Douglas C. Hanson

Topology of the pore-region of a K+ channel revealed by the NMR-derived structures of scorpion toxins

The architecture of the pore-region of a voltage-gated K+ channel, Kv1.3, was probed using four high affinity scorpion toxins as molecular calipers. We established the structural relatedness of these toxins by solving the structures of kaliotoxin and margatoxin and comparing them with the published structure of charybdotoxin; a homology model of noxiustoxin was then developed. Complementary mutagenesis of Kv1.3 and these toxins, combined with electrostatic compliance and thermodynamic mutant cycle analyses, allowed us to identify multiple toxin-channel interactions. Our analyses reveal the existence of a shallow vestibule at the external entrance to the pore. This vestibule is approximately 28-32 A wide at its outer margin, approximately 28-34 A wide at its base, and approximately 4-8 A deep. The pore is 9-14 A wide at its external entrance and tapers to a width of 4-5 A at a depth of approximately 5-7 A from the vestibule. This structural information should directly aid in developing topological models of the pores of related ion channels and facilitate therapeutic drug design.

UK-78,282, a novel piperidine compound that potently blocks the Kv1.3 voltage-gated potassium channel and inhibits human T cell activation

UK‐78,282, a novel piperidine blocker of the T lymphocyte voltage‐gated K+ channel, Kv1.3, was discovered by screening a large compound file using a high‐throughput 86Rb efflux assay. This compound blocks Kv1.3 with a IC50 of ∼200 nM and 1 : 1 stoichiometry. A closely related compound, CP‐190,325, containing a benzyl moiety in place of the benzhydryl in UK‐78,282, is significantly less potent. Three lines of evidence indicate that UK‐78,282 inhibits Kv1.3 in a use‐dependent manner by preferentially blocking and binding to the C‐type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at −50 mV enhances the channels sensitivity to UK‐78,282. Decreasing the number of inactivated channels by exposure to ∼160 mM external K+ decreases the sensitivity to UK‐78,282. Mutations that alter the rate of C‐type inactivation also change the channels sensitivity to UK‐78,282 and there is a direct correlation between τh and IC50 values. Competition experiments suggest that UK‐78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK‐78,282s action on the channel. UK‐78,282 displays marked selectivity for Kv1.3 over several other closely related K+ channels, the only exception being the rapidly inactivating voltage‐gated K+ channel, Kv1.4. UK‐78,282 effectively suppresses human T‐lymphocyte activation.

The pharmacologic regulation of interleukin-1 production: The role of prostaglandins

The role of prostaglandins in the regulation of lipopolysaccharide (LPS)-induced interleukin-1 (IL-1) production by murine C3H/HeN resident peritoneal macrophages was studied. IL-1 production was initially studied in the presence of piroxicam and indomethacin, both inhibitors of prostaglandin biosynthesis. IL-1 was assayed using the IL-1-dependent proliferative response of C3H/HeJ thymocytes. LPS stimulation resulted in 15 to 20 ng/ml of prostaglandin E2 (PGE2) produced in the first hour of culture. IL-1-containing supernatants from drug-treated macrophages at dilutions of up to 1:32 resulted in enhanced thymocyte proliferation compared to control, non-drug-treated cultures and contained less than 2 ng/ml of PGE2. Similar enhancement of proliferation could be obtained by incubating non-drug-treated supernatants with monoclonal anti-PGE2 but not anti-thromboxane B2 (TxB2) antibody. Further dilutions of the drug-treated supernatants gave thymocyte proliferation responses which were indistinguishable from control cultures and, correspondingly, had identical values for IL-1 production. The absence of an effect on IL-1 production was confirmed by quantitation of intracellular IL-1 alpha using goat anti-IL-1 alpha antibody and by quantitation of supernatant IL-1 receptor competition assay. Exogenous PGE2, in the concentration range produced in macrophage supernatants (10-20 ng/ml), directly inhibited IL-1-stimulated thymocyte proliferation. Finally, when macrophages were stimulated with LPS for 24 hr in the presence of added PGE2, thymocyte proliferation was inhibited at the lowest supernatant dilutions, but as the IL-1-containing supernatants were diluted out, the assay curves were indistinguishable from non-PGE2-treated control. Thus, in this system, PGE2 has no effect on IL-1 synthesis, but rather has a direct inhibitory effect on thymocyte proliferation. Nonsteroidal anti-inflammatory drugs are not stimulating IL-1 production but are, in fact, relieving inhibition of the thymocyte IL-1 assay caused by the presence of prostaglandins.

Inhibition of interleukin 1 synthesis by tenidap: A new drug for arthritis

Tenidap is a new antiarthritic drug of novel chemical structure. This study shows the effects of tenidap on the in vitro synthesis of interleukin 1 (IL-1). IL-1 production by murine peritoneal macrophages was induced either by stimulation with lipopolysaccharide (LPS) or by phagocytosis of zymosan. With either stimulus, tenidap inhibited IL-1 production as measured by a quantitative competitive IL-1 receptor binding assay. Approximately 20 ng/mL of IL-1 was produced by 10(6) macrophages in response to LPS and about half that amount was produced in response to zymosan. Fifty percent inhibition of IL-1 production by tenidap was found at 3 microM for both stimuli. Using goat anti-IL-1 alpha and Western blot analysis, the appearance of intracellular 34 kDa pro-IL-1 alpha was inhibited by tenidap down to 3 microM. Tenidap decreased [35S]Met incorporation into cellular protein at 30 microM but not at 10 or 3 microM, indicating selectivity for IL-1 inhibition relative to total protein synthesis. Because tenidap inhibited IL-1 induction by both zymosan and LPS, it must act subsequently to receptor triggering. As the appearance of IL-1 was inhibited both intracellularly and extracellularly, the primary drug effect cannot be on secretion.

Dynamic aspects of antibody structure

The flexible nature of immunoglobulins is clearly demonstrated in electron micrographs of antibody complexes, and serves as the structural basis for the interpretation of time-resolved fluorescence depolarization studies. The extent of the flexibility seen with the electron microscope (EM) will be the initial topic of this review. We will compare rabbit polyclonal and mouse monoclonal antibodies, demonstrating by electron microscopy some interesting differences in flexibilities; the contrast provides new information. A direct approach to immunoglobulin dynamics through time-resolved fluorescence depolarization is discussed in the second part, and a simple theoretical interpretation is elaborated in some detail. Anisotropy decay curves for rabbit, human and mouse IgG antibodies are analysed in the light of this simple theory. Immunoglobulin dynamics, as measured by time-resolved fluorescence depolarization, is interpreted in terms of the flexible structures seen in the electron micrographs. The length of the upper hinge polypeptide is the predominant structural feature which determines both the long correlation time of the anisotropy decay curves and the percentage of closed-hinge dimers seen in the electron micrographs. It is suggested that interactions between residues of the hinge and the C-terminal portions of first constant domains are responsible for the restriction in flexibility associated with short hinge length.

The SAR of UK-78,282: a novel blocker of human T cell Kv1.3 potassium channels

Abstract UK-78,282 was identified in a human T cell 86Rb efflux high-throughput screen of our compound libraries. This compound was found to be a potent and selective blocker of human T cell voltage-gated K+ channels and to inhibit T cell activation. The SAR around UK-78,282 and a general pharmacophore hypothesis are presented in this communication.

Chapter 18. T Lymphocyte Potassium Channel Blockers

Publisher Summary Advances in molecular biology and patch-clamp technology reflect in the discovery of type- n voltage gated potassium channel (Kv1.3) in T lymphocytes. Small-molecule blockers of Kv1.3 inhibit mitogen-induced activation of T cells. As these agents are weak inhibitors and display low selectivity, it is evident that more potent and specific blockers are necessary to convincingly define the role of Kv1.3 in T cell activation. Several such selective high-affinity blockers, both peptide and small-molecule, have been discovered in recent years. It is now thought that Kv1.3 blockers chronically depolarize the T cell membrane, attenuate calcium entry via calcium release-activated calcium (CRAC) channels in the plasma membrane, and consequently abrogate the calcium signaling pathway that is essential for lymphocyte activation. The vital role of Kv1.3 during T cell activation has stimulated a search for potent and selective channel blockers that can be used as immunosuppressive agents. Because of its distinct mechanism and restricted tissue distribution, a Kv1.3 blocker is not likely to initiate the same toxic side-effects of currently used immunosuppressants, such as cyclosporin and FK-506, and therefore may prove useful in the treatment of chronic autoimmune diseases as well as transplantation therapy.