Abstract
To dissect the enzyme inhibitory properties of the immunosuppressive cyclic undecapeptide cyclosporin A (CsA) and gain access to monospecific, non-calcineurin-inhibiting CsA derivatives, [D-Ser8]CsA was subjected to modifications at the D-Ser side chain. Thus, we modified a CsA residue flanking the calcineurin (CaN) and cyclophilin 18 (Cyp18) binding domains of CsA instead of the residues of the CaN binding domain in order to develop a new specificity-determining site within the cyclic peptide. The [O-(NH2 (CH2)5NHC(O)CH2)-D-Ser8]CsA (derivative 9), with an amino group on a tether, exhibits CsA-like inhibition of the peptidyl prolyl cis/trans isomerase activity of Cyp18 with an IC50 value of 3.2 nm, whereas the CaN inhibition by the Cyp18-derivative 9 complex is completely abolished. Consequently, this compound is not able to inhibit the proliferation and cytokine production of activated T cells. Structure-activity relationship studies with a series of [d-Ser(8)]CsA derivatives indicate that the positively charged side chain is an essential requirement for Cyp18-derivative 9 to be ineffective on CaN. Upon protecting the amino group in derivative 9 with the photolabile moiety 2-nitroveratryloxycarbonyl (NVOC), the Cyp18-[O-(NVOC-NH(CH2)5NHC(O)CH2)-D-Ser8]CsA (derivative 11) complex exhibits strong CaN inhibition and shows potent immunosuppressive activity. In stimulated T cells pretreated with derivative 11, a remarkable recovery of transcriptional activation of the nuclear factor of activated T cells (NFAT) has been achieved through light irradiation, as assessed with a NFAT reporter gene assay.
Highlights
Cyclosporin A (CsA)1 is a standard immunosuppressive drug used to prevent allograft rejection after the transplantation of organs or bone marrow [1]
Acidic Modifications—First we investigated the influences of acidic functional groups attached to the D-Ser8 oxygen of [D-Ser8]cyclosporin A (CsA) on both the direct cyclophilin 18 (Cyp18) inhibition and the CaN inhibition by the Cyp18-CsA derivative complex
Negative charges at CsA residue 8 do not have a significant influence on the inhibition of Cyp18 peptidyl-prolyl cis/trans isomerases (PPIases) activity
Summary
The PPIase activity of recombinant human Cyp was measured with a protease-coupled assay on a Hewlett-Packard 8452a diode array spectrophotometer according to Fischer et al [25], with succinyl-AlaPhe-Pro-Phe-4-nitroanilide as the substrate and ␣-chymotrypsin (Merck) as the auxiliary protease in 35 mM HEPES buffer, pH 7.8, at 7 °C. After a 10-min incubation of Cyp (2.5 nM) with an inhibitor of the desired concentration, protease and substrate were added. The final substrate concentration was 40 M. The cis to trans isomerization was monitored at 390 nm. The rate was calculated according to first order reaction. The residual enzymatic activities were plotted versus the inhibitor concentrations and used for calculating the IC50
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