Immunosuppressive activity of [MeBm2t]1-, D-diaminobutyryl-8-, and D-diaminopropyl-8-cyclosporin analogues correlates with inhibition of calcineurin phosphatase activity.
Calcineurin, a Ca2+/calmodulin-dependent phosphatase, has recently been identified as a common target for cyclophilin A-cyclosporin A and FK506 binding protein 12-FK506 complexes. This study has examined the structure activity relationships of cyclosporin A (CsA) and three functionally distinct analogues, [MeBm2t]1-CsA, D-diaminobutyryl-8-CsA (Dab8-CsA), and D-diaminopropyl-8-CsA (Dap8-CsA). Immunosuppressive potency in T cell activation models, NF kappa B activation, and IL-2 mRNA transcription has been compared with analogue affinity for cyclophilin A and inhibition of calcineurin phosphatase activity. CsA, Dap8-CsA, and Dab8-CsA bind to cyclophilin A with a similar affinity (Ki 4 to 5 nM as measured by inhibition of prolyl cis-trans isomerase activity), however, Dap8-CsA and Dab8-CsA inhibit T cell activation less than CsA. Although [MeBm2t]-CsA has weak affinity for cyclophilin A (Ki 540 nM), its immunosuppressive potency is similar to that of CsA. Both cyclophilin A-CsA and cyclophilin A-[MeBm2t]1-CsA complexes inhibit calcineurin phosphatase activity in vitro (Ki 114 and 67 nM, respectively). In Jurkat cells exposed to CsA or the analogues for 2 h, endogenous calcineurin phosphatase activity in cell lysates was inhibited by CsA and [MeBm2t]1-CsA (drug concentrations causing 50% reduction in 32PO4 release of 8 and 55 nM, respectively) in proportion to inhibition of T cell activation, IL-2 mRNA transcription, and NF kappa B activation. Dap8-CsA and Dab8-CsA had a minimal effect on endogenous calcineurin phosphatase activity in Jurkat cell lysates. These findings correlate the functional activity of CsA and structural analogues with calcineurin phosphatase activity and support calcineurin as a target for drug action. The Dap8 and Dab8 modifications of CsA, occurring in residue 8, which is exposed to solvent in the cyclophilin A-CsA complex, appears to significantly alter complex affinity for calcineurin.
Nelson
P A
PA
Vertex Pharmaceuticals Incorporated, Cambridge MA 02139.
Akselband
Y
Y
Kawamura
A
A
Su
M
M
Tung
R D
RD
Rich
D H
DH
Kishore
V
V
Rosborough
S L
SL
DeCenzo
M T
MT
Livingston
D J
DJ
D000591:Amino Acid Isomerases / Q000378:metabolism; D001483:Base Sequence; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors*; D002352:Carrier Proteins / Q000378:metabolism; D002478:Cells, Cultured; D003524:Cyclosporins / Q000494:pharmacology*; D006801:Humans; D007376:Interleukin-2 / Q000096:biosynthesis / Q000235:genetics; D008213:Lymphocyte Activation / Q000187:drug effects; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000494:pharmacology; D019696:Peptidylprolyl Isomerase; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D011090:Polyenes / Q000633:toxicity; D011485:Protein Binding; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D020123:Sirolimus; D013347:Subcellular Fractions / Q000187:drug effects / Q000201:enzymology; D016559:Tacrolimus / Q000633:toxicity; D014158:Transcription, Genetic / Q000235:genetics
D002148:Calmodulin-Binding Proteins; D002352:Carrier Proteins; D003524:Cyclosporins; D007376:Interleukin-2; D046911:Macromolecular Substances; D016328:NF-kappa B; D011090:Polyenes; D012333:RNA, Messenger; C064083:8-hydroxy-6,7-dihydro-(4-(2-butenyl)-4,N-dimethylthreonine)cyclosporin; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D020123:Sirolimus; D016559:Tacrolimus