The long term objective of this research is to prepare inhibitors of collagenase and other metalloenzymes that degrade the macromolecules of the extracellular matrix and to develop these inhibitors into therapeutically useful drugs for the treatment of disease such as rheumatoid and osteoarthritis in which these molecules are broken down at an excessive rate. Other conditions in which such inhibitors might be therapeutically useful are periodontal disease, corneal ulceration and recessive dystrophic epidermolysis bullosa. In addition, collagenases have been implicated in tumor cell metastasis, ovulation, angiogenesis, and other processes requiring cells to overcome barriers to migration. In this application, we propose to use collagenase inhibitors that we have already prepared which are effective in vivo at nanomolar concentrations to characterize collagenases and other metalloproteinases at the molecular level, to characterize some of the pharmacological properties of these inhibitors, and to generate new peptide inhibitors of improved potency, specificity, and pharmacological properties. In keeping with these broad goals, the specific aims of the project are: first, purification of collagenases from pig synovial membranes, neutrophil granules and a human monocyte cell line using affinity techniques that we have developed; irreversible labelling of the active site of these collagenases using inhibitor derivatives and determination of the amino acid sequence of the active site peptides for comparison with other well-characterized zinc proteinases; and investigation of inhibitor specificity with these enzymes. Second, design and synthesis of new peptide inhibitors using conformational analysis, x-ray crystallography and computer graphics techniques coupled both with potency data for currently available compounds as well as newly acquired structure-function data; preparation and evaluation of coordinating amide bond replacements as a new class of collagenase inhibitor. Our third specific aim is determination of inhibitor stability in blood, plasma and tissue homogenates, and in an in vitro model of inflammatory disease using activated macrophages to simulate the in vivo environment of the inhibitor; determination in the mouse of the pharmacokinetic, tissue distribution and biotransformation of one or more collagenase inhibitors in hand as well as any new derivatives with improved properties that may be developed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR039573-03
Application #
3159713
Study Section
Bio-Organic and Natural Products Chemistry Study Section (BNP)
Project Start
1988-07-01
Project End
1991-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
3
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Louisville
Department
Type
Schools of Medicine
DUNS #
City
Louisville
State
KY
Country
United States
Zip Code
40292
Gray, R D; Paterson, C A (1994) Application of peptide-based matrix metalloproteinase inhibitors in corneal ulceration. Ann N Y Acad Sci 732:206-16
Stack, M S; Gray, R D; Pizzo, S V (1993) Modulation of murine B16F10 melanoma plasminogen activator production by a synthetic peptide derived from the laminin A chain. Cancer Res 53:1998-2004
Gray, R D; Darlak, K; Spatola, A F (1992) Design and synthesis of inhibitors of porcine synovial collagenase and gelatinase. Matrix Suppl 1:307-8
Stack, S; Gray, R D; Pizzo, S V (1991) Modulation of plasminogen activation and type IV collagenase activity by a synthetic peptide derived from the laminin A chain. Biochemistry 30:2073-7
Stack, M S; Emberts, C G; Gray, R D (1991) Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin. Arch Biochem Biophys 287:240-9
Stack, M S; Gray, R D (1990) The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase. Arch Biochem Biophys 281:257-63
Burns, F R; Paterson, C A; Gray, R D et al. (1990) Inhibition of Pseudomonas aeruginosa elastase and Pseudomonas keratitis using a thiol-based peptide. Antimicrob Agents Chemother 34:2065-9
Darlak, K; Miller, R B; Stack, M S et al. (1990) Thiol-based inhibitors of mammalian collagenase. Substituted amide and peptide derivatives of the leucine analogue, 2-[(R,S)-mercaptomethyl]-4-methylpentanoic acid. J Biol Chem 265:5199-205
Stack, M S; Gray, R D (1989) Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide. J Biol Chem 264:4277-81