Discipline: Biological Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Session: 1
Brian W. Basinski - Grand Valley State University
Co-Author(s): Co-Author(s): Ahmed Mady, Ph.D., University of Michigan, MI; Nurul Ansari, Ph.D., University of Michigan, MI; Zaneta Nikolovska-Coleska, M.S., Ph.D., University of Michigan, MI
The metalloprotease pregnancy-associated plasma protein-A (PAPP-A), plays a significant role in the IGF system, a cellular pathway that controls metabolism, growth, and cell differentiation. PAPP-A targets and cleaves the insulin-like growth factor binding protein-2 (IGFBP-2) -4, and -5. The IGFBPs bind to and sequester the insulin growth factor (IGF), an endocrine growth hormone. Once the IGFBPs are cleaved they release IGF. Therefore, PAPP-A functions to increase the bioavailability of IGF. IGF binds to and signals the tyrosine kinase receptor named insulin growth factor receptor (IGFR). PAPP-A has been shown to have a role in modulating aging and age-related changes. Gene knock-out of PAPP-A in mice shows increased lifespan and reduced tumorigenicity of cancer. Therefore, the development of small-molecule inhibitors of PAPP-A can be used to recapitulate these effects. Characterized small molecule inhibitors of PAPP-A will lead to the development of chemical tools that will provide insight into the biology and role of PAPP-A in aging.
We discovered a lead compound from a high-throughput screen. From this lead, we synthesized analogs and validate their activity in inhibiting PAPP-A with two orthogonal assays. We used a Quenched Fluorescent Assay which detects PAPP-A activity using an IGFBP-4-derived fluorescent peptide. To determine a range of PAPP-A activity we established the minimum control which contained no PAPP-A and the maximum control which contained PAPP-A, both controls contained no compounds. Additionally, we designed a western blot which detects PAPP-A activity by identifying IGFBP-4 full-length and IGFBP-4 cleaved segments using a polyclonal antibody. The minimum and maximum IGFBP-4 cleavage were determined as controls, where the minimum cleavage reaction contained IGFBP-4 without PAPP-A and the maximum cleavage reaction contained IGFBP-4 with PAPP-A, both controls contained no compounds. PAPP-A activity in the western blot was validated by the introduction of stanniocalcin-1 (STC1), an endogenous inhibitor of PAPP-A, as a positive control. We conclude that this class of compounds is the first to show inhibition of PAPP-A. Further studies are required to understand how the inhibition of PAPP-A enzymatic function modulates aging.
References: Lawrence, J. B.; Oxvig, C.; Overgaard, M. T.; Sottrup-Jensen, L.; Gleich, G. J.; Hays, L. G.; Yates, J. R.; Conover, C. A. The Insulin-like Growth Factor (IGF)-Dependent IGF Binding Protein-4 Protease Secreted by Human Fibroblasts Is Pregnancy-Associated Plasma Protein-A. Proc. Natl. Acad. Sci. U. S. A. 1999, 96 (6), 3149-3153.
Conover, C. A.; Bale, L. K.; Mader, J. R.; Mason, M. A.; Keenan, K. P.; Marler, R. J. Longevity and Age-Related Pathology of Mice Deficient in Pregnancy-Associated Plasma Protein-A. Journals Gerontol. – Ser. A Biol. Sci. Med. Sci. 2010, 65 A (6), 590-599.
Gallagher, E. J.; LeRoith, D. Minireview: IGF, Insulin, and Cancer. Endocrinology 2011, 152 (7), 2546-2551.
Funder Acknowledgement(s): Funder Acknowledgement(s): This research is supported by Glenn Foundation and Calico LifeScience (Z.N-C.) and by the National Science Foundation through the Interdisciplinary REU Program in the Structure and Function of Proteins at the University of Michigan - College of Pharmacy (B.B.).
Faculty Advisor: Dr. Zaneta Nikolovska-Coleska, M.S., Ph.D., zanetan@med.umich.edu
Role: I performed the validation tests on all compounds of a novel class of inhibitors of PAPP-A (~30 compounds total), in three assays. I used a quenched fluorescent assay, this experiment was performed in dose-response (DR) and each compound was tested independently 3 times. I also helped designed a western blot assay (WBA), characterizing PAPP-A activity and optimal concentrations of protein and substrate. With WBA I independently tested all compounds in DR independently 3 times. Finally, I tested all compounds in a cell viability assay, cell line A673.