The homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) of rat liver cytosol is potently inhibited at its active site by nonsteroidal and steroidal antiinflammatory drugs (NSAIDS and SAIDS). It binds prostaglandins with high affinity and may function as a 9-hydroxyprostaglandin dehydrogenase. The enzyme fulfills many of the criteria attributed to a target for antiinflammatory drugs. It has been purified in milligram amounts, crystallized, and its cDNA has been cloned. Since the binding of antiinflammatory drugs at the active site predicts their pharmacological potency, the topography of this site will be elucidated using a multi-faceted approach. To solve the 3-dimensional structure of the enzyme, new crystal forms will be grown which diffract beyond the 6 A degree level. A near full length cDNA for 3 alpha-HSD will be sub-cloned into the sequencing vector pGEM-3 and its sequence determined by dideoxy- sequencing. Knowledge of the primary structure will compliment the mapping of catalytic domains of the enzyme. This will be achieved using a series of suicide substrates based on either 4- nitrobenzaldehyde or Nphenylanthranilic acids. The former series labels the NAD(P)+ binding site and the latter series mimics NSAIDS, and have the potential to alkylate the antiinflammatory drug binding site. By using radiolabeled suicide substrates and peptide mapping procedures, peptides residing at each domain will be identified. Methods which permit the placement of bromoacetylating agents in a positional manner around NSAIDS (indomethacin and meclofenamate) and SAIDS are being developed. These agents will be used to identify amino acids that play a functional importance in the binding of antiinflammatory drugs. To assist in the orientation of prostaglandins at the active site, the 9, 11-and 15-hydroxyprostaglandin dehydrogenase activities will be further characterized. Computer (PROPHET) modeling of bound ligands (NSAIDS, SAIDS and prostaglandins) will lead to the generation of an enzyme excluded volume map, i.e., that volume not occupied by the protein but essential for ligand binding. The relationship, that exists between amino acids identified in the affinity labeling studies and bound ligands, will be revealed by placing these amino acids around the perimeter of this volume map. These studies may provide information for a rational approach to the design of superior antiinflammatory drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM033464-04
Application #
3283217
Study Section
Biochemistry Study Section (BIO)
Project Start
1985-07-01
Project End
1992-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Penning, T M; Abrams, W R; Pawlowski, J E (1991) Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha-bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines;sequence confirmation using nucleotide sequ J Biol Chem 266:8826-34
Pawlowski, J E; Huizinga, M; Penning, T M (1991) Cloning and sequencing of the cDNA for rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase. J Biol Chem 266:8820-5
Penning, T M; Thronton, R; Ricigliano, J W (1991) Clues to the development of mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase: comparison of steroidal and nonsteroidal Michael acceptors and epoxides. Steroids 56:420-7
Penning, T M; Ricigliano, J W (1991) Mechanism based inhibition of hydroxysteroid dehydrogenases. J Enzyme Inhib 5:165-98
Hardy, M P; Gelber, S J; Zhou, Z F et al. (1991) Hormonal control of Leydig cell differentiation. Ann N Y Acad Sci 637:152-63
Pawlowski, J; Huizinga, M; Penning, T M (1991) Isolation and partial characterization of a full-length cDNA clone for 3 alpha-hydroxysteroid dehydrogenase: a potential target enzyme for nonsteroidal anti-inflammatory drugs. Agents Actions 34:289-93
Ricigliano, J W; Penning, T M (1990) Evidence that enzyme-generated aromatic Michael acceptors covalently modify the nucleotide-binding site of 3 alpha-hydroxysteroid dehydrogenase. Biochem J 269:749-55
Ricigliano, J W; Penning, T M (1989) Synthesis and evaluation of non-steroidal mechanism-based inactivators of 3 alpha-hydroxysteroid dehydrogenase. Biochem J 262:139-49
Smithgall, T E; Penning, T M (1988) Electrophoretic and immunochemical characterization of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenases of rat tissues. Biochem J 254:715-21
Penning, T M; Carlson, K E; Sharp, R B (1987) Affinity-labelling of the anti-inflammatory drug and prostaglandin-binding site of 3 alpha-hydroxysteroid dehydrogenase of rat liver cytosol with 17 beta- and 21-bromoacetoxysteroids. Biochem J 245:269-76

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