Cyclosporins:  Structure−Activity Relationships for the Inhibition of the Human MDR1 P-Glycoprotein ABC Transporter

Abstract

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.

PK11195, a peripheral benzodiazepine receptor (pBR) ligand, broadly blocks drug efflux to chemosensitize leukemia and myeloma cells by a pBR-independent, direct transporter-modulating mechanism

“…Therefore, we prepared PMs from DOX40 cells to further characterize PK11195 binding in Pgp-expressing cells and documented that PM fractions contained Pgp but were depleted of mitochondrial inner and outer mitochondrial membrane markers ( Figure 6A). DOX40 PM fractions showed specific 3 H-CSA binding, consistent with documented Pgp binding by CSA, 31,32,[43][44][45][46][47][48][49][50][51] and specific 3 H-PK11195 binding was also reproducibly measured in DOX40 PMs. Specific PK11195 binding that was measured in 8226 PM assays (data not shown) suggests that pBR can be PM-localized, consistent with other reports.…”

“…CSA and many other efflux modulators bind transporters directly. 31,32,[43][44][45][46][47][48][49][50][51] First, we asked whether PK11195 binds Pgp, first by comparing PK11195 binding in 9 primary AML samples for which additional aliquots were available. CSA and PK11195 had increased dye retention in 5 of these samples (Pgp-positive AMLs), whereas 4 samples showed no efflux capacity (Pgp-negative AMLs).…”

Intestinal Ciprofloxacin Efflux: The Role of Breast Cancer Resistance Protein (ABCG2)

“…It is now known that CsA inhibits both P-gp (Loor et al, 2002) and also BCRP, albeit at lower affinity (Gupta et al, 2006;Matsson et al, 2009). Application of high-dose (50 M) CsA abolished net secretory ciprofloxacin flux in bcrp1-MDCKII cells (from 0.24 Ϯ 0.02 to 0.04 Ϯ 0.02 nmol ⅐ cm Ϫ2 ⅐ h Ϫ1 , n ϭ 3; P Ͻ 0.05 versus controls) but inhibited only a fraction of net secretory flux in low passage Caco-2 cell monolayers (from 0.39 Ϯ 0.06 to 0.22 Ϯ 0.04 nmol ⅐ cm Ϫ2 ⅐ h Ϫ1 , n ϭ 3; P Ͻ 0.05).…”

The Immunosuppressant Cyclosporin A Antagonizes Human Formyl Peptide Receptor through Inhibition of Cognate Ligand Binding

“…CsA is much less potent than CsH in inhibiting fMLF-induced superoxide production (11). Furthermore, although more recent studies demonstrated the inhibition of fMLF-induced degranulation by CsA and its analogues, it remains unclear whether the receptor or a signaling pathway was targeted by these cyclosporins (14,29). Several immunosuppressants have been shown to inhibit mast cell degranulation induced by FcRI cross-linking (27,30), an effect apparently mediated through a mechanism shared among these agents and related to their immunosuppressive properties (15).…”