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1.
Chemistry ; 23(25): 6083-6093, 2017 May 02.
Article in English | MEDLINE | ID: mdl-27809361

ABSTRACT

The K-Ras GTPase is a major target in anticancer drug discovery. However, direct interference with signaling by K-Ras has not led to clinically useful drugs yet. Correct localization and signaling by farnesylated K-Ras is regulated by the prenyl binding protein PDEδ. Interfering with binding of PDEδ to K-Ras by means of small molecules provides a novel opportunity to suppress oncogenic signaling. Here we describe the identification and structure-guided development of novel K-Ras-PDEδ inhibitor chemotypes based on pyrrolopyridazinones and pyrazolopyridazinones that bind to the farnesyl binding pocket of PDEδ with low nanomolar affinity. We delineate the structure-property relationship and in vivo pharmacokinetic (PK) and toxicokinetic (Tox) studies for pyrazolopyridazinone-based K-Ras-PDEδ inhibitors. These findings may inspire novel drug discovery efforts aimed at the development of drugs targeting oncogenic Ras.

2.
Nat Commun ; 7: 11360, 2016 Apr 20.
Article in English | MEDLINE | ID: mdl-27094677

ABSTRACT

The prenyl-binding protein PDEδ is crucial for the plasma membrane localization of prenylated Ras. Recently, we have reported that the small-molecule Deltarasin binds to the prenyl-binding pocket of PDEδ, and impairs Ras enrichment at the plasma membrane, thereby affecting the proliferation of KRas-dependent human pancreatic ductal adenocarcinoma cell lines. Here, using structure-based compound design, we have now identified pyrazolopyridazinones as a novel, unrelated chemotype that binds to the prenyl-binding pocket of PDEδ with high affinity, thereby displacing prenylated Ras proteins in cells. Our results show that the new PDEδ inhibitor, named Deltazinone 1, is highly selective, exhibits less unspecific cytotoxicity than the previously reported Deltarasin and demonstrates a high correlation with the phenotypic effect of PDEδ knockdown in a set of human pancreatic cancer cell lines.


Subject(s)
Antineoplastic Agents/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 6/chemistry , Epithelial Cells/drug effects , Gene Expression Regulation, Neoplastic , Phosphodiesterase Inhibitors/chemistry , Proto-Oncogene Proteins p21(ras)/chemistry , Pyrazines/chemistry , Pyrazoles/chemistry , Small Molecule Libraries/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Cyclic Nucleotide Phosphodiesterases, Type 6/genetics , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Expression , Humans , Molecular Docking Simulation , Pancreatic Ducts/drug effects , Pancreatic Ducts/metabolism , Pancreatic Ducts/pathology , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/pharmacology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Pyrazines/chemical synthesis , Pyrazines/pharmacology , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/pharmacology
3.
Stroke ; 44(8): 2324-6, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23686975

ABSTRACT

BACKGROUND AND PURPOSE: We tested the hypothesis that excessive chronic ethanol consumption is associated with more severe ischemic strokes. METHODS: We recruited patients with supratentorial cerebral ischemia within 48 hours of symptom onset. We defined heavy drinkers by a weekly consumption of ethanol of ≥300 g and severe strokes by a National Institutes of Health Stroke Scale score≥6. RESULTS: Of 436 patients, 60 were heavy drinkers. Being a heavy drinker was independently associated with baseline National Institutes of Health Stroke Scale scores≥6 (odds ratio, 2.35; 95% confidence interval, 1.12-5.26; P=0.023) at the logistic regression analysis. This result was not modified with the propensity analysis. CONCLUSION: An excessive chronic ethanol consumption is associated with higher baseline stroke severity.


Subject(s)
Alcohol Drinking/adverse effects , Ethanol/adverse effects , Ischemic Attack, Transient/epidemiology , Severity of Illness Index , Stroke/epidemiology , Acute Disease , Aged , Aged, 80 and over , Alcohol Drinking/epidemiology , Comorbidity , Female , Humans , Interview, Psychological , Ischemic Attack, Transient/physiopathology , Male , Middle Aged , Stroke/physiopathology
4.
Drug Metab Dispos ; 41(3): 568-74, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23230132

ABSTRACT

Ethyl glucuronide (EtG) determination is increasingly used in clinical and forensic toxicology to document ethanol consumption. The enzymes involved in EtG production, as well as potential interactions with common drugs of abuse, have not been extensively studied. Activities of human liver (HLM), kidney (HKM), and intestinal (HIM) microsomes, as well as of 12 major human recombinant UDP-glucuronosyltransferases (UGTs), toward ethanol (50 and 500 mM) were evaluated in vitro using liquid chromatography-tandem mass spectrometry. Enzyme kinetic parameters were determined for pooled microsomes and recombinant UGTs with significant activity. Individual contributions of UGTs were estimated using the relative activity factor approach, proposed for scaling activities obtained with cDNA-expressed enzymes to HLM. Interaction of morphine, codeine, lorazepam, oxazepam, nicotine, cotinine, cannabinol, and cannabidiol (5, 10, 15 mg/l) with ethanol (1.15, 4.6, 11.5 g/l; i.e., 25, 100, 250 mM) glucuronidation was assessed using pooled HLM. Ethanol glucuronidation intrinsic clearance (Cl(int)) was 4 and 12.7 times higher for HLM than for HKM and HIM, respectively. All recombinant UGTs, except UGT1A1, 1A6, and 1A10, produced EtG in detectable amounts. UGT1A9 and 2B7 were the most active enzymes, each accounting for 17 and 33% of HLM Cl(int), respectively. Only cannabinol and cannabidiol significantly affected ethanol glucuronidation. Cannabinol increased ethanol glucuronidation in a concentration-dependent manner, whereas cannabidiol significantly inhibited EtG formation in a noncompetitive manner (IC(50) = 1.17 mg/l; inhibition constant (K(i)) = 3.1 mg/l). UGT1A9 and 2B7 are the main enzymes involved in ethanol glucuronidation. In addition, our results suggest that cannabinol and cannabidiol could significantly alter ethanol glucuronidation.


Subject(s)
Ethanol/metabolism , Glucuronates/metabolism , Glucuronosyltransferase/metabolism , Illicit Drugs/pharmacology , Biotransformation , Cannabidiol/pharmacology , Cannabinol/pharmacology , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Drug Interactions , Enzyme Inhibitors/pharmacology , Fluconazole/pharmacology , Glucuronosyltransferase/antagonists & inhibitors , Humans , Intestines/enzymology , Kidney/enzymology , Kinetics , Microsomes, Liver/enzymology , Niflumic Acid/pharmacology , Recombinant Proteins/metabolism , Substrate Specificity , Tandem Mass Spectrometry , UDP-Glucuronosyltransferase 1A9
5.
PLoS One ; 6(9): e25470, 2011.
Article in English | MEDLINE | ID: mdl-21980470

ABSTRACT

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-limiting step of the kynurenine pathway that is an important component of immunomodulatory and neuromodulatory processes. The IDO1 gene is highly inducible by IFN-γ and TNF-α through interaction with cis-acting regulatory elements of the promoter region. Accordingly, functional polymorphisms in the IDO1 promoter could partly explain the interindividual variability in IDO expression that has been previously documented. METHODOLOGY/PRINCIPAL FINDINGS: A PCR-sequencing strategy, applied to DNA samples from healthy Caucasians, allowed us to identify a VNTR polymorphism in the IDO1 promoter, which correlates significantly with serum tryptophan concentration, controlled partially by IDO activity, in female subjects, but not in males. Although this VNTR does not appear to affect basal or cytokine-induced promoter activity in gene reporter assays, it contains novel cis-acting elements. Three putative LEF-1 binding sites, one being located within the VNTR repeat motif, were predicted in silico and confirmed by chromatin immunoprecipitation. Overexpression of LEF-1 in luciferase assays confirmed an interaction between LEF-1 and the predicted transcription factor binding sites, and modification of the LEF-1 core sequence within the VNTR repeat motif, by site-directed mutagenesis, resulted in an increase in promoter activity. CONCLUSIONS/SIGNIFICANCE: The identification of a VNTR in the IDO1 promoter revealed a cis-acting element interacting with the most downstream factor of the Wnt signaling pathway, suggesting novel mechanisms of regulation of IDO1 expression. These data offer new insights, and suggest further studies, into the role of IDO in various pathological conditions, particularly in cancer where IDO and the Wnt pathway are strongly dysregulated.


Subject(s)
Indoleamine-Pyrrole 2,3,-Dioxygenase/genetics , Lymphoid Enhancer-Binding Factor 1/metabolism , Minisatellite Repeats/genetics , Polymorphism, Genetic/genetics , Response Elements/genetics , Base Sequence , DNA/genetics , DNA/metabolism , Exons/genetics , Female , Gene Expression Regulation, Enzymologic/genetics , Humans , Introns/genetics , Male , Molecular Sequence Data , Transcription, Genetic/genetics , Tryptophan/blood , Young Adult
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