RESUMEN
In the originally published version of this Letter, the authors Arthur F. Kluge, Michael A. Patane and Ce Wang were inadvertently omitted from the author list. Their affiliations are: I-to-D, Inc., PO Box 6177, Lincoln, Massachusetts 01773, USA (A.F.K.); Mitobridge, Inc. 1030 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (M.A.P.); and China Novartis Institutes for BioMedical Research, No. 4218 Jinke Road, Zhangjiang Hi-Tech Park, Pudong District, Shanghai 201203, China (C.W.). These authors contributed to the interpretation of results and design of compounds. In addition, author 'Edward A. Kesicki' was misspelled as 'Ed Kesicki'. These errors have been corrected online.
RESUMEN
The dynamic and reversible acetylation of proteins, catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), is a major epigenetic regulatory mechanism of gene transcription and is associated with multiple diseases. Histone deacetylase inhibitors are currently approved to treat certain cancers, but progress on the development of drug-like histone actyltransferase inhibitors has lagged behind. The histone acetyltransferase paralogues p300 and CREB-binding protein (CBP) are key transcriptional co-activators that are essential for a multitude of cellular processes, and have also been implicated in human pathological conditions (including cancer). Current inhibitors of the p300 and CBP histone acetyltransferase domains, including natural products, bi-substrate analogues and the widely used small molecule C646, lack potency or selectivity. Here, we describe A-485, a potent, selective and drug-like catalytic inhibitor of p300 and CBP. We present a high resolution (1.95 Å) co-crystal structure of a small molecule bound to the catalytic active site of p300 and demonstrate that A-485 competes with acetyl coenzyme A (acetyl-CoA). A-485 selectively inhibited proliferation in lineage-specific tumour types, including several haematological malignancies and androgen receptor-positive prostate cancer. A-485 inhibited the androgen receptor transcriptional program in both androgen-sensitive and castration-resistant prostate cancer and inhibited tumour growth in a castration-resistant xenograft model. These results demonstrate the feasibility of using small molecule inhibitors to selectively target the catalytic activity of histone acetyltransferases, which may provide effective treatments for transcriptional activator-driven malignancies and diseases.
Asunto(s)
Linaje de la Célula , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Histona Acetiltransferasas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Acetilcoenzima A/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Unión Competitiva , Biocatálisis/efectos de los fármacos , Dominio Catalítico/efectos de los fármacos , Línea Celular Tumoral , Linaje de la Célula/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/enzimología , Neoplasias Hematológicas/patología , Compuestos Heterocíclicos de 4 o más Anillos/química , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Humanos , Masculino , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/enzimología , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/enzimología , Neoplasias de la Próstata Resistentes a la Castración/patología , Conformación Proteica , Receptores Androgénicos/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/metabolismoRESUMEN
p300 and CREB-binding protein (CBP) are essential for a multitude of cellular processes. Dysregulation of p300/CBP histone acetyltransferase activity is linked to a broad spectrum of human diseases including cancers. A novel drug-like spirohydantoin (21) has been discovered as a selective orally bioavailable inhibitor of p300/CBP histone acetyltransferase. Lead compound 21 is more potent than the first-in-class lead A-485 in both enzymatic and cellular assays and lacks the off-target inhibition of dopamine and serotonin transporters, that was observed with A-485.
Asunto(s)
Proteína de Unión a CREB/antagonistas & inhibidores , Descubrimiento de Drogas , Proteína p300 Asociada a E1A/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Hidantoínas/farmacología , Compuestos de Espiro/farmacología , Administración Oral , Disponibilidad Biológica , Proteína de Unión a CREB/metabolismo , Relación Dosis-Respuesta a Droga , Proteína p300 Asociada a E1A/metabolismo , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/metabolismo , Humanos , Hidantoínas/administración & dosificación , Hidantoínas/metabolismo , Estructura Molecular , Compuestos de Espiro/administración & dosificación , Compuestos de Espiro/metabolismo , Relación Estructura-ActividadRESUMEN
Protein lysine methyltransferases (PKMTs) regulate diverse physiological processes including transcription and the maintenance of genomic integrity. Genetic studies suggest that the PKMTs SUV420H1 and SUV420H2 facilitate proficient nonhomologous end-joining (NHEJ)-directed DNA repair by catalyzing the di- and trimethylation (me2 and me3, respectively) of lysine 20 on histone 4 (H4K20). Here we report the identification of A-196, a potent and selective inhibitor of SUV420H1 and SUV420H2. Biochemical and co-crystallization analyses demonstrate that A-196 is a substrate-competitive inhibitor of both SUV4-20 enzymes. In cells, A-196 induced a global decrease in H4K20me2 and H4K20me3 and a concomitant increase in H4K20me1. A-196 inhibited 53BP1 foci formation upon ionizing radiation and reduced NHEJ-mediated DNA-break repair but did not affect homology-directed repair. These results demonstrate the role of SUV4-20 enzymatic activity in H4K20 methylation and DNA repair. A-196 represents a first-in-class chemical probe of SUV4-20 to investigate the role of histone methyltransferases in genomic integrity.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Inestabilidad Genómica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , Línea Celular Tumoral , Cristalografía por Rayos X , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Compuestos Heterocíclicos de 4 o más Anillos/química , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación/efectos de los fármacos , Modelos Moleculares , Estructura MolecularRESUMEN
Polycomb repressive complex 2 (PRC2) is a regulator of epigenetic states required for development and homeostasis. PRC2 trimethylates histone H3 at lysine 27 (H3K27me3), which leads to gene silencing, and is dysregulated in many cancers. The embryonic ectoderm development (EED) protein is an essential subunit of PRC2 that has both a scaffolding function and an H3K27me3-binding function. Here we report the identification of A-395, a potent antagonist of the H3K27me3 binding functions of EED. Structural studies demonstrate that A-395 binds to EED in the H3K27me3-binding pocket, thereby preventing allosteric activation of the catalytic activity of PRC2. Phenotypic effects observed in vitro and in vivo are similar to those of known PRC2 enzymatic inhibitors; however, A-395 retains potent activity against cell lines resistant to the catalytic inhibitors. A-395 represents a first-in-class antagonist of PRC2 protein-protein interactions (PPI) for use as a chemical probe to investigate the roles of EED-containing protein complexes.
Asunto(s)
Antineoplásicos/farmacología , Indanos/farmacología , Complejo Represivo Polycomb 2/antagonistas & inhibidores , Sulfonamidas/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Indanos/química , Modelos Moleculares , Estructura Molecular , Complejo Represivo Polycomb 2/química , Complejo Represivo Polycomb 2/metabolismo , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , Sulfonamidas/química , Células Tumorales CultivadasRESUMEN
Herein we report the discovery of a novel series of phosphodiesterase 10A inhibitors. Optimization of a HTS hit (17) resulted in potent, selective, and brain penetrant 23 and 26; both exhibited much lower clearance in vivo and decreased volume of distribution (rat PK) and have thus the potential to inhibit the PDE10A target in vivo at a lower efficacious dose than the reference compound WEB-3.
Asunto(s)
Compuestos Heterocíclicos con 2 Anillos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Hidrolasas Diéster Fosfóricas/metabolismo , Pirazinas/farmacología , Tiazoles/farmacología , Animales , Relación Dosis-Respuesta a Droga , Compuestos Heterocíclicos con 2 Anillos/síntesis química , Compuestos Heterocíclicos con 2 Anillos/química , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/química , Pirazinas/síntesis química , Pirazinas/química , Ratas , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/químicaRESUMEN
Herein we disclose SAR studies of a series of dimethylamino pyrrolidines which we recently reported as novel inhibitors of the PRC2 complex through disruption of EED/H3K27me3 binding. Modification of the indole and benzyl moieties of screening hit 1 provided analogs with substantially improved binding and cellular activities. This work culminated in the identification of compound 2, our nanomolar proof-of-concept (PoC) inhibitor which provided on-target tumor growth inhibition in a mouse xenograft model. X-ray crystal structures of several inhibitors bound in the EED active-site are also discussed.
Asunto(s)
Complejo Represivo Polycomb 2/antagonistas & inhibidores , Complejo Represivo Polycomb 2/metabolismo , Pirrolidinas/farmacología , Sulfonamidas/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Humanos , Ligandos , Ratones , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Complejo Represivo Polycomb 2/química , Unión Proteica , Pirrolidinas/síntesis química , Pirrolidinas/química , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Apolipoprotein E is a 299-residue lipid carrier protein produced in both the liver and the brain. The protein has three major isoforms denoted apoE2, apoE3, and apoE4 which differ at positions 112 and 158 and which occur at different frequencies in the human population. Genome-wide association studies indicate that the possession of two apoE4 alleles is a strong genetic risk factor for late-onset Alzheimer's disease (LOAD). In an attempt to identify a small molecule stabilizer of apoE4 function that may have utility as a therapy for Alzheimer's disease, we carried out an NMR-based fragment screen on the N-terminal domain of apoE4 and identified a benzyl amidine based fragment binder. In addition to NMR, binding was characterized using various other biophysical techniques, and a crystal structure of the bound core was obtained. Core elaboration ultimately yielded a compound that showed activity in an IL-6 and IL-8 cytokine release assay.
Asunto(s)
Apolipoproteína E4/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Amidinas/química , Amidinas/metabolismo , Apolipoproteína E4/química , Apolipoproteína E4/genética , Sitios de Unión , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Liposomas/química , Liposomas/metabolismo , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Temperatura de TransiciónRESUMEN
IDH1 plays a critical role in a number of metabolic processes and serves as a key source of cytosolic NADPH under conditions of cellular stress. However, few inhibitors of wild-type IDH1 have been reported. Here we present the discovery and biochemical characterization of two novel inhibitors of wild-type IDH1. In addition, we present the first ligand-bound crystallographic characterization of these novel small molecule IDH1 binding pockets. Importantly, the NADPH competitive α,ß-unsaturated enone 1 makes a unique covalent linkage through active site H315. As few small molecules have been shown to covalently react with histidine residues, these data support the potential utility of an underutilized strategy for reversible covalent small molecule design.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Histidina , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Isocitrato Deshidrogenasa/química , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Ligandos , Simulación del Acoplamiento Molecular , Mutación , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Adenosine kinase (AK) is an enzyme responsible for converting endogenous adenosine (ADO) to adenosine monophosphate (AMP) in an adenosine triphosphate- (ATP-) dependent manner. The structure of AK consists of two domains, the first a large alpha/beta Rossmann-like nucleotide binding domain that forms the ATP binding site, and a smaller mixed alpha/beta domain, which, in combination with the larger domain, forms the ADO binding site and the site of phosphoryl transfer. AK inhibitors have been under investigation as antinociceptive, antiinflammatory, and anticonvulsant as well as antiinfective agents. In this work, we report the structures of AK in complex with two classes of inhibitors: the first, ADO-like, and the second, a novel alkynylpyrimidine series. The two classes of structures, which contain structurally similar substituents, reveal distinct binding modes in which the AK structure accommodates the inhibitor classes by a 30 degrees rotation of the small domain relative to the large domain. This change in binding mode stabilizes an open and a closed intermediate structural state and provide structural insight into the transition required for catalysis. This results in a significant rearrangement of both the protein active site and the orientation of the alkynylpyrimidine ligand when compared to the observed orientation of nucleosidic inhibitors or substrates.
Asunto(s)
Adenosina Quinasa/antagonistas & inhibidores , Adenosina Quinasa/química , Inhibidores Enzimáticos/química , Morfolinas/química , Pirimidinas/química , Tubercidina/análogos & derivados , Animales , Sitios de Unión , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Toxoplasma/enzimología , Tubercidina/químicaRESUMEN
In a broad genomics analysis to find novel protein targets for antibiotic discovery, MurF was identified as an essential gene product for Streptococcus pneumonia that catalyzes a critical reaction in the biosynthesis of the peptidoglycan in the formation of the cell wall. Lacking close relatives in mammalian biology, MurF presents attractive characteristics as a potential drug target. Initial screening of the Abbott small-molecule compound collection identified several compounds for further validation as pharmaceutical leads. Here we report the integrated efforts of NMR and X-ray crystallography, which reveal the multidomain structure of a MurF-inhibitor complex in a compact conformation that differs dramatically from related structures. The lead molecule is bound in the substrate-binding region and induces domain closure, suggestive of the domain arrangement for the as yet unobserved transition state conformation for MurF enzymes. The results form a basis for directed optimization of the compound lead by structure-based design to explore the suitability of MurF as a pharmaceutical target.
Asunto(s)
Inhibidores Enzimáticos/química , Péptido Sintasas/antagonistas & inhibidores , Péptido Sintasas/química , Streptococcus pneumoniae/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Inhibidores Enzimáticos/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Péptido Sintasas/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología de Secuencia , Especificidad por SustratoRESUMEN
A lack of useful small molecule tools has precluded thorough interrogation of the biological function of SMYD2, a lysine methyltransferase with known tumor-suppressor substrates. Systematic exploration of the structure-activity relationships of a previously known benzoxazinone compound led to the synthesis of A-893, a potent and selective SMYD2 inhibitor (IC50: 2.8 nM). A cocrystal structure reveals the origin of enhanced potency, and effective suppression of p53K370 methylation is observed in a lung carcinoma (A549) cell line.
RESUMEN
A novel series of 4-[(4-cyano-2-arylbenzyloxy)-(3-methyl-3H-imidazol-4-yl)methyl]benzonitriles have been synthesized as selective farnesyltransferase inhibitors using structure-based design. X-ray cocrystal structures of compound 20-FTase-HFP and A313326-FTase-HFP confirmed our initial design. The decreased interaction between the aryl groups and Ser 48 in GGTase-I binding site could be one possible reason to explain the improved selectivity for this new series of FTase inhibitors. Medicinal chemistry efforts led to the discovery of compound 64 with potent cellular activity (EC(50) = 3.5 nM) and outstanding pharmacokinetic profiles in dog (96% bioavailable, 18.4 h oral t(1/2), and 0.19 L/(h x kg) plasma clearance).
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Benzamidas/síntesis química , Imidazoles/síntesis química , Nitrilos/síntesis química , Administración Oral , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/metabolismo , Animales , Benzamidas/farmacocinética , Benzamidas/farmacología , Disponibilidad Biológica , Permeabilidad de la Membrana Celular , Cristalografía por Rayos X , Perros , Diseño de Fármacos , Farnesiltransferasa , Imidazoles/farmacocinética , Imidazoles/farmacología , Modelos Moleculares , Estructura Molecular , Nitrilos/farmacocinética , Nitrilos/farmacología , Relación Estructura-ActividadRESUMEN
Hepatic blockade of glucocorticoid receptors (GR) suppresses glucose production and thus decreases circulating glucose levels, but systemic glucocorticoid antagonism can produce adrenal insufficiency and other undesirable side effects. These hepatic and systemic responses might be dissected, leading to liver-selective pharmacology, when a GR antagonist is linked to a bile acid in an appropriate manner. Bile acid conjugation can be accomplished with a minimal loss of binding affinity for GR. The resultant conjugates remain potent in cell-based functional assays. A novel in vivo assay has been developed to simultaneously evaluate both hepatic and systemic GR blockade; this assay has been used to optimize the nature and site of the linker functionality, as well as the choice of the GR antagonist and the bile acid. This optimization led to the identification of A-348441, which reduces glucose levels and improves lipid profiles in an animal model of diabetes.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/síntesis química , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/síntesis química , Hígado/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Animales , Ácidos y Sales Biliares/química , Sitios de Unión , Hidrocarburos Aromáticos con Puentes/química , Hidrocarburos Aromáticos con Puentes/farmacología , Células CHO , Células Cultivadas , Simulación por Computador , Cricetinae , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 2/sangre , Glucosa/biosíntesis , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/fisiología , Masculino , Ratones , Modelos Moleculares , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Glucocorticoides/metabolismo , Relación Estructura-ActividadRESUMEN
Potent inhibitors of 7,8-dihydroneopterin aldolase (DHNA; EC 4.1.2.25) have been discovered using CrystaLEAD X-ray crystallographic high-throughput screening followed by structure-directed optimization. Screening of a 10 000 compound random library provided several low affinity leads and their corresponding X-ray crystal structures bound to the enzyme. The presence of a common structural feature in each of the leads suggested a strategy for the construction of a directed library of approximately 1000 compounds that were screened for inhibitory activity in a traditional enzyme assay. Several lead compounds with IC(50) values of about 1 microM against DHNA were identified, and crystal structures of their enzyme-bound complexes were obtained by cocrystallization. Structure-directed optimization of one of the leads thus identified afforded potent inhibitors with submicromolar IC(50) values.
Asunto(s)
Aldehído-Liasas/antagonistas & inhibidores , Aldehído-Liasas/química , Benzoatos/química , Inhibidores Enzimáticos/química , Neopterin/química , Pirimidinas/química , Triazoles/química , Benzoatos/síntesis química , Sitios de Unión , Cristalografía por Rayos X , Bases de Datos Factuales , Inhibidores Enzimáticos/síntesis química , Guanina/análogos & derivados , Guanina/síntesis química , Guanina/química , Modelos Moleculares , Estructura Molecular , Purinas/química , Pirimidinas/síntesis química , Relación Estructura-Actividad , Triazoles/síntesis químicaRESUMEN
A dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-Ig™), is engineered to block two targets. Flexibility modulates Fc receptor and complement binding, but could result in undesirable cross-linking of surface antigens and downstream signaling. Understanding the flexibility of parental mAbs is important for designing and retaining functionality of DVD-Ig™ molecules. The architecture and dynamics of a DVD-Ig™ molecule and its parental mAbs was examined using single particle electron microscopy. Hinge angles measured for the DVD-Ig™ molecule were similar to the inner antigen parental mAb. The outer binding domain of the DVD-Ig™ molecule was highly mobile and three-dimensional (3D) analysis showed binding of inner antigen caused the outer domain to fold out of the plane with a major morphological change. Docking high-resolution X-ray structures into the 3D electron microscopy map supports the extraordinary domain flexibility observed in the DVD-Ig™ molecule allowing antigen binding with minimal steric hindrance.
Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Región Variable de Inmunoglobulina/química , Inmunoterapia , Anticuerpos Monoclonales/uso terapéutico , Antígenos/inmunología , Cristalografía por Rayos X , Humanos , Interleucina-12/química , Interleucina-12/inmunología , Interleucina-18/química , Interleucina-18/inmunología , Microscopía Electrónica de Transmisión , Unión Proteica , Ingeniería de Proteínas/métodos , Estructura Terciaria de ProteínaRESUMEN
Several bispecific antibody-based formats have been developed over the past 25 years in an effort to produce a new generation of immunotherapeutics that target two or more disease mechanisms simultaneously. One such format, the dual-variable domain immunoglobulin (DVD-Ig™), combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule. We report the structure of an interleukin (IL)12-IL18 DVD-Ig™ Fab (DFab) fragment with IL18 bound to the inner variable domain (VD) that reveals the remarkable flexibility of the DVD-Ig™ molecule and how the DVD-Ig™ format can function to bind four antigens simultaneously. An understanding of how the inner variable domain retains function is of critical importance for designing DVD-Ig™ molecules, and for better understanding of the flexibility of immunoglobulin variable domains and linkers, which may aid in the design of improved bi- and multi-specific biologics in general.
Asunto(s)
Anticuerpos Biespecíficos/química , Región Variable de Inmunoglobulina/química , Inmunoterapia/métodos , Interleucina-18/química , Anticuerpos Biespecíficos/uso terapéutico , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/uso terapéutico , Especificidad de Anticuerpos , Antígenos/inmunología , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Interleucina-12/inmunología , Interleucina-18/inmunología , Unión Proteica , Ingeniería de Proteínas , Estructura Terciaria de ProteínaRESUMEN
The DVD-Ig (TM) protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.
Asunto(s)
Anticuerpos Monoclonales/química , Diseño de Fármacos , Región Variable de Inmunoglobulina/química , Factor de Necrosis Tumoral alfa/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Humanos , Región Variable de Inmunoglobulina/metabolismo , Cinética , Ligandos , Datos de Secuencia Molecular , Unión Proteica , Ingeniería de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Recombinant human erythropoietin (rHu-EPO) is used to treat anemia by activating the erythropoietin receptor (EPOR) in erythroid progenitor cells, leading to proliferation and differentiation into mature red blood cells. To allow less frequent dosing, a hyperglycosylated version of EPO has been developed with a longer half-life. In principle, an agonistic antibody targeting EPOR would offer an even longer half-life, support robust monthly dosing, and, unlike EPO products, reduce the risk of pure red cell aplasia. The efficiency of signaling and corresponding potency of previously reported antibody mimics are generally suboptimal compared with EPO and not suitable for clinical use. Here we describe a potent, fully human, agonistic antibody (ABT007) targeting EPOR that supports potent, more sustained, and less pulsatile elevation of hematocrit in a human EPOR-expressing transgenic mouse model compared with standard doses of rHu-EPO while requiring less frequent dosing. Resolution of the crystal structure of the EPOR extracellular domain (ECD) complexed to the ABT007 Fab fragment, determined at 0.32 nm, identifies a binding site that is consistent with a novel mechanism of receptor activation based on a unique antibody-imposed conformational change. These results demonstrate that a symmetric molecule can serve as a potent activator of the EPOR.