Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase

Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H]-methyl phenyl pyridinium ([(3)H]-MPP(+)) as the marker ligand and MPP(+), verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The K(d) values calculated from the chromatographic studies correlated with previously reported K(i) values (r(2)=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP(+) and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil.     

Interaction of ibogaine with human α3β4-nicotinic acetylcholine receptors in different conformational states

The interaction of ibogaine and phencyclidine (PCP) with human (h) α3β4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca²⁺ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (±)-epibatidine-induced Ca²⁺ influx in hα3β4 AChRs with ～9-fold higher potency than that for PCP, (b) [³H]ibogaine binds to a single site in the hα3β4 AChR ion channel with relatively high affinity (K_d = 0.46 ± 0.06 μM), and ibogaine inhibits [³H]ibogaine binding to the desensitized hα3β4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [³H]ibogaine binding to the hα3β4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6′) and valine/phenylalanine (position 13′) rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time.     

Development of an immobilized GPR17 receptor stationary phase for binding determination using frontal affinity chromatography coupled to mass spectrometry

A liquid chromatographic stationary phase containing immobilized membranes from cells expressing the P2Y-like receptor GPR17 is described. Cellular membranes from 1321N1 cells transiently transfected with GPR17 vector [GPR17(+)] and from the same cell line transfected with the corresponding empty vector [GPR17(−)] were entrapped on immobilized artificial membrane (IAM) support and packed into 6.6-mm-i.d. glass columns to create GPR17(+)-IAM and GPR17(−)-IAM stationary phases. Frontal chromatography experiments on both GPR17(+)-IAM and GPR17(−)-IAM demonstrated the presence of a specific interaction with GPR17 only in the former that was maximized by increasing the membrane/IAM ratio. GPR17(+)-IAM was used in frontal affinity chromatography experiments to calculate the dissociation constants (K_d) of three ligands—the antagonist cangrelor (formerly AR-C69931MX, a P2Y₁₂/P2Y₁₃ antagonist), MRS2179 (a P2Y₁ receptor antagonist), and the agonist UDP—all of which have been reported to also interact with GPR17. Immobilized GPR17 retained its ability to specifically bind the three analytes, as demonstrated by the agreement of the calculated K_d values with previously reported data. Preliminary ranking experiments suggest the application of GPR17(+)-IAM in ranking affinity studies for the selection of new potential candidates.     

Exploring enantiospecific ligand-protein interactions using cellular membrane affinity chromatography: chiral recognition as a dynamic process

The chiral recognition mechanisms responsible for the enantioselective binding on the alpha3beta4 nicotinic acetylcholine receptor (alpha3 beta4 nAChR) and human organic cation transporter 1 (hOCT1) have been reviewed. The results indicate that chiral recognition on the alpha3beta4 nAChR is a process involving initial tethering of dextromethorphan and levomethorphan at hydrophobic pockets within the central lumen followed by hydrogen bonding interactions favoring dextromethorphan. The second step is the defining enantioselective step. Studies with the hOCT1 indentified four binding sites within the transporter that participated in chiral recognition. Each of the enantiomers of the compounds used in the study interacted with three of these sites, while (R)-verapamil interacted with all four. Chiral recognition arose from the conformational adjustments required to produce optimum interactions. With respect to the prevailing interaction-based models, the data suggest that chiral recognition is a dynamic process and that the static point-based models should be amended to reflect this.     

Initial synthesis and characterization of an immobilized heat shock protein 90 column for online determination of binding affinities

Heat shock protein 90α (Hsp90α) was immobilized on aminopropyl silica via the N terminus to create the Hsp90α(NT) column or via the C terminus to create the Hsp90α(CT) column. Binding to the exposed C terminus on the Hsp90α(NT) column was characterized using frontal chromatography and the C-terminus ligands coumermycin A₁ (CA1) and novobiocin (NOVO). The calculated K_d values were 220 ± 110 nM (CA1) and 100 ± 20 nM (NOVO). Nonlinear chromatography was used to determine the association and dissociation rate constants associated with the NOVO-Hsp90α complex: 22.2 ± 8.8 μM⁻¹ s⁻¹ and 2.7 ± 0.6 s⁻¹, respectively. Binding to the exposed N terminus on the Hsp90α(CT) column was characterized using frontal chromatography. The K_d values of the N-terminus ligands geldanamycin (GM, 90 ± 50 nM), 17-allylamino-17-demethoxygeldanamycin (17-AAG, 210 ± 50 nM), and radicicol (RAD, 20 ± 9 nM) were consistent with previously reported values. The effect of the immobilization on ATPase activity was investigated through the determination of IC₅₀ values for inhibition of ATPase activity on the Hsp90α(CT) column. The IC₅₀ for GM was 2.80 ± 0.18 μM, and the relative IC₅₀ values were 17-AAG > GM > RAD, in agreement with previously reported values and indicating that immobilization had not affected ATPase activity or sensitivity to inhibition.