Type of Document Dissertation Author Hurst, Douglas R URN etd-11142003-183812 Title Inhibition of Membrane Type 1-Matrix Metalloproteinase with Mercaptosulfide Inhibitors Degree Doctor of Philosophy Department Chemistry and Biochemistry, Department of Advisory Committee
Advisor Name Title Qing-Xiang Amy Sang Committee Chair Martin Schwartz Committee Member Randolph Rill Committee Member Thomas Keller Committee Member Keywords
- MMP Inhibitor
- Substrate Specificity
- Molecular Modeling
Date of Defense 2003-11-13 Availability unrestricted AbstractMatrix metalloproteinases (MMPs) are a family of related zinc endopeptidases known to play prominent roles during normal and pathological extracellular matrix (ECM) remodeling events including cancer progression. Membrane type-MMPs (MT-MMPs) are a unique subset tethered to the cell membrane by a transmembrane domain or glycosylphosphatidylinositol (GPI) anchor. Consistent with other MMP family members, MT-MMPs have a propeptide, catalytic domain, linker region, and a hemopexin domain. The first identified MT-MMP, MT1-MMP, has been shown to play a key role in tumor cell invasion and metastasis by complex mechanisms including activation of proMMP-2 and direct hydrolysis of interstitial collagens. MT1-MMP is highly localized at the leading edge of invading cancer cells and may be an attractive target for inhibition. MT1-MMP has already been studied by this laboratory in breast cancer systems, however, the enzyme inhibition kinetics and mechanisms were not investigated.
Synthetic MMP inhibitors (MMPIs) have been in development for more than a decade and have been characterized extensively with collagenase 1 and 2 (MMP-1 and -8), gelatinase A and B (MMP-2 and -9), stromelysin 1 (MMP-3), and matrilysin (MMP-7). Three general requirements have been identified for MMPI potency: a functional group capable of coordinating to Zn(II), at least one functional group participating in hydrogen bonding with the enzyme, and one or more side chains that participate in favorable van der Waals interactions particularly at the S1' pocket. The most potent inhibitors thus far have been achieved with a hydroxamic acid zinc-chelating group. Other functional zinc coordinating groups that are significantly less potent as MMPIs include thiol, carboxylate, phosphinate, and sulfodiimine. Mercaptosulfide inhibitors that have been developed and characterized for several secreted MMP family members exhibit IC50 and Ki values approaching that of similar well-characterized hydroxamates. These inhibitors have not been previously characterized with any MT-MMPs. The purposes of this study were to determine the inhibition profile, investigate mechanisms, and identify requirements for potency of mercaptosulfide inhibitors against the target enzyme, MT1-MMP.
In this study, the detailed mechanisms of inhibition of MT1-MMP were examined with new mercaptosulfide inhibitors. The binding of the inhibitor with the enzyme was identified as reversible and competitive. Stereochemistry at the P1' site favors the absolute R configuration corresponding to the unnatural D amino acid derivative. MT1-MMP was effectively inhibited with a homophenylalanine (homoPhe) side chain at P1', further demonstrating it has a deep S1' pocket. However, a phenylalanine side chain that would be expected to show similar potency was found to be more than 350-fold less potent than the corresponding inhibitor with a leucine side chain. Around the mercaptosulfide zinc coordinating functionality the S,R stereochemistry is favored (Ca,Cb to thiol). A non-prime phthalimido substituent generally did not affect inhibition potency, however, comparison of phthalimidomethyl with phthalimidopropyl showed a 10-fold difference in Ki values in the recently designed pyrrolidine series.
The hemopexin domain is known to influence the collagenolytic activity of collagenases including MT1-MMP by mechanisms that are not fully understood, however, it may not affect the structure of the enzyme active site. To address this hypothesis the substrate specificity and inhibition kinetics of the catalytic domain (cdMT1-MMP) and the transmembrane domain-deleted, soluble ecto domain (DTM-MT1-MMP) were compared. The cdMT1-MMP is catalytically more efficient towards small peptide substrates than DTM-MT1-MMP but the hemopexin domain of MT1-MMP may facilitate the hydrolysis of triple-helical substrates. Type I collagen was hydrolyzed by DTM-MT1-MMP but not cdMT1-MMP. Diastereomeric inhibitor pairs were utilized to demonstrate that the hemopexin domain does not significantly modify active site structure because the ratios of Ki values for the inhibitor pairs were found to correlate between the two enzyme forms with a slope of 1.03. These results suggest that although the hemopexin domain plays a significant role in the hydrolysis of triple-helical substrates, it does not significantly affect inhibition potency by small molecule compounds designed to interact with the active site of the enzyme. Therefore, the catalytic domain of MT1-MMP may be representative for the inhibition studies of the ecto domain.
Although there is structural information of synthetic MMPIs complexed with several MMPs available in the Protein Data Bank, no structural information exists for MMPs complexed with mercaptosulfide inhibitors. This type of peptidomimetic inhibitor is distinct from the hydroxamate peptidomimetics with regard to the zinc coordinating functionality and the preference for P1' D amino acid derivatives. In this study, a model of the mercaptosulfide inhibitors complexed to MT1-MMP is proposed to provide a rational interpretation for the experimental Ki values.
This is the first study to characterize mercaptosulfide inhibitors against MT1-MMP. Mercaptosulfide inhibitors may be used to distinguish MMPs with deep versus shallow S1' pockets. MT1-MMP is a deep pocket MMP and MMP-1 is a shallow pocket MMP. The mercaptosulfide inhibitors with a P1' homoPhe may be used to distinguish the collagenolytic activity of MT1-MMP from MMP-1, two common collagenases, in complex biological systems. Knowledge gained from this study may help with future rational design of MMPIs selectively targeting individual MMPs.
Filename Size Approximate Download Time (Hours:Minutes:Seconds)
28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access 01_drh_dissertation.pdf 5.65 Mb 00:26:10 00:13:27 00:11:46 00:05:53 00:00:30