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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.
FASEB J ; 34(6): 8155-8171, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32342547

RESUMO

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75) demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

3.
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.

4.
J Mol Recognit ; 32(11): e2805, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31423671

RESUMO

Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable "CDR4" loop, formed between ß-strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb, NAT-267, was extended and randomized. From this library, we identified one sdAb, MA-232, bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage-displayed libraries in which the CDR4 and other regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities, we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired VL domain.

5.
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.

6.
Mol Biol Cell ; 30(6): 794-807, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650049

RESUMO

Discriminating among diverse environmental stimuli is critical for organisms to ensure their proper development, homeostasis, and survival. Saccharomyces cerevisiae regulates mating, osmoregulation, and filamentous growth using three different MAPK signaling pathways that share common components and therefore must ensure specificity. The adaptor protein Ste50 activates Ste11p, the MAP3K of all three modules. Its Ras association (RA) domain acts in both hyperosmolar and filamentous growth pathways, but its connection to the mating pathway is unknown. Genetically probing the domain, we found mutants that specifically disrupted mating or HOG-signaling pathways or both. Structurally these residues clustered on the RA domain, forming distinct surfaces with a propensity for protein-protein interactions. GFP fusions of wild-type (WT) and mutant Ste50p show that WT is localized to the shmoo structure and accumulates at the growing shmoo tip. The specifically pheromone response-defective mutants are severely impaired in shmoo formation and fail to localize ste50p, suggesting a failure of association and function of Ste50 mutants in the pheromone-signaling complex. Our results suggest that yeast cells can use differential protein interactions with the Ste50p RA domain to provide specificity of signaling during MAPK pathway activation.


Assuntos
MAP Quinase Quinase Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Ligação a DNA , MAP Quinase Quinase Quinases/fisiologia , Peptídeos/metabolismo , Peptídeos/fisiologia , Feromônios/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Schizosaccharomyces pombe/fisiologia , Transdução de Sinais
7.
PLoS One ; 14(12): e0226593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31891584

RESUMO

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin. These antibodies were ranked for binding via SPR and via flow-cytometry on high-Her2 SKOV3 cells and low-Her2 MCF7 cells, the latter acting as a surrogate for low-Her2 normal cells. A subpanel of variants, representative of different Her2-binding affinities (2 strong, 2 moderate and 3 weak), were further screened via high-content imaging for internalization efficacies in high versus low-Her2 cells. Finally, these antibodies were evaluated in ADC cytotoxicity screening assays (using DM1 and MMAE secondary antibodies) and as antibody-drug conjugates (DM1 and PNU159682). Our results identified specific but weak Her2-binding variants as optimal candidates for developing DM1 and PNU ADCs since they exhibited high potencies (low to sub-nM) in high-Her2 SKOV3 cells and low toxicities in low-Her2 cells. The 2 strong-affinity variants were highly potent in SKOV3 cells but also showed significant toxicities in low-Her2 cells and therefore are predicted to be toxic in normal tissues. Our findings show that pharmacological profiling of an antibody library in multiple binding and functional assays allows for selection of optimal ADCs.


Assuntos
Imunoconjugados/química , Imunoconjugados/farmacologia , Mutação , Receptor ErbB-2/metabolismo , Afinidade de Anticorpos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Doxorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imunoconjugados/genética , Células Jurkat , Células MCF-7 , Receptor ErbB-2/química , Relação Estrutura-Atividade , Trastuzumab/química , Trastuzumab/genética , Trastuzumab/farmacologia
8.
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
9.
Sci Rep ; 8(1): 17361, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478273

RESUMO

The insulin-like growth factor (IGF) axis has been implicated in the progression of malignant disease and identified as a clinically important therapeutic target. Several IGF-1 receptor (IGF-1R) targeting drugs including humanized monoclonal antibodies have advanced to phase II/III clinical trials, but to date, have not progressed to clinical use, due, at least in part, to interference with insulin receptor signalling. We previously reported on the production of a soluble fusion protein consisting of the extracellular domain of human IGF-1R fused to the Fc portion of human IgG1 (first generation IGF-TRAP) that bound human IGF-1 and IGF-2 with a 3 log higher affinity than insulin. We showed that the IGF-TRAP had potent anti-cancer activity in several pre-clinical models of aggressive carcinomas. Here we report on the re-engineering of the IGF-TRAP with the aim of improving physicochemical properties and suitability for clinical applications. We show that cysteine-serine substitutions in the Fc hinge region of IGF-TRAP eliminated high-molecular-weight oligomerized species, while a further addition of a flexible linker, not only improved the pharmacokinetic profile, but also enhanced the therapeutic profile of the IGF-TRAP, as evaluated in an experimental colon carcinoma metastasis model. Dose-response profiles of the modified IGF-TRAPs correlated with their bio-availability profiles, as measured by the IGF kinase-receptor-activation (KIRA) assay, providing a novel, surrogate biomarker for drug efficacy. This study provides a compelling example of structure-based re-engineering of Fc-fusion-based biologics for better manufacturability that also significantly improved pharmacological parameters. It identifies the re-engineered IGF-TRAP as a potent anti-cancer therapeutic.


Assuntos
Antineoplásicos/farmacologia , Carcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Fator de Crescimento Insulin-Like I/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Produtos Biológicos/farmacologia , Biomarcadores Tumorais/metabolismo , Carcinoma/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Feminino , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Fator de Crescimento Insulin-Like II/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor IGF Tipo 1/metabolismo
10.
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
11.
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
12.
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
13.
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
14.
J Biol Chem ; 292(17): 7173-7188, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28228478

RESUMO

The transforming growth factor ß isoforms, TGF-ß1, -ß2, and -ß3, are small secreted homodimeric signaling proteins with essential roles in regulating the adaptive immune system and maintaining the extracellular matrix. However, dysregulation of the TGF-ß pathway is responsible for promoting the progression of several human diseases, including cancer and fibrosis. Despite the known importance of TGF-ßs in promoting disease progression, no inhibitors have been approved for use in humans. Herein, we describe an engineered TGF-ß monomer, lacking the heel helix, a structural motif essential for binding the TGF-ß type I receptor (TßRI) but dispensable for binding the other receptor required for TGF-ß signaling, the TGF-ß type II receptor (TßRII), as an alternative therapeutic modality for blocking TGF-ß signaling in humans. As shown through binding studies and crystallography, the engineered monomer retained the same overall structure of native TGF-ß monomers and bound TßRII in an identical manner. Cell-based luciferase assays showed that the engineered monomer functioned as a dominant negative to inhibit TGF-ß signaling with a Ki of 20-70 nm Investigation of the mechanism showed that the high affinity of the engineered monomer for TßRII, coupled with its reduced ability to non-covalently dimerize and its inability to bind and recruit TßRI, enabled it to bind endogenous TßRII but prevented it from binding and recruiting TßRI to form a signaling complex. Such engineered monomers provide a new avenue to probe and manipulate TGF-ß signaling and may inform similar modifications of other TGF-ß family members.


Assuntos
Engenharia de Proteínas/métodos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Transdução de Sinais , Fator de Crescimento Transformador beta/química , Motivos de Aminoácidos , Animais , Progressão da Doença , Matriz Extracelular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Humanos , Cinética , Camundongos , Ligação Proteica , Dobramento de Proteína , Isoformas de Proteínas , Multimerização Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Solubilidade , Ressonância de Plasmônio de Superfície , Fator de Crescimento Transformador beta/metabolismo , Ultracentrifugação
15.
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
16.
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
17.
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
18.
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
19.
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
20.
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
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