RESUMEN
Pyruvate kinase (PK) is an essential component of cellular metabolism, converting ADP and phosphoenolpyruvate (PEP) to pyruvate in the final step of glycolysis. Of the four unique isoforms of pyruvate kinase, R (PKR) is expressed exclusively in red blood cells and is a tetrameric enzyme that depends on fructose-1,6-bisphosphate (FBP) for activation. PKR deficiency leads to hemolysis of red blood cells resulting in anemia. Activation of PKR in both sickle cell disease and beta-thalassemia patients could lead to improved red blood cell fitness and survival. The discovery of a novel series of substituted urea PKR activators, via the serendipitous identification and diligent characterization of a minor impurity in an High Throughput Screening (HTS) hit will be discussed.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento , Piruvato Quinasa , Piruvato Quinasa/metabolismo , Piruvato Quinasa/antagonistas & inhibidores , Humanos , Descubrimiento de Drogas , Relación Estructura-Actividad , Urea/química , Urea/farmacología , Activadores de Enzimas/farmacología , Activadores de Enzimas/química , Activadores de Enzimas/síntesis química , Estructura Molecular , AnimalesRESUMEN
Plasma kallikrein (pKal) is a serine protease responsible for cleaving high-molecular-weight kininogen to produce the pro-inflammatory peptide, bradykinin. Unregulated pKal activity can lead to hereditary angioedema (HAE) following excess bradykinin release. HAE attacks can lead to a compromised airway that can be life threatening. As there are limited agents for prophylaxis of HAE attacks, there is a high unmet need for a therapeutic agent for regulating pKal with a high degree of specificity. Here we present crystal structures of both full-length and the protease domain of pKal, bound to two very distinct classes of small-molecule inhibitors: compound 1, and BCX4161. Both inhibitors demonstrate low nM inhibitory potency for pKal and varying specificity for related serine proteases. Compound 1 utilizes a surprising mode of interaction and upon binding results in a rearrangement of the binding pocket. Co-crystal structures of pKal describes why this class of small-molecule inhibitor is potent. Lack of conservation in surrounding residues explains the â¼10,000-fold specificity over structurally similar proteases, as shown by in vitro protease inhibition data. Structural information, combined with biochemical and enzymatic analyses, provides a novel scaffold for the design of targeted oral small molecule inhibitors of pKal for treatment of HAE and other diseases resulting from unregulated plasma kallikrein activity.
Asunto(s)
Calicreína Plasmática/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Quininógeno de Alto Peso Molecular/metabolismo , Quininógenos/metabolismo , Calicreína Plasmática/antagonistas & inhibidores , Calicreína Plasmática/metabolismo , Unión Proteica , Conformación Proteica , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
Glycogen is a large polymer of glucose that functions as an important means of storing energy and maintaining glucose homeostasis. Glycogen synthesis and degradation pathways are highly regulated and their dysregulation can contribute to disease. Glycogen storage diseases are a set of disorders that arise from improper glycogen metabolism. Glycogen storage disease II, known as Pompe disease, is caused by a genetic mutation that leads to increased glycogen storage in cells and tissues, resulting in progressive muscle atrophy and respiratory decline for patients. One approach for treating Pompe disease is to reduce glycogen levels by interfering with the glycogen synthesis pathway through glycogen synthase inhibitors. To facilitate the study of glycogen synthase inhibitors in biological samples, such as cultured cells, a high-throughput approach for measuring cellular glycogen was developed. A bioluminescent glycogen detection assay was automated and used to measure the glycogen content in cells grown in 384-well plates. The assay successfully quantified reduced glycogen stores in cells treated with a series of glycogen synthase 1 inhibitors, validating the utility of the assay for drug screening efforts and demonstrating its value for therapy development and glycogen metabolism research.
RESUMEN
Glycogen synthase 1 (GYS1), the rate-limiting enzyme in muscle glycogen synthesis, plays a central role in energy homeostasis and has been proposed as a therapeutic target in multiple glycogen storage diseases. Despite decades of investigation, there are no known potent, selective small-molecule inhibitors of this enzyme. Here, we report the preclinical characterization of MZ-101, a small molecule that potently inhibits GYS1 in vitro and in vivo without inhibiting GYS2, a related isoform essential for synthesizing liver glycogen. Chronic treatment with MZ-101 depleted muscle glycogen and was well tolerated in mice. Pompe disease, a glycogen storage disease caused by mutations in acid α glucosidase (GAA), results in pathological accumulation of glycogen and consequent autophagolysosomal abnormalities, metabolic dysregulation, and muscle atrophy. Enzyme replacement therapy (ERT) with recombinant GAA is the only approved treatment for Pompe disease, but it requires frequent infusions, and efficacy is limited by suboptimal skeletal muscle distribution. In a mouse model of Pompe disease, chronic oral administration of MZ-101 alone reduced glycogen buildup in skeletal muscle with comparable efficacy to ERT. In addition, treatment with MZ-101 in combination with ERT had an additive effect and could normalize muscle glycogen concentrations. Biochemical, metabolomic, and transcriptomic analyses of muscle tissue demonstrated that lowering of glycogen concentrations with MZ-101, alone or in combination with ERT, corrected the cellular pathology in this mouse model. These data suggest that substrate reduction therapy with GYS1 inhibition may be a promising therapeutic approach for Pompe disease and other glycogen storage diseases.
Asunto(s)
Enfermedad del Almacenamiento de Glucógeno Tipo II , Ratones , Animales , Enfermedad del Almacenamiento de Glucógeno Tipo II/tratamiento farmacológico , Glucógeno Sintasa/metabolismo , Glucógeno Sintasa/farmacología , Ratones Noqueados , Glucógeno/metabolismo , Músculo Esquelético/metabolismo , Terapia de Reemplazo Enzimático/métodosRESUMEN
Gamma-Tubulin Small Complex (gamma-TuSC) is the universally-conserved complex in eukaryotes that contains the microtubule (MT) nucleating protein: gamma-tubulin. gamma-TuSC is a heterotetramer with two copies of gamma-tubulin and one copy each of Spc98p and Spc97p. Previously, the structure of gamma-TuSC was determined by single particle electron microscopy (EM) at 25A resolution. gamma-TuSC is Y-shaped with a single flexible arm that could be the key to regulating MT nucleation. EM gold labeling revealed the locations of gamma-tubulin at the top of the Y. In vivo Fluorescence Resonance Energy Transfer (FRET) suggested the relative orientations of Spc98p and Spc97p but did not distinguish which large subunit formed the flexible arm. Here, using fluorescent proteins as covalently attached tags, we used class averages and 3-D random conical tilt reconstructions to confirm the in vivo FRET results, clearly demonstrating that the Spc98p/97p C-termini interact directly with gamma-tubulin. Most significantly we have determined that the flexible arm belongs to Spc98p and our data also suggests that the N-termini of Spc98p and Spc97p are crossed. More generally, our results confirm that despite their small size, covalently-attached fluorescent proteins perform well as subunit labels in single particle EM.