RESUMO
B3GNT2 is responsible for elongation of cell surface long-chain polylactosamine, which influences the regulation of the immune response, making it an attractive target for immunomodulation. In the development of amide containing B3GNT2 inhibitors guided by structure-based drug design, imidazolones were found to successfully serve as amide bioisosteres. This novel imidazolone isosteric strategy alleviated torsional strain of the amide bond on binding to B3GNT2 and improved potency, isoform selectivity, as well as certain physicochemical and pharmacokinetic properties. Herein, we present the synthesis, SAR, X-ray cocrystal structures, and in vivo PK properties of imidazol-4-ones in the context of B3GNT2 inhibition.
Assuntos
Amidas , N-Acetilglucosaminiltransferases , Amidas/farmacologia , Amidas/química , N-Acetilglucosaminiltransferases/metabolismo , Desenho de Fármacos , Relação Estrutura-AtividadeRESUMO
Biologic drug discovery pipelines are designed to deliver protein therapeutics that have exquisite functional potency and selectivity while also manifesting biophysical characteristics suitable for manufacturing, storage, and convenient administration to patients. The ability to use computational methods to predict biophysical properties from protein sequence, potentially in combination with high throughput assays, could decrease timelines and increase the success rates for therapeutic developability engineering by eliminating lengthy and expensive cycles of recombinant protein production and testing. To support development of high-quality predictive models for antibody developability, we designed a sequence-diverse panel of 83 effector functionless IgG1 antibodies displaying a range of biophysical properties, produced and formulated each protein under standard platform conditions, and collected a comprehensive package of analytical data, including in vitro assays and in vivo mouse pharmacokinetics. We used this robust training data set to build machine learning classifier models that can predict complex protein behavior from these data and features derived from predicted and/or experimental structures. Our models predict with 87% accuracy whether viscosity at 150 mg/mL is above or below a threshold of 15 centipoise (cP) and with 75% accuracy whether the area under the plasma drug concentration-time curve (AUC0-672 h) in normal mouse is above or below a threshold of 3.9 × 106 h x ng/mL.
Assuntos
Anticorpos Monoclonais , Descoberta de Drogas , Animais , Camundongos , Anticorpos Monoclonais/química , Simulação por Computador , Proteínas Recombinantes , ViscosidadeRESUMO
Therapeutic antibody development requires discovery of an antibody molecule with desired specificities and drug-like properties. For toxicological studies, a therapeutic antibody must bind the ortholog antigen with a similar affinity to the human target to enable relevant dosing regimens, and antibodies falling short of this affinity design goal may not progress as therapeutic leads. Herein, we report the novel use of mammalian recombination signal sequence (RSS)-directed recombination for complementarity-determining region-targeted protein engineering combined with mammalian display to close the species affinity gap of human interleukin (IL)-13 antibody 731. This fully human antibody has not progressed as a therapeutic in part because of a 400-fold species affinity gap. Using this nonhypothesis-driven affinity maturation method, we generated multiple antibody variants with improved IL-13 affinity, including the highest affinity antibody reported to date (34 fM). Resolution of a cocrystal structure of the optimized antibody with the cynomolgus monkey (or nonhuman primate) IL-13 protein revealed that the RSS-derived mutations introduced multiple successive amino-acid substitutions resulting in a de novo formation of a π-π stacking-based protein-protein interaction between the affinity-matured antibody heavy chain and helix C on IL-13, as well as an introduction of an interface-distant residue, which enhanced the light chain-binding affinity to target. These mutations synergized binding of heavy and light chains to the target protein, resulting in a remarkably tight interaction, and providing a proof of concept for a new method of protein engineering, based on synergizing a mammalian display platform with novel RSS-mediated library generation.
Assuntos
Anticorpos , Interleucina-13 , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Animais , Anticorpos/genética , Anticorpos/imunologia , Afinidade de Anticorpos , Humanos , Interleucina-13/genética , Interleucina-13/imunologia , Macaca fascicularis , Mamíferos , Recombinação GenéticaRESUMO
Bispecific antibodies (Bispecifics) demonstrate exceptional clinical potential to address some of the most complex diseases. However, Bispecific production in a single cell often requires the correct pairing of multiple polypeptide chains for desired assembly. This is a considerable hurdle that hinders the development of many immunoglobulin G (IgG)-like bispecific formats. Our approach focuses on the rational engineering of charged residues to facilitate the chain pairing of distinct heavy chains (HC). Here, we deploy structure-guided protein design to engineer charge pair mutations (CPMs) placed in the CH3-CH3' interface of the fragment crystallizable (Fc) region of an antibody (Ab) to correctly steer heavy chain pairing. When used in combination with our stable effector functionless 2 (SEFL2.2) technology, we observed high pairing efficiency without significant losses in expression yields. Furthermore, we investigate the relationship between CPMs and the sequence diversity in the parental antibodies, proposing a rational strategy to deploy these engineering technologies.
RESUMO
Multiple myeloma (MM) is a hematologic malignancy that is characterized by the accumulation of abnormal plasma cells (PCs) in the bone marrow (BM). Patient outcome may be improved with BiTE (bispecific T-cell engager) molecules, which redirect T cells to lyse tumor cells. B-cell maturation antigen (BCMA) supports PC survival and is highly expressed on MM cells. A half-life extended anti-BCMA BiTE molecule (AMG 701) induced selective cytotoxicity against BCMA-expressing MM cells (average half-maximal effective concentration, 18.8 ± 14.8 pM), T-cell activation, and cytokine release in vitro. In a subcutaneous mouse xenograft model, at all doses tested, AMG 701 completely inhibited tumor formation (P < .001), as well as inhibited growth of established tumors (P ≤ .001) and extended survival in an orthotopic MM model (P ≤ .01). To evaluate AMG 701 bioactivity in cynomolgus monkeys, a PC surface phenotype and specific genes were defined to enable a quantitative digital droplet polymerase chain reaction assay (sensitivity, 0.1%). Dose-dependent pharmacokinetic and pharmacodynamic behavior was observed, with depletion of PC-specific genes reaching 93% in blood and 85% in BM. Combination with a programmed cell death protein 1 (PD-1)-blocking antibody significantly increased AMG 701 potency in vitro. A model of AMG 701 binding to BCMA and CD3 indicates that the distance between the T-cell and target cell membranes (ie, the immunological synapse) is similar to that of the major histocompatibility complex class I molecule binding to a T-cell receptor and suggests that the synapse would not be disrupted by the half-life extending Fc domain. These data support the clinical development of AMG 701.
Assuntos
Anticorpos Biespecíficos , Mieloma Múltiplo , Animais , Complexo CD3 , Macaca fascicularis , Camundongos , Mieloma Múltiplo/tratamento farmacológico , Plasmócitos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is an immune receptor expressed on the surface of microglia, macrophages, dendritic cells, and osteoclasts. The R47H TREM2 variant is a significant risk factor for late-onset Alzheimer's disease (AD), and the molecular basis of R47H TREM2 loss of function is an emerging area of TREM2 biology. Here, we report three high-resolution structures of the extracellular ligand-binding domains (ECDs) of R47H TREM2, apo-WT, and phosphatidylserine (PS)-bound WT TREM2 at 1.8, 2.2, and 2.2 Å, respectively. The structures reveal that Arg47 plays a critical role in maintaining the structural features of the complementarity-determining region 2 (CDR2) loop and the putative positive ligand-interacting surface (PLIS), stabilizing conformations capable of ligand interaction. This is exemplified in the PS-bound structure, in which the CDR2 loop and PLIS drive critical interactions with PS via surfaces that are disrupted in the variant. Together with in vitro and in vivo characterization, our structural findings elucidate the molecular mechanism underlying loss of ligand binding, putative oligomerization, and functional activity of R47H TREM2. They also help unravel how decreased in vitro and in vivo stability of TREM2 contribute to loss of function in disease.
Assuntos
Doença de Alzheimer/genética , Predisposição Genética para Doença , Glicoproteínas de Membrana/química , Proteínas Mutantes/química , Receptores Imunológicos/química , Doença de Alzheimer/patologia , Cristalografia por Raios X , Células Dendríticas/química , Células Dendríticas/patologia , Variação Genética , Humanos , Ligantes , Macrófagos/química , Macrófagos/patologia , Glicoproteínas de Membrana/genética , Microglia/química , Microglia/patologia , Proteínas Mutantes/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Osteoclastos/química , Osteoclastos/patologia , Conformação Proteica , Domínios Proteicos/genética , Receptores Imunológicos/genéticaRESUMO
In this Letter, we report the continued optimization of the N-acyl-2-aminobenzimidazole series, focusing in particular on the N-alkyl substituent and 5-position of the benzimidazole based on the binding mode and the early SAR. These efforts led to the discovery of 16, a highly potent, selective, and orally bioavailable inhibitor of IRAK-4.
Assuntos
Descoberta de Drogas , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Animais , Benzimidazóis/química , Ativação Enzimática/efeitos dos fármacos , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Ratos , Relação Estrutura-AtividadeRESUMO
The discovery, structure-based design, synthesis, and optimization of NIK inhibitors are described. Our work began with an HTS hit, imidazopyridinyl pyrimidinamine 1. We utilized homology modeling and conformational analysis to optimize the indole scaffold leading to the discovery of novel and potent conformationally constrained inhibitors such as compounds 25 and 28. Compounds 25 and 31 were co-crystallized with NIK kinase domain to provide structural insights.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Alcinos/síntese química , Alcinos/química , Alcinos/farmacologia , Aminas/síntese química , Aminas/química , Aminas/farmacologia , Desenho de Fármacos , Células HT29 , Humanos , Ligação de Hidrogênio , Imidazóis/síntese química , Imidazóis/química , Imidazóis/farmacologia , Modelos Moleculares , Inibidores de Proteínas Quinases/síntese química , Proteínas Serina-Treonina Quinases/química , Piridinas/síntese química , Piridinas/química , Piridinas/farmacologia , Pirimidinas/síntese química , Pirimidinas/química , Pirimidinas/farmacologia , Relação Estrutura-Atividade , Quinase Induzida por NF-kappaBRESUMO
Dipeptidyl peptidase IV (DPP-IV) degrades the incretin hormone glucagon-like peptide 1 (GLP-1). Small molecule DPP-IV inhibitors have been used as treatments for type 2 diabetes to improve glucose tolerance. However, each of the marketed small molecule drugs has its own limitation in terms of efficacy and side effects. To search for an alternative strategy of inhibiting DPP-IV activity, we generated a panel of tight binding inhibitory mouse monoclonal antibodies (mAbs) against rat DPP-IV. When tested in vitro, these mAbs partially inhibited the GLP-1 cleavage activity of purified enzyme and rat plasma. To understand the partial inhibition, we solved the co-crystal structure of one of the mAb Fabs (Ab1) in complex with rat DPP-IV. Although Ab1 does not bind at the active site, it partially blocks the side opening, which prevents the large substrates such as GLP-1 from accessing the active site, but not small molecules such as sitagliptin. When Ab1 was tested in vivo, it reduced plasma glucose and increased plasma GLP-1 concentration during an oral glucose tolerance test in rats. Together, we demonstrated the feasibility of using mAbs to inhibit DPP-IV activity and to improve glucose tolerance in a diabetic rat model.
Assuntos
Anticorpos Monoclonais/imunologia , Dipeptidil Peptidase 4/imunologia , Teste de Tolerância a Glucose , Animais , Anticorpos Monoclonais/química , Cristalografia por Raios X , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Ratos , Ratos ZuckerRESUMO
NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5'-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.
Assuntos
Proteínas Serina-Treonina Quinases/química , Tionucleotídeos/química , Apoptose/fisiologia , Linfócitos B/enzimologia , Adesão Celular/fisiologia , Linhagem Celular , Cristalografia por Raios X , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Deleção de Sequência , Tionucleotídeos/metabolismo , Quinase Induzida por NF-kappaBRESUMO
The synthesis and SAR of a series of 4,4-disubstituted cyclohexylbenzamide inhibitors of 11ß-HSD1 are described. Optimization rapidly led to potent, highly selective, and orally bioavailable inhibitors demonstrating efficacy in both rat and non-human primate ex vivo pharmacodynamic models.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Benzamidas/química , Inibidores Enzimáticos/síntese química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Administração Oral , Animais , Benzamidas/síntese química , Benzamidas/farmacocinética , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Macaca fascicularis , Microssomos Hepáticos/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
In this communication, human 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) inhibitory activities of a novel series of diarylsulfones are described. Optimization of this series resulted in several highly potent 11ß-HSD1 inhibitors with excellent pharmacokinetic (PK) properties. Compound (S)-25 showed excellent efficacy in a non-human primate ex vivo pharmacodynamic model.
Assuntos
Inibidores Enzimáticos/síntese química , Sulfonas/síntese química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Animais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Humanos , Farmacocinética , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacocinéticaRESUMO
The discovery and optimization of a series of potent PPARdelta full agonists with partial agonistic activity against PPARgamma is described.
Assuntos
PPAR delta/agonistas , PPAR gama/agonistas , Tiazóis/química , Animais , Simulação por Computador , Cristalografia por Raios X , PPAR delta/metabolismo , PPAR gama/metabolismo , Ratos , Ratos Sprague-Dawley , Tiazóis/síntese química , Tiazóis/farmacologiaRESUMO
The synthesis and SAR of a series of arylsulfonylpiperazine inhibitors of 11beta-HSD1 are described. Optimization rapidly led to potent, selective, and orally bioavailable inhibitors demonstrating efficacy in a cynomolgus monkey ex vivo enzyme inhibition model.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Ácidos Arilsulfônicos/síntese química , Piperazinas/síntese química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Administração Oral , Animais , Ácidos Arilsulfônicos/classificação , Ácidos Arilsulfônicos/farmacologia , Disponibilidade Biológica , Linhagem Celular , Cristalografia por Raios X , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/fisiologia , Humanos , Macaca fascicularis , Camundongos , Piperazinas/classificação , Piperazinas/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Novel 4,4-disubstituted cyclohexylbenzamide inhibitors of 11beta-HSD1 were optimized to account for liabilities relating to in vitro pharmacokinetics, cytotoxicity and protein-related shifts in potency. A representative compound showing favorable in vivo pharmacokinetics was found to be an efficacious inhibitor of 11beta-HSD1 in a rat pharmacodynamic model (ED(50)=10mg/kg).
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Benzamidas/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Animais , Benzamidas/farmacologia , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Macaca fascicularis , Ratos , Ratos Sprague-Dawley , Relação Estrutura-AtividadeRESUMO
Discovery and optimization of a piperidyl benzamide series of 11beta-HSD1 inhibitors is described. This series was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific protein binding, poor solubility, limited in vivo exposure, and in vitro cytotoxicity issues observed with the cyclohexyl benzamide structures. These efforts led to the discovery of piperidyl benzamide 15 which features improved properties over the cyclohexyl benzamide derivatives.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Benzamidas/síntese química , Diabetes Mellitus Tipo 2/tratamento farmacológico , Insulina/metabolismo , Piperidinas/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , Benzamidas/farmacologia , Cristalografia por Raios X/métodos , Desenho de Fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Microssomos/metabolismo , Modelos Químicos , Estrutura Molecular , Solubilidade , Relação Estrutura-AtividadeRESUMO
11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the NADPH dependent interconversion of inactive cortisone to active cortisol. Excess 11beta-HSD1 or cortisol leads to insulin resistance and metabolic syndrome in animal models and in humans. Inhibiting 11beta-HSD1 activity signifies a promising therapeutic strategy in the treatment of Type 2 diabetes and related diseases. Herein, we report two highly potent and selective small molecule inhibitors of human 11beta-HSD1. While compound 1, a sulfonamide, functions as a simple substrate competitive inhibitor, compound 2, a triazole, shows the kinetic profile of a mixed inhibitor. Co-crystal structures reveal that both compounds occupy the 11beta-HSD1 catalytic site, but present distinct molecular interactions with the protein. Strikingly, compound 2 interacts much closer to the cofactor NADP+ and likely modifies its binding. Together, the structural and kinetic analyses demonstrate two distinctive molecular inhibition mechanisms, providing valuable information for future inhibitor design.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Doenças Metabólicas/patologia , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , Sequência de Aminoácidos , Sítios de Ligação , Bioensaio , Catálise , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Cinética , Doenças Metabólicas/enzimologia , Dados de Sequência Molecular , NADP/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Triazóis/química , Triazóis/farmacologiaRESUMO
We report the discovery of potent benzamide inhibitors of 11beta-hydroxysteroid dehydrogenase (11beta-HSD1). The optimization and correlation of in vitro and in vivo metabolic stability will be described. Through modifications to our initial lead 2, we discovered pyridyl compound 13. This compound has a favorable pharmacokinetic profile across three species and showed a dose-dependent decrease in adipose 11beta-HSD1 activity in a monkey ex vivo pharmacodynamic model.
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Benzamidas/administração & dosagem , Benzamidas/síntese química , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/síntese química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/química , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Administração Oral , Animais , Benzamidas/química , Benzamidas/metabolismo , Linhagem Celular , Cristalografia por Raios X , Cães , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Macaca fascicularis , Modelos Animais , Modelos Moleculares , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
High-throughput screening of a small-molecule compound library resulted in the identification of a series of arylsulfonylpiperazines that are potent and selective inhibitors of human 11beta-Hydroxysteroid Dehydrogenase Type 1 (11beta-HSD1). Optimization of the initial lead resulted in the discovery of compound (R)-45 (11beta-HSD1 IC(50)=3nM).
Assuntos
11-beta-Hidroxiesteroide Desidrogenase Tipo 1/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Piperazinas/farmacologia , Compostos de Sulfidrila/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Camundongos , Modelos Moleculares , Estrutura Molecular , Peso Molecular , Piperazinas/síntese química , Piperazinas/química , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Relação Estrutura-Atividade , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/químicaRESUMO
11Beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid action and inhibition of this enzyme is a viable therapeutic strategy for the treatment of type 2 diabetes and the metabolic syndrome. Here, we report a potent and selective 11beta-hydroxysteroid dehydrogenase type 1 inhibitor with a binding mode elucidated from the co-crystal structure with the human 11beta-hydroxysteroid dehydrogenase type 1. The inhibitor is bound to the steroid-binding pocket making contacts with the catalytic center and the solvent channel. The inhibitor binding is facilitated by two direct hydrogen bond interactions involving Tyrosine183 of the catalytic motif Tyr-X-X-X-Lys and Alanine172. In addition, the inhibitor makes many hydrophobic interactions with both the enzyme and the co-factor nicotinamide adenine dinucleotide phosphate (reduced). In lean C57BL/6 mice, the compound inhibited both the in vivo and ex vivo 11beta-hydroxysteroid dehydrogenase type 1 activities in a dose-dependent manner. The inhibitory effects correlate with the plasma compound concentrations, suggesting that there is a clear pharmacokinetic and pharmacodynamic relationship. Moreover, at the same doses used in the pharmacokinetic/pharmacodynamic studies, the inhibitor did not cause the activation of the hypothalamic-pituitary-adrenal axis in an acute mouse model, suggesting that this compound exhibits biological effects with minimal risk of activating the hypothalamic-pituitary-adrenal axis.