RESUMEN
The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.
RESUMEN
Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/genética , Proteínas Inhibidoras de la Apoptosis/genética , Cromatografía en Gel , Reactivos de Enlaces Cruzados , Humanos , Cinética , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteolisis , Espectrometría de Masa por Ionización de Electrospray , Ubiquitina-Proteína Ligasas , Ubiquitinación , Difracción de Rayos XRESUMEN
Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Agammaglobulinemia Tirosina Quinasa , Animales , Células Cultivadas , Ligandos , Poliubiquitina/metabolismo , Ratas , TermodinámicaRESUMEN
Diabetic nephropathy remains an area of high unmet medical need, with current therapies that slow down, but do not prevent, the progression of disease. A reduced phosphorylation state of adenosine monophosphate-activated protein kinase (AMPK) has been correlated with diminished kidney function in both humans and animal models of renal disease. Here, we describe the identification of novel, potent, small molecule activators of AMPK that selectively activate AMPK heterotrimers containing the ß1 subunit. After confirming that human and rodent kidney predominately express AMPK ß1, we explore the effects of pharmacological activation of AMPK in the ZSF1 rat model of diabetic nephropathy. Chronic administration of these direct activators elevates the phosphorylation of AMPK in the kidney, without impacting blood glucose levels, and reduces the progression of proteinuria to a greater degree than the current standard of care, angiotensin-converting enzyme inhibitor ramipril. Further analyses of urine biomarkers and kidney tissue gene expression reveal AMPK activation leads to the modulation of multiple pathways implicated in kidney injury, including cellular hypertrophy, fibrosis, and oxidative stress. These results support the need for further investigation into the potential beneficial effects of AMPK activation in kidney disease.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminopiridinas/farmacología , Nefropatías Diabéticas/tratamiento farmacológico , Activadores de Enzimas/farmacología , Indoles/farmacología , Riñón/efectos de los fármacos , Aminopiridinas/uso terapéutico , Animales , Tamaño de la Célula , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Activación Enzimática , Fibrosis , Humanos , Indoles/uso terapéutico , Isoenzimas/metabolismo , Riñón/metabolismo , Riñón/patología , Pruebas de Función Renal , Macaca fascicularis , Ratones Endogámicos C57BL , Estrés Oxidativo , Fosforilación , Proteinuria/tratamiento farmacológico , Proteinuria/metabolismo , Ratas , Especificidad de la EspecieRESUMEN
In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here, we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC) and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call "3-box" due to its facilitating Cul3 binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3 and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.
Asunto(s)
Proteínas Cullin/química , Proteínas Nucleares/química , Proteínas Represoras/química , Ubiquitina-Proteína Ligasas/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Co-Represoras , Secuencia de Consenso/fisiología , Cristalografía por Rayos X , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Chaperonas Moleculares , Mutación/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , Multimerización de Proteína/fisiología , Estructura Cuaternaria de Proteína/fisiología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiologíaRESUMEN
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
We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.
RESUMEN
Beta-2 microglobulin (beta2m) is a globular protein that self-associates into fibrillar amyloid deposits in patients undergoing hemodialysis therapy. Formation of these beta-sheet-rich assemblies is a fundamental property of polypeptides that can be triggered by diverse conditions. For beta2m, oligomerization into pre-amyloidogenic states occurs in specific response to coordination by Cu2+. Here we report the basis for this self-association at atomic resolution. Metal is not a direct participant in the molecular interface. Rather, binding results in distal alterations enabling the formation of two new surfaces. These interact to form a closed hexameric species. The origins of this include isomerization of a buried and conserved cis-proline previously implicated in the beta2m aggregation pathway. The consequences of this isomerization are evident and reveal a molecular basis for the conversion of this robust monomeric protein into an amyloid-competent state.
Asunto(s)
Amiloide/química , Inmunoglobulinas/química , Microglobulina beta-2/química , Amiloide/metabolismo , Amiloidosis/etiología , Amiloidosis/metabolismo , Cobre/metabolismo , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulinas/metabolismo , Técnicas In Vitro , Modelos Biológicos , Modelos Moleculares , Complejos Multiproteicos/química , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Diálisis Renal/efectos adversos , Microglobulina beta-2/metabolismoRESUMEN
The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a ß-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of ß-2 microglobulin (ß2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between ß-sheets orthogonal to the ß-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric ß2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the ß-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore's ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased.
Asunto(s)
Amiloide/química , Colorantes Fluorescentes/química , Tiazoles/química , Microglobulina beta-2/química , Benzotiazoles , Cristalización , Cristalografía , Fluorescencia , Humanos , Estructura Secundaria de ProteínaRESUMEN
Targeted protein degradation using heterobifunctional chimeras holds the potential to expand target space and grow the druggable proteome. Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Limiting this potential, however, is the remaining need to develop a ligand for the target of interest. While a number of challenging proteins have been successfully targeted by covalent ligands, unless this modification affects form or function, it may lack the ability to drive a biological response. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields. In this work, we employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in targeted protein degradation using Bruton's tyrosine kinase. Our results reveal that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.
Asunto(s)
Inhibición Psicológica , Proteoma , Proteolisis , Ligandos , Agammaglobulinemia Tirosina QuinasaRESUMEN
Glycerol-3-phosphate acyltransferase (GPAT)1 is a mitochondrial outer membrane protein that catalyzes the first step of de novo glycerolipid biosynthesis. Hepatic expression of GPAT1 is linked to liver fat accumulation and the severity of nonalcoholic fatty liver diseases. Here we present the cryo-EM structures of human GPAT1 in substrate analog-bound and product-bound states. The structures reveal an N-terminal acyltransferase domain that harbors important catalytic motifs and a tightly associated C-terminal domain that is critical for proper protein folding. Unexpectedly, GPAT1 has no transmembrane regions as previously proposed but instead associates with the membrane via an amphipathic surface patch and an N-terminal loop-helix region that contains a mitochondrial-targeting signal. Combined structural, computational and functional studies uncover a hydrophobic pathway within GPAT1 for lipid trafficking. The results presented herein lay a framework for rational inhibitor development for GPAT1.
Asunto(s)
Hígado , Membranas Mitocondriales , Humanos , Hígado/metabolismo , Membranas Mitocondriales/metabolismo , Glicerol-3-Fosfato O-Aciltransferasa/química , Glicerol-3-Fosfato O-Aciltransferasa/metabolismo , Secuencia de AminoácidosRESUMEN
Targeted protein degradation is a broad and expanding field aimed at the modulation of protein homeostasis. A focus of this field has been directed toward molecules that hijack the ubiquitin proteasome system with heterobifunctional ligands that recruit a target protein to an E3 ligase to facilitate polyubiquitination and subsequent degradation by the 26S proteasome. Despite the success of these chimeras toward a number of clinically relevant targets, the ultimate breadth and scope of this approach remains uncertain. Here we highlight recent advances in assays and tools available to evaluate targeted protein degradation, including and beyond the study of E3-targeted chimeric ligands. We note several challenges associated with degrader development and discuss various approaches to expanding the protein homeostasis toolbox.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismo , Autofagia/efectos de los fármacos , Descubrimiento de Drogas , Humanos , Lisosomas/metabolismo , Complejo de la Endopetidasa Proteasomal/química , Proteolisis/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Ubiquitina/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Proteolysis-targeting chimeras (PROTACs) and related molecules that induce targeted protein degradation by the ubiquitin-proteasome system represent a new therapeutic modality and are the focus of great interest, owing to potential advantages over traditional occupancy-based inhibitors with respect to dosing, side effects, drug resistance and modulating 'undruggable' targets. However, the technology is still maturing, and the design elements for successful PROTAC-based drugs are currently being elucidated. Importantly, fewer than 10 of the more than 600 E3 ubiquitin ligases have so far been exploited for targeted protein degradation, and expansion of knowledge in this area is a key opportunity. Here, we briefly discuss lessons learned about targeted protein degradation in chemical biology and drug discovery and systematically review the expression profile, domain architecture and chemical tractability of human E3 ligases that could expand the toolbox for PROTAC discovery.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Proteolisis/efectos de los fármacos , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Humanos , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Beta-2 Microglobulin (beta2m) is a small, globular protein, with high solubility under conditions comparable to human serum. A complication of hemodialysis in renal failure patients is the deposition of unmodified beta2m as amyloid fibers. In vitro, exposure of beta2m to equimolar Cu(2+) under near-physiological conditions can result in self-association leading to amyloid fiber formation. Previously, we have shown that the early steps in this process involve a catalyzed structural rearrangement followed by formation of discrete oligomers. These oligomers, however, have a continued requirement for Cu(2+) while mature fibers are resistant to addition of metal chelate. Here, we report that the transition from Cu(2+) dependent to chelate resistant states occurs in the context of small oligomers, dimeric to hexameric in size. These species require Cu(2+) to form, but once generated, do not need metal cation for stability. Importantly, this transition occurs gradually over several days and the resulting oligomers are isolatable and kinetically stable on timescales exceeding weeks. In addition, formation is enhanced by levels of urea similar to those found in hemodialysis patients. Our results are consistent with our hypothesis that transient encounter of full-length wild-type beta2m with transition metal cation at the dialysis membrane interface is causal to dialysis related amyloidosis.
Asunto(s)
Cobre/farmacología , Conformación Proteica/efectos de los fármacos , Microglobulina beta-2/química , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/efectos de los fármacos , Humanos , Pliegue de ProteínaRESUMEN
Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Medición de Intercambio de Deuterio/métodos , Activadores de Enzimas/química , Resonancia por Plasmón de Superficie/métodos , Proteínas Quinasas Activadas por AMP/aislamiento & purificación , Animales , Sitios de Unión , Cristalografía por Rayos X , Medición de Intercambio de Deuterio/instrumentación , Activación Enzimática , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Cinética , Ligandos , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Células Sf9 , Resonancia por Plasmón de Superficie/instrumentaciónRESUMEN
Studies on indole-3-carboxylic acid derivatives as direct activators of human adenosine monophosphate-activated protein kinase (AMPK) α1ß1γ1 isoform have culminated in the identification of PF-06409577 (1), PF-06885249 (2), and PF-06679142 (3) as potential clinical candidates. Compounds 1-3 are primarily cleared in animals and humans via glucuronidation. Herein, we describe the biosynthetic preparation, purification, and structural characterization of the glucuronide conjugates of 1-3. Spectral characterization of the purified glucuronides M1, M2, and M3 indicated that they were acyl glucuronide derivatives. In vitro pharmacological evaluation revealed that all three acyl glucuronides retained selective activation of ß1-containing AMPK isoforms. Inhibition of de novo lipogenesis with representative parent carboxylic acids and their respective acyl glucuronide conjugates in human hepatocytes demonstrated their propensity to activate cellular AMPK. Cocrystallization of the AMPK α1ß1γ1 isoform with 1-3 and M1-M3 provided molecular insights into the structural basis for AMPK activation by the glucuronide conjugates.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Indoles/química , Indoles/metabolismo , Lipogénesis/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/química , Animales , Células Cultivadas , Cristalización/métodos , Activación Enzimática/efectos de los fármacos , Glucurónidos/química , Glucurónidos/metabolismo , Glucurónidos/farmacocinética , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Indoles/farmacología , Macaca fascicularis , Espectroscopía de Resonancia Magnética , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Ratas Wistar , Uridina Difosfato Ácido Glucurónico/farmacologíaRESUMEN
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 AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminopiridinas/farmacología , Activadores de Enzimas/farmacología , Glucosa/metabolismo , Hipoglucemiantes/farmacología , Indoles/farmacología , Aminopiridinas/uso terapéutico , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/uso terapéutico , Femenino , Hipoglucemiantes/uso terapéutico , Indoles/uso terapéutico , Hígado/efectos de los fármacos , Hígado/metabolismo , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismoRESUMEN
AMP-activated protein kinase is a family of heterotrimeric serine/threonine protein kinases that come in twelve different flavors. They serve an essential function in all eukaryotes of conserving cellular energy levels. AMPK complexes are regulated by changes in cellular AMP:ATP or ADP:ATP ratios and by a number of neutraceuticals and some of the widely-used diabetes medications such as metformin and thiazolinonediones. Moreover, biochemical activities of AMPK are tightly regulated by phosphorylation or dephosphorylation by upstream kinases and phosphatases respectively. Efforts are underway in many pharmaceutical companies to discover direct AMPK activators for the treatment of cardiovascular and metabolic diseases such as diabetes, non-alcoholic steatohepatitis (NASH) and diabetic nephropathy. Many advances have been made in the AMPK structural biology arena over the last few years that are beginning to provide detailed molecular insights into the overall topology of these fascinating enzymes and how binding of small molecules elicit subtle conformational changes leading to their activation and protection from dephosphorylation. In the brief review below on AMPK structure and function, we have focused on the recent crystallographic results especially on specific molecular interactions of direct synthetic AMPK activators which lead to biased activation of a sub-family of AMPK isoforms.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Diabetes Mellitus/enzimología , Nefropatías Diabéticas/enzimología , Enfermedad del Hígado Graso no Alcohólico/enzimología , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Adenosina Monofosfato/química , Adenosina Trifosfato/química , Regulación Alostérica , Animales , Diabetes Mellitus/tratamiento farmacológico , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/genética , Nefropatías Diabéticas/patología , Regulación de la Expresión Génica , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/metabolismo , Hipoglucemiantes/uso terapéutico , Modelos Moleculares , Simulación del Acoplamiento Molecular , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Fosforilación , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
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.