camilo rojas - Universidad de los Andes (Colombia) (original) (raw)
Papers by camilo rojas
Biochemical Journal, 2007
The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during ch... more The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2yl)ethyl sulfide]. BPTES is a potent inhibitor of KGA, but not of the liver GA isoform, glutamate dehydrogenase or γglutamyl transpeptidase. Kinetic studies indicate that, with respect to glutamine, BPTES has a K i of approx. 3 µM. Moreover, these studies suggest that BPTES inhibits the allosteric activation caused by phosphate binding and promotes the formation of an inactive complex. Gel-filtration chromatography and sedimentation-velocity analysis were used to examine the effect of BPTES on the phosphate-dependent oligomerization of KGA. This established that BPTES prevents the formation of large phosphate-induced oligomers and instead promotes the formation of a single oligomeric species with distinct physical properties. Sedimentation-equilibrium studies determined that the oligomer produced by BPTES is a stable tetramer. Taken together, the present work indicates that BPTES is a unique and potent inhibitor of rat KGA and elucidates a novel mechanism of inactivation.
Journal of Neurochemistry, 2004
Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective agai... more Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective against long-term neuropathy in diabetic animals. In the current study, we have determined that the GCP II inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) is protective against glucose-induced programmed cell death (PCD) and neurite degeneration in dorsal root ganglion (DRG) neurons in a cell culture model of diabetic neuropathy. In this model, inhibition of caspase activation is mediated through the group II metabotropic glutamate receptor, mGluR3. 2-PMPA neuroprotection is completely reversed by the mGluR3 antagonist (S)-α-ethylglutamic acid (EGLU). In contrast, group I and III mGluR inhibitors have no effect on 2-PMPA neuroprotection. Furthermore, we show that two mGluR3 agonists, the direct agonist (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) and N-acetyl-aspartyl-glutamate (NAAG) provide protection to neurons exposed to high glucose conditions, consistent with the concept that 2-PMPA neuroprotection is mediated by increased NAAG activity. Inhibition of GCP II or mGluR3 may represent a novel mechanism to treat neuronal degeneration under high-glucose conditions.
Bioorganic & Medicinal Chemistry Letters, 2004
In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electro... more In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electrophile-based dipeptidyl peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SAR of these series demonstrate that the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S1 0 binding groups for potency against DPP IV. In addition, both cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series. Stereochemistry at the a-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the ketoazetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for up to 6 h.
Bioorganic & Medicinal Chemistry Letters, 2003
A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptid... more A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1 0 residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxycarbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC 50 value of 220 nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure-activity relationships of GCP II inhibition. #
Steroids, 1995
Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in ch... more Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in cholesterol biosynthesis. One assay detects inhibition of A 5" 7-sterol A T-reductase, the enzyme involved in the conversion of 7-dehydrocholesterol to cholesterol. As'Z-Sterol A T-reductase was inhibited by both RPR 101821, a protonated cyclohexylamine, and BM 15.766, a piperazine derivative, with IC5o values of I p.M. The second assay detects accumulation of any of five intermediates (squalene oxide, squalene dioxide, lanosterol, desmosterol, and 7-dehydrocholesterol) upon inhibition of enzymes catalyzing reactions in the conversion of squalene to cholesterol. In this assay, inhibition data were most accurate when control assays exhibited a conversion of squalene to cholesterol in the order of 50%. The time required to attain 50% conversion of squalene to cholesterol was 6 h. Given a high inhibitor to substrate concentration ratio and the possible values of K i, ko,, and koMfor the reaction between enzymes and inhibitor to form enzyme-inhibitor complexes, it was predicted that in the presence of inhibitors, intermediate accumulation could still be observed after 6 h incubation. The experimental results were in agreement with this prediction. (Steroids 60:475--483, 1995)
Palonosetron Exhibits Unique Molecular Interactions with the 5HT3 Receptor
Anesthesia and Analgesia, 2008
Palonosetron is a 5-HT(3)-receptor antagonist (5-HT(3)-RA) that has been shown to be superior to ... more Palonosetron is a 5-HT(3)-receptor antagonist (5-HT(3)-RA) that has been shown to be superior to other 5-HT(3)-RAs in phase III clinical trials for the prevention of acute, delayed, and overall chemotherapy-induced nausea and vomiting. The improved clinical efficacy of palonosetron may be due, in part, to its more potent binding and longer half-life. However, these attributes alone are not sufficient to explain the results with palonosetron. We sought to elucidate additional differences among 5-HT(3)-RAs that could help explain the observations in the clinic. Receptor site saturation binding experiments were performed with [3H] palonosetron, [3H] granisetron, and [3H] ondansetron to obtain the corresponding Scatchard analyses and Hill coefficients. Diagnostic equilibrium binding experiments and kinetic dissociation experiments were conducted to examine competitive versus potential allosteric interactions between ondansetron, granisetron and palonosetron and the 5-HT(3) receptor. Finally, the long-term effect of the three antagonists on receptor function as measured by Ca2+ influx in HEK 293 cells expressing the 5-HT(3)-receptor was compared. Analyses of binding isotherms using both Scatchard and Hill plots suggested positive cooperativity for palonosetron and simple bimolecular binding for both granisetron and ondansetron. Equilibrium diagnostic tests discriminated differential effects of palonosetron on [3H] ligand binding indicating that palonosetron was an allosteric antagonist whereas granisetron and ondansetron were competitive antagonists. Using dissociation rate strategies, palonosetron was shown to be an allosteric modifier that accelerated the rate of dissociation from the receptor of both granisetron and ondansetron. Differences in the binding mode of palonosetron to the 5-HT(3) receptor were shown to have an impact on receptor function. In these experiments, cells were incubated with each antagonist, followed by infinite dilutions and dissociation for 2.5 h; cells previously incubated with either granisetron or ondansetron showed calcium-ion influx similar to control cells that had not been exposed to a 5-HT(3) receptor antagonist. In contrast, substantial inhibition of calcium-ion influx was observed in cells that had been incubated with palonosetron. Palonosetron exhibited allosteric binding and positive cooperativity when binding to the 5-HT(3) receptor. Palonosetron also triggered functional effects that persisted beyond its binding to the 5-HT(3) receptor at the cell surface. Differences in binding and effects on receptor function may be relevant to the unique beneficial actions of palonosetron. To our knowledge, this is the first report showing palonosetron's interaction with the 5-HT(3) receptor at the molecular level, clearly differentiating it from other 5-HT(3)-RAs.
European Journal of Pharmacology, 2001
. Ž . 2-Phosphonomethyl pentanedioic acid 2-PMPA is a potent and selective inhibitor of glutamate... more . Ž . 2-Phosphonomethyl pentanedioic acid 2-PMPA is a potent and selective inhibitor of glutamate carboxypeptidase II NAALADase , and has shown robust neuroprotective activity in both in vitro and in vivo models of ischemia. In the brain, glutamate carboxypeptidase II Ž .
Azetidine-Based Inhibitors of Dipeptidyl Peptidase IV (DPP IV
Current Topics in Medicinal Chemistry, 2007
The structure-activity relationships of azetidine-based DPP IV inhibitors will be discussed in de... more The structure-activity relationships of azetidine-based DPP IV inhibitors will be discussed in detail in the following review. The azetidine-based DPP IV inhibitors can be divided into three main subtypes, the 2-cyanoazetidines, 3-fluoroazetidines and 2-ketoazetidines. These subtypes have been explored and structure-activity relationships have been established by several groups. Several compounds within each of these subtypes display sub micromolar potency against DPP IV. The most potent cyanoazetidines and ketoazetidines have large, hydrophobic amino acid groups bound to the azetidine nitrogen and display activities below 100 nM. DPP IV inhibition is not sensitive to stereochemistry at the 2-position as both 2-(R)- and 2-(S)-cyano and -keto azetidines display similar inhibitory potencies. While these "warhead"-based cyano- and ketoazetidines have the potential for covalent, bond-forming inhibition, they can also react to internally cyclize into inactive ketopiperazines and dihydroketopyrazine. Thus, chemical instability was also explored for compounds in these two subtypes and certain members of the cyanoazetidine series display aqueous stability comparable to the closely related cyanopyrrolidines. Select 3-fluoroazetidines also display inhibitory potencies below 1 microM without the propensity for cyclization and chemical instability associated with the other subseries.
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](Cheminform, 2003
A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptid... more A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1 0 residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxycarbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC 50 value of 220 nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure-activity relationships of GCP II inhibition. #
Determination of catecholamine permeability coefficients for passive diffusion across phospholipid vesicle membranes
Journal of Membrane Biology, 1981
A convenient catecholamine transport assay has been developed which permits continuous, instantan... more A convenient catecholamine transport assay has been developed which permits continuous, instantaneous monitoring of transmembrane flux. Epinephrine transport has been examined by spectrophotometrically monitoring adrenochrome formation resulting from the passive diffusion of catecholamine into unilamellar phospholipid vesicles containing entrapped potassium ferricyanide. Ferricyanide oxidation of epinephrine under the conditions employed is fast compared to membrane transport, which obviates the need for intravesicular concentration or volume determinations. Epinephrine transport data over a pH 6 to 7 range have been fitted to an integrated rate equation from which a permeability coefficient for neutral epinephrine of 2.7±1.5×10−6 cm/sec has been obtained.
Journal of Medicinal Chemistry, 2008
D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including D-serine, a full a... more D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including D-serine, a full agonist at the glycine site of the NMDA receptor. A series of benzo[d]isoxazol-3-ol derivatives were synthesized and evaluated as DAAO inhibitors. Among them, 5-chlorobenzo[d]isoxazol-3-ol (CBIO) potently inhibited DAAO with an IC50 in the submicromolar range. Oral administration of CBIO in conjunction with D-serine enhanced the plasma and brain levels of D-serine in rats compared to the oral administration of D-serine alone.
Analytical Biochemistry, 2002
Glutamate carboxypeptidase II (GCPII or prostate-specific membrane antigen or NAALADase) is an en... more Glutamate carboxypeptidase II (GCPII or prostate-specific membrane antigen or NAALADase) is an enzyme that catalyzes the hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (G). Inhibitors of GCPII provide neuroprotection in a variety of animal models of central nervous system disorders. Neuroprotection is probably the result of increased NAAG concentrations and decreased levels of excess toxic glutamate. Consequently, GCPII inhibitors could be useful therapeutic agents where increased glutamate levels are the result of increased GCPII activity. Current GCPII in vitro activity assays are cumbersome or have limited sensitivity. In this report we describe a microplate assay to study GCPII inhibition that is most sensitive, efficient, and generates little waste. GCPII turnover number (k cat Þ was 4 s À1 and the binding constant (K m Þ for NAAG and GCPII was 130 nM. The apparent association rate constant for GCPII and NAAG (k cat =K m Þ was 3 Â 10 7 M À1 s À1 . Inhibition studies with the GCPII inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) demonstrated competitive inhibition with a K i ¼ 0:2 nM.
European Journal of Pharmacology, 2010
Palonosetron is a 5-HT 3 receptor antagonist that has demonstrated superiority in preventing both... more Palonosetron is a 5-HT 3 receptor antagonist that has demonstrated superiority in preventing both acute and delayed emesis when compared to older first generation 5-HT 3 receptor antagonists. The objective of this work was to determine if palonosetron exhibits unique molecular interactions with the 5-HT 3 receptor that could provide a scientific rationale for observed clinical efficacy differences. Previously, we showed that palonosetron exhibits allosteric binding and positive cooperativity to the 5-HT 3 receptor in contrast to ondansetron and granisetron which exhibit simple bimolecular binding. The present work shows, through several independent experiments, that palonosetron uniquely triggers 5-HT 3 receptor internalization and induces prolonged inhibition of receptor function. After 24 h incubation followed by dissociation conditions, [ 3 H]palonosetron remained associated with whole cells but not to cell-free membranes (P < 0.001). [ 3 H] Palonosetron's binding to cells was resistant to both protease and acid treatments designed to denature cell surface proteins suggesting that the receptor complex was inside the cells rather than at the surface. Cells pretreated with unlabeled palonosetron subsequently exhibited reduced cell surface 5-HT 3 receptor binding. Palonosetron-triggered receptor internalization was visualized by confocal fluorescence microscopy using cells transfected with 5-HT 3 receptor fused to enhanced cyan fluorescent protein. In contrast, granisetron and ondansetron showed minimal to no effect on receptor internalization or prolonged inhibition of receptor function. These experiments may provide a pharmacological basis for differences noted in published clinical trials comparing palonosetron to other 5-HT 3 receptor antagonists.
New Approaches to Chemotherapy-Induced Nausea and Vomiting: From Neuropharmacology to Clinical Investigations
Cancer Journal, 2006
Nausea and vomiting are considered to be among the most distressing consequences of cytotoxic che... more Nausea and vomiting are considered to be among the most distressing consequences of cytotoxic chemotherapies. Currently, there are several novel 5-HT(3) receptor antagonists for the treatment of chemotherapy-induced nausea and vomiting (CINV), including ondansetron, granisetron, and dolasetron. These agents provide significant improvement in the management of acute emesis but are ineffective at preventing delayed emesis. In 2003, a new 5-HT(3) receptor antagonist, palonosetron HCL (Aloxi), was introduced to the U.S. market. Palonosetron was found to be effective in preventing delayed CINV. Indeed, palonosetron was the first and only 5-HT(3) receptor antagonist approved by the FDA for the prevention of both acute and delayed CINV. More recently, studies on the role of substance P in the emetic process led to the development of aprepitant (Emend) for the prevention of delayed emesis in combination with 5-HT(3) receptor antagonists. Despite these major advances, CINV remains uncontrolled in some patients. Current efforts are focused on treating refractory emesis and include both the clinical evaluation of compounds marketed for other indications and the preclinical evaluation of novel molecules targeting other transmitters in the emetic pathway. Ongoing work in pharmacogenomics has postulated several candidate genes that could be involved in emetic sensitivity and responsiveness to antiemetic therapy. Investigations into the pharmacogenomics of CINV may someday be able to aid in the identification of high risk patients and patients unlikely to respond to conventional therapies.
Pharmacological characterization of AMP 579, a novel adenosine A1/A2 receptor agonist and cardioprotective
Drug Development Research, 1998
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Cardiovascular Drug Reviews, 1996
Key Words: Atherosclerosis-PDGF-receptor tyrosine kinase-Phenolate anion-Phenylaminopyrimidine--Q... more Key Words: Atherosclerosis-PDGF-receptor tyrosine kinase-Phenolate anion-Phenylaminopyrimidine--Quinoline-Quinoxaline-Restenosis-Tyrosine kinase inhibitor.
Brain Research, 2005
Dipeptidyl peptidase IV (DPP IV) is a ubiquitous membrane-bound enzyme that cleaves the two N-ter... more Dipeptidyl peptidase IV (DPP IV) is a ubiquitous membrane-bound enzyme that cleaves the two N-terminal amino acids from peptides with a proline or alanine residue in the second position from the amino end. Potential substrates for DPP IV include several neuropeptides, suggesting a role for DPP IV in neurological processes. We have developed a potent DPP IV inhibitor (IC 50 = 30 nM), 1-(2-Amino-3-methylbutyryl)-azetidine-2-carbonitrile (AMAC), which has shown efficacy in two established models of psychosis: mescaline-induced scratching and amphetamine-induced hyperactivity. In the mescaline-induced scratching model, AMAC treatment before mescaline administration reduced the number of scratching paroxysms by 68% (P < 0.01). The compound showed a dose-dependent effect, inhibiting significantly at 6, 20 and 60 mg/kg (37%, 39% and 68%, respectively). In the amphetamine-induced hyperactivity model, 50 and 60 mg/kg AMAC, given before injection of amphetamine, significantly reduced hyper-locomotion by 65% and 76%, respectively. Additionally, AMAC showed no significant activity in binding assays for 20 receptors thought to be involved in the pathology of schizophrenia, including dopamine, serotonin and glutamate. A structurally similar analog, 1-(2-dimethylamino-3-methyl-butyryl)-azetidine-2-carbonitrile (DAMAC), that does not inhibit DPP IV, was inactive in both models. Taken together, these data suggest that the antipsychotic effects of AMAC are the result of DPP IV inhibition. D
Journal of Neurochemistry, 2002
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number... more Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.
Febs Letters, 2003
D-Serine was previously identi¢ed in mammalian brain and was shown to be a co-agonist at the 'gly... more D-Serine was previously identi¢ed in mammalian brain and was shown to be a co-agonist at the 'glycine' site of the N-methyl-D-aspartate (NMDA)-type receptors. Racemization of serine is catalyzed by serine racemase, a pyridoxal 5P Pphosphate-dependent enzyme expressed mainly in brain and liver. NMDA receptor overactivation has been implicated in a number of pathological conditions and inhibitors of serine racemase are thus potentially interesting targets for therapy. We expressed recombinant mouse serine racemase in insect cells and puri¢ed it to near homogeneity. The enzyme is a non-covalent homodimer in solution and requires divalent cations Mg 2+ , Ca 2+ or Mn 2+ for activity but not for dimerization. In addition to the racemization it also catalyzes speci¢c elimination of L-Ser to pyruvate. D-Serine is eliminated much less e⁄ciently. Both L-serine racemization and elimination activities of serine racemase are of comparable magnitude, display alkaline pH optimum and are negligible below pH 6.5. adenine dinucleotide reduced form; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid; EDTA, ethylenediamine tetraacetic acid FEBS 26899 FEBS Letters 535
Analytical Biochemistry, 2011
D-serine administration has been shown to be effective for the treatment of schizophrenia symptom... more D-serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, D-serine has to be administered at high doses in order to observe clinical effects. This is in large part due to D-serine undergoing oxidation by D-amino acid oxidase (DAAO) before it reaches the brain. Consequently, co-administration of D-serine with a DAAO inhibitor has been suggested as a way to lower the dose of D-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma D-serine levels after oral coadministration. Current HPLC-based methodologies to measure D-serine in plasma are time consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well-format assay to monitor D-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate D-serine from plasma followed by quantitation of D-serine using the DAAO catalyzed reaction. Plasma D-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.
Biochemical Journal, 2007
The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during ch... more The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2yl)ethyl sulfide]. BPTES is a potent inhibitor of KGA, but not of the liver GA isoform, glutamate dehydrogenase or γglutamyl transpeptidase. Kinetic studies indicate that, with respect to glutamine, BPTES has a K i of approx. 3 µM. Moreover, these studies suggest that BPTES inhibits the allosteric activation caused by phosphate binding and promotes the formation of an inactive complex. Gel-filtration chromatography and sedimentation-velocity analysis were used to examine the effect of BPTES on the phosphate-dependent oligomerization of KGA. This established that BPTES prevents the formation of large phosphate-induced oligomers and instead promotes the formation of a single oligomeric species with distinct physical properties. Sedimentation-equilibrium studies determined that the oligomer produced by BPTES is a stable tetramer. Taken together, the present work indicates that BPTES is a unique and potent inhibitor of rat KGA and elucidates a novel mechanism of inactivation.
Journal of Neurochemistry, 2004
Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective agai... more Glutamate carboxypeptidase II (GCP II) inhibition has previously been shown to be protective against long-term neuropathy in diabetic animals. In the current study, we have determined that the GCP II inhibitor 2-(phosphonomethyl) pentanedioic acid (2-PMPA) is protective against glucose-induced programmed cell death (PCD) and neurite degeneration in dorsal root ganglion (DRG) neurons in a cell culture model of diabetic neuropathy. In this model, inhibition of caspase activation is mediated through the group II metabotropic glutamate receptor, mGluR3. 2-PMPA neuroprotection is completely reversed by the mGluR3 antagonist (S)-α-ethylglutamic acid (EGLU). In contrast, group I and III mGluR inhibitors have no effect on 2-PMPA neuroprotection. Furthermore, we show that two mGluR3 agonists, the direct agonist (2R,4R)-4-aminopyrrolidine-2, 4-dicarboxylate (APDC) and N-acetyl-aspartyl-glutamate (NAAG) provide protection to neurons exposed to high glucose conditions, consistent with the concept that 2-PMPA neuroprotection is mediated by increased NAAG activity. Inhibition of GCP II or mGluR3 may represent a novel mechanism to treat neuronal degeneration under high-glucose conditions.
Bioorganic & Medicinal Chemistry Letters, 2004
In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electro... more In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electrophile-based dipeptidyl peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SAR of these series demonstrate that the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S1 0 binding groups for potency against DPP IV. In addition, both cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series. Stereochemistry at the a-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the ketoazetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for up to 6 h.
Bioorganic & Medicinal Chemistry Letters, 2003
A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptid... more A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1 0 residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxycarbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC 50 value of 220 nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure-activity relationships of GCP II inhibition. #
Steroids, 1995
Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in ch... more Two separate enzymatic assays were developed in order to test the selectivity of inhibitors in cholesterol biosynthesis. One assay detects inhibition of A 5" 7-sterol A T-reductase, the enzyme involved in the conversion of 7-dehydrocholesterol to cholesterol. As'Z-Sterol A T-reductase was inhibited by both RPR 101821, a protonated cyclohexylamine, and BM 15.766, a piperazine derivative, with IC5o values of I p.M. The second assay detects accumulation of any of five intermediates (squalene oxide, squalene dioxide, lanosterol, desmosterol, and 7-dehydrocholesterol) upon inhibition of enzymes catalyzing reactions in the conversion of squalene to cholesterol. In this assay, inhibition data were most accurate when control assays exhibited a conversion of squalene to cholesterol in the order of 50%. The time required to attain 50% conversion of squalene to cholesterol was 6 h. Given a high inhibitor to substrate concentration ratio and the possible values of K i, ko,, and koMfor the reaction between enzymes and inhibitor to form enzyme-inhibitor complexes, it was predicted that in the presence of inhibitors, intermediate accumulation could still be observed after 6 h incubation. The experimental results were in agreement with this prediction. (Steroids 60:475--483, 1995)
Palonosetron Exhibits Unique Molecular Interactions with the 5HT3 Receptor
Anesthesia and Analgesia, 2008
Palonosetron is a 5-HT(3)-receptor antagonist (5-HT(3)-RA) that has been shown to be superior to ... more Palonosetron is a 5-HT(3)-receptor antagonist (5-HT(3)-RA) that has been shown to be superior to other 5-HT(3)-RAs in phase III clinical trials for the prevention of acute, delayed, and overall chemotherapy-induced nausea and vomiting. The improved clinical efficacy of palonosetron may be due, in part, to its more potent binding and longer half-life. However, these attributes alone are not sufficient to explain the results with palonosetron. We sought to elucidate additional differences among 5-HT(3)-RAs that could help explain the observations in the clinic. Receptor site saturation binding experiments were performed with [3H] palonosetron, [3H] granisetron, and [3H] ondansetron to obtain the corresponding Scatchard analyses and Hill coefficients. Diagnostic equilibrium binding experiments and kinetic dissociation experiments were conducted to examine competitive versus potential allosteric interactions between ondansetron, granisetron and palonosetron and the 5-HT(3) receptor. Finally, the long-term effect of the three antagonists on receptor function as measured by Ca2+ influx in HEK 293 cells expressing the 5-HT(3)-receptor was compared. Analyses of binding isotherms using both Scatchard and Hill plots suggested positive cooperativity for palonosetron and simple bimolecular binding for both granisetron and ondansetron. Equilibrium diagnostic tests discriminated differential effects of palonosetron on [3H] ligand binding indicating that palonosetron was an allosteric antagonist whereas granisetron and ondansetron were competitive antagonists. Using dissociation rate strategies, palonosetron was shown to be an allosteric modifier that accelerated the rate of dissociation from the receptor of both granisetron and ondansetron. Differences in the binding mode of palonosetron to the 5-HT(3) receptor were shown to have an impact on receptor function. In these experiments, cells were incubated with each antagonist, followed by infinite dilutions and dissociation for 2.5 h; cells previously incubated with either granisetron or ondansetron showed calcium-ion influx similar to control cells that had not been exposed to a 5-HT(3) receptor antagonist. In contrast, substantial inhibition of calcium-ion influx was observed in cells that had been incubated with palonosetron. Palonosetron exhibited allosteric binding and positive cooperativity when binding to the 5-HT(3) receptor. Palonosetron also triggered functional effects that persisted beyond its binding to the 5-HT(3) receptor at the cell surface. Differences in binding and effects on receptor function may be relevant to the unique beneficial actions of palonosetron. To our knowledge, this is the first report showing palonosetron&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s interaction with the 5-HT(3) receptor at the molecular level, clearly differentiating it from other 5-HT(3)-RAs.
European Journal of Pharmacology, 2001
. Ž . 2-Phosphonomethyl pentanedioic acid 2-PMPA is a potent and selective inhibitor of glutamate... more . Ž . 2-Phosphonomethyl pentanedioic acid 2-PMPA is a potent and selective inhibitor of glutamate carboxypeptidase II NAALADase , and has shown robust neuroprotective activity in both in vitro and in vivo models of ischemia. In the brain, glutamate carboxypeptidase II Ž .
Azetidine-Based Inhibitors of Dipeptidyl Peptidase IV (DPP IV
Current Topics in Medicinal Chemistry, 2007
The structure-activity relationships of azetidine-based DPP IV inhibitors will be discussed in de... more The structure-activity relationships of azetidine-based DPP IV inhibitors will be discussed in detail in the following review. The azetidine-based DPP IV inhibitors can be divided into three main subtypes, the 2-cyanoazetidines, 3-fluoroazetidines and 2-ketoazetidines. These subtypes have been explored and structure-activity relationships have been established by several groups. Several compounds within each of these subtypes display sub micromolar potency against DPP IV. The most potent cyanoazetidines and ketoazetidines have large, hydrophobic amino acid groups bound to the azetidine nitrogen and display activities below 100 nM. DPP IV inhibition is not sensitive to stereochemistry at the 2-position as both 2-(R)- and 2-(S)-cyano and -keto azetidines display similar inhibitory potencies. While these &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;warhead&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;-based cyano- and ketoazetidines have the potential for covalent, bond-forming inhibition, they can also react to internally cyclize into inactive ketopiperazines and dihydroketopyrazine. Thus, chemical instability was also explored for compounds in these two subtypes and certain members of the cyanoazetidine series display aqueous stability comparable to the closely related cyanopyrrolidines. Select 3-fluoroazetidines also display inhibitory potencies below 1 microM without the propensity for cyclization and chemical instability associated with the other subseries.
Cheminform, 2003
A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptid... more A series of hydroxamic acids has been prepared as potential inhibitors of glutamate carboxypeptidase II (GCP II). Compounds based on a P1 0 residue (primed-side inhibitors) were more potent than those based on a P1 group (unprimed-side inhibitors). Inhibitory potency of the primed-side GCP II inhibitors was found to be dependent on the number of methylene units between the hydroxamate group and pentanedioic acid. Succinyl hydroxamic acid derivative, 2-(hydroxycarbamoylmethyl)pentanedioic acid, is the most potent GCP II inhibitor with an IC 50 value of 220 nM. The comparison of the results to those of other classes of GCP II inhibitors as well as hydroxamate-based MMP inhibitors provides further insight into the structure-activity relationships of GCP II inhibition. #
Determination of catecholamine permeability coefficients for passive diffusion across phospholipid vesicle membranes
Journal of Membrane Biology, 1981
A convenient catecholamine transport assay has been developed which permits continuous, instantan... more A convenient catecholamine transport assay has been developed which permits continuous, instantaneous monitoring of transmembrane flux. Epinephrine transport has been examined by spectrophotometrically monitoring adrenochrome formation resulting from the passive diffusion of catecholamine into unilamellar phospholipid vesicles containing entrapped potassium ferricyanide. Ferricyanide oxidation of epinephrine under the conditions employed is fast compared to membrane transport, which obviates the need for intravesicular concentration or volume determinations. Epinephrine transport data over a pH 6 to 7 range have been fitted to an integrated rate equation from which a permeability coefficient for neutral epinephrine of 2.7±1.5×10−6 cm/sec has been obtained.
Journal of Medicinal Chemistry, 2008
D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including D-serine, a full a... more D-Amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including D-serine, a full agonist at the glycine site of the NMDA receptor. A series of benzo[d]isoxazol-3-ol derivatives were synthesized and evaluated as DAAO inhibitors. Among them, 5-chlorobenzo[d]isoxazol-3-ol (CBIO) potently inhibited DAAO with an IC50 in the submicromolar range. Oral administration of CBIO in conjunction with D-serine enhanced the plasma and brain levels of D-serine in rats compared to the oral administration of D-serine alone.
Analytical Biochemistry, 2002
Glutamate carboxypeptidase II (GCPII or prostate-specific membrane antigen or NAALADase) is an en... more Glutamate carboxypeptidase II (GCPII or prostate-specific membrane antigen or NAALADase) is an enzyme that catalyzes the hydrolysis of the neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and glutamate (G). Inhibitors of GCPII provide neuroprotection in a variety of animal models of central nervous system disorders. Neuroprotection is probably the result of increased NAAG concentrations and decreased levels of excess toxic glutamate. Consequently, GCPII inhibitors could be useful therapeutic agents where increased glutamate levels are the result of increased GCPII activity. Current GCPII in vitro activity assays are cumbersome or have limited sensitivity. In this report we describe a microplate assay to study GCPII inhibition that is most sensitive, efficient, and generates little waste. GCPII turnover number (k cat Þ was 4 s À1 and the binding constant (K m Þ for NAAG and GCPII was 130 nM. The apparent association rate constant for GCPII and NAAG (k cat =K m Þ was 3 Â 10 7 M À1 s À1 . Inhibition studies with the GCPII inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) demonstrated competitive inhibition with a K i ¼ 0:2 nM.
European Journal of Pharmacology, 2010
Palonosetron is a 5-HT 3 receptor antagonist that has demonstrated superiority in preventing both... more Palonosetron is a 5-HT 3 receptor antagonist that has demonstrated superiority in preventing both acute and delayed emesis when compared to older first generation 5-HT 3 receptor antagonists. The objective of this work was to determine if palonosetron exhibits unique molecular interactions with the 5-HT 3 receptor that could provide a scientific rationale for observed clinical efficacy differences. Previously, we showed that palonosetron exhibits allosteric binding and positive cooperativity to the 5-HT 3 receptor in contrast to ondansetron and granisetron which exhibit simple bimolecular binding. The present work shows, through several independent experiments, that palonosetron uniquely triggers 5-HT 3 receptor internalization and induces prolonged inhibition of receptor function. After 24 h incubation followed by dissociation conditions, [ 3 H]palonosetron remained associated with whole cells but not to cell-free membranes (P < 0.001). [ 3 H] Palonosetron's binding to cells was resistant to both protease and acid treatments designed to denature cell surface proteins suggesting that the receptor complex was inside the cells rather than at the surface. Cells pretreated with unlabeled palonosetron subsequently exhibited reduced cell surface 5-HT 3 receptor binding. Palonosetron-triggered receptor internalization was visualized by confocal fluorescence microscopy using cells transfected with 5-HT 3 receptor fused to enhanced cyan fluorescent protein. In contrast, granisetron and ondansetron showed minimal to no effect on receptor internalization or prolonged inhibition of receptor function. These experiments may provide a pharmacological basis for differences noted in published clinical trials comparing palonosetron to other 5-HT 3 receptor antagonists.
New Approaches to Chemotherapy-Induced Nausea and Vomiting: From Neuropharmacology to Clinical Investigations
Cancer Journal, 2006
Nausea and vomiting are considered to be among the most distressing consequences of cytotoxic che... more Nausea and vomiting are considered to be among the most distressing consequences of cytotoxic chemotherapies. Currently, there are several novel 5-HT(3) receptor antagonists for the treatment of chemotherapy-induced nausea and vomiting (CINV), including ondansetron, granisetron, and dolasetron. These agents provide significant improvement in the management of acute emesis but are ineffective at preventing delayed emesis. In 2003, a new 5-HT(3) receptor antagonist, palonosetron HCL (Aloxi), was introduced to the U.S. market. Palonosetron was found to be effective in preventing delayed CINV. Indeed, palonosetron was the first and only 5-HT(3) receptor antagonist approved by the FDA for the prevention of both acute and delayed CINV. More recently, studies on the role of substance P in the emetic process led to the development of aprepitant (Emend) for the prevention of delayed emesis in combination with 5-HT(3) receptor antagonists. Despite these major advances, CINV remains uncontrolled in some patients. Current efforts are focused on treating refractory emesis and include both the clinical evaluation of compounds marketed for other indications and the preclinical evaluation of novel molecules targeting other transmitters in the emetic pathway. Ongoing work in pharmacogenomics has postulated several candidate genes that could be involved in emetic sensitivity and responsiveness to antiemetic therapy. Investigations into the pharmacogenomics of CINV may someday be able to aid in the identification of high risk patients and patients unlikely to respond to conventional therapies.
Pharmacological characterization of AMP 579, a novel adenosine A1/A2 receptor agonist and cardioprotective
Drug Development Research, 1998
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Cardiovascular Drug Reviews, 1996
Key Words: Atherosclerosis-PDGF-receptor tyrosine kinase-Phenolate anion-Phenylaminopyrimidine--Q... more Key Words: Atherosclerosis-PDGF-receptor tyrosine kinase-Phenolate anion-Phenylaminopyrimidine--Quinoline-Quinoxaline-Restenosis-Tyrosine kinase inhibitor.
Brain Research, 2005
Dipeptidyl peptidase IV (DPP IV) is a ubiquitous membrane-bound enzyme that cleaves the two N-ter... more Dipeptidyl peptidase IV (DPP IV) is a ubiquitous membrane-bound enzyme that cleaves the two N-terminal amino acids from peptides with a proline or alanine residue in the second position from the amino end. Potential substrates for DPP IV include several neuropeptides, suggesting a role for DPP IV in neurological processes. We have developed a potent DPP IV inhibitor (IC 50 = 30 nM), 1-(2-Amino-3-methylbutyryl)-azetidine-2-carbonitrile (AMAC), which has shown efficacy in two established models of psychosis: mescaline-induced scratching and amphetamine-induced hyperactivity. In the mescaline-induced scratching model, AMAC treatment before mescaline administration reduced the number of scratching paroxysms by 68% (P < 0.01). The compound showed a dose-dependent effect, inhibiting significantly at 6, 20 and 60 mg/kg (37%, 39% and 68%, respectively). In the amphetamine-induced hyperactivity model, 50 and 60 mg/kg AMAC, given before injection of amphetamine, significantly reduced hyper-locomotion by 65% and 76%, respectively. Additionally, AMAC showed no significant activity in binding assays for 20 receptors thought to be involved in the pathology of schizophrenia, including dopamine, serotonin and glutamate. A structurally similar analog, 1-(2-dimethylamino-3-methyl-butyryl)-azetidine-2-carbonitrile (DAMAC), that does not inhibit DPP IV, was inactive in both models. Taken together, these data suggest that the antipsychotic effects of AMAC are the result of DPP IV inhibition. D
Journal of Neurochemistry, 2002
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number... more Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. In this paper we report expression of an extracellular portion of human glutamate carboxypeptidase II (amino acids 44–750) in Drosophila Schneider's cells and its purification to homogeneity. A novel assay for hydrolytic activity of recombinant human GCPII (rhGCPII), based on fluorimetric detection of released alpha-amino groups was established, and used for its enzymological characterization. rhGCPII does not show dipeptidylpeptidase IV-like activity assigned to the native form of the enzyme previously. Using a complete set of protected dipeptides, substrate specificity of rhGCPII was elucidated. In addition to the previously described substrates, four novel compounds, Ac-Glu-Met, Ac-Asp-Met and, surprisingly, Ac-Ala-Glu and Ac-Ala-Met were identified as substrates for GCPII, and their respective kinetic constants determined. The glycosylation of rhGCPII was found indispensable for the enzymatic activity.
Febs Letters, 2003
D-Serine was previously identi¢ed in mammalian brain and was shown to be a co-agonist at the 'gly... more D-Serine was previously identi¢ed in mammalian brain and was shown to be a co-agonist at the 'glycine' site of the N-methyl-D-aspartate (NMDA)-type receptors. Racemization of serine is catalyzed by serine racemase, a pyridoxal 5P Pphosphate-dependent enzyme expressed mainly in brain and liver. NMDA receptor overactivation has been implicated in a number of pathological conditions and inhibitors of serine racemase are thus potentially interesting targets for therapy. We expressed recombinant mouse serine racemase in insect cells and puri¢ed it to near homogeneity. The enzyme is a non-covalent homodimer in solution and requires divalent cations Mg 2+ , Ca 2+ or Mn 2+ for activity but not for dimerization. In addition to the racemization it also catalyzes speci¢c elimination of L-Ser to pyruvate. D-Serine is eliminated much less e⁄ciently. Both L-serine racemization and elimination activities of serine racemase are of comparable magnitude, display alkaline pH optimum and are negligible below pH 6.5. adenine dinucleotide reduced form; HEPES, 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid; EDTA, ethylenediamine tetraacetic acid FEBS 26899 FEBS Letters 535
Analytical Biochemistry, 2011
D-serine administration has been shown to be effective for the treatment of schizophrenia symptom... more D-serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, D-serine has to be administered at high doses in order to observe clinical effects. This is in large part due to D-serine undergoing oxidation by D-amino acid oxidase (DAAO) before it reaches the brain. Consequently, co-administration of D-serine with a DAAO inhibitor has been suggested as a way to lower the dose of D-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma D-serine levels after oral coadministration. Current HPLC-based methodologies to measure D-serine in plasma are time consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well-format assay to monitor D-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate D-serine from plasma followed by quantitation of D-serine using the DAAO catalyzed reaction. Plasma D-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.