John A Krawiec

GlaxoSmithKline plc., Londinium, England, Belgium

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Publications (12)75.8 Total impact

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    ABSTRACT: Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.
    Science translational medicine 11/2012; 4(159):159ra148. · 10.76 Impact Factor
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    ABSTRACT: The liver X receptors (LXR) play a key role in cholesterol homeostasis and lipid metabolism. SAR studies around tertiary-amine lead molecule 2, an LXR full agonist, revealed that steric and conformational changes to the acetic acid and propanolamine groups produce dramatic effects on agonist efficacy and potency. The new analogs possess good functional activity, demonstrating the ability to upregulate LXR target genes, as well as promote cholesterol efflux in macrophages.
    Bioorganic & medicinal chemistry letters 09/2009; 19(19):5617-21. · 2.65 Impact Factor
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    ABSTRACT: Endothelial lipase (EL) activity has been implicated in HDL catabolism, vascular inflammation, and atherogenesis, and inhibitors are therefore expected to be useful for the treatment of cardiovascular disease. Sulfonylfuran urea 1 was identified in a high-throughput screening campaign as a potent and non-selective EL inhibitor. A lead optimization effort was undertaken to improve potency and selectivity, and modifications leading to improved LPL selectivity were identified. Radiolabeling studies were undertaken to establish the mechanism of action for these inhibitors, which were ultimately demonstrated to be irreversible inhibitors.
    Bioorganic & medicinal chemistry letters 12/2008; 19(1):27-30. · 2.65 Impact Factor
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    ABSTRACT: Endothelial lipase (EL) is a 482-amino-acid protein from the triglyceride lipase gene family that uses a Ser-His-Asp triad for catalysis. Its expression in endothelial cells and preference for phospholipids rather than triglycerides are unique. Animal models in which it is overexpressed or knocked out indicate EL levels are inversely correlated with high-density lipoprotein cholesterol (HDL-C). HDL-C is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, in which excess cholesterol is effluxed from peripheral tissues for excretion or reabsorption. Thus, EL inhibition in humans is expected to lead to increases in HDL levels and possibly a decrease in cardiovascular disease. To discover inhibitors of EL, a coupled assay for EL has been developed, using its native substrate, HDL. Hydrolysis of HDL by EL yields free fatty acids, which are coupled through acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase to produce the fluorescent species resorufin. This assay was developed into a 5-microL, 1536-well assay format, and a high-throughput screen was executed against the GSK collection. In addition to describing the screening results, novel post-HTS mechanism-of-action studies were developed for EL and applied to 1 of the screening hits as an example.
    Journal of Biomolecular Screening 07/2008; 13(6):468-75. · 2.21 Impact Factor
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    ABSTRACT: The purpose of this study was to determine whether poloxamer 407, a chemical known to increase plasma lipid levels in rodents following parenteral administration, decreased the gene expression of ATP-binding-cassette transporter A1. Using human macrophages cultured with poloxamer 407, there was a significant reduction in the gene expression of ATP-binding-cassette transporter A1; however, there was no effect on the gene expression of either fatty acid synthase or sterol regulatory element binding protein-1. Reduction of ATP-binding-cassette transporter A1 mRNA levels was also observed in both liver and intestine of poloxamer 407-treated rats. When macrophages were cultured with poloxamer 407, the percent of cholesterol effluxed decreased in a concentration-dependent fashion, both in the absence and presence of a synthetic liver X receptor agonist. Lastly, total and unesterified (free) cholesterol concentrations were determined in the liver and 9 peripheral tissues of poloxamer 407- and saline-injected (control) rats. In every tissue, the concentration of total cholesterol for poloxamer 407-treated rats was significantly greater than the corresponding value for controls. Our findings would seem to suggest that the poloxamer 407-mediated reduction in both ATP-binding-cassette transporter A1 gene expression and cellular cholesterol efflux may potentially be one factor that contributes to the accumulation of cholesterol and cholesteryl esters in the liver and 9 peripheral tissues of poloxamer 407-treated rats. Furthermore, the surprising specificity by poloxamer 407 for inhibition of ATP-binding-cassette transporter A1 gene expression over fatty acid synthase and sterol regulatory element binding protein-1 may potentially be due to either disruption of a transcriptional cofactor required for ATP-binding-cassette transporter A1 gene expression, or enhanced turnover of ATP-binding-cassette transporter A1 mRNA.
    European Journal of Pharmacology 06/2006; 536(3):232-40. · 2.59 Impact Factor
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    ABSTRACT: Liver X receptor (LXR) nuclear receptors regulate the expression of genes involved in whole body cholesterol trafficking, including absorption, excretion, catabolism, and cellular efflux, and possess both anti-inflammatory and antidiabetic actions. Accordingly, LXR is considered an appealing drug target for multiple indications. Synthetic LXR agonists demonstrated inhibition of atherosclerosis progression in murine genetic models; however, these and other studies indicated that their major undesired side effect is an increase of plasma and hepatic triglycerides. A significant impediment to extrapolating results with LXR agonists from mouse to humans is the absence in mice of cholesteryl ester transfer protein, a known LXR target gene, and the upregulation in mice but not humans of cholesterol 7alpha-hydroxylase. To better predict the human response to LXR agonism, two synthetic LXR agonists were examined in hamsters and cynomolgus monkeys. In contrast to previously published results in mice, neither LXR agonist increased HDL-cholesterol in hamsters, and similar results were obtained in cynomolgus monkeys. Importantly, in both species, LXR agonists increased LDL-cholesterol, an unfavorable effect not apparent from earlier murine studies. These results reveal additional problems associated with current synthetic LXR agonists and emphasize the importance of profiling compounds in preclinical species with a more human-like LXR response and lipoprotein metabolism.
    The Journal of Lipid Research 11/2005; 46(10):2182-91. · 4.39 Impact Factor
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    ABSTRACT: Substituted 3-(phenylamino)-1H-pyrrole-2,5-diones were identified from a high throughput screen as inducers of human ATP binding cassette transporter A1 expression. Mechanism of action studies led to the identification of GSK3987 as an LXR ligand. GSK3987 recruits the steroid receptor coactivator-1 to human LXRalpha and LXRbeta with EC(50)s of 40 nM, profiles as an LXR agonist in functional assays, and activates LXR though a mechanism that is similar to first generation LXR agonists.
    Journal of Medicinal Chemistry 09/2005; 48(17):5419-22. · 5.61 Impact Factor
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    01/2005;
  • Michael Jaye, John Krawiec
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    ABSTRACT: In the past year, several laboratories taking independent approaches have provided compelling evidence that endothelial lipase, a relatively recent addition to the triglyceride lipase gene family, is a major determinant of HDL metabolism. This review summarizes recent findings from experiments in mice with altered levels of endothelial lipase, from an examination of endothelial lipase catalytic and non-catalytic functions in vitro, and from human genetic studies. An analysis of lipids and lipoproteins in endothelial lipase knockout and transgenic mice and in mice with adenovirus-driven hepatic overexpression of endothelial lipase shows, without exception, that total cholesterol, phospholipid and HDL-cholesterol all vary inversely with the endothelial lipase gene dosage, and primarily depend on endothelial lipase catalytic activity. Endothelial lipase participates in HDL metabolism by promoting the turnover of HDL components and increasing the catabolism of apolipoprotein A-I. The measurement of lipase activity on lipoprotein substrates in vitro demonstrates that endothelial lipase is distinct from other triglyceride lipases in showing the highest activity on HDL. Endothelial lipase gene polymorphisms in humans appear to be associated with HDL-cholesterol or HDL3-cholesterol concentrations. A low HDL-cholesterol level in humans is a risk factor for coronary heart disease. Although not yet demonstrated, it is possible that the action of endothelial lipase on HDL may promote atherogenesis, in which case endothelial lipase may represent an attractive target for pharmaceutical intervention.
    Current Opinion in Lipidology 05/2004; 15(2):183-9. · 5.84 Impact Factor
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    ABSTRACT: Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC50 value of approximately 0.1-0.3 microM), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC50 value of approximately 0.3 microM) versus HDAC3 (IC50 value of approximately 8 microM) and had no inhibitory activity toward HDAC8 (IC50 value >100 microM). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.
    Journal of Pharmacology and Experimental Therapeutics 11/2003; 307(2):720-8. · 3.89 Impact Factor
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    ABSTRACT: High-density lipoprotein (HDL) cholesterol levels are inversely associated with risk of atherosclerotic cardiovascular disease. At least 50% of the variation in HDL cholesterol levels is genetically determined, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism and the HL (LIPC) locus has been associated with variation in HDL cholesterol levels in humans. We describe here the cloning and in vivo functional analysis of a new member of the TG lipase family. In contrast to other family members, this new lipase is synthesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzymatic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology.
    Nature Genetics 05/1999; 21(4):424-8. · 35.21 Impact Factor
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    ABSTRACT: associated with risk of atherosclerotic cardiovascular disease1. At least 50% of the variation in HDL cholesterol levels is geneti- cally determined2,3, but the genes responsible for variation in HDL levels have not been fully elucidated. Lipoprotein lipase (LPL) and hepatic lipase (HL), two members of the triacylglyerol (TG) lipase family, both influence HDL metabolism 2,4-6 and the HL (LIPC) locus has been associated with variation in HDL cho- lesterol levels in humans 7,8 . We describe here the cloning and in vivo functional analysis of a new member of the TG lipase fam- ily. In contrast to other family members, this new lipase is syn- thesized by endothelial cells in vitro and thus has been termed endothelial lipase (encoded by the LIPG gene). EL is expressed in vivo in organs including liver, lung, kidney and placenta, but not in skeletal muscle. In contrast to LPL and HL, EL has a lid of only 19 residues. EL has substantial phospholipase activity, but less triglyceride lipase activity. Overexpression of EL in mice reduced plasma concentrations of HDL cholesterol and its major protein apolipoprotein A-I. The endothelial expression, enzy- matic profile and in vivo effects of EL suggest that it may have a role in lipoprotein metabolism and vascular biology. We isolated a clone containing a unique ORF encoding 500 amino acids from a human placental cDNA library. The primary amino acid sequence was found to be 45% homologous with LPL, 40% with HL and 27% with pancreatic lipase (PL), identify- ing it as a member of the TG lipase family (Fig. 1). It commences with an 18-residue hydrophobic sequence characteristic of a secretory signal peptide, similar to other TG lipases. Cysteine