Affinity

The affinity of a ligand (peptide, small molecule, antibody) for a biological target (receptor, enzyme, nucleic acid) refers to the strength of the reversible non-covalent interaction between the two partners. It is quantified by the dissociation constant Kd, which corresponds to the ligand concentration at which 50 % of binding sites are occupied at equilibrium. The smaller the Kd, the higher the affinity.

Thermodynamically, affinity arises from a balance between enthalpy (ΔH, contributions from hydrogen bonds, electrostatic interactions, van der Waals forces) and entropy (ΔS, desolvation of hydrophobic surfaces, conformational changes of ligand and target). Physiological Kds typically span nanomolar (signaling peptides on their receptors, high-affinity antibodies) to micromolar (ions, metabolites on enzymes). Picomolar affinities characterize highly specific interactions (biotin-streptavidin Kd ≈ 10⁻¹⁴ M, certain matured therapeutic antibodies).

Several derived parameters refine the ligand-target relationship. Ki (inhibition constant) measures the affinity of an antagonist for a target relative to a reference ligand, via the Cheng-Prusoff equation (Ki = IC50 / (1 + [L]/Kd)). Bmax refers to the maximum number of binding sites expressed per unit tissue or cell and is obtained by saturation curve. The Hill number (nH) indicates cooperativity: nH ≈ 1 for a simple interaction, > 1 for positive cooperativity (oxygen binding to hemoglobin, nH ≈ 2.8), < 1 for negative cooperativity.

Experimental methods to measure affinity are numerous. Surface plasmon resonance (SPR, Biacore) and biolayer interferometry (BLI, Octet) measure association (kon) and dissociation (koff) kinetic constants in real time, with Kd = koff/kon. Isothermal titration calorimetry (ITC) directly provides Kd, ΔH, ΔS, and stoichiometry n from a single experiment. Fluorescence anisotropy (FP) suits labeled peptides. Radioligand binding assays (³H, ¹²⁵I) remain a historical reference for membrane receptors. In high-throughput screening, displacement methods (FRET, TR-FRET, AlphaScreen) often replace radioligands.

Pharmacological interpretation requires linking affinity to functional activity. A high-affinity ligand is not necessarily an efficient activator: an antagonist can bind at the agonist's Kd without triggering the intracellular cascade. EC50 (functional effect) therefore differs from Kd (binding). In peptide development, optimizing affinity (SAR, molecular modeling, cryo-EM) must be accompanied by functional assays to confirm the expected biological effect. Receptors with conformational plasticity (GPCR biased signaling) add another dimension: the same ligand can differentially activate G-protein vs β-arrestin pathways depending on its interaction signature.