The progress of diseases often involves the dysregulation of cellular signaling events leading to altered abnormal cellular phenotypes. Cancerous diseases for instance are often characterized by an increase of abnormal cell division and proliferation due to enhanced release of growth factors such as epidermal growth factor (EGF) or tumor necrosis factor ? (TNF-?). These growth factors are expressed as pro-proteins, which are recognized, cleaved and thereby activated by membrane-anchored metalloproteases (sheddases) such as a disintegrin and metalloproteases (ADAM's). Therefore, ADAMs have emerged as targets for a number of pathophysiological conditions such as cancer and neurological disorders. ADAM's are closely related to matrix metalloproteases (MMP's), which are validated targets in anti-cancer drug discovery. Although very potent MMP inhibitors have been developed, their use in cancer therapy has been hampered by a lack of efficacy and side effects in clinical trials, which are partially attributed to cross-inhibition of other zinc-dependent sheddases such as ADAM's. Thus, recent research has focused on the development of selective inhibitors addressing only the enzyme directly related to the pathophysiological condition. Most of the MMP and ADAM inhibitors in development harbor a strongly chelating hydroxamic acid (hydroxamate) group binding a zinc atom in the catalytic site, thereby blocking shedding activity. Research of such compounds has abated as they have been found to act in a relatively promiscuous manner. Using alternative chelating motifs could offer a better way of modulating binding strength and finally, binding selectivity, which would significantly improve the usefulness of metalloprotease inhibitors in biochemical and biomedical research as well as cancer therapy. While ADAM17, also known as TNF-? converting enzyme (TACE), is probably the most prominent ADAM explored as a target for the treatment of lung and breast cancer and inflammatory diseases such as rheumatoid arthritis, other ADAM's such as ADAM12 are much less characterized potential targets for therapeutic purposes. Therefore, the specific aim of this proposal is to screen the Molecular Libraries Small Molecule Repository (MLSMR) for ADAM12 inhibitors focusing on the discovery of compounds with chelating motifs other than the traditional hydroxamate group in the downstream secondary screening and medicinal chemistry efforts in order to minimize cross-inhibition of other ADAM's. A selectivity panel of five ADAM proteases related to ADAM12 will allow to assess binding selectivity in both biochemical and cell-based assays monitoring the shedding of natural ADAM substrates, thereby generating a comprehensive data set on the inhibitory profile of biomedically interesting ADAM proteases. The primary high-throughput screening will employ a fluorescence-based assay measuring the cleavage of a quenched substrate peptide. This assay has recently been miniaturized to 1536- well plate format demonstrating the robustness required for an unattended robotic screening of the MLSMR compound library and a sufficient performance to ensure data quality for subsequent analysis. Cross-validated compounds derived after primary and secondary assays will be further developed to enhance potency and selectivity for ADAM12. !
ADAM proteases are validated targets in drug discovery as shown by the efforts undertaken in designing inhibitors against ADAM17 (TACE). ADAM12 represents another protease involved in the maturation of growth factors and their receptors and has been shown to be over-expressed in tumors. Currently, the development of selective ADAM inhibitors is hampered by the high degree of structural similarity among MMP, ADAM and ADAMTS proteases. This project aims to identify ADAM12 specific small-molecule inhibitors that we hope can further be developed into useful tools for both biomedical and clinical research, most optimally complementing current efforts in cancer treatment and therapy.!
