preferred method to exhaustively explore the translation-rotation space at a fine grid resolution, albeit with the trade-off of approximating force fields with correlation functions. This work presents a direct search alternative that samples the states in Cartesian space at the same resolution and computational cost as standard FFT methods. Operating in real space allows the use of standard force field functional forms used in typical non-FFT methods as well as the implementation of strategies for focused exploration of conformational flexibility. Currently, a few misplaced side chains can cause docking programs to fail. This work specifically addresses the problem of side chain rearrangements upon complex formation. Based on the observation that most side chains retain their unbound conformation upon binding, each rigidly docked pose is initially scored ignoring up to a limited number of side chain overlaps which are resolved in subsequent repacking and minimization steps. On test systems where side chains are altered and backbones held in their bound state, this implementation provides significantly better native pose recovery and higher quality (lower RMSD) predictions when compared with five of the most popular docking programs. The method is implemented in the software program ProPOSE (Protein Pose Optimization by Systematic Enumeration).


Assuntos
Técnicas de Química Analítica/métodos , Simulação por Computador , Proteínas/química , Software , Complexo Antígeno-Anticorpo/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica
7.
Sci Rep ; 8(1): 2260, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29396522

RESUMO

Assisted Design of Antibody and Protein Therapeutics (ADAPT) is an affinity maturation platform interleaving predictions and testing that was previously validated on monoclonal antibodies (mAbs). This study expands the applicability of ADAPT to single-domain antibodies (sdAbs), a promising class of recombinant antibody-based biologics. As a test case, we used the camelid sdAb A26.8, a VHH that binds Clostridium difficile toxin A (TcdA) relatively weakly but displays a reasonable level of TcdA neutralization. ADAPT-guided A26.8 affinity maturation resulted in an improvement of one order of magnitude by point mutations only, reaching an equilibrium dissociation constant (KD) of 2 nM, with the best binding mutants having similar or improved stabilities relative to the parent sdAb. This affinity improvement generated a 6-fold enhancement of efficacy at the cellular level; the A26.8 double-mutant T56R,T103R neutralizes TcdA cytotoxicity with an IC50 of 12 nM. The designed mutants with increased affinities are predicted to establish novel electrostatic interactions with the antigen. Almost full additivity of mutation effects is observed, except for positively charged residues introduced at adjacent positions. Furthermore, analysis of false-positive predictions points to general directions for improving the ADAPT platform. ADAPT guided the efficacy enhancement of an anti-toxin sdAb, an alternative therapeutic modality for C. difficile.


Assuntos
Anticorpos Neutralizantes/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Produtos Biológicos/metabolismo , Desenho de Fármacos , Enterotoxinas/antagonistas & inibidores , Fatores Imunológicos/metabolismo , Anticorpos de Domínio Único/metabolismo , Animais , Anticorpos Neutralizantes/genética , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Fatores Imunológicos/genética , Concentração Inibidora 50 , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anticorpos de Domínio Único/genética , Células Vero
8.
J Comput Aided Mol Des ; 32(1): 143-150, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28983727

RESUMO

The Farnesoid X receptor (FXR) exhibits significant backbone movement in response to the binding of various ligands and can be a challenge for pose prediction algorithms. As part of the D3R Grand Challenge 2, we tested Wilma-SIE, a rigid-protein docking method, on a set of 36 FXR ligands for which the crystal structures had originally been blinded. These ligands covered several classes of compounds. To overcome the rigid protein limitations of the method, we used an ensemble of publicly available structures for FXR from the PDB. The use of the ensemble allowed Wilma-SIE to predict poses with average and median RMSDs of 2.3 and 1.4 Å, respectively. It was quite clear, however, that had we used a single structure for the receptor the success rate would have been much lower. The most successful predictions were obtained on chemical classes for which one or more crystal structures of the receptor bound to a molecule of the same class was available. In the absence of a crystal structure for the class, observing a consensus binding mode for the ligands of the class using one or more receptor structures of other classes seemed to be indicative of a reasonable pose prediction. Affinity prediction proved to be more challenging with generally poor correlation with experimental IC50s (Kendall tau ~ 0.3). Even when the 36 crystal structures were used the accuracy of the predicted affinities was not appreciably improved. A possible cause of difficulty is the internal energy strain arising from conformational differences in the receptor across complexes, which may need to be properly estimated and incorporated into the SIE scoring function.


Assuntos
Descoberta de Drogas , Simulação de Acoplamento Molecular , Receptores Citoplasmáticos e Nucleares/metabolismo , Software , Sítios de Ligação , Desenho Assistido por Computador , Cristalografia por Raios X , Bases de Dados de Proteínas , Desenho de Fármacos , Humanos , Ligantes , Ligação Proteica , Conformação Proteica , Receptores Citoplasmáticos e Nucleares/química , Termodinâmica
9.
PLoS One ; 12(7): e0181490, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28750054

RESUMO

Effective biologic therapeutics require binding affinities that are fine-tuned to their disease-related molecular target. The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform aids in the selection of mutants that improve/modulate the affinity of antibodies and other biologics. It uses a consensus z-score from three scoring functions and interleaves computational predictions with experimental validation, significantly enhancing the robustness of the design and selection of mutants. The platform was tested on three antibody Fab-antigen systems that spanned a wide range of initial binding affinities: bH1-VEGF-A (44 nM), bH1-HER2 (3.6 nM) and Herceptin-HER2 (0.058 nM). Novel triple mutants were obtained that exhibited 104-, 46- and 32-fold improvements in binding affinity for each system, respectively. Moreover, for all three antibody-antigen systems over 90% of all the intermediate single and double mutants that were designed and tested showed higher affinities than the parent sequence. The contributions of the individual mutants to the change in binding affinity appear to be roughly additive when combined to form double and triple mutants. The new interactions introduced by the affinity-enhancing mutants included long-range electrostatics as well as short-range nonpolar interactions. This diversity in the types of new interactions formed by the mutants was reflected in SPR kinetics that showed that the enhancements in affinities arose from increasing on-rates, decreasing off-rates or a combination of the two effects, depending on the mutation. ADAPT is a very focused search of sequence space and required only 20-30 mutants for each system to be made and tested to achieve the affinity enhancements mentioned above.


Assuntos
Anticorpos/uso terapêutico , Desenho de Fármacos , Proteínas Recombinantes/uso terapêutico , Afinidade de Anticorpos/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Modelos Moleculares , Mutação/genética , Ressonância de Plasmônio de Superfície , Termodinâmica , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
PLoS One ; 11(9): e0163113, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27631624

RESUMO

Staphylococcal protein A (SpA) and streptococcal protein G (SpG) affinity chromatography are the gold standards for purifying monoclonal antibodies (mAbs) in therapeutic applications. However, camelid VHH single-domain Abs (sdAbs or VHHs) are not bound by SpG and only sporadically bound by SpA. Currently, VHHs require affinity tag-based purification, which limits their therapeutic potential and adds considerable complexity and cost to their production. Here we describe a simple and rapid mutagenesis-based approach designed to confer SpA binding upon a priori non-SpA-binding VHHs. We show that SpA binding of VHHs is determined primarily by the same set of residues as in human mAbs, albeit with an unexpected degree of tolerance to substitutions at certain core and non-core positions and some limited dependence on at least one residue outside the SpA interface, and that SpA binding could be successfully introduced into five VHHs against three different targets with no adverse effects on expression yield or antigen binding. Next-generation sequencing of llama, alpaca and dromedary VHH repertoires suggested that species differences in SpA binding may result from frequency variation in specific deleterious polymorphisms, especially Ile57. Thus, the SpA binding phenotype of camelid VHHs can be easily modulated to take advantage of tag-less purification techniques, although the frequency with which this is required may depend on the source species.


Assuntos
Camelídeos Americanos/imunologia , Engenharia de Proteínas , Anticorpos de Domínio Único/genética , Proteína Estafilocócica A/genética , Animais
11.
J Chem Inf Model ; 56(7): 1292-303, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27367467

RESUMO

Affinity modulation of antibodies and antibody fragments of therapeutic value is often required in order to improve their clinical efficacies. Virtual affinity maturation has the potential to quickly focus on the critical hotspot residues without the combinatorial explosion problem of conventional display and library approaches. However, this requires a binding affinity scoring function that is capable of ranking single-point mutations of a starting antibody. We focus here on assessing the solvated interaction energy (SIE) function that was originally developed for and is widely applied to scoring of protein-ligand binding affinities. To this end, we assembled a structure-function data set called Single-Point Mutant Antibody Binding (SiPMAB) comprising several antibody-antigen systems suitable for this assessment, i.e., based on high-resolution crystal structures for the parent antibodies and coupled with high-quality binding affinity measurements for sets of single-point antibody mutants in each system. Using this data set, we tested the SIE function with several mutation protocols based on the popular methods SCWRL, Rosetta, and FoldX. We found that the SIE function coupled with a protocol limited to sampling only the mutated side chain can reasonably predict relative binding affinities with a Spearman rank-order correlation coefficient of about 0.6, outperforming more aggressive sampling protocols. Importantly, this performance is maintained for each of the seven system-specific component subsets as well as for other relevant subsets including non-alanine and charge-altering mutations. The transferability and enrichment in affinity-improving mutants can be further enhanced using consensus ranking over multiple methods, including the SIE, Talaris, and FOLDEF energy functions. The knowledge gained from this study can lead to successful prospective applications of virtual affinity maturation.


Assuntos
Anticorpos/imunologia , Afinidade de Anticorpos , Antígenos/imunologia , Biologia Computacional/métodos , Solventes/química , Antígenos/química , Antígenos/genética , Bases de Dados de Proteínas , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Relação Estrutura-Atividade , Termodinâmica
12.
Biochemistry ; 55(16): 2319-31, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27031688

RESUMO

To study the mechanism of ligating nicked RNA strands, we conducted molecular dynamics simulations of Trypanosoma brucei RNA editing ligases L1 and L2 complexed with double-stranded RNA (dsRNA) fragments. In each resulting model, a Mg(2+) ion coordinates the 5'-PO4 of the nicked nucleotide and the 3'-OH of the terminal nucleotide for a nucleophilic reaction consistent with the postulated step 3 chemistry of the ligation mechanism. Moreover, coordination of the 3'-OH to the Mg(2+) ion may lower its pKa, thereby rendering it a more effective nucleophile as an oxyanion. Thus, Mg(2+) may play a twofold role: bringing the reactants into the proximity of each other and activating the nucleophile. We also conducted solvated interaction energy calculations to explore whether ligation specificities can be correlated to ligase-dsRNA binding affinity changes. The calculated dsRNA binding affinities are stronger for both L1 and L2 when the terminal nucleotide is changed from cytosine to guanine, in line with their experimentally measured ligation specificities. Because the ligation mechanism is also influenced by interactions of the ligase with partner proteins from the editosome subcomplex, we also modeled the structure of the RNA-bound L2 in complex with the oligonucleotide binding (OB) domain of largest editosome interacting protein A1. The resulting L2-dsRNA-A1 model, which is consistent with mutagenesis and binding data recorded to date, provides the first atomic-level glimpse of plausible interactions around the RNA ligation site in the presence of an OB domain presented in-trans to a nucleic acid ligase.


Assuntos
Ligases/metabolismo , Proteínas de Protozoários/metabolismo , Edição de RNA , RNA de Protozoário/metabolismo , Trypanosoma brucei brucei/metabolismo , Humanos , Ligases/química , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , Mapas de Interação de Proteínas , Proteínas de Protozoários/química , RNA de Protozoário/química , Termodinâmica , Trypanosoma brucei brucei/química , Tripanossomíase Africana/parasitologia
13.
J Chem Inf Model ; 56(6): 955-64, 2016 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-26282162

RESUMO

Prospective assessments of the Wilma-SIE (solvated interaction energy) platform for ligand docking and ranking were performed during the 2013 and 2014 editions of the Community Structure-Activity Resource (CSAR) blind challenge. Diverse targets like a steroid-binding protein, a serine protease (factor Xa), a tyrosine kinase (Syk), and a nucleotide methyltransferase (TrmD) were included. Pose selection was achieved with high precision; in all 24 tests Wilma-SIE top-ranked the native pose among carefully generated sets of decoy conformations. Good separation for the native pose was also observed indicating robustness in pose scoring. Cross-docking was also accomplished with high accuracy for the various systems, with ligand median-RMSD values around 1 Å from the crystal structures. Larger deviations were occasionally obtained due to the rigid-target approach even if multiple target structures were used. Affinity ranking of congeneric ligands after cross-docking was reasonable for three of the four systems, with Spearman ranking coefficients around 0.6. Poor affinity ranking for FXa is possibly due to missing structural domains, which are present during measurements. Assignment of protonation states is critical for affinity scoring with the SIE function, as shown here for the Syk system. Including the FiSH model improved cross-docking but worsened affinity predictions, pointing to the need for further fine-tuning of this newer solvation model. The consistently strong performance of the Wilma-SIE platform in recent CSAR and SAMPL blind challenges validates its applicability for virtual screening on a broad range of molecular targets.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Proteínas/química , Proteínas/metabolismo , Relação Estrutura-Atividade
14.
Antimicrob Agents Chemother ; 58(12): 7430-40, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25267679

RESUMO

Helicobacter pylori is motile by means of polar flagella, and this motility has been shown to play a critical role in pathogenicity. The major structural flagellin proteins have been shown to be glycosylated with the nonulosonate sugar, pseudaminic acid (Pse). This glycan is unique to microorganisms, and the process of flagellin glycosylation is required for H. pylori flagellar assembly and consequent motility. As such, the Pse biosynthetic pathway offers considerable potential as an antivirulence drug target, especially since motility is required for H. pylori colonization and persistence in the host. This report describes screening the five Pse biosynthetic enzymes for small-molecule inhibitors using both high-throughput screening (HTS) and in silico (virtual screening [VS]) approaches. Using a 100,000-compound library, 1,773 hits that exhibited a 40% threshold inhibition at a 10 µM concentration were identified by HTS. In addition, VS efforts using a 1.6-million compound library directed at two pathway enzymes identified 80 hits, 4 of which exhibited reasonable inhibition at a 10 µM concentration in vitro. Further secondary screening which identified 320 unique molecular structures or validated hits was performed. Following kinetic studies and structure-activity relationship (SAR) analysis of selected inhibitors from our refined list of 320 compounds, we demonstrated that three inhibitors with 50% inhibitory concentrations (IC50s) of approximately 14 µM, which belonged to a distinct chemical cluster, were able to penetrate the Gram-negative cell membrane and prevent formation of flagella.


Assuntos
Antibacterianos/farmacologia , Flagelos/efeitos dos fármacos , Flagelina/antagonistas & inibidores , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/patogenicidade , Bibliotecas de Moléculas Pequenas/farmacologia , Açúcares Ácidos/metabolismo , Antibacterianos/química , Transporte Biológico , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Descoberta de Drogas , Flagelos/genética , Flagelos/metabolismo , Flagelina/biossíntese , Flagelina/genética , Expressão Gênica , Glicosilação/efeitos dos fármacos , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Ensaios de Triagem em Larga Escala , Simulação de Acoplamento Molecular , Movimento/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Interface Usuário-Computador , Virulência
15.
J Med Chem ; 57(10): 4368-81, 2014 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-24779610

RESUMO

To determine if the conjugation of a small receptor ligand to a peptidic carrier to potentially facilitate transport across the blood-brain barrier (BBB) by "molecular Trojan horse" transcytosis is feasible, we synthesized several transport peptide-fallypride fusion molecules as model systems and determined their binding affinities to the hD2 receptor. Although they were affected by conjugation, the binding affinities were found to be still in the nanomolar range (between 1.5 and 64.2 nM). In addition, homology modeling of the receptor and docking studies for the most potent compounds were performed, elucidating the binding modes of the fusion molecules and the structure elements contributing to the observed high receptor binding. Furthermore, no interaction between the hybrid compounds and P-gp, the main excretory transporter of the BBB, was found. From these results, it can be inferred that the approach to deliver small neuroreceptor ligands across the BBB by transport peptide carriers is feasible.


Assuntos
Benzamidas/química , Proteínas de Transporte/química , Antagonistas de Dopamina/síntese química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Sequência de Aminoácidos , Benzamidas/farmacocinética , Transporte Biológico , Barreira Hematoencefálica , Antagonistas de Dopamina/farmacocinética , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Receptores de Dopamina D2/metabolismo , Receptores da Transferrina/metabolismo , Transcitose
16.
J Comput Aided Mol Des ; 28(4): 417-27, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24474162

RESUMO

We continued prospective assessments of the Wilma-solvated interaction energy (SIE) platform for pose prediction, binding affinity prediction, and virtual screening on the challenging SAMPL4 data sets including the HIV-integrase inhibitor and two host-guest systems. New features of the docking algorithm and scoring function are tested here prospectively for the first time. Wilma-SIE provides good correlations with actual binding affinities over a wide range of binding affinities that includes strong binders as in the case of SAMPL4 host-guest systems. Absolute binding affinities are also reproduced with appropriate training of the scoring function on available data sets or from comparative estimation of the change in target's vibrational entropy. Even when binding modes are known, SIE predictions lack correlation with experimental affinities within dynamic ranges below 2 kcal/mol as in the case of HIV-integrase ligands, but they correctly signaled the narrowness of the dynamic range. Using a common protein structure for all ligands can reduce the noise, while incorporating a more sophisticated solvation treatment improves absolute predictions. The HIV-integrase virtual screening data set consists of promiscuous weak binders with relatively high flexibility and thus it falls outside of the applicability domain of the Wilma-SIE docking platform. Despite these difficulties, unbiased docking around three known binding sites of the enzyme resulted in over a third of ligands being docked within 2 Å from their actual poses and over half of the ligands docked in the correct site, leading to better-than-random virtual screening results.


Assuntos
Inibidores de Integrase de HIV/farmacologia , Integrase de HIV/metabolismo , HIV/enzimologia , Simulação de Acoplamento Molecular , Sítios de Ligação , Desenho Assistido por Computador , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/enzimologia , Infecções por HIV/virologia , Integrase de HIV/química , Inibidores de Integrase de HIV/química , Humanos , Ligantes , Ligação Proteica , Termodinâmica
17.
Curr Pharm Des ; 20(20): 3266-80, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-23947651

RESUMO

Water plays an active role in many fundamental phenomena in cellular systems such as molecular recognition, folding and conformational equilibria, reaction kinetics and phase partitioning. Hence, our ability to account for the energetics of these processes is highly dependent on the models we use for calculating solvation effects. For example, theoretical prediction of protein-ligand binding modes (i.e., docking) and binding affinities (i.e., scoring) requires an accurate description of the change in hydration that accompanies solute binding. In this review, we discuss the challenges of constructing solvation models that capture these effects, with an emphasis on continuum models and on more recent developments in the field. In our discussion of methods, relatively greater attention will be given to boundary element solutions to the Poisson equation and to nonpolar solvation models, two areas that have become increasingly important but are likely to be less familiar to many readers. The other focus will be upon the trending efforts for evaluating solvation models in order to uncover limitations, biases, and potentially attractive directions for their improvement and applicability. The prospective and retrospective performance of a variety of solvation models in the SAMPL blind challenges will be discussed in detail. After just a few years, these benchmarking exercises have already had a tangible effect in guiding the improvement of solvation models.


Assuntos
Teoria Quântica , Água/química , Modelos Químicos , Reprodutibilidade dos Testes , Solubilidade
18.
J Phys Chem B ; 116(23): 6872-9, 2012 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-22432509

RESUMO

We explore the use of exhaustive docking as an alternative to Monte Carlo and molecular dynamics sampling for the direct integration of the partition function for protein-ligand binding. We enumerate feasible poses for the ligand and calculate the Boltzmann factor contribution of each pose to the partition function. From the partition function, the free energy, enthalpy, and entropy can be derived. All our calculations are done with a continuum solvation model that includes solving the Poisson equation. In contrast to Monte Carlo and molecular dynamics simulations, exhaustive docking avoids (within the limitations of a discrete sampling) the question of "Have we run long enough?" due to its deterministic complete enumeration of states. We tested the method on the T4 lysozyme L99A mutant, which has a nonpolar cavity that can accommodate a number of small molecules. We tested two electrostatic models. Model 1 used a solute dielectric of 2.25 for the complex apoprotein and free ligand and 78.5 for the solvent. Model 2 used a solute dielectric of 2.25 for the complex and apoprotein but 1.0 for the free ligand. For our test set of eight molecules, we obtain a reasonable correlation with a Pearson r(2) = 0.66 using model 1. The trend in binding affinity ranking is generally preserved with a Kendall τ = 0.64 and Spearman ρ = 0.83. With model 2, the correlation is improved with a Pearson r(2) = 0.83, Kendall τ = 0.93, and Spearman ρ = 0.98. This suggests that the energy function and sampling method adequately captured most of the thermodynamics of binding of the nonpolar ligands to T4 lysozyme L99A.


Assuntos
Muramidase/química , Termodinâmica , Bacteriófago T4/enzimologia , Sítios de Ligação , Ligantes , Modelos Moleculares , Simulação de Dinâmica Molecular , Método de Monte Carlo , Muramidase/genética , Muramidase/metabolismo , Eletricidade Estática
19.
Methods Mol Biol ; 819: 295-303, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22183544

RESUMO

The solvated interaction energy (SIE) is an end-point, physics-based scoring function for predicting ligand-binding affinities. It supplements the force-field interaction energy with the desolvation cost of binding. Parameters such as the solute dielectric constant, Born radii, a cavity term and an overall scaling coefficient and additive constant have been previously calibrated against a training set of 99 protein-ligand complexes. We describe the application of the method to estimating binding free energies from molecular dynamics trajectories of protein-ligand binding complexes.


Assuntos
Biologia Computacional/métodos , Proteínas/metabolismo , Solventes/química , Simulação de Dinâmica Molecular , Ligação Proteica , Termodinâmica
20.
J Comput Aided Mol Des ; 26(5): 661-7, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22190141

RESUMO

Next-generation solvation models are devised to mimic the accuracy and generality of explicit solvation models at the speed of current popular implicit solvation models. One such method is the first-shell of hydration (FiSH) continuum model that was trained on hydration energetics from LIE calculations and molecular dynamics simulations in explicit solvent. Here we tested prospectively the FiSH model on the SAMPL-3 hydration data set that zooms in the effect of chlorination on solvation. We compare these FiSH predictions with those from retrospective LIE calculations. We find that neither FiSH nor LIE can reproduce well the absolute values and the trend of hydration free energies in the biphenyl and dioxin aromatic chlorination series. Some of the hypotheses behind this performance are discussed and tested. The LIE explicit-solvent model shows some improvement relative to the FiSH continuum model, and we correct a systematic deviation in the continuum van der Waals term of FiSH associated with aromatic Cl atom type.


Assuntos
Hidrocarbonetos Clorados/química , Modelos Químicos , Simulação de Dinâmica Molecular , Termodinâmica , Ligantes , Solubilidade , Solventes/química , Água/química
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