Publications by authors named "Kay Ahn"

24 Publications

  • Page 1 of 1

Discovery of PF-06835919: A Potent Inhibitor of Ketohexokinase (KHK) for the Treatment of Metabolic Disorders Driven by the Overconsumption of Fructose.

J Med Chem 2020 11 27;63(22):13546-13560. Epub 2020 Sep 27.

Pfizer Inc. Medicine Design, 1 Portland Street, Cambridge, Massachusetts 02139, United States.

Increased fructose consumption and its subsequent metabolism have been implicated in metabolic disorders such as nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH) and insulin resistance. Ketohexokinase (KHK) converts fructose to fructose-1-phosphate (F1P) in the first step of the metabolic cascade. Herein we report the discovery of a first-in-class KHK inhibitor, PF-06835919 (), currently in phase 2 clinical trials. The discovery of was built upon our originally reported, fragment-derived lead and the recognition of an alternative, rotated binding mode upon changing the ribose-pocket binding moiety from a pyrrolidinyl to an azetidinyl ring system. This new binding mode enabled efficient exploration of the vector directed at the Arg-108 residue, leading to the identification of highly potent 3-azabicyclo[3.1.0]hexane acetic acid-based KHK inhibitors by combined use of parallel medicinal chemistry and structure-based drug design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c00944DOI Listing
November 2020

Assessing IRAK4 Functions in ABC DLBCL by IRAK4 Kinase Inhibition and Protein Degradation.

Cell Chem Biol 2020 12 3;27(12):1500-1509.e13. Epub 2020 Sep 3.

Medicinal Chemistry, Janssen (China) Research & Development Center, Shanghai 201210, China. Electronic address:

The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2020.08.010DOI Listing
December 2020

Pharmacologic Characterization of JNJ-42226314, [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone, a Reversible, Selective, and Potent Monoacylglycerol Lipase Inhibitor.

J Pharmacol Exp Ther 2020 03 9;372(3):339-353. Epub 2019 Dec 9.

Janssen Research & Development, LLC, San Diego, California.

The serine hydrolase monoacylglycerol lipase (MAGL) is the rate-limiting enzyme responsible for the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. Inhibition of 2-AG degradation leads to elevation of 2-AG, the most abundant endogenous agonist of the cannabinoid receptors (CBs) CB1 and CB2. Activation of these receptors has demonstrated beneficial effects on mood, appetite, pain, and inflammation. Therefore, MAGL inhibitors have the potential to produce therapeutic effects in a vast array of complex human diseases. The present report describes the pharmacologic characterization of [1-(4-fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone (JNJ-42226314), a reversible and highly selective MAGL inhibitor. JNJ-42226314 inhibits MAGL in a competitive mode with respect to the 2-AG substrate. In rodent brain, the compound time- and dose-dependently bound to MAGL, indirectly led to CB1 occupancy by raising 2-AG levels, and raised norepinephrine levels in cortex. In vivo, the compound exhibited antinociceptive efficacy in both the rat complete Freund's adjuvant-induced radiant heat hypersensitivity and chronic constriction injury-induced cold hypersensitivity models of inflammatory and neuropathic pain, respectively. Though 30 mg/kg induced hippocampal synaptic depression, altered sleep onset, and decreased electroencephalogram gamma power, 3 mg/kg still provided approximately 80% enzyme occupancy, significantly increased 2-AG and norepinephrine levels, and produced neuropathic antinociception without synaptic depression or decreased gamma power. Thus, it is anticipated that the profile exhibited by this compound will allow for precise modulation of 2-AG levels in vivo, supporting potential therapeutic application in several central nervous system disorders. SIGNIFICANCE STATEMENT: Potentiation of endocannabinoid signaling activity via inhibition of the serine hydrolase monoacylglycerol lipase (MAGL) is an appealing strategy in the development of treatments for several disorders, including ones related to mood, pain, and inflammation. [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone is presented in this report to be a novel, potent, selective, and reversible noncovalent MAGL inhibitor that demonstrates dose-dependent enhancement of the major endocannabinoid 2-arachidonoylglycerol as well as efficacy in models of neuropathic and inflammatory pain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/jpet.119.262139DOI Listing
March 2020

Mechanistic Characterization of Long Residence Time Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2).

Biochemistry 2018 12 7;57(51):6997-7010. Epub 2018 Dec 7.

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triacylglycerol (TAG) synthesis. Genetic knockdown or pharmacological inhibition of DGAT2 leads to a decrease in very-low-density lipoprotein TAG secretion and hepatic lipid levels in rodents, indicating DGAT2 may represent an attractive therapeutic target for treatment of hyperlipidemia and hepatic steatosis. We have previously described potent and selective imidazopyridine DGAT2 inhibitors with high oral bioavailability. However, the detailed mechanism of DGAT2 inhibition has not been reported. Herein, we describe imidazopyridines represented by PF-06424439 (1) and 2 as long residence time inhibitors of DGAT2. We demonstrate that 1 and 2 are slowly reversible, time-dependent inhibitors, which inhibit DGAT2 in a noncompetitive mode with respect to the acyl-CoA substrate. Detailed kinetic analysis demonstrated that 1 and 2 inhibit DGAT2 in a two-step binding mechanism, in which the initial enzyme-inhibitor complex (EI) undergoes an isomerization step resulting in a much higher affinity complex (EI*) with overall apparent inhibition constants ( K* values) of 16.7 and 16.0 nM for 1 and 2, respectively. The EI* complex dissociates with dissociation half-lives of 1.2 and 1.0 h for 1 and 2, respectively. A binding assay utilizing I-labeled imidazopyridine demonstrated that the level of imidazopyridine binding to DGAT2 mutant enzymes, H161A and H163A, dramatically decreased to 11-17% of that of the wild-type enzyme, indicating that these residues are critical for imidazopyridines to bind to DGAT2. Taken together, imidazopyridines may thus represent a promising lead series for the development of DGAT2 inhibitors that display an unprecedented combination of potency, selectivity, and in vivo efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.8b01096DOI Listing
December 2018

Discovery of Fragment-Derived Small Molecules for in Vivo Inhibition of Ketohexokinase (KHK).

J Med Chem 2017 09 11;60(18):7835-7849. Epub 2017 Sep 11.

Medicine Design, Pfizer Inc. , 1 Portland Street, Cambridge, Massachusetts 02139, United States.

Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.7b00947DOI Listing
September 2017

Discovery of Inactive Conformation-Selective Kinase Inhibitors by Utilizing Cascade Assays.

Biochemistry 2017 08 14;56(34):4449-4456. Epub 2017 Jun 14.

Molecular & Cellular Pharmacology, Lead Discovery, Janssen Research and Development, LLC , Spring House, Pennsylvania 19477, United States.

Achieving selectivity across the human kinome is a major hurdle in kinase inhibitor drug discovery. Targeting inactive (vs active) kinase conformations offers advantages in achieving selectivity because of their more diversified structures. Discovery of inactive conformation-selective inhibitors, however, has been hampered partly by the lack of general assay methods. Herein, we show that such inhibitors can be discovered by utilizing kinase cascade assays. This type of assay is initiated with the target kinase in its unphosphorylated, inactive conformation, which is activated during the assay. Inactive conformation-selective inhibitors stabilize the inactive kinase, block activation, and yield reduced kinase activity. We investigate the properties of the assay by mathematical modeling, as well as by proof-of-concept experiments using the BRAF-MEK1 cascade. This study demonstrates effective identification of inactive conformation-selective inhibitors by cascade assays, reveals key factors that impact results, and provides guidelines for successful cascade assay development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.7b00521DOI Listing
August 2017

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism.

ACS Chem Biol 2016 09 19;11(9):2529-40. Epub 2016 Jul 19.

Cardiovascular, Metabolic, and Endocrine Diseases (CVMED) Research Unit, Pfizer Inc. , 610 Main Street, Cambridge, Massachusetts 02139, United States.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acschembio.6b00266DOI Listing
September 2016

Discovery of 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999): A Highly Selective Mechanism-Based Myeloperoxidase Inhibitor for the Treatment of Cardiovascular Diseases.

J Med Chem 2015 Nov 28;58(21):8513-28. Epub 2015 Oct 28.

Worldwide Research and Development, Pfizer, Inc., Groton, Connecticut 06340, United States.

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Clinical evidence suggests a causal role for MPO in various autoimmune and inflammatory disorders including vasculitis and cardiovascular and Parkinson's diseases, implying that MPO inhibitors may represent a therapeutic treatment option. Herein, we present the design, synthesis, and preclinical evaluation of N1-substituted-6-arylthiouracils as potent and selective inhibitors of MPO. Inhibition proceeded in a time-dependent manner by a covalent, irreversible mechanism, which was dependent upon MPO catalysis, consistent with mechanism-based inactivation. N1-Substituted-6-arylthiouracils exhibited low partition ratios and high selectivity for MPO over thyroid peroxidase and cytochrome P450 isoforms. N1-Substituted-6-arylthiouracils also demonstrated inhibition of MPO activity in lipopolysaccharide-stimulated human whole blood. Robust inhibition of plasma MPO activity was demonstrated with the lead compound 2-(6-(5-chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide (PF-06282999, 8) upon oral administration to lipopolysaccharide-treated cynomolgus monkeys. On the basis of its pharmacological and pharmacokinetic profile, PF-06282999 has been advanced to first-in-human pharmacokinetic and safety studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b00963DOI Listing
November 2015

Discovery and Optimization of Imidazopyridine-Based Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2).

J Med Chem 2015 Sep;58(18):7173-85

Worldwide Medicinal Chemistry, ‡Cardiovascular, Metabolic and Endocrine Diseases Research Unit, and §Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research & Development , Cambridge, Massachusetts 02139, United States.

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b01006DOI Listing
September 2015

Discovery of Selective Small Molecule Inhibitors of Monoacylglycerol Acyltransferase 3.

J Med Chem 2015 Sep 8;58(18):7164-72. Epub 2015 Sep 8.

Worldwide Medicinal Chemistry, ‡Cardiovascular, Metabolic and Endocrine Diseases Research Unit, and §Pharmacokinetics, Dynamics and Metabolism, Pfizer Worldwide Research & Development , Cambridge, Massachusetts 02139, United States.

Inhibition of triacylglycerol (TAG) biosynthetic enzymes has been suggested as a promising strategy to treat insulin resistance, diabetes, dyslipidemia, and hepatic steatosis. Monoacylglycerol acyltransferase 3 (MGAT3) is an integral membrane enzyme that catalyzes the acylation of both monoacylglycerol (MAG) and diacylglycerol (DAG) to generate DAG and TAG, respectively. Herein, we report the discovery and characterization of the first selective small molecule inhibitors of MGAT3. Isoindoline-5-sulfonamide (6f, PF-06471553) selectively inhibits MGAT3 with high in vitro potency and cell efficacy. Because the gene encoding MGAT3 (MOGAT3) is found only in higher mammals and humans, but not in rodents, a transgenic mouse model expressing the complete human MOGAT3 was used to characterize the effects of 6f in vivo. In the presence of a combination of diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2) inhibitors, an oral administration of 6f exhibited inhibition of the incorporation of deuterium-labeled glycerol into TAG in this mouse model. The availability of a potent and selective chemical tool and a humanized mouse model described in this report should facilitate further dissection of the physiological function of MGAT3 and its role in lipid homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.5b01008DOI Listing
September 2015

PF-1355, a mechanism-based myeloperoxidase inhibitor, prevents immune complex vasculitis and anti-glomerular basement membrane glomerulonephritis.

J Pharmacol Exp Ther 2015 May 19;353(2):288-98. Epub 2015 Feb 19.

Pfizer World Wide Research and Development, Cambridge, Massachusetts (W.Z., R.R., C.S., C.C., A.S., J.W., K.A., A.K., D.S., T.S.M., Y.Z., B.K., L.B.); Department of Pathology, University of Michigan, Ann Arbor, Michigan (R.W., K.J.); and Pfizer World Wide Research and Development, Groton, Connecticut (P.D.B., C.O., W.B., T.K., T.C., S.B.)

Small vessel vasculitis is a life-threatening condition and patients typically present with renal and pulmonary injury. Disease pathogenesis is associated with neutrophil accumulation, activation, and oxidative damage, the latter being driven in large part by myeloperoxidase (MPO), which generates hypochlorous acid among other oxidants. MPO has been associated with vasculitis, disseminated vascular inflammation typically involving pulmonary and renal microvasculature and often resulting in critical consequences. MPO contributes to vascular injury by 1) catabolizing nitric oxide, impairing vasomotor function; 2) causing oxidative damage to lipoproteins and endothelial cells, leading to atherosclerosis; and 3) stimulating formation of neutrophil extracellular traps, resulting in vessel occlusion and thrombosis. Here we report a selective 2-thiouracil mechanism-based MPO inhibitor (PF-1355 [2-(6-(2,5-dimethoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamide) and demonstrate that MPO is a critical mediator of vasculitis in mouse disease models. A pharmacokinetic/pharmacodynamic response model of PF-1355 exposure in relation with MPO activity was derived from mouse peritonitis. The contribution of MPO activity to vasculitis was then examined in an immune complex model of pulmonary disease. Oral administration of PF-1355 reduced plasma MPO activity, vascular edema, neutrophil recruitment, and elevated circulating cytokines. In a model of anti-glomerular basement membrane disease, formerly known as Goodpasture disease, albuminuria and chronic renal dysfunction were completely suppressed by PF-1355 treatment. This study shows that MPO activity is critical in driving immune complex vasculitis and provides confidence in testing the hypothesis that MPO inhibition will provide benefit in treating human vasculitic diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/jpet.114.221788DOI Listing
May 2015

Mechanistic characterization of a 2-thioxanthine myeloperoxidase inhibitor and selectivity assessment utilizing click chemistry--activity-based protein profiling.

Biochemistry 2013 Dec 9;52(51):9187-201. Epub 2013 Dec 9.

Cardiovascular and Metabolic Diseases Research Unit and ‡Medicinal Chemistry, Pfizer Worldwide Research and Development , Cambridge, Massachusetts 02139, United States.

Myeloperoxidase (MPO) is a heme peroxidase that catalyzes the production of hypochlorous acid. Despite a high level of interest in MPO as a therapeutic target, there have been limited reports about MPO inhibitors that are suitable for evaluating MPO in pharmacological studies. 2-Thioxanthine, 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (A), has recently been reported to inhibit MPO by covalently modifying the heme prosthetic group. Here we report a detailed mechanistic characterization demonstrating that A possesses all the distinguishing features of a mechanism-based inactivator. A is a time-dependent MPO inhibitor and displays saturable inactivation kinetics consistent with a two-step mechanism of inactivation and a potency (k(inact)/K(I) ratio) of 8450 ± 780 M⁻¹ s⁻¹. MPO inactivation by A is dependent on MPO catalysis and is protected by substrate. A reduces MPO compound I to compound II with a second-order rate constant of (0.801 ± 0.056) × 10⁶ M⁻¹ s⁻¹, and its irreversible inactivation of MPO occurs prior to release of the activated inhibitory species. Despite its relatively high selectivity against a broad panel of more than 100 individual targets, including enzymes, receptors, transporters, and ion channels, we demonstrate that A labels multiple other protein targets in the presence of MPO. By synthesizing an alkyne analogue of A and utilizing click chemistry-activity-based protein profiling, we present that the MPO-activated inhibitory species can diffuse away to covalently modify other proteins, as reflected by the relatively high partition ratio of A, which we determined to be 15.6. This study highlights critical methods that can guide the discovery and development of next-generation MPO inhibitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi401354dDOI Listing
December 2013

The synthesis and in vivo evaluation of [18F]PF-9811: a novel PET ligand for imaging brain fatty acid amide hydrolase (FAAH).

Nucl Med Biol 2012 Oct 8;39(7):1058-67. Epub 2012 May 8.

Pfizer Worldwide Research and Development, Pfizer Inc., Eastern Point Road, Mail Stop 8274-1342, Groton, CT 06340, USA.

Introduction: Fatty acid amide hydrolase (FAAH) is responsible for the enzymatic degradation of the fatty acid amide family of signaling lipids, including the endogenous cannabinoid (endocannabinoid) anandamide. The involvement of the endocannabinoid system in pain and other nervous system disorders has made FAAH an attractive target for drug development. Companion molecular imaging probes are needed, however, to assess FAAH inhibition in the nervous system in vivo. We report here the synthesis and in vivo evaluation of [(18)F]PF-9811, a novel PET ligand for non-invasive imaging of FAAH in the brain.

Methods: The potency and selectivity of unlabeled PF-9811 were determined by activity-based protein profiling (ABPP) both in vitro and in vivo. [(18)F]PF-9811 was synthesized in a 3-step, one-pot reaction sequence, followed by HPLC purification. Biological evaluation was performed by biodistribution and dynamic PET imaging studies in male rats. The specificity of [(18)F]PF-9811 uptake was evaluated by pre-administration of PF-04457845, a potent and selective FAAH inhibitor, 1h prior to radiotracer injection.

Results: Biodistribution studies show good uptake (SUV~0.8 at 90 min) of [(18)F]PF-9811 in rat brain, with significant reduction of the radiotracer in all brain regions (37%-73% at 90 min) in blocking experiments. Dynamic PET imaging experiments in rat confirmed the heterogeneous uptake of [(18)F]PF-9811 in brain regions with high FAAH enzymatic activity, as well as statistically significant reductions in signal following pre-administration of the blocking compound PF-04457845.

Conclusions: [(18)F]PF-9811 is a promising PET imaging agent for FAAH. Biodistribution and PET imaging experiments show that the tracer has good uptake in brain, regional heterogeneity, and specific binding as determined by blocking experiments with the highly potent and selective FAAH inhibitor, PF-04457845.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.nucmedbio.2012.03.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3611965PMC
October 2012

Evidence for regulated monoacylglycerol acyltransferase expression and activity in human liver.

J Lipid Res 2012 May 6;53(5):990-999. Epub 2012 Mar 6.

Center for Human Nutrition, Washington University School of Medicine, St. Louis, MO;. Electronic address:

Intrahepatic lipid accumulation is extremely common in obese subjects and is associated with the development of insulin resistance and diabetes. Hepatic diacylglycerol and triacylglycerol synthesis predominantly occurs through acylation of glycerol-3-phosphate. However, an alternative pathway for synthesizing diacylglycerol from monoacylglycerol acyltransferases (MGAT) could also contribute to hepatic glyceride pools. MGAT activity and the expression of the three genes encoding MGAT enzymes (MOGAT1, MOGAT2, and MOGAT3) were determined in liver biopsies from obese human subjects before and after gastric bypass surgery. MOGAT expression was also assessed in liver of subjects with nonalcoholic fatty liver disease (NAFLD) or control livers. All MOGAT genes were expressed in liver, and hepatic MGAT activity was readily detectable in liver lysates. The hepatic expression of MOGAT3 was highly correlated with MGAT activity, whereas MOGAT1 and MOGAT2 expression was not, and knockdown of MOGAT3 expression attenuated MGAT activity in a liver-derived cell line. Marked weight loss following gastric bypass surgery was associated with a significant reduction in MOGAT2 and MOGAT3 expression, which were also overexpressed in NAFLD subjects. These data suggest that the MGAT pathway is active and dynamically regulated in human liver and could be an important target for pharmacologic intervention for the treatment of obesity-related insulin resistance and NAFLD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1194/jlr.P025536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329399PMC
May 2012

Deconstruction of activity-dependent covalent modification of heme in human neutrophil myeloperoxidase by multistage mass spectrometry (MS(4)).

Biochemistry 2012 Mar 1;51(10):2065-77. Epub 2012 Mar 1.

Pfizer Worldwide Research, Groton, Connecticut 06340, United States.

Myeloperoxidase (MPO) is known to be inactivated and covalently modified by treatment with hydrogen peroxide and agents similar to 3-(2-ethoxypropyl)-2-thioxo-2,3-dihydro-1H-purin-6(9H)-one (1), a 254.08 Da derivative of 2-thioxanthine. Peptide mapping by liquid chromatography and mass spectrometry detected modification by 1 in a labile peptide-heme-peptide fragment of the enzyme, accompanied by a mass increase of 252.08 Da. The loss of two hydrogen atoms was consistent with mechanism-based oxidative coupling. Multistage mass spectrometry (MS(4)) of the modified fragment in an ion trap/Orbitrap spectrometer demonstrated that 1 was coupled directly to heme. Use of a 10 amu window delivered the full isotopic envelope of each precursor ion to collision-induced dissociation, preserving definitive isotopic profiles for iron-containing fragments through successive steps of multistage mass spectrometry. Iron isotope signatures and accurate mass measurements supported the structural assignments. Crystallographic analysis confirmed linkage between the methyl substituent of the heme pyrrole D ring and the sulfur atom of 1. The final orientation of 1 perpendicular to the plane of the heme ring suggested a mechanism consisting of two consecutive one-electron oxidations of 1 by MPO. Multistage mass spectrometry using stage-specific collision energies permits stepwise deconstruction of modifications of heme enzymes containing covalent links between the heme group and the polypeptide chain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi201872jDOI Listing
March 2012

Discovery of PF-04457845: A Highly Potent, Orally Bioavailable, and Selective Urea FAAH Inhibitor.

ACS Med Chem Lett 2011 Feb;2(2):91-96

Pfizer Worldwide Research and Development, Groton, Connecticut 06340, United States.

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic and anti-inflammatory phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for the treatment of inflammatory pain and other nervous system disorders. Herein, we report the discovery and characterization of a highly efficacious and selective FAAH inhibitor PF-04457845 (23). Compound 23 inhibits FAAH by a covalent, irreversible mechanism involving carbamylation of the active-site serine nucleophile of FAAH with high in vitro potency (k(inact)/K(i) and IC(50) values of 40300 M(-1) s(-1) and 7.2 nM, respectively, for human FAAH). Compound 23 has exquisite selectivity for FAAH relative to other members of the serine hydrolase superfamily as demonstrated by competitive activity-based protein profiling. Oral administration of 23 at 0.1 mg/kg results in efficacy comparable to that of naproxen at 10 mg/kg in a rat model of inflammatory pain. Compound 23 is being evaluated in human clinical trials.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ml100190tDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109749PMC
February 2011

Mechanistic and pharmacological characterization of PF-04457845: a highly potent and selective fatty acid amide hydrolase inhibitor that reduces inflammatory and noninflammatory pain.

J Pharmacol Exp Ther 2011 Jul 19;338(1):114-24. Epub 2011 Apr 19.

Pfizer Worldwide Research and Development, Groton, Connecticut, USA.

The endogenous cannabinoid (endocannabinoid) anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH). Pharmacological blockade of FAAH has emerged as a potentially attractive strategy for augmenting endocannabinoid signaling and retaining the beneficial effects of cannabinoid receptor activation, while avoiding the undesirable side effects, such as weight gain and impairments in cognition and motor control, observed with direct cannabinoid receptor 1 agonists. Here, we report the detailed mechanistic and pharmacological characterization of N-pyridazin-3-yl-4-(3-{[5-(trifluoromethyl)pyridin-2-yl]oxy}benzylidene)piperidine-1-carboxamide (PF-04457845), a highly efficacious and selective FAAH inhibitor. Mechanistic studies confirm that PF-04457845 is a time-dependent, covalent FAAH inhibitor that carbamylates FAAH's catalytic serine nucleophile. PF-04457845 inhibits human FAAH with high potency (k(inact)/K(i) = 40,300 M(-1)s(-1); IC(50) = 7.2 nM) and is exquisitely selective in vivo as determined by activity-based protein profiling. Oral administration of PF-04457845 produced potent antinociceptive effects in both inflammatory [complete Freund's adjuvant (CFA)] and noninflammatory (monosodium iodoacetate) pain models in rats, with a minimum effective dose of 0.1 mg/kg (CFA model). PF-04457845 displayed a long duration of action as a single oral administration at 1 mg/kg showed in vivo efficacy for 24 h with a concomitant near-complete inhibition of FAAH activity and maximal sustained elevation of anandamide in brain. Significantly, PF-04457845-treated mice at 10 mg/kg elicited no effect in motility, catalepsy, and body temperature. Based on its exceptional selectivity and in vivo efficacy, combined with long duration of action and optimal pharmacokinetic properties, PF-04457845 is a clinical candidate for the treatment of pain and other nervous system disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/jpet.111.180257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3126636PMC
July 2011

Fatty acid amide hydrolase as a potential therapeutic target for the treatment of pain and CNS disorders.

Expert Opin Drug Discov 2009 Jul;4(7):763-784

Pfizer Global Research and Development, Groton/New London Laboratories, Groton, CT 06340.

BACKGROUND: Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes without showing the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. OBJECTIVES: This review highlights advances in the development of FAAH inhibitors of different mechanistic classes and their in vivo efficacy. Also highlighted are advances in technology for the in vitro and in vivo selectivity assessment of FAAH inhibitors employing activity-based protein profiling (ABPP) and click chemistry-ABPP, respectively. Recent reports on structure-based drug design for human FAAH generated by protein engineering using interspecies active site conversion are also discussed. METHODS: The literature searches of Medline and SciFinder databases were used. CONCLUSIONS: There has been tremendous progress in our understanding in FAAH and development of FAAH inhibitors with in vivo efficacy, selectivity, and drug like pharmacokinetic properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1517/17460440903018857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2882713PMC
July 2009

SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

J Biol Chem 2010 Mar 8;285(11):8340-51. Epub 2010 Jan 8.

Department of Cardiovascular, Pfizer Global Research and Development, Groton, Connecticut 06340, USA.

Sirtuins catalyze NAD(+)-dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophore-containing peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M109.088682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2832984PMC
March 2010

Discovery and characterization of a highly selective FAAH inhibitor that reduces inflammatory pain.

Chem Biol 2009 Apr;16(4):411-20

Pfizer Global Research and Development, Groton, CT 06340, USA.

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.chembiol.2009.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2692831PMC
April 2009

Benzothiophene piperazine and piperidine urea inhibitors of fatty acid amide hydrolase (FAAH).

Bioorg Med Chem Lett 2009 May 24;19(10):2865-9. Epub 2009 Mar 24.

Pfizer Global Research and Development, Ann Arbor, MI 48105, USA.

The synthesis and structure-activity relationships (SAR) of a series of benzothiophene piperazine and piperidine urea FAAH inhibitors is described. These compounds inhibit FAAH by covalently modifying the enzyme's active site serine nucleophile. Activity-based protein profiling (ABPP) revealed that these urea inhibitors were completely selective for FAAH relative to other mammalian serine hydrolases. Several compounds showed in vivo activity in a rat complete Freund's adjuvant (CFA) model of inflammatory pain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2009.03.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150822PMC
May 2009

Structure-guided inhibitor design for human FAAH by interspecies active site conversion.

Proc Natl Acad Sci U S A 2008 Sep 27;105(35):12820-4. Epub 2008 Aug 27.

The Skaggs Institute for Chemical Biology, La Jolla, CA 92037, USA.

The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-A crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.0806121105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2529035PMC
September 2008

Enzymatic pathways that regulate endocannabinoid signaling in the nervous system.

Chem Rev 2008 May 23;108(5):1687-707. Epub 2008 Apr 23.

Pfizer Global Research and Development, Groton, Connecticut 06340, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cr0782067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3150828PMC
May 2008

The design and synthesis of human branched-chain amino acid aminotransferase inhibitors for treatment of neurodegenerative diseases.

Bioorg Med Chem Lett 2006 May 6;16(9):2337-40. Epub 2005 Sep 6.

Pfizer Global Research and Development, Ann Arbor, MI, USA.

The inhibition of the cytosolic isoenzyme BCAT that is expressed specifically in neuronal tissue is likely to be useful for the treatment of neurodegenerative and other neurological disorders where glutamatergic mechanisms are implicated. Compound 2 exhibited an IC50 of 0.8 microM in the hBCATc assays; it is an active and selective inhibitor. Inhibitor 2 also blocked calcium influx into neuronal cells following inhibition of glutamate uptake, and demonstrated neuroprotective efficacy in vivo. SAR, pharmacology, and the crystal structure of hBCATc with inhibitor 2 are described.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmcl.2005.07.058DOI Listing
May 2006