▪ Abstract In the mid to late 1980s, studies were published that provided the first evidence for the existence of glutamate receptors that are not ligand-gated cation channels but are coupled to effector systems through GTP-binding proteins. Since those initial reports, tremendous progress has been made in characterizing these metabotropic glutamate receptors (mGluRs), including cloning and characterization of cDNA that encodes a family of eight mGluR subtypes, several of which have multiple splice variants. Also, tremendous progress has been made in developing new highly selective mGluR agonists and antagonists and toward determining the physiologic roles of the mGluRs in mammalian brain. These findings have exciting implications for drug development and suggest that the mGluRs provide a novel target for development of therepeutic agents that could have a significant impact on neuropharmacology.

Pharmacology and functions of metabotropic glutamate receptors.

The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.

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Glutamate Receptor Ion Channels: Structure, Regulation, and Function

Microglial cells are the resident macrophages in the central nervous system. These cells of mesodermal/mesenchymal origin migrate into all regions of the central nervous system, disseminate through the brain parenchyma, and acquire a specific ramified morphological phenotype termed "resting microglia." Recent studies indicate that even in the normal brain, microglia have highly motile processes by which they scan their territorial domains. By a large number of signaling pathways they can communicate with macroglial cells and neurons and with cells of the immune system. Likewise, microglial cells express receptors classically described for brain-specific communication such as neurotransmitter receptors and those first discovered as immune cell-specific such as for cytokines. Microglial cells are considered the most susceptible sensors of brain pathology. Upon any detection of signs for brain lesions or nervous system dysfunction, microglial cells undergo a complex, multistage activation process that converts them into the "activated microglial cell." This cell form has the capacity to release a large number of substances that can act detrimental or beneficial for the surrounding cells. Activated microglial cells can migrate to the site of injury, proliferate, and phagocytose cells and cellular compartments.

Physiology of Microglia

Voltage-gated Ca(2+) channels mediate Ca(2+) entry into cells in response to membrane depolarization. Electrophysiological studies reveal different Ca(2+) currents designated L-, N-, P-, Q-, R-, and T-type. The high-voltage-activated Ca(2+) channels that have been characterized biochemically are complexes of a pore-forming alpha1 subunit of approximately 190-250 kDa; a transmembrane, disulfide-linked complex of alpha2 and delta subunits; an intracellular beta subunit; and in some cases a transmembrane gamma subunit. Ten alpha1 subunits, four alpha2delta complexes, four beta subunits, and two gamma subunits are known. The Cav1 family of alpha1 subunits conduct L-type Ca(2+) currents, which initiate muscle contraction, endocrine secretion, and gene transcription, and are regulated primarily by second messenger-activated protein phosphorylation pathways. The Cav2 family of alpha1 subunits conduct N-type, P/Q-type, and R-type Ca(2+) currents, which initiate rapid synaptic transmission and are regulated primarily by direct interaction with G proteins and SNARE proteins and secondarily by protein phosphorylation. The Cav3 family of alpha1 subunits conduct T-type Ca(2+) currents, which are activated and inactivated more rapidly and at more negative membrane potentials than other Ca(2+) current types. The distinct structures and patterns of regulation of these three families of Ca(2+) channels provide a flexible array of Ca(2+) entry pathways in response to changes in membrane potential and a range of possibilities for regulation of Ca(2+) entry by second messenger pathways and interacting proteins.

Structure and regulation of voltage-gated Ca2+ channels.

In electrically nonexcitable cells, Ca2+ influx is essential for regulating a host of kinetically distinct processes involving exocytosis, enzyme control, gene regulation, cell growth and prolifera...

Store-operated calcium channels.

Genetic markers associated with Alzheimer's disease are provided. Also provided are methods of determining the presence or absence in a subject of one or more polymorphisms associated with Alzheimer's disease and methods of determining the level of risk for Alzheimer's disease in a subject. Further provided are nucleic acid compositions and kits for use in determining the presence or absence in a subject of one or more polymorphisms associated with Alzheimer's disease and kits for determining the level of risk for Alzheimer's disease in a subject.

Genetic markers for Alzheimer's disease and methods using the same

In accordance with the present invention, it has been discovered that compounds which modulate the γ-secretase enzyme to make more of the shorter, less toxic and less aggregation-prone Aβ peptides (such as Aβ37 and Aβ38), while making less of the longer and more toxic and aggregation-prone AB peptides (such as AB40 and AB42) are useful as gamma-secretase modulators. In addition, these GSM compounds have further been discovered to have the selective property of modulating the formation of various AB peptides, while not inhibiting the overall activity of the γ-secretase enzyme. Thus, such compounds do not impede other critical functions of the γ-secretase enzyme, such as generating fragments from Notch that appear to control gene expression and cell differentiation. Therefore, in accordance with the present invention, there are provided screening assays useful for determining whether test compounds have GSM activity; accordingly, invention assays facilitate the identification of new gamma-secretase modulators. Such screening assays can be performed in a variety of ways, e.g., by assessing whether test compounds: lower the level of Aβ42 peptide,increase the level of Aβ37 and/or Aβ38 peptides, have substantially no effect on the processing of other γ-secretase substrates, and/or interact with at least one component of the γ-secretase complex. Also provided in accordance with the present invention are compounds having gamma-secretase modulatory activity as identified by any of the methods described herein; methods for producing formulations useful for modulating gamma-secretase activity, as well as the resulting formulations; methods for modulating gamma-secretase activity employing compounds identified according to invention methods and/or invention formulations, and the like.

Gamma-secretase modulatory compounds, methods for identifying same, and uses therefor

Calcium-release activated calcium (CRAC) channel inhibitors represent a new class of oral immunomodulatory agents with a potential safety profile suitable for chronic dosing to treat autoimmune disorders. Calcium entry through CRAC channels is a critical step in the functional responses of T cells and mast cells. Patients with mutations in CRAC channels are known to have a form of severe combined immunodeficiency, wherein the adaptive immune response is suppressed without major impairment of other organ systems. CalciMedica has discovered several novel small molecules that selectively inhibit CRAC channels, and is currently developing the first of these inhibitors, CM2489, for the treatment of psoriasis. We describe here the properties of CM3457, another selective CRAC channel inhibitor that is structurally distinct from CM2489. CM3457 potently inhibits CRAC channels, Th1, Th2 and Th17-derived cytokine production, T cell proliferation, and mast cell degranulation. Once-daily oral dosing of CM3457 significantly inhibits inflammation and joint histopathology in a rat collagen-induced arthritis model. CM3457 also inhibits lung inflammation and eosinophilia, as well as improves lung function, in a rat OVA-induced asthma model. These data provide evidence that CM3457 may be an effective therapeutic in the treatment of autoimmune disorders and asthma.

CM3457, a potent and selective oral CRAC channel inhibitor, suppresses T and mast cell function and is efficacious in rat models of arthritis and asthma (72.3)

The presence of specific receptors for vasoactive intestinal peptide (VIP) in numerous cell and membrane preparations has been well documented.’ The binding of VIP to cell-surface receptors correlates with the stimulation of adenylate cyclase activity and elevation of intracellular concentration of CAMP, leading to the biological effects of the peptide.2 Hence, structural and functional characterization of VIP receptors will be integral to understanding the molecular mechanism of action of VIP. Our studies have focused on the characterization of VIP receptors from rat lung. There is evidence that the lung may be an important target organ for the actions of VIP based on the demonstration of VIP innervation associated with airways and pulmonary vessels,’ bronchodilatory effects of VIP in humans,’ and its relaxant effects on pulmonary vascular smooth muscle.’ In this study, we describe characterization of VIP receptors from rat lung membranes both in the membrane-bound and detergent-solubilized states. In the former case, rat lung membrane proteins were covalently labeled with [‘2’I]VIP using a bifunctional cross-linker and a 55-kDa species was identified as a ligand-binding unit of rat lung VIP receptors. Cross-linking studies have been reported in greater detail elsewhere.6 In the latter case, [ 12’I]VIP-labeled, membrane-bound receptors were solubilized in Triton X-100 and studied by gel filtration and sucrose density gradient sedimentation, yielding an apparent molecular weight of 270,000 for the [ 12’I]VIPreceptor complex in Triton X-100 solution.

Structural characterization of vasoactive intestinal peptide receptors from rat lung membranes.

This invention relates to a process of recombinant DNA technology and means for producing polypeptides having Interleukin-2 (IL-2) activity in Pichia pastoris (P. pastoris) yeast cells. In another aspect, the invention relates to a process for extracting a polypeptide having IL-2 activity from P. pastoris cells. IL-2 is a naturally-occurring polypeptide that has a great potential in treatment of immunological disorders, bacterial or viral infections and other severe clinical conditions.

Gonul Velicelebi

Papers

Genetic markers for Alzheimer's disease and methods using the same

Gamma-secretase modulatory compounds, methods for identifying same, and uses therefor

CM3457, a potent and selective oral CRAC channel inhibitor, suppresses T and mast cell function and is efficacious in rat models of arthritis and asthma (72.3)

Structural characterization of vasoactive intestinal peptide receptors from rat lung membranes.

Production of interleukin-2 polypeptides in pichia pastoris yeast cells