RESUMEN
Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that optimizes the peptide cargo of major histocompatibility class I (MHC-I) molecules and regulates adaptive immunity. It has unusual substrate selectivity for length and sequence, resulting in poorly understood effects on the cellular immunopeptidome. To understand substrate selection by ERAP1, we solved 2 crystal structures of the enzyme with bound transition-state pseudopeptide analogs at 1.68 Å and 1.72 Å. Both peptides have their N terminus bound at the active site and extend away along a large internal cavity, interacting with shallow pockets that can influence selectivity. The longer peptide is disordered through the central region of the cavity and has its C terminus bound in an allosteric pocket of domain IV that features a carboxypeptidase-like structural motif. These structures, along with enzymatic and computational analyses, explain how ERAP1 can select peptides based on length while retaining the broad sequence-specificity necessary for its biological function.
RESUMEN
A series of DNA gyrase inhibitors were designed based on the X-ray structure of a parent thiophene scaffold with the objective to improve biochemical and whole-cell antibacterial activity, while reducing cardiac ion channel activity. The binding mode and overall design hypothesis of one series was confirmed with a co-crystal structure with DNA gyrase. Although some analogs retained both biochemical activity and whole-cell antibacterial activity, we were unable to significantly improve the activity of the series and analogs retained activity against the cardiac ion channels, therefore we stopped optimization efforts.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , Girasa de ADN/metabolismo , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Inhibidores de Topoisomerasa II/farmacología , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Línea Celular , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Noqueados , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/químicaRESUMEN
The discovery and lead optimisation of a novel series of SYK inhibitors is described. These were optimised for SYK potency and selectivity against Aurora B. Compounds were profiled in a human skin penetration study to identify a suitable candidate molecule for pre-clinical development. Compound 44 (GSK2646264) was selected for progression and is currently in Phase I clinical trials.
Asunto(s)
Antiinflamatorios/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Piel/efectos de los fármacos , Quinasa Syk/antagonistas & inhibidores , Administración Tópica , Antiinflamatorios/administración & dosificación , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Dominio Catalítico , Línea Celular Tumoral , Humanos , Simulación del Acoplamiento Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/administración & dosificación , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , Quinasa Syk/químicaRESUMEN
A potent VEGF inhibitor with novel antibody architecture and antigen binding mode has been developed. The molecule, hereafter referred to as VEGF dual dAb (domain antibody), was evaluated in vitro for binding to VEGF and for potency in VEGF-driven models and compared with other anti-VEGF biologics that have been used in ocular anti-angiogenic therapeutic regimes. VEGF dual dAb is more potent than bevacizumab and ranibizumab for VEGF binding, inhibition of VEGF receptor binding assays (RBAs), and VEGF-driven in vitro models of angiogenesis and displays comparable inhibition to aflibercept (Eylea). VEGF dual dAb is dimeric, and each monomer contains two distinct anti-VEGF domain antibodies attached via linkers to a human IgG1 Fc domain. Mechanistically, the enhanced in vitro potency of VEGF dual dAb, in comparison to other anti-VEGF biologics, can be explained by increased binding stoichiometry. A consistent model of the target engagement has been built based on the x-ray complexes of each of the two isolated domain antibodies with the VEGF antigen.
Asunto(s)
Bevacizumab/farmacología , Ranibizumab/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/farmacología , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Dominio Único/farmacología , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/metabolismo , Inhibidores de la Angiogénesis/química , Inhibidores de la Angiogénesis/farmacología , Animales , Cristalografía por Rayos X , Descubrimiento de Drogas , Células HEK293 , Humanos , Modelos Moleculares , Anticuerpos de Dominio Único/química , Porcinos , Factor A de Crecimiento Endotelial Vascular/químicaRESUMEN
The optimisation of the azanaphthyridine series of Spleen Tyrosine Kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over hERG activity. A good pharmacokinetic profile was achieved by modification of the pKa. Morpholine compound 32 is a potent SYK inhibitor showing moderate selectivity, good oral bioavailability and good efficacy in the rat Arthus model but demonstrated a genotoxic potential in the Ames assay.
Asunto(s)
Naftiridinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Administración Oral , Animales , Disponibilidad Biológica , Cristalografía por Rayos X , Humanos , Pruebas de Mutagenicidad , Naftiridinas/administración & dosificación , Naftiridinas/farmacocinética , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
5-Aryl-4-carboxamide-1,3-oxazoles are a novel, potent and selective series of GSK-3 inhibitors. The optimization of the series to yield compounds with cell activity and brain permeability is described.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Oxazoles/química , Oxazoles/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Animales , Trastorno Bipolar/tratamiento farmacológico , Encéfalo/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Modelos Moleculares , Oxazoles/síntesis química , Oxazoles/farmacocinética , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
The Janus family of tyrosine kinases (JAK1, JAK2, JAK3, and TYK2) play an essential role in the receptor signaling of cytokines that have been implicated in the pathogenesis of severe asthma, and there is emerging interest in the development of small-molecule-inhaled JAK inhibitors as treatments. Here, we describe the optimization of a quinazoline series of JAK inhibitors and the results of mouse lung pharmacokinetic (PK) studies where only low concentrations of parent compound were observed. Subsequent investigations revealed that the low exposure was due to metabolism by aldehyde oxidase (AO), so we sought to identify quinazolines that were not metabolized by AO. We found that specific substituents at the quinazoline 2-position prevented AO metabolism and this was rationalized through computational docking studies in the AO binding site, but they compromised kinome selectivity. Results presented here highlight that AO metabolism is a potential issue in the lung.
Asunto(s)
Aldehído Oxidasa/metabolismo , Inhibidores de las Cinasas Janus/farmacocinética , Pulmón/metabolismo , Administración Intranasal , Administración Intravenosa , Animales , Sitios de Unión , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inhibidores de las Cinasas Janus/administración & dosificación , Inhibidores de las Cinasas Janus/síntesis química , Hígado/metabolismo , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Simulación del Acoplamiento Molecular , Quinazolinas/síntesis química , Quinazolinas/farmacocinética , Quinazolinas/farmacología , Relación Estructura-ActividadRESUMEN
The discovery of a novel series of 2-(4-pyridyl)thienopyridinone GSK-3ß inhibitors is reported. X-ray crystallography reveals its binding mode and enables rationalization of the SAR. The initial optimization of the template for improved cellular activity and predicted CNS penetration is also presented.
Asunto(s)
Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Tiofenos/farmacología , Línea Celular , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Glucógeno Sintasa Quinasa 3 beta , Humanos , Modelos Moleculares , Estructura Molecular , Permeabilidad/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridonas/síntesis química , Piridonas/química , Estereoisomerismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/químicaRESUMEN
The lead optimisation of the diaminopyrimidine carboxamide series of spleen tyrosine kinase inhibitors is described. The medicinal chemistry strategy was focused on optimising the human whole blood activity whilst achieving a sufficient margin over liability kinases and hERG activity. GSK143 is a potent and highly selective SYK inhibitor showing good efficacy in the rat Arthus model.
Asunto(s)
Compuestos de Anilina/química , Compuestos de Anilina/uso terapéutico , Antiinflamatorios/química , Antiinflamatorios/uso terapéutico , Reacción de Arthus/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/química , Pirimidinas/uso terapéutico , Administración Oral , Compuestos de Anilina/farmacología , Animales , Antiinflamatorios/farmacología , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Ratas , Relación Estructura-Actividad , Quinasa SykRESUMEN
Following the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective phosphodiesterase 4B inhibitors, [Hamblin, J. N.; Angell, T.; Ballentine, S., et al. Bioorg. Med. Chem. Lett.2008, 18, 4237] the SAR of the 5-position was investigated further. A range of substituted heterocycles showed good potencies against PDE4. Optimisation using X-ray crystallography and computational modelling led to the discovery of 16, with sub-nM inhibition of LPS-induced TNF-α production from isolated human peripheral blood mononuclear cells.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Compuestos Heterocíclicos/química , Inhibidores de Fosfodiesterasa/química , Pirazoles/química , Piridinas/química , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Humanos , Modelos Químicos , Modelos Moleculares , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/farmacología , Estructura Terciaria de Proteína , Pirazoles/síntesis química , Pirazoles/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
ER aminopeptidase 1 (ERAP1) is an intracellular enzyme that generates antigenic peptides and is an emerging target for cancer immunotherapy and the control of autoimmunity. ERAP1 inhibitors described previously target the active site and are limited in selectivity, minimizing their clinical potential. To address this, we targeted the regulatory site of ERAP1 using a high-throughput screen and discovered a small molecule hit that is highly selective for ERAP1. (4aR,5S,6R,8S,8aR)-5-(2-(Furan-3-yl)ethyl)-8-hydroxy-5,6,8a-trimethyl-3,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carboxylic acid is a natural product found in Dodonaea viscosa that constitutes a submicromolar, highly selective, and cell-active modulator of ERAP1. Although the compound activates hydrolysis of small model substrates, it is a competitive inhibitor for physiologically relevant longer peptides. Crystallographic analysis confirmed that the compound targets the regulatory site of the enzyme that normally binds the C-terminus of the peptide substrate. Our findings constitute a novel starting point for the development of selective ERAP1 modulators that have potential for further clinical development.
Asunto(s)
Aminopeptidasas/antagonistas & inhibidores , Presentación de Antígeno/efectos de los fármacos , Diterpenos de Tipo Clerodano/farmacología , Epítopos/metabolismo , Péptidos/metabolismo , Inhibidores de Proteasas/farmacología , Sitio Alostérico , Aminopeptidasas/química , Aminopeptidasas/metabolismo , Animales , Dominio Catalítico , Cristalografía por Rayos X , Diterpenos de Tipo Clerodano/química , Diterpenos de Tipo Clerodano/metabolismo , Activadores de Enzimas/química , Activadores de Enzimas/metabolismo , Activadores de Enzimas/farmacología , Epítopos/química , Células HeLa , Humanos , Ratones , Antígenos de Histocompatibilidad Menor/química , Antígenos de Histocompatibilidad Menor/metabolismo , Péptidos/química , Inhibidores de Proteasas/química , Inhibidores de Proteasas/metabolismo , Unión Proteica , Proteolisis/efectos de los fármacosRESUMEN
Crystallography-driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of a series of quinoline derivatives that were highly potent and selective inhibitors of PDE4 with a good pharmacokinetic profile in the rat. Quinolines 43 and 48 have potential as oral medicines for the treatment of COPD.
Asunto(s)
Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/administración & dosificación , Inhibidores de Fosfodiesterasa/química , Quinolinas/administración & dosificación , Quinolinas/química , Administración Oral , Animales , Bovinos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Humanos , Masculino , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Crystallography driven optimisation of a lead derived from similarity searching of the GSK compound collection resulted in the discovery of quinoline-3-carboxamides as highly potent and selective inhibitors of phosphodiesterase 4B. This series has been optimized to GSK256066, a potent PDE4B inhibitor which also inhibits LPS induced production of TNF-alpha from isolated human peripheral blood mononuclear cells with a pIC(50) of 11.1. GSK256066 also has a suitable profile for inhaled dosing.
Asunto(s)
Antiinflamatorios/química , Inhibidores de Fosfodiesterasa 4 , Inhibidores de Fosfodiesterasa/química , Quinolinas/química , Administración por Inhalación , Animales , Antiinflamatorios/síntesis química , Antiinflamatorios/farmacocinética , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Inhibidores de Fosfodiesterasa/síntesis química , Inhibidores de Fosfodiesterasa/farmacocinética , Quinolinas/síntesis química , Quinolinas/farmacocinética , Ratas , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A PDE4B over 4D-selective inhibitor programme was initiated to capitalise on the recently discovered predominance of the PDE4B subtype in inflammatory cell regulation. The SAR of a tetrahydrobenzothiophene (THBT) series did not agree with either of two proposed docking modes in the 4B binding site. A subsequent X-ray co-crystal structure determination revealed that the THBT ligand displaces the Gln-443 residue, invariably ligand-anchoring in previous PDE4 co-crystal structures, and even shifts helix-15 by 1-2A. For the first time, several residues of the C-terminus previously proposed to be involved in subtype selectivity are resolved and three of them extend into the ligand binding site potentially allowing for selective drug design.
Asunto(s)
Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/metabolismo , Inhibidores de Fosfodiesterasa 4 , Tiofenos/química , Tiofenos/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/genética , Humanos , Modelos Moleculares , Estructura Molecular , Mutación , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that helps generate peptides presented by Major Histocompatibility Complex Class I (MHC class I) molecules and is an emerging target for immunotherapy applications. Despite almost two decades of research on ERAP1, lack of high-resolution crystal structures has hampered drug-development efforts. By optimizing the protein construct, we obtained a high-resolution (1.60 Å) crystal structure of the closed-conformation of ERAP1 with a potent phosphinic pseudopeptide inhibitor bound in its active site. The structure provides key insight on the mechanism of inhibition as well as selectivity toward homologous enzymes and allows detailed mapping of the internal cavity of the enzyme that accommodates peptide-substrates. Bis-tris propane and malic acid molecules, found bound in pockets in the internal cavity, reveal potential druggable secondary binding sites. The ability to obtain high-resolution crystal structures of ERAP1 removes a major bottleneck in the development of compounds that regulate its activity and will greatly accelerate drug-discovery efforts.
RESUMEN
Optimisation of a high-throughput screening hit resulted in the discovery of 4-(substituted amino)-1-alkyl-pyrazolo[3,4-b]pyridine-5-carboxamides as potent and selective inhibitors of Phosphodiesterase 4 (PDE4). Herein, we describe early SAR studies around this novel template highlighting preferred substituents and rationalization of SAR through X-ray crystal structures of analogues bound to the PDE4 active site. Pyrazolopyridine 20a was found to be a potent and selective PDE4 inhibitor which also inhibits LPS induced TNF-alpha production from isolated human peripheral blood mononuclear cells and has an encouraging rat PK profile suitable for oral dosing.
Asunto(s)
Química Farmacéutica/métodos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4/química , Inhibidores de Fosfodiesterasa 4 , Pirazoles/síntesis química , Pirazoles/farmacocinética , Piridinas/síntesis química , Piridinas/farmacocinética , Aminopiridinas/farmacología , Animales , Benzamidas/farmacología , Cristalografía por Rayos X/métodos , Ciclopropanos/farmacología , Diseño de Fármacos , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Modelos Biológicos , Modelos Químicos , Pirrolidinonas/química , Ratas , Rolipram/farmacología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Tetrahydropyran derivative 1 was discovered in a high-throughput screening campaign to find new inhibitors of mycobacterial InhA. Following initial in-vitro profiling, a structure-activity relationship study was initiated and a focused library of analogs was synthesized and evaluated. This yielded compound 42 with improved antimycobacterial activity and low cytotoxicity. Additionally, the crystal structure of InhA in complex with inhibitor 1 was resolved, to reveal the binding mode and provide hints for further optimization.
Asunto(s)
Antituberculosos/química , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Piranos/química , Piranos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Humanos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Relación Estructura-Actividad , Tuberculosis/tratamiento farmacológico , Tuberculosis/microbiologíaRESUMEN
Phenotypic screens for bactericidal compounds are starting to yield promising hits against tuberculosis. In this regard, whole-genome sequencing of spontaneous resistant mutants generated against an indazole sulfonamide (GSK3011724A) identifies several specific single-nucleotide polymorphisms in the essential Mycobacterium tuberculosis ß-ketoacyl synthase (kas) A gene. Here, this genomic-based target assignment is confirmed by biochemical assays, chemical proteomics and structural resolution of a KasA-GSK3011724A complex by X-ray crystallography. Finally, M. tuberculosis GSK3011724A-resistant mutants increase the in vitro minimum inhibitory concentration and the in vivo 99% effective dose in mice, establishing in vitro and in vivo target engagement. Surprisingly, the lack of target engagement of the related ß-ketoacyl synthases (FabH and KasB) suggests a different mode of inhibition when compared with other Kas inhibitors of fatty acid biosynthesis in bacteria. These results clearly identify KasA as the biological target of GSK3011724A and validate this enzyme for further drug discovery efforts against tuberculosis.
Asunto(s)
3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/genética , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Indazoles/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Sulfonamidas/farmacología , Tuberculosis Pulmonar/tratamiento farmacológico , Animales , Farmacorresistencia Bacteriana/genética , Femenino , Ratones , Ratones Endogámicos C57BL , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/genética , Polimorfismo de Nucleótido Simple/genética , Tuberculosis Pulmonar/microbiología , Tuberculosis Pulmonar/prevención & controlRESUMEN
Tuberculosis (TB) is one of the world's oldest and deadliest diseases, killing a person every 20 s. InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, is the target of the frontline antitubercular drug isoniazid (INH). Compounds that directly target InhA and do not require activation by mycobacterial catalase peroxidase KatG are promising candidates for treating infections caused by INH resistant strains. The application of the encoded library technology (ELT) to the discovery of direct InhA inhibitors yielded compound 7 endowed with good enzymatic potency but with low antitubercular potency. This work reports the hit identification, the selected strategy for potency optimization, the structure-activity relationships of a hundred analogues synthesized, and the results of the in vivo efficacy studies performed with the lead compound 65.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Descubrimiento de Drogas , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/metabolismo , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The identification and exploration of a novel, potent and selective series of N-(3-cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)amide inhibitors of JNK2 and JNK3 kinases is described. Compounds 5a and 11a were identified as potent inhibitors of JNK3 (pIC50 6.7 and 6.6, respectively), with essentially equal potency against JNK2 (pIC50 6.5). Selectivity within the mitogen-activated protein kinase (MAPK) family, against JNK1, p38alpha and ERK2, was observed for the series. X-ray crystallography of 5e and 8a in JNK3 revealed a unique binding mode, with the 3-cyano substituent forming an H-bond acceptor interaction with the hinge region of the ATP-binding site.