Ranking High Affinity Ligands of Low Solubility by NMR Spectroscopy

Abstract

C ommon wisdom holds that the experimental measurement of ligand/receptor binding parameters requires concentrations of both partners of the order of the dissociation constant (K D ) to be determined. Whereas this puts NMR spectroscopy in a favorable position to determine the interaction parameters for many biologically relevant molecular interactions, pharmacological ligands often have K D values in the nanomolar range. Fluorescence spectroscopy with its superior sensitivity seems better suited to characterize such ligands but does require fluorescence of the ligand or the receptor to change upon interaction. Low solubility in water is another recurrent issue when characterizing the binding properties of pharmacological ligands, whereby the addition of inorganic solvent and their effect on the protein and protein binding have to be carefully controlled.Within a given series of (un)related ligands, one typically looks for a relative ranking of the molecules and potentially for information on the identity of the binding site. Competition experiments are the most obvious choice for such a ranking. We propose here the use of NMR spectroscopy in a competition experiment that gives simultaneous relative K D values for the ligands and also qualitative information on the binding site. We demonstrate the method by ranking the binding of the N-methyl D-Ala3/N-ethyl Val4 cyclosporin A (named Alisporivir) toward the human cyclophilin A (CypA) as compared to its mother compound cyclosporin A (CsA) and two chemical intermediates, the N-ethyl Val4 Cs (EthV4Cs) and the N-methyl Val4 Cs (MeV4Cs). Alisporivir is a nonimmunosuppressive analogue of CsA ( Figure S1 in the Supporting Information) that presents better antiviral properties than CsA inhibiting HCV subgenomic replicon replication.1,2 Ranking the ligands for CypA binding might help to establish whether differential affinity can explain the biological observation of Alisporivir's superior antiviral character.When complexing the protein with one or the other ligand, we had previously noticed 3 that the differential chemical shift changes, that is, between CypAÀCsA and CypAÀAlisporivir, were very limited and localized but clearly could be observed for a couple of resonance peaks (Figure 1 and Figure A 1:1 CsA:Alisporivir mixture from individual 5 mg/mL stock solutions in chloroform of both undecapeptides was directly deposited in a standard NMR tube. The chloroform was evaporated under nitrogen flux, leaving a white solid layer containing both ligands at the glass interface.15 N-labeled CypA protein solution was introduced in this tube, and sequential [ 1 H, 15 N]-HSQC spectra were recorded. Whereas the resonances corresponding to the free CypA protein were initially the only ones detected, novel resonances that had been assigned to both CypA complexes 3 gradually appeared (Figure 1). Integration of these peaks in a series of HSQC spectra showed a linear decrease of the free CypA peaks, reflecting the complete loading of the protein with the ligands. Peak integrals corresp...

Dissociation Kinetics of a Binary Complex in Solution by Protein Displacement

“…For both CsA and Alisporivir, ligand binding induces distinct chemical shifts that thereby act as indicators of both the apo and holo proteins 20. The crystal structure of CsA‐bound Cyclophilin A shows two lysine residues in or near the CsA binding site,12 and we previously found that Alisporivir binds in a very similar manner 24.…”

“…We recently have used the differential NMR spectra of CypA in its apo form or in its complex with CsA and/or Alisporivir to obtain a relative K D value for both ligands without addition of any co‐solvent 20. The latter molecule, currently in advanced clinical trials against the hepatitis C virus, is only subtly different from CsA (Figure S1 in the Supporting Information), but the few molecular changes lead to a better antiviral effect even in a cell model where the question of immunosuppression is without relevance 21–23.…”

X-ray structure of alisporivir in complex with cyclophilin A at 1.5 Å resolution

“…Cyclophilin inhibitors interfere with this interaction and consequently alter the HCV RNA-replication process. The fivefold to tenfold higher efficacy of alisporivir versus CsA in a cellular model, however, does not directly correlate with the relative equilibrium dissociation constants (K d ) of the ligands for CypA, as we previously showed that K d (CypA-CsA) ' 1.4 Â K d (CypA-ALV) (Landrieu et al, 2011). Taking into account a K d (CypA-CsA) value of 11 nM, as measured by Liu et al (2006), this would give a K d (CypA-ALV) of $7-8 nM.…”

Dissociation Kinetics of a Binary Complex in Solution by Protein Displacement

“…For both CsA and Alisporivir, ligand binding induces distinct chemical shifts that thereby act as indicators of both the apo and holo proteins. [20] The crystal structure of CsAbound Cyclophilin A shows two lysine residues in or near the CsA binding site, [12] and we previously found that Alisporivir binds in a very similar manner. [24] We therefore produced for this study a selectively 15 N-Lys-labeled Cyclophilin sample, and found that both the Lys82 and Lys125 can be used to distinguish both protein forms ( Figure S2 in the Supporting Information).…”

“…We recently have used the differential NMR spectra of CypA in its apo form or in its complex with CsA and/or Alisporivir to obtain a relative K D value for both ligands without addition of any co-solvent. [20] The latter molecule, currently in advanced clinical trials against the hepatitis C virus, is only subtly different from CsA ( Figure S1 in the Supporting Information), but the few molecular changes lead to a better antiviral effect even in a cell model where the question of immunosuppression is without relevance. [21][22][23] The difference in equilibrium dissociation constant of both drugs towards CypA that we found thereby seems small to explain the enhanced in vivo efficacy of Alisporivir.…”