Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.

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We report the synthesis of a series of phosphinic acid dipeptide analogues, NH2CH(R1)PO(OH)CH2CH(R2)CO2H, related to DAla-DAla. The best of these compounds are potent, essentially irreversible inhibitors of DAla-DAla ligase, and their preferred stereochemistry was shown by chiral synthesis of (1(S)-aminoethyl)(2(R)-carboxy-1-n-propyl)phosphinic acid, 12b, and by X-ray crystallography of its derivative benzyl [1(S)-[(benzyloxycarbonyl)-amino]ethyl](2(R)-carbomethoxy-1-propyl) phosphinate, 13, to correspond to the stereochemical configuration of DAla-DAla at both centers. A mechanism for the inhibition of DAla-DAla ligase by these compounds is proposed to involve an ATP-dependent formation of phosphorylated inhibitor within the enzyme's active site. The antibacterial activities of these compounds are modest although their spectra include both Gram-positive and Gram-negative susceptible organisms. The best antibacterial activity was shown by (1(S)-aminoethyl) [2-carboxy-2(R)-(methylthio)-1-ethyl]phosphinic acid, 3e, whose MIC's range from 4-128 micrograms/mL on nine of a panel of 11 bacterial organisms. Combination of one of the more active phosphinic acids 12b with the alanine racemase inhibitor fluoro-D-alanine enhances the antibacterial spectrum of the latter on several strains of bacteria and inhibits fluoro-D-alanine's self-reversal, which normally occurs at concentrations several fold higher than its MIC level. This inhibition of fluoro-D-alanine self-reversal is consistent with an involvement of DAla-DAla ligase inhibition in the antibacterial activity of these compounds.

The voltage-gated potassium channel, K(v)1.3, is a novel target for development of immunosuppressants. Using a functional (86)Rb(+) efflux assay, a new class of high-affinity K(v)1.3 inhibitors has been identified. The initial active in this series, 4-phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1-yl]cyclohexanone (PAC), which is representative of a disubstituted cyclohexyl (DSC) template, displays a K(i) of ca. 300 nM and a Hill coefficient near 2 in the flux assay and in voltage clamp recordings of K(v)1.3 channels in human T-lymphocytes. PAC displays excellent specificity as it only blocks members of the K(v)1 family of potassium channels but does not affect many other types of ion channels, receptors, or enzyme systems. Block of K(v)1.3 by DSC analogues occurs with a well-defined structure-activity relationship. Substitution at the C-1 ketone of PAC generates trans (down) and cis (up) isomer pairs. Whereas many DSC derivatives do not display selectivity in their interaction with different K(v)1.x channels, trans DSC derivatives distinguish between K(v)1.x channels based on their rates of C-type inactivation. DSC analogues reversibly inhibit the Ca(2+)-dependent pathway of T cell activation in in vitro assays. Together, these data suggest that DSC derivatives represent a new class of immunosuppressant agents and that specific interactions of trans DSC analogues with channel conformations related to C-type inactivation may permit development of selective K(v)1.3 channel inhibitors useful for the safe treatment of autoimmune diseases.