RESUMEN
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (ß and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1ß1γ1, α1ß2γ1, α1ß2γ3, α2ß1γ1, α2ß2γ1 and α2ß2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were â¼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr(172/174)). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with ß1-containing heterotrimers, ß2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Adenosina Monofosfato/química , Regulación Alostérica , Compuestos de Bifenilo , Activación Enzimática , Activadores de Enzimas/química , Pruebas de Enzimas , Humanos , Isoenzimas/química , Cinética , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Pironas/química , Proteínas Recombinantes/química , Tiofenos/químicaRESUMEN
Lysosomal acid lipase (LAL) is a serine hydrolase which hydrolyzes cholesteryl ester and triglycerides delivered to the lysosomes into free cholesterol and free fatty acids. Mutations in the LAL gene (LIPA) result in accumulation of triglycerides and cholesterol esters in various tissues of the body, leading to pathological conditions such as Wolman's disease (WD) and cholesteryl ester storage disease (CESD). CESD patients homozygous for His295Tyr (H295Y) mutation have less than 5% of normal LAL activity. To shed light on the molecular basis for this loss-of-function phenotype, we have generated the recombinant H295Y enzyme and studied its biophysical and biochemical properties. No significant differences were observed in the expression levels or glycosylation patterns between the mutant and the wild type LAL. However, the H295Y mutant displayed only residual enzymatic activity (<5%) compared to the wild type. While wild type LAL is mostly a monomer at pH 5.0, the vast majority H295Y exists as a high molecular soluble aggregate. Besides, the H295Y mutant has a 20°C lower melting temperature compared to the wild type. Transient expression studies in WD fibroblasts showed that mutation of His295 to other amino acids resulted in a significant loss of enzymatic activity. A homology model of LAL revealed that His295 is located on an α-helix of the cap domain and could be important for tethering it to its core domain. The observed loss-of-function phenotype in CESD patients might arise from a combination of protein destabilization and the shift to a non-functional soluble aggregate.
Asunto(s)
Lisosomas/enzimología , Esterol Esterasa/genética , Enfermedad de Wolman/enzimología , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Ésteres del Colesterol/química , Ésteres del Colesterol/metabolismo , Clonación Molecular , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Glicosilación , Humanos , Cinética , Metabolismo de los Lípidos , Lisosomas/patología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Plásmidos/química , Plásmidos/metabolismo , Agregado de Proteínas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Células Sf9 , Spodoptera , Esterol Esterasa/aislamiento & purificación , Esterol Esterasa/metabolismo , Enfermedad de Wolman/genética , Enfermedad de Wolman/patologíaRESUMEN
ITK (interleukin-2-inducible T-cell kinase) is a critical component of signal transduction in T-cells and has a well-validated role in their proliferation, cytokine release and chemotaxis. ITK is an attractive target for the treatment of T-cell-mediated inflammatory diseases. In the present study we describe the discovery of kinase inhibitors that preferentially bind to an allosteric pocket of ITK. The novel ITK allosteric site was characterized by NMR, surface plasmon resonance, isothermal titration calorimetry, enzymology and X-ray crystallography. Initial screening hits bound to both the allosteric pocket and the ATP site. Successful lead optimization was achieved by improving the contribution of the allosteric component to the overall inhibition. NMR competition experiments demonstrated that the dual-site binders showed higher affinity for the allosteric site compared with the ATP site. Moreover, an optimized inhibitor displayed non-competitive inhibition with respect to ATP as shown by steady-state enzyme kinetics. The activity of the isolated kinase domain and auto-activation of the full-length enzyme were inhibited with similar potency. However, inhibition of the activated full-length enzyme was weaker, presumably because the allosteric site is altered when ITK becomes activated. An optimized lead showed exquisite kinome selectivity and is efficacious in human whole blood and proximal cell-based assays.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Regulación Alostérica , Sitio Alostérico , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Conformación Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Resonancia por Plasmón de SuperficieRESUMEN
The role of ADAM-8 in cancer and inflammatory diseases such as allergy, arthritis and asthma makes it an attractive target for drug development. Therefore, the catalytic domain of human ADAM-8 was expressed, purified and crystallized in complex with a hydroxamic acid inhibitor, batimastat. The crystal structure of the enzyme-inhibitor complex was refined to 2.1 Å resolution. ADAM-8 has an overall fold similar to those of other ADAM members, including a central five-stranded ß-sheet and a catalytic Zn(2+) ion. However, unique differences within the S1' binding loop of ADAM-8 are observed which might be exploited to confer specificity and selectivity to ADAM-8 competitive inhibitors for the treatment of diseases involving this enzyme.
Asunto(s)
Proteínas ADAM/química , Dominio Catalítico , Proteínas de la Membrana/química , Fenilalanina/análogos & derivados , Inhibidores de Proteasas/química , Tiofenos/química , Proteínas ADAM/metabolismo , Humanos , Ligandos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , Inhibidores de Proteasas/metabolismo , Unión Proteica , Desplegamiento Proteico , Tiofenos/metabolismoRESUMEN
Multi-drug-resistant forms of the Gram-negative pathogen Acinetobacter baumannii are an emerging threat to human health and further complicate the general problem of treating serious bacterial infections. Meeting this challenge requires an improved understanding of the relationships between the structures of major therapeutic targets in this organism and the activity levels exhibited against it by different antibiotics. Here we report the first crystal structures of A. baumannii penicillin-binding proteins (PBPs) covalently inactivated by four ß-lactam antibiotics. We also relate the results to kinetic, biophysical, and computational data. The structure of the class A protein PBP1a was solved in apo form and for its covalent conjugates with benzyl penicillin, imipenem, aztreonam, and the siderophore-conjugated monocarbam MC-1. It included a novel domain genetically spliced into a surface loop of the transpeptidase domain that contains three conserved loops. Also reported here is the first high-resolution structure of the A. baumannii class B enzyme PBP3 in apo form. Comparison of this structure with that of MC-1-derivatized PBP3 of Pseudomonas aeruginosa identified differences between these orthologous proteins in A. baumannii and P. aeruginosa. Thermodynamic analyses indicated that desolvation effects in the PBP3 ligand-binding sites contributed significantly to the thermal stability of the enzyme-antibiotic covalent complexes. Across a significant range of values, they correlated well with results from studies of inactivation kinetics and the protein structures. The structural, biophysical, and computational data help rationalize differences in the functional performance of antibiotics against different protein targets and can be used to guide the design of future agents.
Asunto(s)
Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/farmacología , beta-Lactamas/farmacología , Acinetobacter baumannii/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Cromatografía Liquida , Cristalización , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia MolecularRESUMEN
Imidazo[1,5-a]quinoxalines were synthesized that function as irreversible Bruton's tyrosine kinase (BTK) inhibitors. The syntheses and SAR of this series of compounds are presented as well as the X-ray crystal structure of the lead compound 36 in complex with a gate-keeper variant of ITK enzyme. The lead compound showed good in vivo efficacy in preclinical RA models.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinoxalinas/farmacología , Agammaglobulinemia Tirosina Quinasa , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinoxalinas/químicaRESUMEN
Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.
Asunto(s)
MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Tiofenos/química , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Diseño de Fármacos , Humanos , MAP Quinasa Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Tiofenos/síntesis química , Tiofenos/farmacologíaRESUMEN
Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.
Asunto(s)
MAP Quinasa Quinasa 2/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Tiofenos/química , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 2 Dependiente de la Ciclina/metabolismo , Humanos , MAP Quinasa Quinasa 2/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/farmacologíaRESUMEN
The inhibition of PKC-zeta has been proposed to be a potential drug target for immune and inflammatory diseases. A series of 2-(6-phenyl-1H indazol-3-yl)-1H-benzo[d]imidazoles with initial high crossover to CDK-2 has been optimized to afford potent and selective inhibitors of protein kinase c-zeta (PKC-zeta). The determination of the crystal structures of key inhibitor:CDK-2 complexes informed the design and analysis of the series. The most selective and potent analog was identified by variation of the aryl substituent at the 6-position of the indazole template to give a 4-NH(2) derivative. The analog displays good selectivity over other PKC isoforms (alpha, betaII, gamma, delta, epsilon, mu, theta, eta and iota/lambda) and CDK-2, however it displays marginal selectivity against a panel of other kinases (37 profiled).
Asunto(s)
Bencimidazoles/síntesis química , Química Farmacéutica/métodos , Inhibidores Enzimáticos/farmacología , Imidazoles/síntesis química , Proteína Quinasa C/química , Proteína Quinasa C/aislamiento & purificación , Bencimidazoles/farmacología , Cristalografía por Rayos X , Ciclina A/química , Quinasa 2 Dependiente de la Ciclina/metabolismo , Diseño de Fármacos , Humanos , Imidazoles/farmacología , Concentración 50 Inhibidora , Modelos Químicos , Modelos Moleculares , Conformación Molecular , Isoformas de ProteínasRESUMEN
Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic pi-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H(2) (PGH(2)), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH(2) in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.
Asunto(s)
Inhibidores Enzimáticos/química , Oxidorreductasas Intramoleculares/química , Microsomas/enzimología , Secuencia de Aminoácidos , Biopolímeros , Inhibidores Enzimáticos/farmacología , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Prostaglandina-E Sintasas , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Potent covalent inhibitors of Bruton's tyrosine kinase (BTK) based on an aminopyrazole carboxamide scaffold have been identified. Compared to acrylamide-based covalent reactive groups leading to irreversible protein adducts, cyanamide-based reversible-covalent inhibitors provided the highest combined BTK potency and EGFR selectivity. The cyanamide covalent mechanism with BTK was confirmed through enzyme kinetic, NMR, MS, and X-ray crystallographic studies. The lead cyanamide-based inhibitors demonstrated excellent kinome selectivity and rat pharmacokinetic properties.
RESUMEN
In light of accumulating evidence that aggressive LDL-lowering therapy may offer increased protection against coronary heart disease, we undertook the design and synthesis of a novel series of HMG-CoA reductase inhibitors based upon a substituted pyrazole template. Optimizing this series using both structure-based design and molecular property considerations afforded a class of highly efficacious and hepatoselective inhibitors resulting in the identification of (3 R,5 R)-7-[2-(4-fluoro-phenyl)-4-isopropyl-5-(4-methyl-benzylcarbamoyl)-2 H-pyrazol-3-yl]-3,5-dihydroxy-heptanoic (PF-3052334) as a candidate for the treatment of hypercholesterolemia.
Asunto(s)
Ácidos Heptanoicos/síntesis química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/síntesis química , Hipercolesterolemia/tratamiento farmacológico , Hígado/efectos de los fármacos , Pirazoles/síntesis química , Animales , LDL-Colesterol/biosíntesis , LDL-Colesterol/sangre , Cricetinae , Cobayas , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ácidos Heptanoicos/química , Ácidos Heptanoicos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Técnicas In Vitro , Hígado/metabolismo , Masculino , Mesocricetus , Células Musculares/efectos de los fármacos , Células Musculares/metabolismo , Pirazoles/química , Pirazoles/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Optimization of the pharmacokinetic (PK) properties of a series of activators of adenosine monophosphate-activated protein kinase (AMPK) is described. Derivatives of the previously described 5-aryl-indole-3-carboxylic acid clinical candidate (1) were examined with the goal of reducing glucuronidation rate and minimizing renal excretion. Compounds 10 (PF-06679142) and 14 (PF-06685249) exhibited robust activation of AMPK in rat kidneys as well as desirable oral absorption, low plasma clearance, and negligible renal clearance in preclinical species. A correlation of in vivo renal clearance in rats with in vitro uptake by human and rat renal organic anion transporters (human OAT/rat Oat) was identified. Variation of polar functional groups was critical to mitigate active renal clearance mediated by the Oat3 transporter. Modification of either the 6-chloroindole core to a 4,6-difluoroindole or the 5-phenyl substituent to a substituted 5-(3-pyridyl) group provided improved metabolic stability while minimizing propensity for active transport by OAT3.
Asunto(s)
Proteínas Quinasas Activadas por AMP/efectos de los fármacos , Activadores de Enzimas/síntesis química , Activadores de Enzimas/farmacología , Indoles/síntesis química , Indoles/farmacología , Animales , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/farmacocinética , Humanos , Indoles/farmacocinética , Absorción Intestinal , Riñón/efectos de los fármacos , Riñón/enzimología , Masculino , Modelos Moleculares , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
The nuclear hormone receptor retinoic acid receptor-related orphan C2 (RORC2, also known as RORγt) is a promising target for the treatment of autoimmune diseases. A small molecule, inverse agonist of the receptor is anticipated to reduce production of IL-17, a key proinflammatory cytokine. Through a high-throughput screening approach, we identified a molecule displaying promising binding affinity for RORC2, inhibition of IL-17 production in Th17 cells, and selectivity against the related RORA and RORB receptor isoforms. Lead optimization to improve the potency and metabolic stability of this hit focused on two key design strategies, namely, iterative optimization driven by increasing lipophilic efficiency and structure-guided conformational restriction to achieve optimal ground state energetics and maximize receptor residence time. This approach successfully identified 3-cyano- N-(3-(1-isobutyrylpiperidin-4-yl)-1-methyl-4-(trifluoromethyl)-1 H-pyrrolo[2,3- b]pyridin-5-yl)benzamide as a potent and selective RORC2 inverse agonist, demonstrating good metabolic stability, oral bioavailability, and the ability to reduce IL-17 levels and skin inflammation in a preclinical in vivo animal model upon oral administration.
Asunto(s)
Diseño de Fármacos , Agonismo Inverso de Drogas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Piridinas/administración & dosificación , Piridinas/farmacología , Administración Oral , Animales , Disponibilidad Biológica , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Piridinas/farmacocinética , Células Th17/efectos de los fármacos , Células Th17/metabolismoRESUMEN
Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Ciclobutanos/farmacología , Janus Quinasa 1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Pirroles/farmacología , Sulfonamidas/farmacología , Animales , Artritis Experimental/tratamiento farmacológico , Ciclobutanos/química , Ciclobutanos/farmacocinética , Ciclobutanos/uso terapéutico , Perros , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Janus Quinasa 1/química , Janus Quinasa 2/antagonistas & inhibidores , Modelos Moleculares , Conformación Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/uso terapéutico , Pirroles/química , Pirroles/farmacocinética , Pirroles/uso terapéutico , Ratas , Especificidad por Sustrato , Sulfonamidas/química , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapéutico , Distribución TisularRESUMEN
A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Piridinas/síntesis química , Pirroles/síntesis química , Enfermedad Aguda , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Cristalografía por Rayos X , Humanos , Inflamación/tratamiento farmacológico , Péptidos y Proteínas de Señalización Intracelular , Modelos Moleculares , Proteínas Serina-Treonina Quinasas/química , Piridinas/química , Piridinas/farmacología , Pirroles/química , Pirroles/farmacología , Ratas , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Células U937RESUMEN
Crystals of phosphorylated JAK1 kinase domain were initially generated in complex with nucleotide (ADP) and magnesium. The tightly bound Mg2+-ADP at the ATP-binding site proved recalcitrant to ligand displacement. Addition of a molar excess of EDTA helped to dislodge the divalent metal ion, promoting the release of ADP and allowing facile exchange with ATP-competitive small-molecule ligands. Many kinases require the presence of a stabilizing ligand in the ATP site for crystallization. This procedure could be useful for developing co-crystallization systems with an exchangeable ligand to enable structure-based drug design of other protein kinases.
Asunto(s)
Adenosina Difosfato/química , Adenosina Trifosfato/química , Cristalización/métodos , Ácido Edético/química , Janus Quinasa 1/química , Magnesio/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Sitios de Unión , Cationes Bivalentes , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Humanos , Janus Quinasa 1/genética , Janus Quinasa 1/metabolismo , Magnesio/metabolismo , Modelos Moleculares , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , SpodopteraRESUMEN
Matrix metalloproteinase-13 (MMP-13) is a zinc-dependent protease responsible for the cleavage of type II collagen, the major structural protein of articular cartilage. Degradation of this cartilage matrix leads to the development of osteoarthritis. We previously have described highly potent and selective carboxylic acid containing MMP-13 inhibitors; however, nephrotoxicity in preclinical toxicology species precluded development. The accumulation of compound in the kidneys mediated by human organic anion transporter 3 (hOAT3) was hypothesized as a contributing factor for the finding. Herein we report our efforts to optimize the MMP-13 potency and pharmacokinetic properties of non-carboxylic acid leads resulting in the identification of compound 43a lacking the previously observed preclinical toxicology at comparable exposures.
Asunto(s)
Metaloproteinasa 13 de la Matriz/efectos de los fármacos , Inhibidores de la Metaloproteinasa de la Matriz/síntesis química , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Osteoartritis/tratamiento farmacológico , Pirimidinas/síntesis química , Pirimidinas/farmacología , Tetrazoles/síntesis química , Tetrazoles/farmacología , Animales , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Colagenasas/efectos de los fármacos , Perros , Diseño de Fármacos , Humanos , Riñón/metabolismo , Macaca fascicularis , Masculino , Inhibidores de la Metaloproteinasa de la Matriz/toxicidad , Modelos Moleculares , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Unión Proteica , Ratas , Ratas Sprague-Dawley , Relación Estructura-ActividadRESUMEN
Adenosine monophosphate-activated protein kinase (AMPK) is a protein kinase involved in maintaining energy homeostasis within cells. On the basis of human genetic association data, AMPK activators were pursued for the treatment of diabetic nephropathy. Identification of an indazole amide high throughput screening (HTS) hit followed by truncation to its minimal pharmacophore provided an indazole acid lead compound. Optimization of the core and aryl appendage improved oral absorption and culminated in the identification of indole acid, PF-06409577 (7). Compound 7 was advanced to first-in-human trials for the treatment of diabetic nephropathy.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Nefropatías Diabéticas/tratamiento farmacológico , Activadores de Enzimas/química , Indoles/química , Administración Oral , Adsorción , Animales , Cristalografía por Rayos X , Perros , Activadores de Enzimas/síntesis química , Activadores de Enzimas/farmacocinética , Activadores de Enzimas/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Indazoles/síntesis química , Indazoles/química , Indazoles/farmacología , Indoles/síntesis química , Indoles/farmacocinética , Indoles/farmacología , Inyecciones Intravenosas , Macaca fascicularis , Masculino , Modelos Moleculares , Conformación Proteica , RatasRESUMEN
Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug-target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from Pseudomonas aeruginosa and characterize the reactivity of lactivicin and ß-lactam core structures. Results from drug sensitivity studies with ß-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.