Affinity Screening Using Competitive Binding with Fluorine‐19 Hyperpolarized Ligands

Abstract

Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (KD) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand to the binding pocket of interest is known.

Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization

“…The average K D,c values are also in good agreement with previously reported values (benzamidine, 16 ± 2 μM; benzylamine, 218 ± 43 μM at 50 mM Tris, pH 8.0, 304 K). 15 Figure 4 further indicates that for the data sets obtained here, the group fitting method (open symbols) provided values of K D,c located within the confidence intervals of the individual fits, close to the individual values of the more accurate channel. If the data sets comprise no optimal sets of concentrations, the group fitting may be applied effectively to obtain a more reliable K D,c value.…”

“…It was previously shown that the ratio of these differences, here designated as α, is equal to the ratio of p b under competitive binding to that under binding without competition. 15 In Figure 3, it is apparent that the R 2,obs values from Channel 1 with benzamidine (Figure 3a) and Channel 2 with benzylamine (Figure 3d) appear mid-way between the two limits, which are reflected by the values of α close to 0.5. This indicates a partial displacement of the bound reporters due to binding competition.…”

“…, α ≈ 0.5. 15,26 Hence, the multiplexed detection in both cases includes one set of concentrations expected to allow for reliable determination of K D,c .…”

“…S2−S6). 15 Alternatively, the K D,c value was determined by fitting to the CPMG data sets from both channels at the same time. For this purpose, the decay rate of R 2,obs was expressed as a function of p b given by Eq.…”

“…A method to circumvent the need for titrations, based on the relaxation measurement of displaced reporter ligands, 11–14 has been recently developed for use with D-DNP in our group. 15 Even in this case, the measurement of relaxation parameters at two or three different concentrations can be desirable. DNP instruments that are capable of polarizing multiple samples have been developed.…”