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.

cDNA encoding murine FK506-binding protein (FKBP): nucleotide and deduced amino acid sequence.

A FKBP cDNA encoding murine FK506 binding protein (FKBP) has been cloned, and its complete nucleotide sequence has been determined. The open reading frame within the 1556-bp cDNA segment encodes an 108 amino acid (aa) protein that differs from the human FKBP by three aa and from the bovine FKBP by five aa. Molecular modeling of the protein places the aa substitutions at positions not directly involved in drug binding or interaction with the potential drug target protein, calcineurin A.

A negative regulator of mitosis in Aspergillus is a putative membrane-spanning protein.

The temperature-sensitive cell cycle mutation bimE7 of Aspergillus nidulans causes cells to become blocked in mitosis at a restrictive temperature. Previous work has shown that this mitotic block is induced even when cells are arrested in the S or G2 phase. The mitotic block is also observed in cells carrying a null mutation in bimE, obtained by molecular disruption of the gene (Osmani, S.A., Engle, D.B., Doonan, J.H., and Morris, N.R. (1988) Cell 52, 241-251), indicating that a lack of bimE function is responsible for the phenotype. We have cloned the bimE gene by complementation of the mutant phenotype and have isolated and sequenced its corresponding cDNA. The gene product is encoded by a 6.5-7-kilobase mRNA. The deduced amino acid sequence suggests a protein with three transmembrane domains. The sequence contains numerous potential N-glycosylation sites and several putative cAMP-dependent phosphorylation sites. No homologous protein sequences were found in the common data bases. The bimE gene product is a novel component in the regulation of mitosis.