Pseudoprolines (YPro)
:
Targeting Bioactive Conformations of Cyclosporine Analogues
Luc Patiny, Michael Keller, Torsten Wöhr, Jean-François Guichou and Manfred Mutter
Institute of Organic Chemistry, University of Lausanne, BCH-Dorigny, CH-1015 Lausanne, Switzerland, Tel.: +41-21-692-3950; fax: +41-21-692-3955; e-mail: manfred.mutter@ico.unil.ch
Received: 15 August 2001 / Uploaded 22 August 2001
Abstract : The direct insertion of a 2'-C-disubstituted pseudoproline (YPro) system at Thr2 of cyclosporin C (CsC) results in strong conformational constrains upon the peptide backbone. A 1-2 cis-amide bond containing conformer shows differential binding affinities to its receptors cyclophilin A and calcineurin A, indicating alternative binding modes compared to the native molecule.
Pseudoprolines (YPro) have been introduced as proline mimetics of tailored structural and functional properties[1] (Scheme 1). In particular, 2'-C-disubstituted ring structures derived from Ser, Thr or Cys strongly enhance the unique features of Pro in inducing cis-amide bonds into the peptide backbone, thus serving as solubilizing, structure disrupting protection technique in peptide synthesis[2] as well as a general tool for targeting molecular recognition processes[3]. Here, we investigate the direct insertion of 2'-C-disubstituted YPro systems into bioactive peptides of higher complexity, i.e. cyclosporine C (CsC) and its effect upon the structural and biological profiles of the resulting CsC derivatives.
Scheme 1
CsC (II, scheme 2) differs from the well known immunosuppressive analogue CsA (I) by the presence of a functional amino acid at position 2, i.e. a threonine (Thr2) residue replacing the amino butyric acid (Abu2) in CsA. Whereas the biological and pharmacokinetic properties of CsC and CsA were shown to be very similar[4], the presence of an OH group at Thr2 together with a non-methylated amide bond between residues 1 and 2 makes CsC a most attractive candidate for probing the YPro concept[1,2,3] in structure-function studies. Despite of the considerable steric constrains involved in the cyclocondensation reaction of CsC with the acetone derived acetal (Scheme 2), the direct insertion of a 2'-C-disubstituted oxazolidine system into CsC proceeds very selectively in acceptable yields. Most notably, the alternative cyclization of the OH group at residue 1 (Bmt) resulting in a 6-membered ring has not been observed under the given experimental conditions, possibly due to the steric hindrance of the Bmt side chain*.
Scheme 2
The YPro-containing target molecule III proved to be stable (> 1 month at 37°C) in foetal bovine serum/methanol (v/v 1/1) and showed a half-life of ca 83 minutes in a 1M HClaq/acetonitrile mixture (v/v 1/1) and a half-life of 17 hours in a 0.1M HClaq/acetonitrile mixture (v/v 1/1) as monitored by analytical HPLC (Figure 1). Under the hydrolysis conditions, the formed CsC slowly converts to the isoCsC[5].
Figure 1. Hydrolysis of III in HClaq 0.1M/acetonitrile (v/v : 1/1).
The impact of the insertion of a 2’-C-disubstituted YPro into CsC upon the conformational properties was investigated by NMR spectroscopy. Due to the pronounced hydrophobicity of the target molecule, d6-DMSO was used as solvent mimicking closely the native aqueous conditions[6]. Surprisingly, only two major conformers at ratio 55:45 were observed by 1H NMR, in contrast to the numerous conformers found for CsC (Figure 2).
Figure 2. 1H NMR of the NH region of II (left) and III (right).
In harmony with systematic studies on model dipeptides of type Xaa-YPro[7], the presence of two substituents at 2'-C of YPro induces a 1-2 cis amide bond in both conformers as shown by ROESY experiments (Figure 3). The major difference of the two conformers originates from amide bond 3-4, which was found to be trans in the major and cis in the minor conformer, whereas the remaining amide bonds are exclusively trans.
Figure 3. ROESY of III. The major conformer has 1-2 and 3-4 cis amide bonds (NOE 2Ha-1Hb and 3Ha-4Ha). The minor conformer shows only one 1-2 cis amide bond (NOE 2'Ha-1'Hb, no NOE between 3'Ha and 4'Ha).
The observed increase of conformational constrain in combination with the presence of cis-amide bonds induced by the YPro system should have a dramatic effect upon the biological activity of III, as the amide bonds in the bioactive conformation of CsC are postulated to be all-trans[8]. Most surprisingly, the YPro containing derivative III shows even slightly increased binding affinity to its receptor cyclophilin A (CypA) compared to the native compounds CsC (II) or CsA (I) (Table 1)[9]. In contrast, the binding affinity of the binary complex (CypA - III) towards calcineurin is reduced by a factor of 160, as indicated by the IL-2 reporter gene assay[10].
Table 1. Relative biological activity to CsA, i.e. IC50(analogue)/IC50(CsA)
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CsA (cyclosporin) |
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CsC |
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Thr(YMe,Mepro)2CsC |
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Obviously, the induction of a cis amide bond at position 1-2 of CsC does not interfer with its binding capacity to CypA, pointing to alternative binding modes of YPro-containing CsC to its receptor. The indicated structure-function relationship of this novel class of CsC derivatives will be of considerable impact for the design of non-immunosuppresive, anti-viral compounds.
Acknowledgments
We are grateful to Prof. U. Ruegg for collaborating and A. Carrupt from the University of Lausanne for performing the biological tests. This work was supported by the Swiss National Science Foundation.
References