RESUMEN
Inositol hexakisphosphate kinases (IP6Ks) have been studied for their role in glucose homeostasis, metabolic disease, fatty liver disease, chronic kidney disease, neurological development, and psychiatric disease. IP6Ks phosphorylate inositol hexakisphosphate (IP6) to the pyrophosphate, 5-diphosphoinositol-1,2,3,4,6-pentakisphosphate (5-IP7). Most of the currently known potent IP6K inhibitors contain a critical carboxylic acid which limits blood-brain barrier (BBB) penetration. In this work, the synthesis and testing of a variety of carboxylic acid isosteres resulted in several new compounds with improved BBB penetration. The most promising compound has an IP6K1 IC50 of 16 nM with an improved brain/plasma ratio and a favorable pharmacokinetic profile. This series of brain penetrant compounds may be used to investigate the role of IP6Ks in CNS disorders.
Asunto(s)
Barrera Hematoencefálica , Fosfotransferasas (Aceptor del Grupo Fosfato) , Barrera Hematoencefálica/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Animales , Humanos , Relación Estructura-Actividad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ratones , Masculino , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Ácidos Carboxílicos/síntesis química , RatasRESUMEN
Inositol polyphosphates (IPs) are a group of inositol metabolites that act as secondary messengers for external signalling cues. They play various physiological roles such as insulin release, telomere length maintenance, cell metabolism, and aging. Inositol hexakisphosphate kinase 2 (IP6K2) is a key enzyme that produces 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-IP7), which influences the early stages of glucose-induced exocytosis. Therefore, regulation of IP6Ks may serve as a promising strategy for treating diseases such as diabetes and obesity. In this study, we designed, synthesised, and evaluated flavonoid-based compounds as new inhibitors of IP6K2. Structure-activity relationship studies identified compound 20s as the most potent IP6K2 inhibitor with an IC50 value of 0.55 µM, making it 5-fold more potent than quercetin, the reported flavonoid-based IP6K2 inhibitor. Compound 20s showed higher inhibitory potency against IP6K2 than IP6K1 and IP6K3. Compound 20s can be utilised as a hit compound for further structural modifications of IP6K2 inhibitors.
Asunto(s)
Inhibidores Enzimáticos , Flavonoides , Insulina , Fosfotransferasas (Aceptor del Grupo Fosfato) , Flavonoides/farmacología , Inositol , Transducción de Señal , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacologíaRESUMEN
Inorganic polyphosphate (polyP) has been implicated in an astonishing array of biological functions, ranging from phosphorus storage to molecular chaperone activity to bacterial virulence. In bacteria, polyP is synthesized by polyphosphate kinase (PPK) enzymes, which are broadly subdivided into two families: PPK1 and PPK2. While both enzyme families are capable of catalyzing polyP synthesis, PPK1s preferentially synthesize polyP from nucleoside triphosphates, and PPK2s preferentially consume polyP to phosphorylate nucleoside mono- or diphosphates. Importantly, many pathogenic bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii encode at least one of each PPK1 and PPK2, suggesting these enzymes may be attractive targets for antibacterial drugs. Although the majority of bacterial polyP studies to date have focused on PPK1s, PPK2 enzymes have also begun to emerge as important regulators of bacterial physiology and downstream virulence. In this review, we specifically examine the contributions of PPK2s to bacterial polyP homeostasis. Beginning with a survey of the structures and functions of biochemically characterized PPK2s, we summarize the roles of PPK2s in the bacterial cell, with a particular emphasis on virulence phenotypes. Furthermore, we outline recent progress on developing drugs that inhibit PPK2 enzymes and discuss this strategy as a novel means of combatting bacterial infections.
Asunto(s)
Bacterias/enzimología , Bacterias/patogenicidad , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Polifosfatos/química , Polifosfatos/metabolismo , Virulencia , Factores de Virulencia/metabolismoRESUMEN
Inositol hexakisphosphate kinase (IP6K) is an important mammalian enzyme involved in various biological processes such as insulin signalling and blood clotting. Recent analyses on drug metabolism and pharmacokinetic properties on TNP (N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine), a pan-IP6K inhibitor, have suggested that it may inhibit cytochrome P450 (CYP450) enzymes and induce unwanted drug-drug interactions in the liver. In this study, we confirmed that TNP inhibits CYP3A4 in type I binding mode more selectively than the other CYP450 isoforms. In an effort to find novel purine-based IP6K inhibitors with minimal CYP3A4 inhibition, we designed and synthesised 15 TNP analogs. Structure-activity relationship and biochemical studies, including ADP-Glo kinase assay and quantification of cell-based IP7 production, showed that compound 9 dramatically reduced CYP3A4 inhibition while retaining IP6K-inhibitory activity. Compound 9 can be a tool molecule for structural optimisation of purine-based IP6K inhibitors.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Estructura Molecular , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Relación Estructura-ActividadRESUMEN
Circulating phosphate levels are tightly controlled within a narrow range in mammals. By using a novel small-molecule inhibitor, we show that the enzymatic activity of inositol hexakisphosphate kinases (IP6K) is essential for phosphate regulation in vivo. IP6K inhibition suppressed XPR1, a phosphate exporter, thereby decreasing cellular phosphate export, which resulted in increased intracellular ATP levels. The in vivo inhibition of IP6K decreased plasma phosphate levels without inhibiting gut intake or kidney reuptake of phosphate, demonstrating a pivotal role of IP6K-regulated cellular phosphate export on circulating phosphate levels. IP6K inhibition-induced decrease in intracellular inositol pyrophosphate, an enzymatic product of IP6K, was correlated with phosphate changes. Chronic IP6K inhibition alleviated hyperphosphataemia, increased kidney ATP, and improved kidney functions in chronic kidney disease rats. Our results demonstrate that the enzymatic activity of IP6K regulates circulating phosphate and intracellular ATP and suggest that IP6K inhibition is a potential novel treatment strategy against hyperphosphataemia.
Asunto(s)
Fosfatos/sangre , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Homeostasis/efectos de los fármacos , Humanos , Hiperfosfatemia/tratamiento farmacológico , Fosfatos de Inositol/metabolismo , Mamíferos , Fosfatos/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Ratas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Virales/genética , Receptores Virales/metabolismo , Insuficiencia Renal Crónica/tratamiento farmacológico , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of nosocomial infections, which are becoming increasingly difficult to treat due to antibiotic resistance. Polyphosphate (polyP) plays a key role in P. aeruginosa virulence, stress response, and antibiotic tolerance, suggesting an attractive drug target. Here, we show that the small molecule gallein disrupts polyphosphate homeostasis by inhibiting all members of both polyphosphate kinase (PPK) families (PPK1 and PPK2) encoded by P. aeruginosa, demonstrating dual-specificity PPK inhibition for the first time. Inhibitor treatment phenocopied ppk deletion to reduce cellular polyP accumulation and attenuate biofilm formation, motility, and pyoverdine and pyocyanin production. Most importantly, gallein attenuated P. aeruginosa virulence in a Caenorhabditis elegans infection model and synergized with antibiotics while exhibiting negligible toxicity toward the nematodes or HEK293T cells, suggesting our discovery of dual-specificity PPK inhibitors as a promising starting point for the development of new antivirulence therapeutics. IMPORTANCE Many priority bacterial pathogens such as P. aeruginosa encode both PPK1 and PPK2 enzymes to maintain polyphosphate homeostasis. While PPK1 and PPK2 have distinct structures and catalytic mechanisms, they are both capable of synthesizing and consuming polyphosphate; thus, PPK2 enzymes can compensate for the loss of PPK1 and vice versa. In this study, we identified the small molecule gallein as a dual-specificity inhibitor of both PPK1 and PPK2 enzyme families in P. aeruginosa. Inhibitor treatment reduced cellular polyP in wild-type (WT), Δppk1, and Δppk2 strains to levels that were on par with the Δppk1 Δppk2A Δppk2B Δppk2C knockout control. Treatment also attenuated biofilm formation, motility, toxin production, and virulence to a similar extent, thereby elucidating a hitherto-undocumented role of PPK2 enzymes in P. aeruginosa virulence phenotypes. This work therefore establishes PPK2s, in addition to PPK1, as valuable drug targets in P. aeruginosa and provides a favorable starting molecule for future inhibitor design efforts.
Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/patogenicidad , Xantenos/farmacología , Animales , Antibacterianos/uso terapéutico , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/microbiología , Inhibidores Enzimáticos/uso terapéutico , Células HEK293 , Humanos , Fenotipo , Fosfotransferasas (Aceptor del Grupo Fosfato)/clasificación , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Virulencia/efectos de los fármacos , Xantenos/uso terapéuticoRESUMEN
Inositol and its phosphate metabolites play a pivotal role in several biochemical pathways and gene expression regulation: inositol pyrophosphates (PP-IPs) have been increasingly appreciated as key signaling modulators. Fluctuations in their intracellular levels hugely impact the transfer of phosphates and the phosphorylation status of several target proteins. Pharmacological modulation of the proteins associated with PP-IP activities has proved to be beneficial in various pathological settings. IP7 has been extensively studied and found to play a key role in pathways associated with PP-IP activities. Three inositol hexakisphosphate kinase (IP6K) isoforms regulate IP7 synthesis in mammals. Genomic deletion or enzymic inhibition of IP6K1 has been shown to reduce cell invasiveness and migration capacity, protecting against chemical-induced carcinogenesis. IP6K1 could therefore be a useful target in anticancer treatment. Here, we summarize the current understanding that established IP6K1 and the other IP6K isoforms as possible targets for cancer therapy. However, it will be necessary to determine whether pharmacological inhibition of IP6K is safe enough to begin clinical study. The development of safe and selective inhibitors of IP6K isoforms is required to minimize undesirable effects.
Asunto(s)
Antineoplásicos/uso terapéutico , Sistemas de Liberación de Medicamentos , Inhibidores Enzimáticos/uso terapéutico , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Antineoplásicos/química , Carcinogénesis/inducido químicamente , Carcinogénesis/efectos de los fármacos , Carcinogénesis/metabolismo , Carcinogénesis/patología , Inhibidores Enzimáticos/química , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/inducido químicamente , Neoplasias/enzimología , Neoplasias/patología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Thiamine pyrophosphate (TPP) is an essential cofactor for various pivotal cellular processes in all living organisms, including bacteria. Thiamine biosynthesis occurs in bacteria but not in humans; therefore, the enzymes in this pathway are attractive targets for antibiotic development. Among these enzymes, thiamine monophosphate kinase (ThiL) catalyzes the final step of this pathway, phosphorylating thiamine monophosphate to produce TPP. Here, we extensively investigated ThiL in Pseudomonas aeruginosa, a major pathogen responsible for hospital-acquired infections. We demonstrate that thiL deletion abolishes not only thiamine biosynthesis but also thiamine salvage capability and results in growth defects of the ΔthiL strain even in the presence of thiamine derivatives, except for TPP. Most importantly, the pathogenesis of the ΔthiL strain was markedly attenuated, compared with that of WT cells, with lower inflammatory cytokine induction and 103-104-fold decreased bacterial loads in an in vivo infection model in which the intracellular TPP level was in the submicromolar range. To validate P. aeruginosa ThiL (PaThiL) as a drug target, we further characterized its biochemical properties, determining a Vmax of 4.0 ± 0.2 nmol·min-1 and Km values of 111 ± 8 and 8.0 ± 3.5 µm for ATP and thiamine monophosphate, respectively. An in vitro small-molecule screening assay identified PaThiL inhibitors including WAY213613, a noncompetitive inhibitor with a Ki value of 13.4 ± 2.3 µm and potential antibacterial activity against P. aeruginosa These comprehensive biological and biochemical results indicate that PaThiL represents a potential drug target for the development of an augmented repertoire of antibiotics against P. aeruginosa.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato) , Pseudomonas aeruginosa/enzimología , Tiamina/biosíntesis , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Pseudomonas aeruginosa/genéticaRESUMEN
In mammals, a family of three inositol hexakisphosphate kinases (IP6Ks) synthesizes the inositol pyrophosphate 5-IP7 from IP6. Genetic deletion of Ip6k1 protects mice from high fat diet induced obesity, insulin resistance and fatty liver. IP6K1 generated 5-IP7 promotes insulin secretion from pancreatic ß-cells, whereas it reduces insulin signaling in metabolic tissues by inhibiting the protein kinase Akt. Thus, IP6K1 promotes high fat diet induced hyperinsulinemia and insulin resistance in mice while its deletion has the opposite effects. IP6K1 also promotes fat accumulation in the adipose tissue by inhibiting the protein kinase AMPK mediated energy expenditure. Genetic deletion of Ip6k3 protects mice from age induced fat accumulation and insulin resistance. Accordingly, the pan IP6K inhibitor TNP [N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine] ameliorates obesity, insulin resistance and fatty liver in diet induced obese mice by improving Akt and AMPK mediated insulin sensitivity and energy expenditure. TNP also protects mice from bone loss, myocardial infarction and ischemia reperfusion injury. Thus, the IP6K pathway is a potential target in obesity and other metabolic diseases. Here, we summarize the studies that established IP6Ks as a potential target in metabolic diseases. Further studies will reveal whether inhibition of this pathway has similar pleiotropic benefits on metabolic health of humans.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Enfermedades Metabólicas/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Animales , Metabolismo Energético/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Humanos , Fosfatos de Inositol/metabolismo , Enfermedades Metabólicas/tratamiento farmacológico , Enfermedades Metabólicas/metabolismo , Ratones , Terapia Molecular Dirigida , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Ácido Fítico/metabolismoRESUMEN
Inositol hexakisphosphate kinases (IP6Ks) have been increasingly studied as therapeutically interesting enzymes. IP6K isoform specific knock-outs have been used to successfully explore inositol pyrophosphate physiology and related pathologies. A pan-IP6K inhibitor, N2-(m-trifluorobenzyl)-N6-(p-nitrobenzyl) purine (TNP), has been used to confirm phenotypes observed in genetic knock-out experiments; however, it suffers by having modest potency and poor solubility making it difficult to handle for in vitro applications in the absence of DMSO. Moreover, TNP's pan-IP6K inhibitory profile does not inform which IP6K isoform is responsible for which phenotypes. In this report we describe a series of purine-based isoform specific IP6K1 inhibitors. The lead compound was identified after multiple rounds of SAR and has been found to selectively inhibit IP6K1 over IP6K2 or IP6K3 using biochemical and biophysical approaches. It also boasts increased solubility and IP6K1 potency over TNP. These new compounds are useful tools for additional assay development and exploration of IP6K1 specific biology.
Asunto(s)
Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Humanos , Isoformas de Proteínas , Relación Estructura-ActividadRESUMEN
Dietary flavonoids inhibit certain protein kinases and phospholipid kinases by competing for their ATP-binding sites. These nucleotide pockets have structural elements that are well-conserved in two human small-molecule kinases, inositol hexakisphosphate kinase (IP6K) and inositol polyphosphate multikinase (IPMK), which synthesize multifunctional inositol phosphate cell signals. Herein, we demonstrate that both kinases are inhibited by quercetin and 16 related flavonoids; IP6K is the preferred target. Relative inhibitory activities were rationalized by X-ray analysis of kinase/flavonoid crystal structures; this detailed structure-activity analysis revealed hydrophobic and polar ligand/protein interactions, the degree of flexibility of key amino acid side chains, and the importance of water molecules. The seven most potent IP6K inhibitors were incubated with intact HCT116 cells at concentrations of 2.5 µM; diosmetin was the most selective and effective IP6K inhibitor (>70% reduction in activity). Our data can instruct on pharmacophore properties to assist the future development of inositol phosphate kinase inhibitors. Finally, we propose that dietary flavonoids may inhibit IP6K activity in cells that line the gastrointestinal tract.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Quercetina/farmacología , Sitios de Unión , Cristalografía por Rayos X , Células HCT116 , Humanos , Fosfatos de Inositol/metabolismo , Estructura Molecular , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quercetina/química , Quercetina/metabolismo , Relación Estructura-ActividadRESUMEN
Bisphosphonic acids, which are structural analogs of pyrophosphate, constitute a class of compounds with very high potential for the construction of effective inhibitors of enzymes operating on oligo- and polyphosphates. The bisphosphonate-based methodology was applied for the discovery of inhibitors of two families of polyphosphate kinases (PPK1 and PPK2). Screening of thirty-two structurally diverse bisphosphonic acids and related compounds revealed several micromolar inhibitors of both enzymes. Importantly, selectivity of bisphosphonates could be achieved by application of the appropriate side chain.
Asunto(s)
Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Isoenzimas/antagonistas & inhibidores , Nucleótidos/metabolismo , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Polifosfatos/metabolismoRESUMEN
Importance: Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare fatal premature aging disease. There is no approved treatment. Objective: To evaluate the association of monotherapy using the protein farnesyltransferase inhibitor lonafarnib with mortality rate in children with HGPS. Design, Setting, and Participants: Cohort study comparing contemporaneous (birth date ≥1991) untreated patients with HGPS matched with treated patients by age, sex, and continent of residency using conditional Cox proportional hazards regression. Treatment cohorts included patients from 2 single-group, single-site clinical trials (ProLon1 [n = 27; completed] and ProLon2 [n = 36; ongoing]). Untreated patients originated from a separate natural history study (n = 103). The cutoff date for patient follow-up was January 1, 2018. Exposure: Treated patients received oral lonafarnib (150 mg/m2) twice daily. Untreated patients received no clinical trial medications. Main Outcomes and Measures: The primary outcome was mortality. The primary analysis compared treated patients from the first lonafarnib trial with matched untreated patients. A secondary analysis compared the combined cohorts from both lonafarnib trials with matched untreated patients. Results: Among untreated and treated patients (n = 258) from 6 continents, 123 (47.7%) were female; 141 (54.7%) had a known genotype, of which 125 (88.7%) were classic (c.1824C>T in LMNA). When identified (n = 73), the primary cause of death was heart failure (79.4%). The median treatment duration was 2.2 years. Median age at start of follow-up was 8.4 (interquartile range [IQR], 4.8-9.5) years in the first trial cohort and 6.5 (IQR, 3.7-9.0) years in the combined cohort. There was 1 death (3.7%) among 27 patients in the first trial group and there were 9 deaths (33.3%) among 27 patients in the matched untreated group. Treatment was associated with a lower mortality rate (hazard ratio, 0.12; 95% CI, 0.01-0.93; P = .04). In the combined cohort, there were 4 deaths (6.3%) among 63 patients in the treated group and 17 deaths (27.0%) among 63 patients in the matched untreated group (hazard ratio, 0.23; 95% CI, 0.06-0.90; P = .04). Conclusions and Relevance: Among patients with HGPS, lonafarnib monotherapy, compared with no treatment, was associated with a lower mortality rate after 2.2 years of follow-up. Study interpretation is limited by its observational design.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Piperidinas/uso terapéutico , Progeria/tratamiento farmacológico , Piridinas/uso terapéutico , Adolescente , Adulto , Causas de Muerte , Niño , Estudios de Cohortes , Femenino , Humanos , Estimación de Kaplan-Meier , Lamina Tipo A/biosíntesis , Lamina Tipo A/metabolismo , Masculino , Progeria/genética , Progeria/mortalidad , Procesamiento Proteico-Postraduccional , Adulto JovenRESUMEN
Inositol hexakisphosphate kinases (IP6Ks) regulate a myriad of cellular processes, not only through their catalytic activity (which synthesizes InsP7, a multifunctional inositol pyrophosphate signaling molecule) but also through protein-protein interactions. To further study the enzymatic function and distinguish between these different mechanisms, specific inhibitors that target IP6K catalytic activity are required. Only one IP6K inhibitor is commonly used: N2-( m-(trifluoromethyl)benzyl) N6-( p-nitrobenzyl)purine (TNP). TNP is, however, compromised by weak potency, inability to distinguish between IP6K isoenzymes, off-target activities, and poor pharmacokinetic properties. Herein, we describe a new inhibitor discovery strategy, based on the high degree of structural conservation of the nucleotide-binding sites of IP6Ks and protein kinases; we screened for novel IP6K2 inhibitors using a focused set of compounds with features known, or computationally predicted, to target nucleotide binding by protein kinases. We developed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay of adenosine diphosphate (ADP) formation from adenosine triphosphate (ATP). Novel hit compounds for IP6K2 were identified and validated with dose-response curves and an orthogonal assay. None of these inhibitors affected another inositol pyrophosphate kinase, PPIP5K. Our screening strategy offers multiple IP6K2 inhibitors for future development and optimization. This approach will be applicable to inhibitor discovery campaigns for other inositol phosphate kinases.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas , Ensayos de Selección de Medicamentos Antitumorales/métodos , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Fosfotransferasas (Aceptor del Grupo Fosfato)/química , Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
Inorganic polyphosphate (polyP) is present in all living forms of life. Studied mainly in prokaryotes, polyP and its associated enzymes are vital in diverse metabolic activities, in some structural functions, and most importantly in stress responses. Bacterial species, including many pathogens, encode a homolog of a major polyP synthesis enzyme, Poly Phosphate Kinase (PPK) with 2 different genes coding for PPK1 and PPK2. Genetic deletion of the ppk1 gene leads to reduced polyP levels and the consequent loss of virulence and stress adaptation responses. This far, no PPK1 homolog has been identified in higher-order eukaryotes, and, therefore, PPK1 represents a novel target for chemotherapy. The aim of the current study is to investigate PPK1 from Escherichia coli with comprehensive understanding of the enzyme's structure and binding sites, which were used to design pharmacophores and screen a library of compounds for potential discovery of selective PPK1 inhibitors. Verification of the resultant inhibitors activities was conducted using a combination of mutagenic and chemical biological approaches. The metabolic phenotypic maps of the wild type E. coli (WT) and ppk1 knockout mutant were generated and compared with the metabolic map of the chemically inhibited WT. In addition, biofilm formation ability was measured in WT, ppk1 knockout mutant, and the chemically inhibited WT. The results demonstrated that chemical inhibition of PPK1, with the designed inhibitors, was equivalent to gene deletion in altering specific metabolic pathways, changing the metabolic fingerprint, and suppressing the ability of E. coli to form a biofilm.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Biopelículas , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , VirulenciaRESUMEN
OBJECTIVE: This study aims to identify serum microRNAs (miRNAs) related to ovarian cancer. STUDY DESIGN: MiRNA profiling data (GSE79943) were generated from the Gene Expression Omnibus, including 3 serum samples from healthy individuals and 4/3/16/6 serum samples from patients with ovarian cancer stage I/II/III/IV. Differentially expressed miRNAs (DEmiRNAs) were identified between controls and ovarian cancer stage I/II/III/IV by using limma package (p-value <0.05 and |log2 fold change| ≥0.5). miRWALK2.0 database was used to find experiment-validated targets of DEmiRNAs, and CTD database was utilized to screen known genes related to ovarian cancer. clusterProfiler package was used to perform pathway enrichment analysis of DEmiRNAs. Targets of DEmiRNAs were validated by using GSE40595, involving 8 normal ovarian stroma, 31 ovarian cancer stroma, 6 human ovarian surface epthelium, and 32 ovarian tumor epthelial component. RESULTS: Between stage I/II/III/IV and control, 39/143/29/39 DEmiRNAs were identified, which were regarded as key miRNAs. Between 4 DEmiRNA sets, 15 common DEmiRNAs were identified (e.g. up-regulated hsa-miR-1181 and hsa-miR-4314). Hsa-miR-1181 participated in "Jak-STAT signaling pathway" and "miRNAs in cancer"; hsa-miR-4314 took part in cancer-related pathways. STAT3 and KRAS, known marker genes of ovarian cancer, were targeted by hsa-miR-1181 and hsa-miR-4314, respectively. Besides, FOXP1 was targeted by hsa-miR-1181; FOXP1-AS1 and FOXP1-IT1 were down-regulated in ovarian cancer. GRWD1, IP6K1, and NEGR1 were targeted by hsa-miR-4314; GRWD1, IP6K1, and NEGR1 were down-regulated in ovarian tumor. CONCLUSION: MiR-1181 and miR-4314 might promote ovarian tumorigenesis via down-regulating FOXP1 and GRWD1/IP6K1/NEGR1, respectively. In addition, the 15 common DEmiRNAs might provide directions for ovarian cancer diagnosis.
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Biomarcadores de Tumor/sangre , Regulación Neoplásica de la Expresión Génica , MicroARNs/sangre , Neoplasias Ováricas/sangre , Adulto , Biomarcadores de Tumor/química , Biomarcadores de Tumor/metabolismo , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Moléculas de Adhesión Celular Neuronal/antagonistas & inhibidores , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Biología Computacional , Bases de Datos Genéticas , Regulación hacia Abajo , Femenino , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Perfilación de la Expresión Génica , Humanos , MicroARNs/química , MicroARNs/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadificación de Neoplasias , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Ovario/citología , Ovario/metabolismo , Ovario/patología , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Proyectos Piloto , ARN Neoplásico/sangre , ARN Neoplásico/química , ARN Neoplásico/metabolismo , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Células del Estroma/citología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic ß-cells and is important for insulin exocytosis. To better understand IP7 regulation in ß-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high Km for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wortmannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (U73122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic ß-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7's role in cellular signal transduction is likely to have been underestimated.
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Adenosina Trifosfato/metabolismo , Inhibidores Enzimáticos/farmacología , Fosfatos de Inositol/antagonistas & inhibidores , Células Secretoras de Insulina/efectos de los fármacos , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/antagonistas & inhibidores , Androstadienos/farmacología , Animales , Arsenicales/farmacología , Línea Celular , Cromonas/farmacología , Cricetulus , Estrenos/farmacología , Expresión Génica , Humanos , Fosfatos de Inositol/metabolismo , Insulina/biosíntesis , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Morfolinas/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Pirrolidinonas/farmacología , Receptor de Insulina/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Succinimidas/farmacología , Triazoles/farmacología , WortmaninaRESUMEN
Methicillin resistant Staphylococcus aureus (MRSA) is resistant to known antibiotics and has become a great challenge for healthcare professionals, therefore new molecules are needed to manage this situation. In this study, new lead molecules 4-Amino-5-(2-Hydroxyphenyl)-1,2,4-Triazol-3-Thione (U1) and4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol(U1A Schiff base) were synthesized by fusion method that showed promising antibacterial activity (U1A: 26mm and U1: 14mm) against MRSA.FT-IR and NMR were used for structural characterization of these derivatives and their toxicity properties were assessed by Lipinski's rule of 5. New potential drug targets of this bacterium were also identified by comparative and subtraction genomics techniques. In particular, octanoyl-[GcvH]: protein N-octanoyl transferase and phosphor mevalonate kinase were used as potential targets in AutoDock Vina studies. This study can provide a framework to find potential drug targets for other pathogenic microorganisms that can successfully be docked with compound U1 and U1A.
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Antibacterianos/síntesis química , Antibacterianos/farmacología , Diseño Asistido por Computadora , Diseño de Fármacos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Triazoles/síntesis química , Triazoles/farmacología , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Antibacterianos/farmacocinética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Espectroscopía de Resonancia Magnética , Staphylococcus aureus Resistente a Meticilina/enzimología , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Simulación del Acoplamiento Molecular , Estructura Molecular , Terapia Molecular Dirigida/métodos , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , Relación Estructura-Actividad , Triazoles/farmacocinéticaRESUMEN
Mesenchymal stem/stromal cells (MSCs) are the predominant source of bone and adipose tissue in adult bone marrow and play a critical role in skeletal homeostasis. Age-induced changes in bone marrow favor adipogenesis over osteogenesis leading to skeletal involution and increased marrow adiposity so pathways that prevent MSC aging are potential therapeutic targets for treating age-related bone diseases. Here, we show that inositol hexakisphosphate kinase 1 (Ip6k1) deletion in mice increases MSC yields from marrow and enhances cell growth and survival ex vivo. In response to the appropriate stimuli, Ip6k1-/- versus Ip6k1+/+ MSCs also exhibit enhanced osteogenesis and hematopoiesis-supporting activity and reduced adipogenic differentiation. Mechanistic-based studies revealed that Ip6k1-/- MSCs express higher MDM2 and lower p53 protein levels resulting in lower intrinsic mitochondrial reactive oxygen species (ROS) levels as compared to Ip6k1+/+ MSCs, but both populations upregulate mitochondrial ROS to similar extents in response to oxygen-induced stress. Finally, we show that mice fed a high fat diet exhibit reduced trabecular bone volume, and that pharmacological inhibition of IP6K1 using a pan-IP6K inhibitor largely reversed this phenotype while increasing MSC yields from bone marrow. Together, these findings reveal an important role for IP6K1 in regulating MSC fitness and differentiation fate. Unlike therapeutic interventions that target peroxisome proliferator-activated receptor gamma and leptin receptor activity, which yield detrimental side effects including increased fracture risk and altered feeding behavior, respectively, inhibition of IP6K1 maintains insulin sensitivity and prevents obesity while preserving bone integrity. Therefore, IP6K1 inhibitors may represent more effective insulin sensitizers due to their bone sparing properties. Stem Cells 2017;35:1973-1983.
Asunto(s)
Dieta Alta en Grasa , Células Madre Mesenquimatosas/enzimología , Músculo Esquelético/patología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Adipogénesis , Animales , Médula Ósea/metabolismo , Proliferación Celular , Supervivencia Celular , Eliminación de Gen , Hematopoyesis , Células Madre Mesenquimatosas/metabolismo , Ratones , Osteogénesis , Estrés Oxidativo , Fosfotransferasas (Aceptor del Grupo Fosfato)/antagonistas & inhibidores , Fosfotransferasas (Aceptor del Grupo Fosfato)/deficienciaRESUMEN
Inositol hexakisphosphate kinase 1 (IP6K1), which generates 5-diphosphoinositol pentakisphosphate (5-IP7), physiologically mediates numerous functions. We report that IP6K1 deletion leads to brain malformation and abnormalities of neuronal migration. IP6K1 physiologically associates with α-actinin and localizes to focal adhesions. IP6K1 deletion disrupts α-actinin's intracellular localization and function. The IP6K1 deleted cells display substantial decreases of stress fiber formation and impaired cell migration and spreading. Regulation of α-actinin by IP6K1 requires its kinase activity. Deletion of IP6K1 abolishes α-actinin tyrosine phosphorylation, which is known to be regulated by focal adhesion kinase (FAK). FAK phosphorylation is substantially decreased in IP6K1 deleted cells. 5-IP7, a product of IP6K1, promotes FAK autophosphorylation. Pharmacologic inhibition of IP6K by TNP [N2-(m-Trifluorobenzyl), N6-(p-nitrobenzyl)purine] recapitulates the phenotype of IP6K1 deletion. These findings establish that IP6K1 physiologically regulates neuronal migration by binding to α-actinin and influencing phosphorylation of both FAK and α-actinin through its product 5-IP7.