RESUMO
Generative biology combines artificial intelligence (AI), advanced life sciences technologies, and automation to revolutionize the process of designing novel biomolecules with prescribed properties, giving drug discoverers the ability to escape the limitations of biology during the design of next-generation protein therapeutics. Significant hurdles remain, namely: (i) the inherently complex nature of drug discovery, (ii) the bewildering number of promising computational and experimental techniques that have emerged in the past several years, and (iii) the limited availability of relevant protein sequence-function data for drug-like molecules. There is a need to focus on computational methods that will be most practically effective for protein drug discovery and on building experimental platforms to generate the data most appropriate for these methods. Here, we discuss recent advances in computational and experimental life sciences that are most crucial for impacting the pace and success of protein drug discovery.
Assuntos
Inteligência Artificial , Descoberta de Drogas , Humanos , Descoberta de Drogas/métodos , BiologiaRESUMO
BACKGROUND: The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted. METHODS: Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells. FINDINGS: We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion. CONCLUSIONS: Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases. FUNDING: This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.
Assuntos
Barreira Hematoencefálica , Transcitose , Barreira Hematoencefálica/metabolismo , Transcitose/fisiologia , Receptores da Transferrina , Transporte Biológico/fisiologia , AnticorposRESUMO
Pramlintide is an equipotent amylin analogue that reduces food intake and body weight in obese subjects and has been clinically approved as an adjunctive therapy for the treatment of adult diabetic patients. However, due to its extremely short half-life in vivo, a regimen of multiple daily administrations is required for achieving clinical effectiveness. Herein is described the development of prototypical long-acting pramlintide bioconjugates, in which pramlintide's disulfide-linked macrocycle was replaced by a cyclic thioether motif. This modification enabled stable chemical conjugation to a half-life extending antibody. In contrast to pramlintide (t1/2 < 0.75 h), bioconjugates 35 and 38 have terminal half-lives of â¼2 days in mice and attain significant exposure levels that are maintained up to 7 days. Single dose subcutaneous administration of 35 in lean mice, given 18-20 h prior to oral acetaminophen (AAP) administration, significantly reduced gastric emptying (as determined by plasma AAP levels). In a separate study, similar administration of 35 in fasted lean mice effected a reduction in food intake for up to 48 h. These data are consistent with durable amylinomimetic responses and provide the basis for further development of such long-acting amylinomimetic conjugates for the potential treatment of obesity and associated pathologies.
Assuntos
Agonistas dos Receptores da Amilina , Agonistas dos Receptores da Amilina/farmacologia , Agonistas dos Receptores da Amilina/uso terapêutico , Amiloide , Animais , Peso Corporal , Humanos , Hipoglicemiantes/uso terapêutico , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Camundongos , Obesidade/induzido quimicamente , Obesidade/tratamento farmacológicoRESUMO
The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables avidity-driven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementarity-determining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant diet-induced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.
Assuntos
Anticorpos Monoclonais , Ingestão de Alimentos/efeitos dos fármacos , Obesidade , Oxintomodulina , Peptídeos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Cisteína/química , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Obesidade/sangue , Obesidade/tratamento farmacológico , Oxintomodulina/química , Oxintomodulina/farmacocinética , Oxintomodulina/farmacologia , Peptídeos/química , Peptídeos/farmacocinética , Peptídeos/farmacologiaRESUMO
T-cell-mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein-coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Mieloma Múltiplo/terapia , Receptores Acoplados a Proteínas G/imunologia , Linfócitos T/efeitos dos fármacos , Animais , Anticorpos Biespecíficos/imunologia , Antineoplásicos Imunológicos/imunologia , Linhagem Celular Tumoral , Citotoxicidade Imunológica/efeitos dos fármacos , Feminino , Humanos , Imunoterapia , Camundongos Endogâmicos BALB C , Mieloma Múltiplo/imunologia , Linfócitos T/imunologiaRESUMO
Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer's disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility.
Assuntos
Inibidores Enzimáticos/síntese química , Compostos Heterocíclicos/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Animais , Cristalografia por Raios X , Cães , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos/farmacologia , Humanos , Concentração Inibidora 50 , Peso Molecular , Ratos , SolubilidadeRESUMO
Inhibition of signal transduction downstream of the IL-23 receptor represents an intriguing approach to the treatment of autoimmunity. Using a chemogenomics approach marrying kinome-wide inhibitory profiles of a compound library with the cellular activity against an IL-23-stimulated transcriptional response in T lymphocytes, a class of inhibitors was identified that bind to and stabilize the pseudokinase domain of the Janus kinase tyrosine kinase 2 (Tyk2), resulting in blockade of receptor-mediated activation of the adjacent catalytic domain. These Tyk2 pseudokinase domain stabilizers were also shown to inhibit Tyk2-dependent signaling through the Type I interferon receptor but not Tyk2-independent signaling and transcriptional cellular assays, including stimulation through the receptors for IL-2 (JAK1- and JAK3-dependent) and thrombopoietin (JAK2-dependent), demonstrating the high functional selectivity of this approach. A crystal structure of the pseudokinase domain liganded with a representative example showed the compound bound to a site analogous to the ATP-binding site in catalytic kinases with features consistent with high ligand selectivity. The results support a model where the pseudokinase domain regulates activation of the catalytic domain by forming receptor-regulated inhibitory interactions. Tyk2 pseudokinase stabilizers, therefore, represent a novel approach to the design of potent and selective agents for the treatment of autoimmunity.
Assuntos
Modelos Moleculares , Transdução de Sinais , Linfócitos T/enzimologia , TYK2 Quinase/química , Cristalografia por Raios X , Estabilidade Enzimática , Humanos , Janus Quinase 1/genética , Janus Quinase 1/metabolismo , Janus Quinase 3/genética , Janus Quinase 3/metabolismo , Estrutura Terciária de Proteína , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/metabolismo , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , TYK2 Quinase/genéticaRESUMO
Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants' ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.
Assuntos
Benchmarking , Mapeamento de Interação de Proteínas/normas , Ressonância de Plasmônio de Superfície/normas , Proteínas de Bactérias/química , Ligação Competitiva , Humanos , Proteínas Imobilizadas/química , Interferometria/normas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Padrões de Referência , Ribonucleases/antagonistas & inibidores , Ribonucleases/química , Espectrometria de Massas por Ionização por Electrospray/normasRESUMO
Obtaining diffraction quality crystals is frequently an iterative process which traditionally has involved screening large numbers of crystallization conditions. Due to advances in high-throughput gene engineering, recombinant expression, and purification, the protein of interest has now become one of the many variables routinely investigated during crystallization trials. As such, construct design is a critical step in the path toward successful crystallization. In this chapter will we address construct design strategies frequently employed to improve the solution and crystallization behavior of proteins. Topics covered include choosing a recombinant expression system and reducing disorder through truncations and surface mutagenesis. Also covered are strategies to reduce heterogeneity from posttranslational modifications, impurities, and aggregation.
Assuntos
Engenharia Genética , Vetores Genéticos/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/biossínteseRESUMO
Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.
Assuntos
Antivirais/farmacologia , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Cristalografia por Raios X , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Hidrodinâmica , Camundongos , Modelos Moleculares , Nucleoproteínas/ultraestrutura , Orthomyxoviridae/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Bibliotecas de Moléculas Pequenas/uso terapêutico , SoluçõesRESUMO
Despite the availability of numerous gene fusion systems, recombinant protein expression in Escherichia coli remains difficult. Establishing the best fusion partner for difficult-to-express proteins remains empirical. To determine which fusion tags are best suited for difficult-to-express proteins, a comparative analysis of the newly described SUMO fusion system with a variety of commonly used fusion systems was completed. For this study, three model proteins, enhanced green fluorescent protein (eGFP), matrix metalloprotease-13 (MMP13), and myostatin (growth differentiating factor-8, GDF8), were fused to the C termini of maltose-binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), NUS A, ubiquitin (Ub), and SUMO tags. These constructs were expressed in E. coli and evaluated for expression and solubility. As expected, the fusion tags varied in their ability to produce tractable quantities of soluble eGFP, MMP13, and GDF8. SUMO and NUS A fusions enhanced expression and solubility of recombinant proteins most dramatically. The ease at which SUMO and NUS A fusion tags were removed from their partner proteins was then determined. SUMO fusions are cleaved by the natural SUMO protease, while an AcTEV protease site had to be engineered between NUS A and its partner protein. A kinetic analysis showed that the SUMO and AcTEV proteases had similar KM values, but SUMO protease had a 25-fold higher kcat than AcTEV protease, indicating a more catalytically efficient enzyme. Taken together, these results demonstrate that SUMO is superior to commonly used fusion tags in enhancing expression and solubility with the distinction of generating recombinant protein with native sequences.
Assuntos
Clonagem Molecular/métodos , Fusão Gênica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteína SUMO-1/biossíntese , Proteína SUMO-1/genética , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Endopeptidases/biossíntese , Endopeptidases/química , Endopeptidases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Proteínas Recombinantes de Fusão/química , Proteína SUMO-1/química , SolubilidadeRESUMO
The demands of structural and functional genomics for large quantities of soluble, properly folded proteins in heterologous hosts have been aided by advancements in the field of protein production and purification. Escherichia coli, the preferred host for recombinant protein expression, presents many challenges which must be surmounted in order to over-express heterologous proteins. These challenges include the proteolytic degradation of target proteins, protein misfolding, poor solubility, and the necessity for good purification methodologies. Gene fusion technologies have been able to improve heterologous expression by overcoming many of these challenges. The ability of gene fusions to improve expression, solubility, purification, and decrease proteolytic degradation will be discussed in this review. The main disadvantage, cleaving the protein fusion, will also be addressed. Focus will be given to the newly described SUMO fusion system and the improvements that this technology has advanced over traditional gene fusion systems.
Assuntos
Fusão Gênica/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/biossíntese , Escherichia coli/metabolismo , Expressão Gênica , Dobramento de ProteínaRESUMO
Heparan sulfate (HS) plays essential roles in assisting herpes simplex virus infection and other biological processes. The biosynthesis of HS includes numerous specialized sulfotransferases that generate a variety of sulfated saccharide sequences, conferring the selectivity of biological functions of HS. We report a structural study of human HS 3-O-sulfotransferase isoform 3 (3-OST-3), a key sulfotransferase that transfers a sulfuryl group to a specific glucosamine in HS generating an entry receptor for herpes simplex virus 1. We have obtained the crystal structure of 3-OST-3 at 1.95 A in a ternary complex with 3'-phosphoadenosine 5'-phosphate and a tetrasaccharide substrate. Mutational analyses were also performed on the residues involved in the binding of the substrate. Residues Gln255 and Lys368 are essential for the sulfotransferase activity and lie within hydrogen bonding distances to the carboxyl and sulfo groups of the uronic acid unit. These residues participate in the substrate recognition of 3-OST-3. This structure provides atomic level evidence for delineating the substrate recognition and catalytic mechanism for 3-OST-3.
Assuntos
Herpesvirus Humano 1/metabolismo , Sulfotransferases/química , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Cristalografia por Raios X , Análise Mutacional de DNA , Glutamina/química , Humanos , Ligação de Hidrogênio , Íons , Cinética , Lisina/química , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos/química , Plasmídeos/metabolismo , Polissacarídeos/química , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
The 3-O-sulfation of glucosamine by heparan sulfate 3-O-sulfotransferase-1 (3-OST-1) is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). In this paper, we present evidence of a conformational change that occurs in 3-OST-1 upon binding to heparan sulfate. The intrinsic fluorescence of 3-OST-1 was increased in the presence of HS, suggesting a conformational change. This apparent conformational change was further investigated using differential chemical modification of 3-OST-1 to measure the solvent accessibility of the lysine residues. 3-OST-1 was treated with acetic anhydride in either the presence or absence of HS using both acetic anhydride and hexadeuterioacetic anhydride under nondenaturing and denaturing conditions, respectively. The relative reactivity of the lysine residues to acetylation and [2H] acetylation in the presence or absence of HS was analyzed by measuring the ratio of acetylated and deuterioacetylated peptides using matrix-assisted laser desorption ionization mass spectrometry. The solvent accessibilities of the lysine residues were altered differentially depending on their location. In particular, we observed a group of lysine residues in the C-terminus of 3-OST-1 that become more solvent accessible when 3-OST-1 binds to HS. This observation indicates that a conformational change could be occurring during substrate binding. A truncated mutant of 3-OST-1 that lacked this C-terminal region was expressed and found to exhibit a 200-fold reduction in sulfotransferase activity. The results from this study will contribute to our understanding of the interactions between 3-OSTs and HS.
Assuntos
Heparitina Sulfato/metabolismo , Sulfotransferases/química , Sulfotransferases/metabolismo , Substituição de Aminoácidos/genética , Animais , Ativação Enzimática/genética , Heparitina Sulfato/química , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Camundongos , Modelos Moleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/genética , Conformação Proteica , Alinhamento de Sequência , Espectrometria de Fluorescência , Especificidade por Substrato/genética , Sulfotransferases/genéticaRESUMO
Heparan sulfate interacts with antithrombin, a protease inhibitor, to regulate blood coagulation. Heparan sulfate 3-O-sulfotransferase isoform 1 performs the crucial last step modification in the biosynthesis of anticoagulant heparan sulfate. This enzyme transfers the sulfuryl group (SO(3)) from 3'-phosphoadenosine 5'-phosphosulfate to the 3-OH position of a glucosamine residue to form the 3-O-sulfo glucosamine, a structural motif critical for binding of heparan sulfate to antithrombin. In this study, we report the crystal structure of 3-O-sulfotransferase isoform 1 at 2.5-A resolution in a binary complex with 3'-phosphoadenosine 5'-phosphate. This structure reveals residues critical for 3'-phosphoadenosine 5'-phosphosulfate binding and suggests residues required for the binding of heparan sulfate. In addition, site-directed mutagenesis analyses suggest that residues Arg-67, Lys-68, Arg-72, Glu-90, His-92, Asp-95, Lys-123, and Arg-276 are essential for enzymatic activity. Among these essential amino acid residues, we find that residues Arg-67, Arg-72, His-92, and Asp-95 are conserved in heparan sulfate 3-O-sulfotransferases but not in heparan N-deacetylase/N-sulfotransferase, suggesting a role for these residues in conferring substrate specificity. Results from this study provide information essential for understanding the biosynthesis of anticoagulant heparan sulfate and the general mechanism of action of heparan sulfate sulfotransferases.