Cyclosporin H Is a Potent and Selective Formyl Peptide Receptor Antagonist

he cyclic undecapeptide, cyclosporin (Cs) H, is a potent inhibitor of FMLP-induced superoxide anion (02-) formation in human neutrophils. We studied the effects of CsH in comparison with those of N-tbutoxycarbonyl-~-phenylalanyl-~-leucyl-~-phenyialanyl-~-ieucyi-~-phenylalanine IBocPLPLP), a well known formyl peptide receptor antagonist, and of other Cs on activation of Nh,2'-O-dibutyryl adenosine 3:5'-monophosphatedifferentiated HL-60 cells and human erythroleukemia cells (HEL cells). CsH inibited FMLP binding in HL-60 membranes with a Ki (inhibition constant) of 0.10 pM. CsH inhibited activation by FMLP of high affinity GTPase (the enzymatic activity of a-subunits of heterotrimeric regulatory guanine nucleotide-binding proteins) in HL-60 membranes with a Ki of 0.79 pM. CsH inhibited the stimulatory effects of FMLP on cytosolic CaL+ concentration ([CaZ'],), 02formation, and P-glucuronidase release with K, values of 0.08, 0.24, and 0.45 pM, respectively. BocPLPLP was 14-fold less potent than CsH in inhibiting FMLP binding and 4to 6-fold less potent than CsH in inhibiting FMLP-induced GTP hydrolysis, rises in'[Ca'+],, 02-formation, and 0-glucuronidase release. CsAreduced FMLP-induced 02formation by 20%, but CsB, CsC, CsD, and CsE did not. CsA, CsB, CsC, CsD, and CsE did not affect FMLP-induced rises in [Ca'+l,. BocPLPLP inhibited leukotriene B4-induced rises in [Ca'+Ii with a K,of 0.33 pM, whereas CsH showed no inhibitory effect. CsH and BocPLPLP did not inhibit the rises in [Ca2+], induced by several other stimuli in HL-60 cells and HEL cells. Our results show that 1) CsH is a more potent formyl peptide receptor antagonist than BocPLPLP; 2) unlike BocPLPLP, CsH is selective; and 3) N-methyl-o-valine which is present at position 11 of the amino acid sequence of CsH but not of other Cs is crucial for FMLP antagonism. Journal of Immunology, 1993, 150: 4591. T he chemotactic peptide, FMLP, binds to formyl peptide receptors in human and rabbit neutrophils and differentiated HL-60 leukemic cells and activates pertussis toxin-sensitive G-proteins' which possess high affinity GTPase activity (for review, see Refs. 1 4 ) . G-proteins mediate activation of phospholipase C with subReceived for publication July 17, 1992. Accepted for publicatlon February 4, 1993. The costs of publication of this article were defrayed in part by the payment oi page charges. This article must therefore be hereby marked ndverrisernenr in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and Fonds der Chemlschen Industrie. I This work was supported by grants of the Deutsche Forschungsjiemernschatt tirr Pharrnakologie, Freie Universitat Berlln, Thielsllee 69-73, D-1000 Berlln ' Address rorrespondence and wprint requests to Dr. Roland Seifert, lnstitut 33, Federal Republlc of Germany sequent activation of protein kinase C and increase in [Ca2+li (1 -4) . In addition, FMLP stimulates NADPH oxidase-catalyzed 0 2 formation and P-glucuronidase release from azurophilic granules (1-4). Rises in [Ca"], are evident with FMLP at much lower concentrations than activation of GTPase, 02formation, and enzyme release (1-6). Tertiary butoxycarbonyl analogs of FMLP are well known competitive antagonists at formyl peptide receptors and inhibit chemotactic peptide-induced cell activation