Department of Pharmacology
The Johns Hopkins University
725 N. Wolfe Street
Hunterian Building, Room 516
Baltimore, MD 21205
1994 Searle Scholar
- Chemical and molecular biological approaches to elucidating mechanisms of intracellular signal transduction pathways, particularly those of the immune and neural systems.
- Identification, purification and characterization of molecular targets for small organic ligands including natural products and synthetic drugs as a means of uncovering novel signaling molecules.
- Study of the cellular functions of newly-identified molecules in signal transduction, including their subcellular localization and their interactions with upstream and downstream signaling molecules.
- Study of the molecular recognition between ligands and their receptors through modification of the ligands by chemical synthesis and alteration of receptors by mutagenesis.
A molecular understanding of intracellular signal transduction pathways in general, and those involved in T cell activation in particular, is the major objective of our current research program. A combination of techniques from synthetic organic chemistry, protein biochemistry, molecular and cell biology is applied to achieve such an objective. The molecular tools to be used include small ligands such as natural products and synthetic drugs.
Thalidomide as a probe to study mechanisms of immunoregulation, sleep/wake regulation and embryogenesis.
Thalidomide is a synthetic drug originally used as a sleeping pill in the late 1950's and early 1960's owing to its ability to induce natural REM sleep. It was later found to be a teratogen, causing congenital birth defects in tens of thousands of babies. Most recently, it was shown to have immunosuppressive activities, being most effective in treating graft- versus-host disease. Although much work has been carried out in the past thirty years to unravel the mode of action of this mysterious drug, its molecular target remains elusive. Of the three physiological processes affected by thalidomide, those of the immune, the neural and the embryo systems, the immune system is the best studied. Therefore, current efforts are being focused on the identification of both cellular and molecular targets for thalidomide in lymphocytes. The effect of thalidomide in various cellular assays including mixed lymphocyte reaction, mouse spleen cell proliferation in response to mitogens and alloantigens are being systematically investigated. We have synthesized a series of derivatives of thalidomide to establish a structure/function profile of these analogs in T cell activation assays, guiding future derivatizations for making affinity matrices and radioactive photoaffinity labels of the drug. These will be used to detect potential receptors for the drug from thymus extracts. Eventually we hope to clone and sequence the genomic and complementary DNA of the thalidomide target and study its cellular functions. The thalidomide target, in turn, may lead to the identification of more signaling molecules involved in the regulation of the immune response, sleep/wake cycle and embryogenesis.
Intracellular signal transduction pathways mediated by the protein phosphatase calcineurin.
The immunosuppressant cyclosporin A (CsA), widely used in organ transplants, and its functional analog, FK506, have been applied as tools to elucidate the mechanism of T cell receptor-mediated signal transduction during T cell activation. These two drugs, through binding to their immediate intracellular receptors called immunophilins, bind to and inhibit the enzymatic activity of the calcium, calmodulin-dependent protein phosphatase calcineurin. Calcineurin is now known to play pivotal roles in many intracellular signaling pathways. In helper T cells, it transmits the signal from the T cell receptor into the nucleus leading to the transcription of cytokine genes such as interleukin 2. In cytotoxic T cells and mast cells, on the other hand, activation of calcineurin leads to the exocytosis of secretory granules. The divergence of outcome of different signaling pathways mediated by calcineurin implicates different downstream signaling molecules. We are currently focusing on identifying the substrates of calcineurin in different signaling pathways. In addition, calcineurin is composed of multiple subunits with several distinct structural motifs. We are also investigating the mechanism of catalysis by the catalytic domain of calcineurin and cellular regulation of calcineurin through its structural motifs.