Fields, Gregg B.

Membrane type 1 matrix metalloproteinase (MT1-MMP)/(MMP-14) is a type I transmembrane metalloproteinase involved in degradation of extracellular matrix (ECM) and non-ECM substrates. MT1-MMP facilitates cell migration, and is associated with multiple physiological processes, such as morphogenesis, skeletal development, and wound healing. However, MT1-MMP overexpression causes cancer cell invasion, metastasis, and growth. Therefore, MT1-MMP activities should be regulated either through endogenous tissue inhibitors of MMPs (TIMPs) or novel synthetic MT1-MMP inhibitors. The specific aim of this thesis work is the synthesis and evaluation of novel MT1-MMP C6 and C12 hit inhibitors obtained from one-bead one-compound (OBOC) libraries in order to aid in the discovery and development of effective treatments for many cancers, including melanoma, small cell lung cancer, head and neck carcinoma, bladder cancer, breast cancer, etc.

Using flow cytometry and two forms of hypoxic induction, chemical (CoCl2) and gaseous (Tri-gas chamber), this study highlighted the advantages of quantitative analyses of MMP-14 expression (intracellularly/extracellularly) for one primary (BxPC-3) and one metastatic (HPAF-II) pancreatic cancer line. No significant changes in MMP-14 expression were observed for pancreatic cancer lines using CoCl2. Increased expression levels of MMP-14 were observed using tri-gas incubation maintaining oxygen levels at 2% intracellularly for HPAF-II, but not BxPC-3. A combination of 2D/3D cell culturing techniques were also used to examine changes in cellular behavior/morphology after hypoxic exposure and a 24-hour reoxygenation cycle. BxPC-3 cells showed a greater propensity toward oxidative damage caused by reoxygenation using 2D culturing techniques. Using 3D biomimetic culturing techniques, reoxygenated BxPC-3 cells did not undergo significant apoptosis or necrosis. These results suggest that changes in cellular metabolism and behavior depend on both phenotype and culture scaffolding.

Various key players have been implicated in the development of pancreatic cancer, among these is a member of the matrix metalloproteinase (MMP) family of proteolytic enzymes, MMP 14. This enzyme’s proteolytic activities have been implicated in cancer proliferation, invasion, and metastasis; however, little is known about its non-proteolytic and/or intracellular roles. Furthermore, research to date has focused on in vitro cell culture conditions under normoxic conditions, yet cancer exists physiologically under hypoxic conditions. Under physiological hypoxic conditions members of the MMP family have been associated with altered cellular behavior. Thus, there is a need to elucidate MMP-14’s roles under both normoxic and hypoxic conditions. This study seeks to: (1) characterize the expression of MMP-14 in representative pancreatic cancer cell lines in relation to other cancer associated MMPs; (2) elucidate the impact of hypoxic conditions on MMP-14 expression and/or functionality; and (3) monitor the differences in MMP-14 at both the gene and protein expression levels.

Capable of reshaping the extracellular matrix, matrix metalloproteinases (MMPs) are of enormous consequence to human health. The pathologies of cancers and diseases of the skeletal, central nervous, and cardiovascular systems often owe to the overactivity of MMPs. While efforts to produce therapeutic inhibitors have been largely unsuccessful, triple-helical peptide inhibitors (THPIs) of MMPs show tremendous potential. The synthesis of phosphinic pseudodipeptide building blocks needed for THPIs is entirely replicable and convenient. Here we replicate a crucial step in the synthesis, the cascade bis-deprotection, and formation of Fmoc-amine. The procedure’s feasibility is demonstrated through a 77% yield of the Fmoc-Gly-Val phosphinic pseudodipeptide building block to be incorporated into THPIs of the gelatinases. In the future, it is hoped that such procedures will culminate in large-scale production of refined THPIs, enabling in-depth biochemical studies, further optimization, clinical trials, and novel therapeutics.

Matrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating lesions in both experimental autoimmune encephalopathy (EAE) and multiple sclerosis patients suggesting its possible role in pathological events associated with autoimmune neurodegenerative processes. In addition, MMP-28 has been linked to modulation of immune response and activation of macrophages which presents another role of this enzyme in autoimmune pathologies. In the study described herein, MMP-28 has been shown to affect myelin composition and appearance, mitochondrial protein content, and vesicular transport proteins. Moreover, the decrease in myelin basic protein quantity observed in healthy MMP-28KO animals affected the myelin staining intensity in various brain regions including corpus callous. Cellular energetic studies did not reveal differences in mitochondrial function in MMP-28KO animals and no difference in reactive oxygen species was observed. In the EAE model, MMP-28 deletion increased the occurrence of atypical form of EAE characterized by increased inflammation of arbor vitae of the brain. In addition, MMP-28 deletion decreased the inflammatory infiltrates present in brains obtained from EAE animals. Lastly, MMP-28 has been shown to affect cellular energetics and activation of bone marrow derived macrophages during the initial stages and after 24 h activation. In addition, MMP-28 deletion increased proinflammatory cytokines and receptors CD86 and iNOS found in M1 polarized macrophages.

Matrix Metalloproteinase-13 (MMP-13) belongs to a large family of proteolytic enzymes which are characterized by their ability to degrade the extracellular matrix components. MMP-13 appears to have a critical role in tumor invasion and metastasis. In this study, several fluorogenic probes specific for MMP-13 were designed and characterized. These synthesized probes could be modified with chelators to be applied for imaging MMP-13 in breast cancer and/or multiple myeloma models. The activity and selectivity of MMP-13 and other MMPs against these probes were studied through two approaches. It was found that these probes were cleaved by all MMPs, but MMP-13 showed the highest activity and selectivity towards these peptides.

Collagen is the major structural scaffold in the body and serves as barrier between tissues, and thus its turnover is tightly regulated. Collagen triple-helical structure renders it resistant to general proteolysis. Several proteases are capable of cleaving the triplehelical regions of collagen, including several mammalian matrix metalloproteinases (MMPs) and bacterial collagenases. MMP-mediated collagenolysis is associated with numerous diseases and some bacterial collagenases have found clinical application use due to their efficiency in the hydrolysis of the collagen triple-helix. A selective Förster resonance energy transfer triple-helical peptide (fTHP) probe for monitoring the activity of Clostridial collagenase has been developed. The fTHP [sequence: Gly-mep-Flp-(Glyvi Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] was stable at 37 °C and was efficiently hydrolyzed by bacterial collagenase (kcat/KM = 25,000 s -1 M-1) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The bacterial collagenase fTHP assay can be utilized in applications where specific activity towards triple-helical collagen needs to be evaluated, such as isolation of cells from various tissues. An fTHP scaffold was also utilized to evaluate the sequence preferences of eight MMPs. Residues spanning from P3 to P11 investigated using a positional scanning synthetic combinatorial library. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. The results of this study show that the P10 11 substrate play an important role in the collagenase-substrate interactions and that modifying these residues can drastically affect the affinity of MMPs towards THP substrates. The identified sequence preferences of MMPs will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery.

Matrix Metalloproteinase-14 (MMP-14) functions as a protease that assists in the digestion of type 1 collagen, a significant component of the extracellular matrix (ECM). MMP-14 may be essential in the metastases and invasion of cancers like lung, melanoma, and colon as well as diseases of the connective tissue. The progression of certain cancers may be constricted by developing specific enzymatic inhibitors that can target the action of this enzyme. In order to study the role of MMP-14 mediated invasion, we focused on the creation of a human breast adenocarcinoma (MCF-7) cell line stably overexpressing the MMP-14 protein. This cell line will be used for future diagnostic research of cell-based MMP-14 kinetic activity in order to eventually develop an effective and specific inhibitor for this protease.

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play an important role in tumor growth and invasion (Gialeli, 2010). They can degrade cell-adhesion molecules that allow for cell-cell and cell-extracellular matrix interactions, which is necessary for cancer cells to degrade physical barriers (Gialeli, 2010). The presence of MMPs in cancer cells help promote cell invasion. In this project, melanoma cell lines WM115 (primary) and WM266-4 (metastatic) were used to conduct the invasion assay. The invasion assay was designed and optimized to test the ability of the cell to cut and pass through the collagen membrane. Next, a general MMP inhibitor was tested for inhibition of invasion. Varying inhibitor concentrations were used to see the effects. Following that, a more specific inhibitor targeting MT-1 MMP was tested with WM266-4 cells and inhibition of invasion was observed. MT1-MMP was the target of the inhibitor because of its involvement in cell invasion and its ability to cleave the collagen membrane.

Matrix metalloproteinases MMPs are a family of proteolytic enzymes that mediate the degradation of
various components of the extracellular matrix. Their functions are essential for normal physiological
processes such as wound healing, but their dysregulation is associated with various pathologies
including autoimmune diseases such as Multiple Sclerosis MS. Experimental Autoimmune
Encephalomyelitis EAE is a well-established murine model of MS that is mediated by CD4 T-cells.
These cells penetrate the blood-brain-barrier BBB, recruit other immune cells, initiate destruction of the
myelin sheath, and cause axonal loss. MMP-9 is a hallmark enzyme in progression of MS that is
required for penetration of the BBB and generation of autoantigens in EAE. In addition, recent studies
have demonstrated that MMP-9 contributes to normal intracellular function of various cell types
including antigen activated T-cells; however, the intracellular role of MMP-9 in immune cell activation
during EAE pathogenesis is not known. In this study, we used a highly selective MMP-9 triple-helical
peptide inhibitor THPI that is a phosphinate transition state analog to examine antigen specific T-cell
responses. We found that selective inhibition of MMP-9 can mitigate pathogenic T-cell activity and
cellular trafficking as well as the clinical severity of EAE, suggesting that selective MMP-9 inhibition in
MS can be a potent therapeutic option.