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Our laboratory works on the elucidation of eukaryotic cell signal transduction pathways, their role in vertebrate cell function and pathophysiology, and the use of this knowledge of structure, function and mechanism to identify potential therapeutic targets and develop novel therapeutics. Current research has a major focus on the role of protein phosphorylation pathways in disease onset and progression and their potential as drug discovery targets. An overview of two major areas in the laboratory is provided below and selected publications for more details are listed.
The role of calmodulin (CaM) mediated signal transduction pathways in physiology and pathophysiology.
Historically, our research in the area of intracellular signal transduction pathways has used an interdisciplinary approach with a focus on gaining insight into in vivo function, viewing organisms as multivariable complex biological systems in which to test hypotheses following the maxim of in vivo veritas. For example, our approach to the discovery of calmodulin (CaM) as a unique macromolecular chemical entity candidate for intracellular integration of calcium signaling pathways used approaches that we would now call proteomics-based. We focused on purification of a protein that had the appropriate physical properties and activities to fulfill a potential biological signal transduction role, for which there were no identified players at the time. Our development of CaM-affinity chromatography was for the purpose of stepping further into the molecular mechanisms by identifying binding partners, viewing binding as a necessary but not sufficient event for function in signal transduction. Our mapping of one of the first CaM recognition regions for an enzyme and providing a molecular precedent for signal transduction cross-talk among protein phosphorylation pathways were driven by a desire to place macromolecular binding and recognition events into a functional pathways context. This work led to elucidation of a molecular mechanism of CaM:enzyme recognition and protein kinase regulation that remains applicable to multiple CaM-regulated enzymes. As we moved into more complex biological systems analysis, we adopted newly emerging technologies in order to understand how CaM and a CaM-regulated enzyme could be encoded, expressed, regulated, and assembled into a calcium signal transduction complex. Along the way, we made several serendipitous discoveries as a result of taking an empirical leap into chemical dissection of such a complex system. More recently, we have used interdisciplinary efforts that combine the use of integrative (in vivo) chemical biology and molecular genetics to gain insight into how landmark CaM-regulated protein kinases are involved in physiology and pathophysiology, and which of these intertwined complex networks are amenable to small molecule intervention. These insights, in turn, provided the starting point for an integrative drug discovery campaign, where we continue the interdisciplinary study of in vivo function in vertebrate biology, but with the focused goal of addressing unmet medical needs.
Integrative drug discovery and the development of novel therapeutics for unmet medical needs and attenuation of disease progression.
The success rate for discovery and development of new drugs has been a continuing problem for most of the past three decades. Further, most drugs are palliative (relief of disease symptoms). Drugs that alter disease progression are rare. The United States Food and Drug Administration (FDA) and National Institutes of Health (NIH) requested that academia and industry address this crisis by exploring strategies to improve efficiency and innovation in the drug discovery and development process, especially in major areas of unmet medical need. The outcome from meetings and discussions with our colleagues in industry and at government agencies to identify how we could respond to the need for a more effective process that would also facilitate innovation was the evolution of “smart chemistry” approaches to early drug discovery.
The “smart chemistry” approach mines the information found in chemical and pharmacological databases to help identify physical properties common among small molecules that turn out to be good drugs, and proposes testable hypotheses about what common themes among small molecules with different structures make them more drug-like. The identification of common themes and the use of emerging computational tools aids in the design of new molecules. We then select diverse representatives from the collection of virtual molecules, and make them using established synthesis methods or new synthetic production schemes that we develop. The resultant series of novel molecules are then assembled into small, highly focused sets of special collections called chemical libraries. The libraries are searched for biologically useful structures, in a process called biological screening. Active molecules are called hits. Hits must be subjected to additional synthetic chemistry diversification called medicinal chemistry refinement in order to improve activity or physical properties compatible with biological systems. This recursive cycle of chemical synthesis and biological activity evaluation is at the heart of the drug discovery process. The “smart chemistry” approach to discovery allows a better fit between biology and chemistry at both the virtual molecule design phase and in the assembly line phase of synthetic production and testing, making possible rapid discovery of novel compounds with potential for development into new therapeutics. “Smart chemistry” considers the potential for bioavailability and safety as well as efficacy in the early design and synthesis stages of discovery, and uses early experimental outcomes of the biological and physical chemistry testing to drive the medicinal chemistry refinement phase. The fewer number and higher bioavailability potential of compounds emerging from the “smart chemistry” approach make in vivo biological testing a practical goal for early synthetic work evaluation.
We are using the “smart chemistry” approach integrated with “smart biology” screens for rapid discovery of novel small molecules with potential use in targeting pathophysiology progression related to diseases ranging from neurological disorders, cancer, inflammatory conditions, cardiovascular and pulmonary disease. A proof-of-concept is our recent discovery and development of novel small molecule compounds that selectively attenuate the increased production of proteins called proinflammatory cytokines, which can cause tissue injury and disease when produced in excess. A similar approach is used for the design, synthesis and evaluation of selective protein kinase inhibitors targeting a series of intracellular, calmodulin-regulated, protein phopshorylation pathways that function as regulators of cell death or tissue barrier homeostasis. We ultimately hope to find, by targeting pathophysiology mechanisms which contribute to disease progression, a series of novel small molecules with potential to be effective against a variety of disorders.
Publications:
Munoz L, Ralay Ranaivo H, Roy SM, Hu W, Craft JM, McNamara LK, Wing Chico L, Van Eldik L, and Watterson DM (2007) A novel p38a MAPK inhibitor suppresses brain proinflammatory cytokine up-regulation and attenuates synaptic dysfunction and behavioral deficits in an Alzheimer’s disease mouse model. J Neuroinflammation 4: 21.
Reynoso R, Perrin RM, Breslin JW, Daines DA, Watson KD, Watterson DM, Wu MH and Yuan S (2007) A role for long chain myosin light chain kinase (MLCK-210) in microvascular hyperpermeability during severe burns. Shock, 28:589-595.
Somera-Molena KC, Robin B, Somera CA, Anderson C, Koh S, Behanna HA, Van Eldik LJ, Watterson DM and Wainwright MS (2007) Glial activation links early-life seizures and long-term neurologic dysfunction: evidence using a small molecule inhibitor of pro-inflammatory cytokine upregulation. Epilepsia 48: 1785-1800.
Rossi JL, Velentza AV, Steinhorn DM, Watterson DM and Wainwright MS (2007) MLCK 210 gene knockout or kinase inhibition preserves lung function following endotoxin-induced lung injury in mice. Amer J Physiol Lung Cell Mol Physiol 292: L1327-L1334.
Hu W, Ralay Ranaivo H, Roy SM, Behanna HA, Wing LK, Munoz L, Guo L, Van Eldik LJ and Watterson DM (2007) Development of a novel therapeutic suppressor of brain proinflammatory cytokine up-regulation tthat attenuates synaptic dysfunction and behavioral deficits, Bioorg Med Chem Lett 17:414-418.
Ralay Ranaivo H, Carusio N, Wangensteen R, Ohlmann P, Loichot C, Tesse A, Chalupsky K, Lobysheva I, Haiech J, Watterson DM, and Andriantsitohaina R (2007) Protection against endotoxic shock as a consequence of reduced nitrosative stress in MLCK210 null mice. Amer J Pathol 170: 439-446.
Schumacher AM, Velentza AV, Watterson DM, and Dresios J (2006) Death associated protein kinase phosphorylates mammalian ribosomal protein S6 and reduces protein synthesis. Biochem. 45:13614-13621
Ralay Ranaivo H, Craft JM, Hu W, Guo L, Wing LK, Van Eldik LJ and Watterson DM (2006) Glia as a therapeutic target: selective suppression of human Abeta-induced upregulation of brain proinflammatory cytokine production attenuates neurodegeneration. J Neurosci 26:662-670.
Wing LK, Behanna HA, Van Eldik LJ, Watterson DM and Ralay Ranaivo H (2006) De novo and molecular target-independent discovery of orally bioavailable lead compounds for neurological disorders. Curr Alz Res, 3:205-214.
Behanna HA, Watterson DM and Ralay Ranaivo H (2006) Development of a novel bioavailable inhibitor of the calmodulin-regulated protein kinase MLCK: a lead compound that attenuates vascular leak. Bioch. Biophy. Acta, 1763:1266-1274.
Clayburgh DR, Barrett RA, Tang Y, Meddings JB, Van Eldik LJ, Watterson DM, Clarke LL, Mrsny RJ and Turner JR (2005) Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo. J Clin Invest 115:2702-2715.
Schumacher AM, Schavocky JP, Velentza AV, Mirzoeva S and Watterson DM (2004) A calmodulin regulated protein kinase linked to neuronal survival is a substrate for the calmodulin regulated death associated protein kinase. Biochem. 43: 8116-8124.
Velentza AV, Wainwright MS, Zasadzki M, Mirzoeva S, Schumacher AM, Haiech J, Focia PJ, Egli M and Watterson DM. (2003) An aminopyridazine-based inhibitor of a pro-apoptotic protein kinase attenuates hypoxia-ischemia induced acute brain injury. Bioorganic & Medicinal Chemistry Letters. 13:3465-3470.
Wainwright, M.S, J. Rossi, J. Schavocky, S. Crawford, D. Steinhorn, A. V. Velentza, M. Zasadzki, V. Shirinsky, Y. Jia, J. Haiech, L. J. Van Eldik, & D. M. Watterson. (2003) Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment. Proceedings of the National Academy of Sciences, U.S.A. 100:6233-6238.
Schumacher A, Velentza A and Watterson DM (2002) Death associated protein kinase as a potential therapeutic target. Expert Opin.Ther.Targets 6(4):497-506.
