Pyrrolopyrazinedione-based inhibitors of human hormone-sensitive lipase

Journal of Medicinal Chemistry

Volumn 46, Issue 7, 2003, Pages 1120-1122

  Slee, Deborah H ^a   Bhat, Abhijit S ^a   Nguyen, Truc N ^a   Kish, Mary ^a   Lundeen, Katy ^a   Newman, Michael J ^a   McConnell, Stephen J ^a  

^a Ontogen Corporation   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHOLESTEROL OLEATE; ENZYME INHIBITOR; GLUCOSE; LIPID; PYRAZINE DERIVATIVE; PYRROLIDINE DERIVATIVE; TRIACYLGLYCEROL LIPASE;

ARTICLE; CARBON NUCLEAR MAGNETIC RESONANCE; CYCLIZATION; DIABETES MELLITUS; ENZYME ACTIVITY; ENZYME ASSAY; ENZYME INHIBITION; HUMAN; LIPID METABOLISM; LIQUID CHROMATOGRAPHY; MASS SPECTROMETRY; PROTON NUCLEAR MAGNETIC RESONANCE;

ANIMALS; CELL LINE; CHOLESTEROL ESTERASE; DATABASES, FACTUAL; DRUG EVALUATION, PRECLINICAL; ENZYME INHIBITORS; HUMANS; INSECTS; PYRAZINES; STRUCTURE-ACTIVITY RELATIONSHIP;

EID: 0037468883     PISSN: 00222623     EISSN: None     Source Type: Journal    
DOI: 10.1021/jm020460y     Document Type: Article

References (14)

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11. Cloning and protein purification: The full-length human HSL gene was cloned by PCR from human adipocyte cDNA (Clontech). A PCR fragment containing the full-length human HSL gene was cut with the restriction enzymes Eco RI and Bgl II and ligated into the plasmid pAcHLT-A (PharMingen), a vector suitable for His6-tagged expression in the baculovirus/insect cell system. The sequence of the human HSL gene was confirmed to be 100% correct compared to the human HSL sequence from genbank (accession # 896474). This HSL cDNA/pAcHLT-A construct and BaculoGold DNA were cotransfected into Sf9 insect cells according to manufacturer instructions (PharMingen).
12. For large-scale production of recombinant human HSL, Sf9 cells were grown in suspension culture in TNM-FH medium. Cells were infected with a recombinant high titer viral stock at a multiplicity of infection (MOI) between 5 and 10 and incubated at 27 °C for 60 h. Cells were harvested by centrifugation at 2500g for 5 min. Pellets were processed in 500 mL batches and lysed in 25 mL lysis buffer (0.25 M sucrose, 10 mM 2-mercaptoethanol + protease inhibitor cocktail (Sigma P-8849, used at 1: 100 dilution) using 10 strokes of a dounce homogenizer. 1% CHAPS was added followed by sonication on ice (8x, 30 s pulses, Branson 450 sonifier). Insoluble material was removed by centrifugation at 10 000g for 30 min at 4 °C. To this 25 mL of buffer A (100 mM sodium phosphate, pH 8.0, 600 mM NaCl, 20% glycerol, 10 mM 2-mercaptoethanol + protease inhibitor cocktail was added. This material was loaded onto a 2 mL Ni-NTA column and washed with 20 bed volumes of wash buffer (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 10% glycerol, 0.3% CHAPS + protease inhibitor cocktail). HSL protein was eluted with 8 mL of wash buffer + 60 mM imidizole. Protein was concentrated to 4 mL using a stirred-cell concentrator with 30 000 molecular weight cutoff (Amicon). Protein was then dialyzed against 3 × 1 L changes of 50 mM phosphate, pH 8.0, 0.03% CHAPS, 1 mM EDTA, 1 mM DTT, 10% glycerol. Protein determination was accomplished by BCA assay (Pierce).
13. Osterlund, T.; Danielsson, B.; Degerman, E.; Contreras, J. A.; Edgren, G.; Davis, R. C.; Schotz, M. C.; Holm, C. Domain-structure analysis of recombinant rat hormone-sensitive lipase. Biochem. J. 1996, 319, 411-420.
14. Each 100 uL reaction contained 10 ng of HSL protein, 20 mM KPO4, pH 7.4, 1 mM EDTA, 0.1% Triton X-100, 5 mM PNPB, 5% DMSO or test compound. Reactions were incubated for 1 h at 27 °C and immediately read at OD 400 nM.