Mammalian mitochondria possess an inner membrane channel, the permeability transition pore (MTP), which can be inhibited by nanomolar concentrations of cyclosporin (CS) A. The molecular basis for MTP inhibition by CSA remains unclear. Mitochondria also possess a matrix cyclophilin (CyP) with a unique N-terminal sequence (CyP-M). To test the hypothesis that it interacts with the MTP, we have studied the interactions of CyP-M with rat liver mitochondria by Western blotting with a specific antibody against its unique N terminus. Although sonication in isotonic sucrose at pH 7.4 releases a large proportion of CyP-M, a sizeable CyP-M fraction sediments with submitochondrial particles at 150,000 ؋ g. We show that the interactions of this CyP-M pool with submitochondrial particles are disrupted (i) by the addition of CSA, which inhibits the pore, but not of CSH, which does not, and (ii) by acidic pH condition, which also leads to selective inhibition of the MTP; furthermore, we show that the effect of acidic pH on CyP-M binding is prevented by diethylpyrocarbonate, which fully prevents the inhibitory effect of H ؉ on the MTP (Nicolli, A., Petronilli, V., and Bernardi, P. (1993) Biochemistry 32, 4461-4465). These data suggest that CyP-M binding is involved in opening of the MTP and that pore inhibition by CSA and protons may be due to unbinding of CyP-M from its putative binding site on the MTP. A role for CyP-M in MTP regulation is also supported by a study with a series of CSA derivatives with graded affinity for CyP. We show that with each derivative the potency at inhibition of the peptidylprolyl cis-transisomerase activity of CyP-M purified to homogeneity is similar to that displayed at inhibition of MTP opening, relative to that displayed by CSA. Decreased binding to CyP-M (but not CyP-A) and decreased efficiency at MTP inhibition is obtained by substitutions in position 8 while a 4-substituted, nonimmunosuppressive derivative is as effective as the native CSA molecule, indicating that calcineurin is not involved in MTP inhibition by CSA.
The transport of a fluorescent cyclosporin analogue was measured in killifish (Fundulus heteroclitus) proximal tubules by means of epifluorescence microscopy and digital image analysis. Renal cells rapidly accumulated the cyclosporin analogue from the medium and attained steady state within 60 min; luminal fluorescence increased over the first 60-90 min. At steady state, luminal fluorescence intensity was two to three times higher than cellular. Cellular fluorescence intensity was a linear function of medium substrate concentration and was not affected by any treatment used. In contrast, luminal fluorescence exhibited a saturable component as the medium concentration of the cyclosporin was increased. Secretion into the lumen was blocked by metabolic inhibitors, vanadate, other cyclosporins, such as cyclosporin A and cyclosporin G, and substrates for P-glycoprotein (verapamil, vinblastine, and quinine) but not by substrates for the renal organic anion or organic cation transport systems, such as p-aminohippurate or tetraethylammonium. The data are consistent with the fluorescent cyclosporin analogue entering proximal tubule cells by simple diffusion and then being pumped into the tubular lumen by P-glycoprotein.
Cyclosporins, in particular the nonimmunosuppressive derivative SDZ NIM 811, exhibit potent anti-human immunodeficiency virus type 1 (HIV-1) activity in vitro. SDZ NIM 811 interferes at two stages of the viral replication cycle: (i) translocation of the preintegration complex to the nucleus and (ii) production of infectious virus particles. Immunosuppressive activity is not correlated with anti-HIV-1 activity of cyclosporins. However, binding to cyclophilin A, the major cellular receptor protein of cyclosporins, is a prerequisite for HIV inhibition: all structural changes of the cyclosporin A molecule leading to loss of affinity to cyclophilin abolished the antiviral effect. Cyclosporin derivatives did not interact directly with HIV-1 proteins; cyclophilin was the only detectable receptor protein for antivirally active cyclosporins. There is no evidence that inhibition of HIV occurs via a gain of function of cyclophilin in the presence of cyclosporins: the complex of cyclophilin A with SDZ NIM 811 does not bind to calcineurin or to any other viral or cellular proteins under conditions in which calcineurin binding to the cyclophilin A-cyclosporin A complex is easily detectable. Thus, the loss of function caused by binding of cyclosporins to cyclophilin seems to be sufficient for the anti-HIV effect. Cyclophilin A was demonstrated to bind to HIV-1 p24 gag , and the formation of complexes was blocked by cyclosporins with 50% inhibitory concentrations of about 0.7 M. HIV-2 and simian immunodeficiency virus are only weakly or not at all inhibited by cyclosporins. For gag-encoded proteins derived from HIV-1, HIV-2, or simian immunodeficiency virus particles, cyclophilin-binding capacity correlated with sensitivity of the viruses to inhibition by cyclosporins. Cyclophilin A also binds to HIV-1 proteins other than gag-encoded proteins, namely, p17 gag , Nef, Vif, and gp120 env ; the biological significance of these interactions is questionable. We conclude that HIV-1 Gag-cyclophilin A interaction may be essential in HIV-1 replication, and interference with this interaction may be the molecular basis for the antiviral activity of cyclosporins.
Cyclosporine is a new immunosuppressive drug which was first marketed in 1983 under the trade name Sandimmune®. This compound, an innovation in selective immune modulation, was isolated from a fungal culture and characterized as a cyclic undecapeptide containing a novel amino acid together with several N‐methylated amino acids. The new amino acid (4R)‐4‐[(E)‐2‐butenyl]‐4,N‐dimethyl‐L‐threonine (MeBmt) was the only unknown amino acid of cyclosporine and there had previously been no means for its isolation. For this reason and because it seemed possible that MeBmt could play a significant role in determining the pharmacological activity of cyclosporine, its synthesis in enantiomerically pure form was undertaken. The next step was the development of a total synthesis of cycloporine, which appeared attractive, not only for its intrinsic worth, but also as an important tool for investigating the relationships between structure and immunosuppressive activity. Essential for the immunosuppression are the amino acids MeBmt, Abu, Sar, and MeVal in the positions 1, 2, 3 and 11, but probably also still larger parts of the molecule. Such information could be valuable for finding new chemical leads or drugs with a new activity profile.
The conformation of [D-MeSe?-D-Ser-(O-Gly)*]CS, a water soluble cyclosporin derivative, has been determined in (D,)DMSO and in water using NMR. In these polar solvents the conformation is identical and very similar to the structure found in the cyclophilin-cyclosporin complex. However, it differs significantly from its conformation in deuterated chloroform. This demonstrates unambiguously that the large structure change is induced primarily by the polar solvent rather than by complex formation with cyclophilin.
Although several cytosolic proteins including calmodulin and cyclophilin have been shown to bind cyclosporine, the direct involvement of these proteins in the immunosuppressive activity of cyclosporine remains to be established. In the present study, a quantitative immunoassay for cyclophilin was developed which made it possible to compare its relative affinity for cyclosporine and any of its analogues. The binding of cyclophilin to cyclosporine coated on a solid phase was revealed by anti-cyclophilin rabbit antiserum followed by antiglobulin-enzyme conjugate. This reaction could be inhibited by addition of free cyclosporine or certain cyclosporine analogues. By studying the binding of cyclophilin to more than fifty cyclosporine derivatives modified singly on each of the eleven amino acid residues, it could be shown that cyclophilin binds to the residues of cyclosporine known to be critical for its immunosuppressive activity. These data identify cyclophilin as a highly discriminating stereospecific binding protein for cyclosporine.
The heptapeptide H‐MeBmt‐Abu‐Sar‐MeLeu‐Val‐MeLeu‐Ala‐OBzl (20) was synthesized for coupling with the previously described cyclosporine tetrapeptide sequence Boc‐D‐Ala‐MeLeu‐MeVal‐OH (21). The product of the coupling, the undecapeptide Boc‐D‐Ala‐MeLeu‐MeLeu‐MeVal‐MeBmt‐abu‐Sar‐MeLeu‐Val‐MeLeu‐Ala‐OBzl (22), was then deprotected and cyclized to cyclosporine (1). The tetrapeptide diastereoisomer Boc‐D‐ala‐MeLeu‐MeLeu‐D‐MeVAl‐OH (23) could also be used as a starting material to produce selectively the desired undecapeptide 22. In this case, the N‐methyl‐D‐valine unit, was selectively isomerized to the L‐from by using the appropriate condensing agent. The diastereoisomeric undecapeptide Boc‐D‐ala‐MeLeu‐MeLeu‐D‐MeVal‐MeBmt‐Abu‐Sar‐MeLeu‐Val‐MeLeuAla‐OBzl (24) was also synthesized starting from 21 by using the mixed pivalic anhydride method to selectively invert the configuration of the N‐methyl‐L‐valine. The structure of the undecapeptide 24 was confirmed by deprotection and cyclization to ‘cyclosporin H’, a natural product known to have the structure [D‐MeVal11]cyclosporine (2).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.