RESUMEN
Aspartate transcarbamoylase (ATC) is the first committed step in de novo pyrimidine biosynthesis in eukaryotes and plants. A potent transition state analog of human ATCase (PALA) has previously been assessed in clinical trials for the treatment of cancer, but was ultimately unsuccessful. Additionally, inhibition of this pathway has been proposed to be a target to suppress cell proliferation in E. coli, the malarial parasite and tuberculosis. In this manuscript we screened a 70-member library of ATC inhibitors developed against the malarial and tubercular ATCases for inhibitors of the human ATC. Four compounds showed low nanomolar inhibition (IC50 30-120â nM) in an inâ vitro activity assay. These compounds significantly outperform PALA, which has a triphasic inhibition response under identical conditions, in which significant activity remains at PALA concentrations above 10â µM. Evidence for a druggable allosteric pocket in human ATC is provided by both inâ vitro enzyme kinetic, homology modeling and in silico docking. These compounds also suppress the proliferation of U2OS osteoblastoma cells by promoting cell cycle arrest in G0/G1 phase. This report provides the first evidence for an allosteric pocket in human ATC, which greatly enhances its druggability and demonstrates the potential of this series in cancer therapy.
Asunto(s)
Aspartato Carbamoiltransferasa , Proliferación Celular , Inhibidores Enzimáticos , Osteosarcoma , Humanos , Proliferación Celular/efectos de los fármacos , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/patología , Osteosarcoma/metabolismo , Aspartato Carbamoiltransferasa/antagonistas & inhibidores , Aspartato Carbamoiltransferasa/metabolismo , Aspartato Carbamoiltransferasa/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Bibliotecas de Moléculas Pequeñas/síntesis química , Regulación Alostérica/efectos de los fármacos , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento Molecular , Estructura Molecular , Ensayos de Selección de Medicamentos Antitumorales , Línea Celular Tumoral , Neoplasias Óseas/tratamiento farmacológico , Neoplasias Óseas/patología , Neoplasias Óseas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismoRESUMEN
Aspartate transcarbamoylase (ATCase) plays a key role in the second step of de novo pyrimidine biosynthesis in eukaryotes and has been proposed to be a target to suppress cell proliferation in E. coli, human cells and the malarial parasite. We hypothesized that a library of ATCase inhibitors developed for malarial ATCase (PfATCase) may also contain inhibitors of the tubercular ATCase and provide a similar inhibition of cellular proliferation. Of the 70 compounds screened, 10 showed single-digit micromolar inhibition in an inâ vitro activity assay and were tested for their effect on M.â tuberculosis cell growth in culture. The most promising compound demonstrated a MIC90 of 4â µM. A model of MtbATCase was generated using the experimental coordinates of PfATCase. In silico docking experiments showed this compound can occupy a similar allosteric pocket on MtbATCase to that seen on PfATCase, explaining the observed species selectivity seen for this compound series.
Asunto(s)
Escherichia coli , Mycobacterium tuberculosis , Humanos , Ácido AspárticoRESUMEN
The discovery and development of new drugs against malaria remain urgent. Aspartate transcarbamoylase (ATC) has been suggested to be a promising target for antimalarial drug development. Here, we describe a series of small-molecule inhibitors of P. falciparum ATC with low nanomolar binding affinities that selectively bind to a previously unreported allosteric pocket, thereby inhibiting ATC activation. We demonstrate that the buried allosteric pocket is located close to the traditional ATC active site and that reported compounds maintain the active site of PfATC in its low substrate affinity/low activity conformation. These compounds inhibit parasite growth in blood stage cultures at single digit micromolar concentrations, whereas limited effects were seen against human normal lymphocytes. To our knowledge, this series represent the first PfATC-specific allosteric inhibitors.
Asunto(s)
Antimaláricos , Malaria Falciparum , Humanos , Antimaláricos/farmacología , Antimaláricos/química , Plasmodium falciparum , Ácido Aspártico/metabolismo , Dominio CatalíticoRESUMEN
Malate dehydrogenases (MDHs) sustain tumor growth and carbon metabolism by pathogens including Plasmodium falciparum. However, clinical success of MDH inhibitors is absent, as current small molecule approaches targeting the active site are unselective. The presence of an allosteric binding site at oligomeric interface allows the development of more specific inhibitors. To this end we performed a differential NMR-based screening of 1500 fragments to identify fragments that bind at the oligomeric interface. Subsequent biophysical and biochemical experiments of an identified fragment indicate an allosteric mechanism of 4-(3,4-difluorophenyl) thiazol-2-amine (4DT) inhibition by impacting the formation of the active site loop, located >30 Å from the 4DT binding site. Further characterization of the more tractable homolog 4-phenylthiazol-2-amine (4PA) and 16 other derivatives are also reported. These data pave the way for downstream development of more selective molecules by utilizing the oligomeric interfaces showing higher species sequence divergence than the MDH active site.
Asunto(s)
Malato Deshidrogenasa/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Sitios de Unión , Dominio Catalítico , Malato Deshidrogenasa/química , Modelos Moleculares , Plasmodium falciparum/química , Proteínas Protozoarias/químicaRESUMEN
Asthma is a respiratory disease that currently affects around 300 million people worldwide and is defined by coughing, shortness of breath, wheezing, mucus overproduction, chest tightness, and expiratory airflow limitation. Increased levels of interleukin 17 (IL-17) have been observed in sputum, nasal and bronchial biopsies, and serum of patients with asthma compared to healthy controls. Patients with higher levels of IL-17 have a more severe asthma phenotype. Biologics are available for T helper 2 (Th2)-high asthmatics, but the Th17-high subpopulation has a relatively low response to these treatments, rendering it a rather severe asthma phenotype to treat. Several experimental models suggest that targeting the IL-17 pathway may be beneficial in asthma. Moreover, as increased activation of the Th17/IL-17 axis is correlated with reduced inhaled corticosteroids (ICS) sensitivity, targeting the IL-17 pathway might reverse ICS unresponsiveness. In this review, we present and discuss the current knowledge on the role of IL-17 in asthma and its interaction with the Th2 pathway, focusing on the rationale for therapeutic targeting of the IL-17 pathway.
RESUMEN
Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.
Asunto(s)
Antimaláricos , Aspartato Carbamoiltransferasa/genética , Naftiridinas/farmacología , Plasmodium falciparum , Proteínas Protozoarias/genética , Antimaláricos/farmacología , Ácido Aspártico , Plasmodium falciparum/enzimología , Plasmodium falciparum/genéticaRESUMEN
The appearance of multi-drug resistant strains of malaria poses a major challenge to human health and validated drug targets are urgently required. To define a protein's function in vivo and thereby validate it as a drug target, highly specific tools are required that modify protein function with minimal cross-reactivity. While modern genetic approaches often offer the desired level of target specificity, applying these techniques is frequently challenging-particularly in the most dangerous malaria parasite, Plasmodium falciparum. Our hypothesis is that such challenges can be addressed by incorporating mutant proteins within oligomeric protein complexes of the target organism in vivo. In this manuscript, we provide data to support our hypothesis by demonstrating that recombinant expression of mutant proteins within P. falciparum leverages the native protein oligomeric state to influence protein function in vivo, thereby providing a rapid validation of potential drug targets. Our data show that interference with aspartate metabolism in vivo leads to a significant hindrance in parasite survival and strongly suggest that enzymes integral to aspartate metabolism are promising targets for the discovery of novel antimalarials.
RESUMEN
Antimicrobial resistance resulting in ineffective treatment of infectious diseases is an increasing global problem, particularly in infections with pathogenic bacteria. In some bacteria, such as Streptococcus pyogenes, the pathogenicity is strongly linked to the attachment of virulence factors. Their attachment to the cellular membrane is a transpeptidation reaction, catalyzed by sortase enzymes. As such, sortases pose an interesting target for the development of new antivirulence strategies that could yield novel antimicrobial drugs. Using the substitution-oriented fragment screening (SOS) approach, we discovered a potent and specific inhibitor (C10) of sortase A from S. pyogenes. The inhibitor C10 showed high specificity towards S. pyogenes sortase A, with an IC50 value of 10⯵M and a Kd of 60⯵M. We envision that this inhibitor could be employed as a starting point for further exploration of sortase's potential as therapeutic target for antimicrobial drug development.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Streptococcus pyogenes/efectos de los fármacos , Aminoaciltransferasas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Cinética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Streptococcus pyogenes/enzimología , Relación Estructura-ActividadRESUMEN
Aspartate transcarbamoylase catalyzes the second step of de-novo pyrimidine biosynthesis. As malarial parasites lack pyrimidine salvage machinery and rely on de-novo production for growth and proliferation, this pathway is a target for drug discovery. Previously, an apo crystal structure of aspartate transcarbamoylase from Plasmodium falciparum (PfATC) in its T-state has been reported. Here we present crystal structures of PfATC in the liganded R-state as well as in complex with the novel inhibitor, 2,3-napthalenediol, identified by high-throughput screening. Our data shows that 2,3-napthalediol binds in close proximity to the active site, implying an allosteric mechanism of inhibition. Furthermore, we report biophysical characterization of 2,3-napthalenediol. These data provide a promising starting point for structure based drug design targeting PfATC and malarial de-novo pyrimidine biosynthesis.
Asunto(s)
Antiparasitarios/química , Antiparasitarios/farmacología , Aspartato Carbamoiltransferasa/antagonistas & inhibidores , Plasmodium falciparum/enzimología , Aspartato Carbamoiltransferasa/química , Aspartato Carbamoiltransferasa/metabolismo , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Simulación del Acoplamiento Molecular , Plasmodium falciparum/química , Plasmodium falciparum/efectos de los fármacosRESUMEN
Refolding of proteins derived from inclusion bodies is very promising as it can provide a reliable source of target proteins of high purity. However, inclusion body-based protein production is often limited by the lack of techniques for the detection of correctly refolded protein. Thus, the selection of the refolding conditions is mostly achieved using trial and error approaches and is thus a time-consuming process. In this study, we use the latest developments in the differential scanning fluorimetry guided refolding approach as an analytical method to detect correctly refolded protein. We describe a systematic buffer screen that contains a 96-well primary pH-refolding screen in conjunction with a secondary additive screen. Our research demonstrates that this approach could be applied for determining refolding conditions for several proteins. In addition, it revealed which "helper" molecules, such as arginine and additives are essential. Four different proteins: HA-RBD, MDM2, IL-17A and PD-L1 were used to validate our refolding approach. Our systematic protocol evaluates the impact of the "helper" molecules, the pH, buffer system and time on the protein refolding process in a high-throughput fashion. Finally, we demonstrate that refolding time and a secondary thermal shift assay buffer screen are critical factors for improving refolding efficiency.
Asunto(s)
Replegamiento Proteico , Proteínas/química , Tampones (Química) , Cromatografía en Gel , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación Proteica , Desnaturalización Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
The majority of modern anticancer approaches target DNA/protein targets involved in tumour-cell proliferation. Such approaches have a major drawback, as nonproliferating cancer cells remain unaffected and may cause relapse or remission. Human coatomer protein complex I (COPI) subunit ζ (Copζ), a component of the coat protein involved in cell apoptosis and intracellular trafficking, has recently been proposed as a potential anticancer drug target. Previous studies have shown that two different isoforms of the Copζ subunit exist in mammalian cells. While normal cells express both Copζ1 and Copζ2 isoforms, various types of tumour cells display a loss of Copζ2 expression and rely solely on Copζ1 for growth and survival. Subsequent knockdown of Copζ1 results in specific inhibition of both proliferating and dormant tumour-cell populations, with no adverse growth effects on normal cells. Therefore, a Copζ1-targeting therapy was proposed to bypass the problem of dormant cancer cells that are resistant to conventional antiproliferative drugs, which is the major cause of tumour relapse. In order to aid in structure-based inhibitor design, a crystal structure is required. In this article, the recombinant expression, purification, crystallization and crystal structure of Copζ1, as well as the expression and purification of Copζ2, are reported.