We report highly potent, selective, and low cost bifunctional acetylcholinesterase (AChE) inhibitors developed by our two-step prototype optimization strategy utilizing computer modeling of ligand docking with target proteins: 1) identify low affinity sites normally missed by x-ray crystallography; and 2) design bifunctional analogs capable of simultaneous binding at the computer-determined low affinity site and the x-rayidentified high affinity site. Applying this strategy to 9-amino-1,2,3,4-tetrahydroacridine (THA), a drug for Alzheimer's disease, we obtained alkylene linked bis-THA analogs. These analogs were up to 10,000-fold more selective and 1,000-fold more potent than THA in inhibiting rat AChE and yet required one simple reaction to synthesize. Additionally, alkylene linked benzyl-THA analogs were developed to examine the specificity of the docking-derived low affinity THA peripheral site in AChE. The present work and our previous computational studies strongly suggest that a low affinity THA peripheral site exists in AChE. This peripheral site provides a structural basis for design of improved cholinesterase ligands for treating Alzheimer's disease and for other health-related purposes.

Kit is a receptor tyrosine kinase (RTK) that binds stem cell factor. This receptor ligand combination is important for normal hematopoiesis, as well as pigmentation, gut function, and reproduction. Structurally, Kit has both an extracellular and intracellular region. The intracellular region is comprised of a juxtamembrane domain (JMD), a kinase domain, a kinase insert, and a carboxyl tail. Inappropriate expression or activation of Kit is associated with a variety of diseases in humans. Activating mutations in Kit have been identified primarily in the JMD and the second part of the kinase domain and have been associated with gastrointestinal stromal cell tumors and mastocytosis, respectively. There are also reports of activating mutations in some forms of germ cell tumors and core binding factor leukemias. Since the cloning of the Kit ligand in the early 1990s, there has been an explo sion of information relating to the mechanism of action of normal forms of Kit as well as activated mutants. This is important because understanding this RTK at the biochemical level could assist in the development of therapeutics to treat primary and secondary defects in the tissues that require Kit. Furthermore, understanding the mechanisms mediating transformation of cells by activated Kit mutants will help in the design of interventions for human disease associated with these mutations. The objective of this review is to summarize what is known about normal and oncogenic forms of Kit. We will place particular emphasis on recent developments in understanding the mechanisms of action of normal and activated forms of this RTK and its association with human disease, particularly in hematopoietic cells.

G-protein-gated inwardly rectifying K؉ (GIRK) channels are widely expressed in the brain and are activated by at least eight different neurotransmitters. As K ؉ channels, they drive the transmembrane potential toward E K when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits (GIRK1-4 or Kir 3.1-4), with GIRK1 being the most unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However, more recent data indicate that native channels can be either homo-or heterotetrameric complexes composed of several GIRK subunit combinations. The functional implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK channels formed by the co-assembly of GIRK2 and GIRK3, the most abundant GIRK subunits found in the mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at the single channel level was an ϳ5-fold lower sensitivity to activation by G␤␥. Complexes containing only GIRK2 and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels composed of GIRK2 and GIRK3 subunits exist in brain.