RESUMO
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
Fibroblast growth factor 21 (FGF21), an endocrine hormone in the FGF family, plays a critical role in regulating metabolic homeostasis and has emerged as a therapeutic target for metabolic diseases, including Type 2 diabetes mellitus. FGF21 functions through a receptor complex that consists of an FGF receptor (FGFR) and a co-receptor ß-Klotho. Here, we identify and biochemically and structurally characterize 39F7, a high-affinity agonistic monoclonal antibody (mAb) against ß-Klotho that mimics FGF21 function. The co-crystal structure of ß-Klotho KL1 domain in complex with 39F7 Fab revealed that the recognition of 39F7 is centered on Trp-295 of ß-Klotho in a FGF21 noncompetitive manner. KL1 adopts a (ß/α)8 TIM barrel fold which resembles that of ß-glycosylceramidase, but lacks molecular features for enzymatic activity, suggesting that KL1 functions as a scaffold protein instead. In vitro characterization demonstrated that, although 39F7 does not compete with FGF21, it is specific for ß-Klotho/FGFR1c activation. Furthermore, the agonistic activity of 39F7 required the full IgG molecule to be bivalent, suggesting that 39F7 functions by promoting receptor/co-receptor dimerization. Supported by negative stain EM analysis of full-length ß-Klotho, we propose a molecular model wherein the agonistic antibody 39F7 acts in a ß-Klotho- and FGFR1c-dependent manner, mimicking FGF21 activity. More importantly, 39F7 offers promising therapeutic potential in the axis of FGF21 signaling as an antibody therapy alternative to FGF21 analogs for treatment of metabolic diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Membrana/imunologia , Animais , Afinidade de Anticorpos , Células CHO , Cricetulus , Cristalografia por Raios X , Humanos , Proteínas Klotho , Proteínas de Membrana/agonistas , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Microscopia Eletrônica , Ligação Proteica , Conformação Proteica , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de SinaisRESUMO
Small molecules and antibodies each have advantages and limitations as therapeutics. Here, we present for the first time to our knowledge, the structure-guided design of "chemibodies" as small molecule-antibody hybrids that offer dual recognition of a single target by both a small molecule and an antibody, using DPP-IV enzyme as a proof of concept study. Biochemical characterization demonstrates that the chemibodies present superior DPP-IV inhibition compared to either small molecule or antibody component alone. We validated our design by successfully solving a co-crystal structure of a chemibody in complex with DPP-IV, confirming specific binding of the small molecule portion at the interior catalytic site and the Fab portion at the protein surface. The discovery of chemibodies presents considerable potential for novel therapeutics that harness the power of both small molecule and antibody modalities to achieve superior specificity, potency, and pharmacokinetic properties.
Assuntos
Dipeptidil Peptidase 4/química , Inibidores Enzimáticos/síntese química , Imunoconjugados/farmacologia , Animais , Anticorpos Monoclonais/química , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Imunoconjugados/química , Modelos Moleculares , Ratos , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
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
Retinol-Binding Protein 4 (RBP4) is a plasma protein that transports retinol (vitamin A) from the liver to peripheral tissues. This Letter highlights our efforts in discovering the first, to our knowledge, non-retinoid small molecules that bind to RBP4 at the retinol site and reduce serum RBP4 levels in mice, by disrupting the interaction between RBP4 and transthyretin (TTR), a plasma protein that binds RBP4 and protects it from renal excretion. Potent compounds were discovered and optimized quickly from high-throughput screen (HTS) hits utilizing a structure-based approach. Inhibitor co-crystal X-ray structures revealed unique disruptions of RBP4-TTR interactions by our compounds through induced loop conformational changes instead of steric hindrance exemplified by fenretinide. When administered to mice, A1120, a representative compound in the series, showed concentration-dependent retinol and RBP4 lowering.
Assuntos
Descoberta de Drogas , Proteínas Plasmáticas de Ligação ao Retinol/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Ligantes , Masculino , Camundongos , Modelos Moleculares , Estrutura Molecular , Ratos , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Vitamina A/sangueRESUMO
Sphingosine kinase 1 (SphK1) is a lipid kinase that catalyzes the conversion of sphingosine to sphingosine-1-phosphate (S1P), which has been shown to play a role in lymphocyte trafficking, angiogenesis, and response to apoptotic stimuli. As a central enzyme in modulating the S1P levels in cells, SphK1 emerges as an important regulator for diverse cellular functions and a potential target for drug discovery. Here, we present the crystal structures of human SphK1 in the apo form and in complexes with a substrate sphingosine-like lipid, ADP, and an inhibitor at 2.0-2.3 Å resolution. The SphK1 structures reveal a two-domain architecture in which its catalytic site is located in the cleft between the two domains and a hydrophobic lipid-binding pocket is buried in the C-terminal domain. Comparative analysis of these structures with mutagenesis and kinetic studies provides insight into how SphK1 recognizes the lipid substrate and catalyzes ATP-dependent phosphorylation.
Assuntos
Lisofosfolipídeos/química , Esfingosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Catálise , Cristalografia por Raios X , Humanos , Cinética , Lisofosfolipídeos/metabolismo , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Esfingosina/química , Esfingosina/metabolismo , Especificidade por SubstratoRESUMO
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 eukaryotic initiation factor 4E (eIF4E) plays a central role in the initiation of gene translation and subsequent protein synthesis by binding the 5' terminal mRNA cap structure. We designed and synthesized a series of novel compounds that display potent binding affinity against eIF4E despite their lack of a ribose moiety, phosphate, and positive charge as present in m7-GMP. The biochemical activity of compound 33 is 95 nM for eIF4E in an SPA binding assay. More importantly, the compound has an IC(50) of 2.5 µM for inhibiting cap-dependent mRNA translation in a rabbit reticular cell extract assay (RRL-IVT). This series of potent, truncated analogues could serve as a promising new starting point toward the design of neutral eIF4E inhibitors with physicochemical properties suitable for cellular activity assessment.
Assuntos
Fator de Iniciação 4E em Eucariotos/metabolismo , Guanina/análogos & derivados , Guanosina Monofosfato/análogos & derivados , Guanosina Monofosfato/farmacologia , Organofosfonatos/síntese química , Capuzes de RNA/metabolismo , Animais , Cristalografia por Raios X , Desenho de Fármacos , Fator de Iniciação 4E em Eucariotos/química , Guanina/síntese química , Guanina/farmacologia , Guanosina Monofosfato/síntese química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Organofosfonatos/farmacologia , Ácidos Fosforosos , Biossíntese de Proteínas/efeitos dos fármacos , Capuzes de RNA/química , Coelhos , Reticulócitos/efeitos dos fármacos , Reticulócitos/metabolismo , Relação Estrutura-AtividadeRESUMO
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
Retinol-binding protein 4 (RBP4) transports retinol from the liver to extrahepatic tissues, and RBP4 lowering is reported to improve insulin sensitivity in mice. We have identified A1120, a high affinity (K(i) = 8.3 nm) non-retinoid ligand for RBP4, which disrupts the interaction between RBP4 and its binding partner transthyretin. Analysis of the RBP4-A1120 co-crystal structure reveals that A1120 induces critical conformational changes at the RBP4-transthyretin interface. Administration of A1120 to mice lowers serum RBP4 and retinol levels but, unexpectedly, does not improve insulin sensitivity. In addition, we show that Rpb4(-/-) mice display normal insulin sensitivity and are not protected from high fat diet-induced insulin resistance. We conclude that lowering RBP4 levels does not improve insulin sensitivity in mice. Therefore, RBP4 lowering may not be an effective strategy for treating diabetes.