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Cyclic undecapeptide cyclo-[MeBmt(1)-Abu(2)-MeGly(3)-MeLeu(4)-Val(5)-MeLeu(6)-Ala(7)-D-Ala(8)-MeLeu(9)-MeLeu(10)-MeVal(11)], the immunosuppressive and antifungal antibiotic cyclosporin A (CsA), was reported to interfere with the MDR1 P-glycoprotein (Pgp), a transmembranous adenosine 5'-triphosphate binding cassette (ABC) transporter with phospholipid flippase or "hydrophobic vacuum cleaner" properties that mediate multidrug resistance (MDR) of cancer cells. By use of photoaffinity-labeled cyclosporins and membranes from Pgp-expressing cells, it was recently shown that in vitro, Pgp molecules could bind a large cyclosporin domain involving residues 4-9 as well as the side chain of residue 1. Tumor cell MDR can also be reversed by a product more distantly related to cyclosporin with the structure [Thr(2), Leu(5), D-Hiv(8), Leu(10)]-CsA (SDZ 214-103). In a standardized assay that measures Pgp function in vivo (on intact live cells) by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells, SDZ 214-103 was found to be one of the most active Pgp inhibitors among naturally occurring cyclosporins, with an IC(50) of 1.6 microM in an assay where CsA gives an IC(50) of 3.4 microM. Using the in vivo assay, 60, mostly natural, cyclosporin analogues were analyzed to establish structure-activity relationships (SAR). Our SAR are compatible with the in vitro-defined Pgp binding domain model and further disclose that in vivo Pgp inhibition is favored by larger hydrophobic side chains on cyclosporin residues 1, 4, 6, and 8 and a smaller one on residue 7, although with no effect on the residue 5 side chain; moreover, larger hydrophobic side chains on other residues 2, 3, 10, and 11 (outside the in vitro-defined Pgp binding domain) also favor the eventual inhibition of Pgp function. The N-desmethylation of any of the seven N-methylated amides, as naturally occurring in numerous cyclosporins, regularly leads to a decreased Pgp inhibitory activity (Pgp-InhA), up to its abrogation if it occurs at residues 4 and 9. Nevertheless, despite unfavorable use of [Thr(2)] and [Leu(10)] residues, all [D-Hiv(8)] analogues whose lead is SDZ 214-103 show a large Pgp-InhA. The SAR for Pgp inhibition by cyclosporins are thus very complex. Because CsA and SDZ 214-103 show largely different conformations when free in solution, but remarkably similar ones when bound to the cytosolic cyclophilins, SAR for Pgp inhibition must similarly include requirements for occurrence of suitable conformers for insertion in the cell membrane, sufficient conformational plasticity for gaining access to Pgp binding sites, and an adequate conformer structure there to achieve such binding with a high enough affinity and possibly escape from sequestration on cyclophilins.

The human formylpeptide receptor (FPR) is a seven-transmembranous G-protein-coupled receptor (7TM-GPCR) for chemotactic peptides of bacterial origins, possibly involved in the recruitment and activation of neutrophils in various inflammatory diseases of mucosal epithelia. Mutational analyses suggest that interactions of formylated peptides with FPR occur on the outer exoplasmic leaflet/domains of the plasma membrane. The immunosuppressive and antifungal antibiotic cyclic undecapeptide cyclosporin A (CsA; cyclo-[MeBmt(1)-Abu(2)-MeGly(3)-MeLeu(4)-Val(5)-MeLeu(6)-Ala(7)-D-Ala(8)-MeLeu(9)-MeLeu(10)-MeVal(11)]) and some tested analogues such as [Ala(2)]-CsA, [Thr(2)]-CsA, [Val(2)]-CsA, and [Nva(2)]-CsA were able of inhibiting the binding of formylpeptides to the FPR, with [D-MeVal(11)]-CsA (CsH) being much more active than the other analogues. CsH is devoid of immunosuppressive and antifungal activities, and its large potency for human FPR inhibition is of inverse agonism origin. Formylpeptide binding to FPR-expressing cells does not only induce chemotaxis; it also causes a rapid release of granule enzymes in the extracellular medium, allowing the easy monitoring of any inhibition of FPR function "in vivo" (with intact live cells). With such an assay, CsH was confirmed to be the most potent FPR inhibitory cyclosporin, although a far related immunosuppressive cyclosporin analogue, FR901459 ([Thr(2), Leu(5), Leu(10)]-CsA), was found to display a high FPR inhibitory activity (FPR-InhA). To establish structure-activity relationships (SAR) for FPR function inhibition, 59 cyclosporins were now studied by this standardized assay (with differentiated human leukemic cell line HL-60 as FPR-expressing cells and with N-acetyl-beta-D-glucosaminidase release as read-out). These SAR confirmed the low FPR-InhA of classical cyclosporins, where such activity was only seldom found: the most active ones ([Thr(2), Ile(5)]-CsA, [aMeIle(11)]-CsA, and [MeAla(11)]-CsA) remained 3-10-fold less potent than CsH. In contrast, the SAR disclosed that N(10)-desmethylated cyclosporins were particularly prone to display a large FPR-InhA: their most potent one was a [Thr(2), Gly(3), Leu(5), D-Hiv(8), Leu(10)]-CsA, found to be only 2-4-fold less active than [D-MeVal(11)]-CsA (CsH), with which it shows six differences out of 11 residues. Because the free conformations of both CsH and N(10)-desmethylated cyclosporins differ from those of "classical" (N(10)-methylated, [L-MeVal(11)]-using) cyclosporins, these potent FPR inhibitory cyclosporins probably bind to FPR pharmacophores for which classical cyclosporins show little affinity. Moreover, because the conformations of the N(10)-desmethylated cyclosporins widely differ from the CsH one, they probably bind to different pharmacophores on the FPR molecules.

Cyclic depsipeptide cyclo-[D-Hmp(1)-L-MeVal(2)-L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle+ ++(6)-L-MeVal(7)-L-Leu(8)-L-betaHOMeVal(9)], the antifungal antibiotic aureobasidin A (AbA), was reported to interfere with ATP-binding cassette (ABC) transporters in yeast and mammalian cells, particularly the MDR1 P-glycoprotein (Pgp), a transmembrane phospholipid flippase or "hydrophobic vacuum cleaner" that mediates multidrug resistance (MDR) of cancer cells. In a standardized assay that measures Pgp function by the Pgp-mediated efflux of the calcein-AM Pgp substrate and uses human lymphoblastoid MDR-CEM (VBL(100)) cells as highly resistant Pgp-expressing cells and the cyclic undecapeptide cyclosporin A (CsA) as a reference MDR-reversing agent (IC(50) of 3.4 microM), AbA was found to be a more active Pgp inhibitor (IC(50) of 2.3 microM). Out of seven natural analogues and 18 chemical derivatives of AbA, several were shown to display even more potent Pgp-inhibitory activity. The Pgp-inhibitory activity was increased about 2-fold by some minor modifications such as those found in the naturally occurring aureobasidins AbB ([D-Hiv(1)]-AbA), AbC ([Val(6)]-AbA), and AbD [gammaHOMeVal(9)]-AbA). The replacement of the [Phe(3)-MePhe(4)-Pro(5)] tripeptide by an 8-aminocaprylic acid or the N(7)()-desmethylation of MeVal(7) led to only a 3.3-fold decreased capacity to inhibit Pgp function, suggesting that the Pgp inhibitory potential of aureobasidins, though favored by the establishment of an antiparallel beta-sheet between the [D-Hmp(1)-L-MeVal(2)-L-Phe(3)] and [L-aIle(6)-L-MeVal(7)-L-Leu(8)-] tripeptides, does not critically depend on the occurrence of the [L-Phe(3)-L-MePhe(4)-L-Pro(5)-L-aIle(6)] type II' beta-turn secondary structure. In contrast, the most potent Pgp inhibitors were found among AbA analogues with [betaHO-MeVal(9)] residue alterations, with some data suggesting a negative impact of the [L-Leu(8)-L-betaHOMeVal(9)-D-Hmp(1)] gamma-turn secondary structure on Pgp inhibitory potential. The [2,3-dehydro-MeVal(9)]-AbA was the most potent Pgp inhibitory aureobasidin, being 13-fold more potent than AbA and 19-fold more potent (on a molar basis) than CsA. Finally, there was no correlation between the SAR for the human MDR1 Pgp inhibition and the SAR for Saccharomyces cerevisiae antifungal activity, which is mediated by an inositol phosphoceramide synthase activity.

By sequestering cytosolic calcineurin into a molecular complex with cyclophilin and its consequent T-cell dysfunction, some cyclosporins, such as CsA and FR901459 ([Thr2-Leu5-Leu10]-CsA), display potent immunosuppressive activity. Independently on this property, cyclosporins may display one or more other biological activities mediated by interaction with cell surface glycoproteins. Several cyclosporins inhibit the function of human MDRI-encoded P-glycoprotein (Pgp), a flippase known to cause cancer multidrug resistance, but also expressed by some normal immunocompetent cells and by normal epithelial cells which control drug bioavailability in vivo. CsA is known to be a potent Pgp inhibitor with a 3.2 microM IC50 in an assay where the most potent derivative SDZ PSC 833 gives a 0.49 microM IC50. FR901459 is now shown to be a good Pgp inhibitor, being 2-fold weaker only (IC50 of 6 microM) than CsA. Some cyclosporins may also inhibit the function of the human FPR1-encoded formyl peptide receptor (FPR), a chemotactic receptor whose absence is known to impair antibacterial immunity. Yet this inhibition is very weak for all, but one of them, CsH, whose 0.15 micro/M IC50 makes it a much more potent FPR inhibitor than CsA (IC50 >10 microM in the same assay). FR901459 is now shown to be a very potent inhibitor of FPR function (IC50 of 0.6 microM). Since CsH shows little Pgp-inhibitory activity and has no known immunosuppressive activity, FR901459 displays a unique pharmacological profile: like CsA, it inhibits T-cell function; less than CsA, it can inhibit Pgp function on selected leukocyte subsets and on epithelial barriers known to control drug bioavailability; however, much more efficiently than CsA, it can inhibit the FPR function, a receptor involved in some leukocytic inflammatory responses to chemotactic peptides.