Orosomucoid binding induced amplification of inherent chirality of the immunosuppressant drug sirolimus

Abstract

The circular dichroism (CD) activity of the lactone and enone groups are displayed below 250 nm. The asymmetrically perturbed n→π * transitions of the 9-, 26-, and 32-ones give rise to a negative CD band centered around 300 nm (Fig. 1). 12 The weak, π→π * of origin contribution of the triene moiety is suppressed by the more intense carbonyl Cotton effects (CEs) and can only be seen as unresolved vibrational peaks on the short-wavelength side of the n→π * feature. 12 The presence of the AAG in the sample solution, however, deeply alters this chiroptical profile amplifying the CD amplitudes in the absorption region of the triene chromophore (Fig. 1). The molar dichroic absorption coefficient (∆ε) measured in protein-free buffer solution at 275 nm increases by ten-fold and the anisotropy factor (g = ∆ε/ε) between 260-280 nm raises also by an order of magnitude (~10 -4 → ~10 -3 ). Concomitantly, the g max value of the n→π * CE is bathochromically shifted by 5 nm (Fig. 2). The excitation energy of n→π * transition of the carbonyl chromophore sensitively depends on the polarity of the medium. The binding of sirolimus to its the pharmacological target protein named FKBP is also accompanied with some modest CD and UV spectroscopic changes that refer to a more planar and rigid conformation of the triene moiety adopted at the binding site. 12It is to be noted that the related immunosuppressive drug tacrolimus lacks the triene component. Despite of its AAG association, 17 the intrinsic n→π * CE of tacrolimus shows no alteration in protein solution (Fig. S1, ESI) pointing out the importance of the triene chromophore in the CD spectroscopic detection of the binding interaction. properties. [20][21][22] Therefore, the interaction of sirolimus with the separated genetic variants of AAG has also been studied. The F1/S variant induced very similar CD changes and UV shift as seen with the native sample (Fig. 3). In marked contrast, the A form caused a much smaller intensity gain of the π→π * CE and only a minor red shift of the absorption maxima. Overall, it is shown for the first time that the conformational chirality of the triene chromophore allows the chiroptical sensing and evaluation of sirolimus-AAG interactions providing a basis to use this methodology for studying related compounds, too (e.g., temsirolimus, everolimus, ridaforolimus).

Comment on “Multiple Independent Binding Sites for Small-Molecule Inhibitors on the Oncoprotein c-Myc”

“…To obtain insight into possible mechanisms of ligand binding induced CD, the molar circular dichroic absorption coefficient (Δε in M −1 cm −1 ) of the extrinsic ellipticity band has to be determined . To estimate this parameter, the exact molar concentrations of the interacting partners are required.…”

“…To obtain insight into possible mechanisms of ligand binding induced CD, the molar circular dichroic absorption coefficient (Δε in M −1 cm −1 ) of the extrinsic ellipticity band has to be determined. 2 To estimate this parameter, the exact molar concentrations of the interacting partners are required. These were not specified in the paper 1 but were in the Ph.D. dissertation of one co-author 3 showing that the inhibitor was used in a 5-fold molar excess (100 μM) in relation to the c-Myc peptides (20 μM).…”