Synthesis and neurotrophic activity of nonimmunosuppressant cyclosporin A derivatives

In order to exploit cyclophilin as a potential target for neurological drug design, we demonstrate in this presentation that several nonimmunosuppressant analogues of cyclosporin A, modified at the various positions in the 'effector' domain, are equipotent nerve growth agents compared to cyclosporin A. Our results suggest that neurotrophic activity of cyclosporin A and its derivatives resides in the binding domain, and binding to cyclophilin and/or inhibiting rotamase activity may be a necessity for neurotrophic effects of cyclophilin ligands.     

The NMR structure of cyclosporin A bound to cyclophilin in aqueous solution

Cyclosporin A bound to the presumed receptor protein cyclophilin was studied in aqueous solution at pH 6.0 by nuclear magnetic resonance spectroscopy using uniform 15N- or 13C-labeling of cyclosporin A and heteronuclear spectral editing techniques. Sequence-specific assignments were obtained for all but one of the cyclosporin A proton resonances. With an input of 108 intramolecular NOEs and four vicinal 3JHN alpha coupling constants, the three-dimensional structure of cyclosporin A bound to cyclophilin was calculated with the distance geometry program DISMAN, and the structures resulting from 181 converged calculations were energy refined with the program FANTOM. A group of 120 conformers was selected on the basis of the residual constraint violations and energy criteria to represent the solution structure. The average of the pairwise root-mean-square distances calculated for the backbone atoms of the 120 structures was 0.58 A. The structure represents a novel conformation of cyclosporin A, for which the backbone conformation is significantly different from the previously reported structures in single crystals and in chloroform solution. The structure has all peptide bonds in the trans form, contains no elements of regular secondary structure and no intramolecular hydrogen bonds, and exposes nearly all polar groups to its environment. The root-mean-square distance between the backbone atoms of the crystal structure of cyclosporin A and the mean of the 120 conformers representing the NMR structure of cyclosporin A bound to cyclophilin is 2.5 A.     

Molecular modeling studies in the complex between cyclophilin and cyclosporin A.

The structure of the complex between cyclophilin and cyclosporin A is predicted by combining X-ray crystallographic and NMR spectroscopic data using molecular modeling. The drug was placed at the receptor site using a directed docking procedure in which an impulse is imparted to a pre-oriented ligand along an established path. Both ligand and receptor atoms are flexible during the procedure. Two conformers of the MeBMT side chain are shown to result in similar ligand-receptor interaction energies. The models for the drug-receptor complex appear consistent with known experimental data and provide a significant opportunity for the design of compounds with enhanced therapeutic value.     

Crystal structure of the complex of brugia malayi cyclophilin and cyclosporin A

The resistance of the human parasite Brugia malayi to the antiparasitic activity of cyclosporin A (CsA) may arise from the presence of cyclophilins with relatively low affinity for the drug. The structure of the complex of B. malayi cyclophilin (BmCYP-1) and CsA, with eight independent copies in the asymmetric unit, has been determined at a resolution of 2.7 A. The low affinity of BmCYP-1 for CsA arises from incomplete preorganization of the binding site so that the formation of a hydrogen bond between His132 of BmCYP-1 and N-methylleucine 9 of CsA is associated with a shift in the backbone of approximately 1 A in this region.     

The 3D structure of a cyclosporin analogue in water is nearly identical to the cyclophilin-bound cyclosporin conformation

The conformation of [ -MeSer³- -Ser-(O-Gly⁸]CS, a water soluble cyclosporin derivative, has been determined in (D₆)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.     

Effects of repeated injection of cyclosporin A on pentylenetetrazol-induced convulsion and cyclophilin mRNA levels in rat brain

To investigate the relationship between the immune system and convulsions in an animal model, we examined the effects of repeated administration with the immunosuppressant cyclosporin A on pentylenetetrazol (PTZ)-induced convulsions and the changes in the mRNA expression of its binding protein cyclophilin in the rat brain. The consecutive administration of cyclosporin A (5 mg/kg s.c., 14 days) significantly aggravated the severity of convulsions induced with PTZ 75 mg/kg i.p. Furthermore, it down-regulated the levels of cyclophilin mRNA in several brain regions and inhibited the PTZ-induced increase of hippocampal cyclophilin mRNA. Compared with the group without PTZ pretreatment or the group treated with chronic vehicle administration after the PTZ-preinjection, chronic cyclosporin A administration after the initial injection of PTZ apparently aggravated convulsions after the second PTZ injection. Interestingly, the increase in hippocampal cyclophilin mRNA observed after a single PTZ injection was not found after the second PTZ injection in the group with PTZ pretreatment. Therefore, these findings suggest that cyclosporin A administered peripherally can affect the central nervous system, and that an immune response associated with the first convulsive episode plays a key role in severity during subsequent attacks.     

NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.     

Solution structure of cyclosporin A and a nonimmunosuppressive analog bound to fully deuterated cyclophilin.

A simple strategy involving 1H nuclear magnetic resonance (NMR) spectroscopy and complete protein deuteration was used to determine the structures of two receptor-bound drugs. A potent immunosuppressive, cyclosporin A (CsA) binds tightly to the ubiquitous and highly conserved 17.7-kDa immunophilin, cyclophilin (CyP). Fully deuterated CyP was produced by overexpressing the human CyP gene in Escherichia coli grown on deuterated algal hydrolysate in 98% D2O. As only the CsA molecule is protonated in the CsA-CyP complex, we were able to make a complete sequential assignment of the bound drug using standard two-dimensional proton NMR experiments. The structure determination was accomplished using dynamical simulated annealing calculations with a total of 124 NMR-derived distance and torsion angle restraints. Aside from binding CsA, CyP also acts as a peptidyl-prolyl cis-trans isomerase. Thus, much importance had been ascribed to the cis peptide bond present in the structures reported for free CsA in organic solvents and in crystal studies. Interestingly, CyP-bound CsA exists in an all-trans conformation with no detectable elements of regular secondary structure and no intramolecular hydrogen bonds. A nonactive CsA analog, MeAla6-CsA, was studied using the same CyP deuteration strategy. In addition to structural elucidation of the two bound drugs, we were able to differentiate between the bound and surface-exposed residues of the drugs and also validate our previous hypothesis that the single CyP tryptophan is located in the CsA-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

1H NMR studies on bovine cyclophilin: preliminary structural characterization of this specific cyclosporin A binding protein

High-field 1H NMR spectroscopy has been used to study the conformation of the cytosolic cyclosporin A binding protein cyclophilin. For the drug-free form of cyclophilin, spectral editing methods in conjunction with a pH titration were used to identify all four His residues present in the protein, and two-dimensional COSY and RELAY spectroscopy was used to elucidate the scalar connectivities in the aromatic and upfield methyl regions of the spectrum. From these scalar connectivities, it was possible to distinguish between inter- and intraresidue dipolar interactions within the aromatic and upfield methyl regions of cyclophilin in the NOESY spectrum. The results of this analysis showed extensive interresidue cross-relaxation among and between these latter spectral regions indicative of the proximal relationships of several of these residues and the presence of a hydrophobic core within cyclophilin.     

Substitution in position 3 of cyclosporin A abolishes the cyclophilin-mediated gain-of-function mechanism but not immunosuppression

Binary complex formation between the immunosuppressive drug cyclosporin A (CsA) and cyclophilin 18 is the prerequisite for the ability of CsA to inhibit the protein phosphatase activity of calcineurin, a central mediator of antigen-receptor signaling. We show here that several CsA derivatives substituted in position 3 can inhibit calcineurin without prior formation of a complex with cyclophilin 18. [Methylsarcosine(3)]CsA was shown to inhibit calcineurin, either in its free form with an IC(50) value of 10 microm, or in its complex form with cyclophilin 18 with an IC(50) of 500 nm. [Dimethylaminoethylthiosarcosine(3)]CsA ([Dat-Sar(3)]CsA) was found to inhibit calcineurin on its own, with an IC(50) value of 1.0 microm, but was not able to inhibit calcineurin after forming the [Dat-Sar(3)]CsA-cyclophilin 18 binary complex. Despite their different inhibitory properties, both CsA and [Dat-Sar(3)]CsA suppressed T cell proliferation and cytokine production mainly through blocking NFAT activation and interleukin-2 gene expression. Furthermore, to demonstrate that [Dat-Sar(3)]CsA can inhibit calcineurin in a cyclophilin-independent manner in vivo, we tested its effect in a Saccharomyces cerevisiae strain (Delta12), in which all the 12 cyclophilins and FKBPs were deleted. [Dat-Sar(3)]CsA, but not CsA, bypassed the requirement for cellular cyclophilins and caused growth inhibition in the salt-stressed Delta12 strain.     

Synapsin associates with cyclophilin B in an ATP- and cyclosporin A-dependent manner

Immunophilins are ubiquitous enzymes responsible for proline isomerisation during protein synthesis and for the chaperoning of several membrane proteins. These activities can be blocked by the immunosuppressants cyclosporin A, FK506 and rapamycin. It has been shown that all three immunosuppressants have neurotrophic activity and can modulate neurotransmitter release, but the molecular basis of these effects is currently unknown. Here, we show that synapsin I, a synaptic vesicle-associated protein, can be purified from Torpedo cholinergic synaptosomes through its affinity to cyclophilin B, an immunophilin that is particularly abundant in brain. The interaction is direct and conserved in mammals, and shows a dissociation constant of about 0.5 microM in vitro. The binding between the two proteins can be disrupted by cyclosporin A and inhibited by physiological concentrations of ATP. Furthermore, cyclophilin B co-localizes with synapsin I in rat synaptic vesicle fractions and its levels in synaptic vesicle-containing fractions are decreased in synapsin knockout mice. These results suggest that immunophilins are involved in the complex protein networks operating at the presynaptic level and implicate the interaction between cyclophilin B and synapsins in presynaptic function.     

Derivatives of cyclosporin at position 2 as probes for cyclophilin

Based on the structure of the cyclosporin A / cyclophilin complex, a series of 2-substituted cyclosporins have been synthesisted as cyclophilin ligands. Their in vitro evaluation demonstrates that although there is a large lipophlic pocket offering potential for affinity improvement, minor steric interactions or conformer distribution in aqueous solution play a dramatic role.     

Modeling conformational changes in cyclosporin A.

NMR and X-ray structures for the immunosuppressant cyclosporin A (CsA) reveal a remarkable difference between the unbound (free) conformation in organic solvents and the conformation bound to cyclophilin. We have performed computer simulations of the molecular dynamics of CsA under a variety of conditions and confirmed the stability of these two conformations at room temperature in water and in vacuum. However, when the free conformation was modeled in vacuum at 600 K, a transition pathway leading to the bound conformation was observed. This involved a change in the cis MeLeu-9 peptide bond to a trans conformation and the movement of the side chains forming the dominant hydrophobic cluster (residues MeBmt-1, MeLeu-4, MeLeu-6, and MeLeu-10) to the opposite side of the plane formed by the backbone atoms in the molecular ring. The final conformation had a backbone RMS deviation from the bound conformation of 0.53 A and was as stable in dynamics simulations as the bound conformation. Our calculations allowed us to make a detailed analysis of a transition pathway between the free and the bound conformations of CsA and to identify two distinct regions of coordinated movement in CsA, both of which underwent transitions independently.     

Two distinct regions of cyclophilin B are involved in the recognition of a functional receptor and of glycosaminoglycans on T lymphocytes

Cyclophilin B is a cyclosporin A-binding protein exhibiting peptidyl-prolyl cis/trans isomerase activity. We have previously shown that it interacts with two types of binding sites on T lymphocytes. The type I sites correspond to specific functional receptors and the type II sites to sulfated glycosaminoglycans. The interactions of cyclophilin B with type I and type II sites are reduced in the presence of cyclosporin A and of a synthetic peptide mimicking the N-terminal part of cyclophilin B, respectively, suggesting that the protein possesses two distinct binding regions. In this study, we intended to characterize the areas of cyclophilin B involved in the interactions with binding sites present on Jurkat cells. The use of cyclophilin B mutants modified in the N-terminal region demonstrated that the 3Lys-Lys-Lys5 and 14Tyr-Phe-Asp16 clusters are probably solely required for the interactions with the type II sites. We further engineered mutants of the conserved central core of cyclophilin B, which bears the catalytic and the cyclosporin A binding sites as an approach to localize the binding regions for the type I sites. The enzymatic activity of cyclophilin B was dramatically reduced after substitution of the Arg62 and Phe67 residues, whereas the cyclosporin A binding activity was destroyed by mutation of the Trp128 residue and strongly decreased after modification of the Phe67 residue. Only the substitution of the Trp128 residue reduced the binding of the resulting cyclophilin B mutant to type I binding sites. The catalytic site of cyclophilin B therefore did not seem to be essential for cellular binding and the cyclosporin A binding site appeared to be partially involved in the binding to type I sites.     

NMR studies of [U-C]cyclosporin A bound to human cyclophilin B

NMR data (¹H and ¹³C chemical shifts, NOEs) on [[U-¹³C]cyclosporin A bound to cyclophilin B were compared to previously published data on the [U-¹³C]CsA/CyPA complex [Fesik et al., (1991) Biochemistry 30, 6574–6583]. Despite only 64% sequence identity between CyPA and CyPB, the conformation and active site environment of CsA when bound to CyPA and CyPB are nearly identical as judged by the similarity of the NMR data.     

Structural Basis of Cyclophilin B Binding by the Calnexin/Calreticulin P-domain

Little is known about how chaperones in the endoplasmic reticulum are organized into complexes to assist in the proper folding of secreted proteins. One notable exception is the complex of ERp57 and calnexin that functions as part the calnexin cycle to direct disulfide bond formation in N-glycoproteins. Here, we report three new complexes composed of the peptidyl prolyl cis-trans-isomerase cyclophilin B and any of the lectin chaperones: calnexin, calreticulin, or calmegin. The 1.7 Å crystal structure of cyclophilin with the proline-rich P-domain of calmegin reveals that binding is mediated by the same surface that binds ERp57. We used NMR titrations and mutagenesis to measure low micromolar binding of cyclophilin to all three lectin chaperones and identify essential interfacial residues. The immunosuppressant cyclosporin A did not affect complex formation, confirming the functional independence of the P-domain binding and proline isomerization sites of cyclophilin. Our results reveal the P-domain functions as a unique protein-protein interaction domain and implicate a peptidyl prolyl isomerase as a new element in the calnexin cycle.     

Cyclophilin A and calcineurin functions investigated by gene inactivation, cyclosporin A inhibition and cDNA arrays approaches in the phytopathogenic fungus Botrytis cinerea

Calcineurin phosphatase and cyclophilin A are cellular components involved in fungal morphogenesis and virulence. Their roles were investigated in the phytopathogenic fungus Botrytis cinerea using gene inactivation, drug inhibition and cDNA macroarrays approaches. First, the BCP1 gene coding for cyclophilin A was identified and inactivated by homologous recombination. The bcp1Delta null mutant obtained was still able to develop infection structures but was altered in symptom development on bean and tomato leaves. Opposite to this, calcineurin inhibition using cyclosporin A (CsA) modified hyphal morphology and prevented infection structure formation. CsA drug pattern signature on macroarrays allowed the identification of 18 calcineurin-dependent (CND) genes among 2839 B. cinerea genes. Among the co-regulated CND genes, three were shown to be organized as a physical cluster that could be involved in secondary metabolism. The signature of BCP1 inactivation on macroarrays allowed the identification of only three BCP1 cyclophilin-dependent (CPD) genes that were different from CND genes. Finally, no CsA drug pattern signature was observed in the bcp1Delta null mutant which provided a molecular target validation of the drug.     

Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus (HIV) type 1: interference with HIV protein-cyclophilin A interactions.

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 p24gag, and the formation of complexes was blocked by cyclosporins with 50% inhibitory concentrations of about 0.7 microM. 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, p17gag, Nef, Vif, and gp120env; 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.     

1H, 13C and 15N backbone assignments of cyclophilin when bound to cyclosporin A (CsA) and preliminary structural characterization of the CsA binding site.

The backbone 1H, 13C and 15N chemical shifts of cyclophilin (CyP) when bound to cyclosporin A (CsA) have been assigned from heteronuclear two- and three-dimensional NMR experiments involving selectively 15N- and uniformly 15N- and 15N,13C-labeled cyclophilin. From an analysis of the 1H and 15N chemical shifts of CyP that change upon binding to CsA and from CyP/CsA NOEs, we have determined the regions of cyclophilin involved in binding to CsA.     

Two new cyclophilins from Fusarium sambucinum and Aspergillus niger: resistance of cyclophilin/cyclosporin A complexes against proteolysis.

Two new peptidyl-prolyl-cis/trans-isomerases were purified to homogeneity from Fusarium sambucinum and Aspergillus niger. They belong to the class of cyclosporin A binding proteins (cyclophilins) and have molecular masses of about 18 kDa. As has been shown for other cyclophilins, the isomerase activity of the enzymes is inhibited by cyclosporin A in the nanomolar range. Furthermore binding of cyclosporin A prevents proteolytic digestion of the cyclophilin/cyclosporin complexes by the endoproteases GluC, LysC and alpha-chymotrypsin, in contrast to the free cyclophilins, which are readily cleaved by these proteases. We could also observe this protection for cyclophilins from sheep thymus and from the cyclosporin producing fungus Tolypocladium inflatum.