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 24,000-dalton polypeptide that binds strongly and can be specifically crosslinked to the 5'-terminal cap structure m7GpppN in eukaryotic mRNAs has been detected in protein synthesis initiation factor preparations [Proc. Nat. Acad. Sci. USA (1978) 75, 4843-4847]. This polypeptide has been purified to apparent homogeneity by one chromatographic passage through an affinity resin prepared by coupling the levulinic acid 02',3'-acetal of'm7GDP to AH-Sepharose 4B. Translation A striking structural feature of most eukaryotic mRNAs that distinguishes them from prokaryotic mRNAs is the 5'-terminal cap," m7GpppN (1). This modification occurs on nascent pre-mRNA chains and is conserved as a stabilizing element in mature mRNA molecules (2-5). Eukaryotic and prokaryotic mRNAs are also functionally distinct. In contrast to prokaryotic protein synthesis, which initiates at multiple internal AUG codons in polycistronic mRNA, translation of many if not all eukaryotic mRNAs begins at a single site, the 5'-proximal AUG triplet. Recently, a scanning mechanism has been proposed to explain how eukaryotic ribosomes select the initiator codon (6, 7). In this model the small ribosomal subunit binds initially at or near the 5' terminus and only subsequently repositions at the adjacent AUG as the large subunit joins to begin peptide bond synthesis.Results of numerous studies have shown that the 5'-terminal cap promotes translation by facilitating initiation complex formation (1). It has been suggested further that the cap is recognized as part of the mechanism of ribosome attachment to mRNA (8). Consistent with this suggestion, eukaryotic initiation factor preparations contain a polypeptide of apparent molecular weight 24,000 that interacts specifically with the cap and can be crosslinked to the 5'-m7G of oxidized mRNA (9). This cap-binding protein was found in less than stoichiometric amounts in purified eIF-3 and eIF-4B (9), factors involved in the binding of natural mRNAs to initiation complexes (10). The same factors added in excess to reticulocyte lysate increased the weak translational activity of chemically decapped vesicular stomatitis virus mRNAs by severalfold to a level of about 50% of that of the corresponding capped mRNAs (11). Sucrose gradient sedimentation separated this stimulatory activity from eIF-3 and eIF-4B but it copurified with the cap-crosslinking 24,000-dalton polypeptide (11). These findings as well as other reports (12-15) suggest a regulatory role for eIF-4B and possibly other eukaryotic initiation factors. However, because the purified eIF-4B used in previous studies contained, in addition MATERIALS AND METHODS Preparation of Affinity Resins. Synthesis of levulinic acid acetals of m7GDP and GDP was carried out by a modification of the procedure of Seela and Waldek (16). The products were lyophilized and stored at -70°C in 5.6-,umol aliquots corresponding to 50 A2s units and 75 A2M units for the m7GDP and GDP derivatives, respectively. Immobilization of the nucleotide derivatives by coupling to resin ...

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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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.