Metalloproteinases--Inhibitors

The proteolytic activities of the ADAM (a disintegrin and metalloproteinase),
ADAMTS (a disintegrin and metalloproteinases with thrombospondin motifs) and
MMP (matrix metalloproteinase) protein families play important roles in normal and
multiple pathological conditions. These metalloproteases have potential implications
in the degradation of the extracellular matrix and in the processing of bioactive
molecules. Under pathological conditions these proteases are involved in many
diverse processes from tumor cell migration to cartilage destruction in rheumatoid
arthritis. In the present study, the gene expression levels of six ADAMs, eight MMPs,
and four ADAMTSs were analyzed by Real Time PCR. RNA was isolated from
multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic
normal tissue and tumor samples. This method allowed for detected changes in mRNA expressiOn of the individual metalloproteainase genes to be compared
between normal and metastatic states, and also between tissue and cultured cells.
Substantial differences have been observed in the level of ADAM and MMP mRNA
expression between tissue and cell lines. In general, the level of expression is several
folds higher in cultured cells compared to the isogenic tissue they are derived from.
Protein microarrays were utilized in order to evaluate the correlations between MMP
and TIMP mRNA copy numbers and protein abundance in cell culture. In several
cases, distinct differences were observed regarding the localization of the proteins
examined. In order to determine if the metalloprotease genes that were elevated at the
level of RNA expression produce functional proteins, the foundations of an in situ
FRET assay have been established. This will greatly aid in a better understanding of
the behavior of metallopeptidases in a cellular context.

Alterations in activities of one family of proteases, the metzincins have been implicated
in an array of physiological and pathological processes. In the present study, metzincin
inhibitors were developed by utilizing topologically constrained peptides and
pseudopeptides. The endothelin-family framework was used to develop a disulfideconstrained
topology. This framework was chosen due to its three-dimensional similarity
with a family of endogenous metzincin inhibitors, the tissue inhibitors of
metalloproteases (TIMPs). The collagenous triple-helix was chosen as a second
framework, because only a subset of proteolytic enzymes have the capacity to bind and
hydrolyze a triple-helix. Both templates were successfully modified to generate an array
of inhibitors. These inhibitors displayed subnanomolar to micromolar apparent Ki values,
while being moderately selective metzincin inhibitors. In both cases the threedimensional
structure was determined to be important for activity. This work encourages
the further development of both frameworks as metzincin inhibitors.

Tissue Inhibitors of Metalloproteinases (TIMPs) are produced by a wide variety of cell types. Similar to matrixins, the expression of TIMPs in the tissue is also controlled during tissue remodeling and physiological conditions to maintain a balance in the metabolism of extracellular matrix. Disruption of this balance can result in diseases associated with uncontrolled turnover of matrix, such as arthritis, cancer, and cardiovascular disease. Some of the biological processes TIMPs participate in include: regulation of cell morphology and organ morphogenesis, inhibition of angiogenesis, steroidogenesis, and tissue remodeling. One major function of TIMPs is inhibition of Matrix Metaloproteinase (MMPs). This project used bioinformatic techniques to identify two Caenorhabditis elegans TIMP cDNA cloned by BLAST searching of EST database. These TIMP cDNA were amplified, cloned and expressed, proteins were purified. Kinetic studies were carried out to evaluate the inhibitory activities against various MMPs and TACE. This research project will provide some insight on the function of C. elegans TIMPs.