Fast NMR Methods for Measuring in the Direct and/or Competition Mode the Dissociation Constants of Chemical Fragments Interacting with a Receptor

Abstract

Ligand‐based NMR screening represents a powerful method in fragment‐based drug discovery for the identification of chemical matter interacting with the receptor of interest. The large dynamic range of these methods allows the detection of weakly binding ligands. However, the methodology has not been extensively used for quantifying the strength of these interactions. This knowledge is important for ranking fragments according to their binding strength and for prioritizing structure‐based and medicinal chemistry activities. Rapid NMR methods for measuring the dissociation constant in direct and competition modes are presented here. The theory underpinning these methods are presented, along with their application to the measurement of the binding affinities of several ligands of the heat shock protein 90.

Unraveling Sugar Binding Modes to DC-SIGN by Employing Fluorinated Carbohydrates

“…Control experiments varying the 180° pulse repetition frequency using different τ delays between 0.1 and 20 ms for 4F-Man in the presence of DC-SIGN showed the presence of chemical exchange. This fact hampers the estimation of quantitative binding affinities from the observed T 2 values [24,25]. In any case, in order to minimize this effect short τ delays (2 ms) were used with the monosaccharide mixture.…”

Metal Chelation Therapy and Parkinson’s Disease: A Critical Review on the Thermodynamics of Complex Formation between Relevant Metal Ions and Promising or Established Drugs

“…Equations to correct K d values, by taking into account the effect of a competing ligand (e.g., the buffer) and of the different pH, have been proposed [316]. Standardized conditions to measure the K d of metal-protein complexes are also being proposed [317][318][319], and this should lead to more reproducible results, thus eventually allowing a more reliable comparison among K d numbers. Table S2 (Supplementary Materials) reports the metal-ligand speciation available in the literature for the ligands listed in Table 2 (rows) and the relevant metal ions, i.e., Cu(II), Cu(I), Fe(III), Fe(II), Mn(II), and Zn(II) (columns).…”

19F NMR viewed through two different lenses: ligand-observed and protein-observed19F NMR applications for fragment-based drug discovery

“…62 These experiments have been prominently used for FBDD, and as such are extensively reviewed. 4,[16][17][18] In recent years several enabling methodologies have been developed that further the field, including creation of unique fluorinated fragment libraries, 63 broadband protocols for expanded mixtures, 64 quantitative methodology for measuring dissociation constants, 65,66 multi-dimensional NMR experiments (e.g., COSY, 67 ILOE 62 ), and screening protocols with both cellular lysates and intact cells. 68,69 Many of these advances will be highlighted here.…”

“…Another reported quantification method using direct FAXS relies on a ligand titration to quantify K d . 66 This methodology only works for molecules with low binding affinities and conditions where the total ligand concentration is approximately equal to the concentration of free ligand, i.e. using low protein concentration.…”