Corticotropin-releasing factor (CRF) receptor antagonists are under preclinical and clinical investigation for stress-related disorders. In this study the impact of receptor-ligand binding kinetics on CRF 1 receptor antagonist pharmacology was investigated by measuring the association rate constant (k 1 ), dissociation rate constant (k Ϫ1 ), and kinetically derived affinity at 37°C. Three aspects of antagonist pharmacology were reevaluated: comparative binding activity of advanced compounds, in vivo efficacy, and structure-activity relationships. Twelve lead compounds, with little previously noted difference of affinity, varied substantially in their kinetic binding activity with a 510-fold range of kinetically derived affinity (k Ϫ1 /k 1 ), 170-fold range of k Ϫ1 , and 13-fold range of k 1 . The k Ϫ1 values indicated previous affinity measurements were not close to equilibrium, resulting in compression of the measured affinity range. Dissociation was exceptionally slow for three ligands (k Ϫ1 t 1/2 of 1.6 -7.2 h at 37°C). Differences of binding behavior were consistent with in vivo pharmacodynamics (suppression of adrenocorticotropin in adrenalectomized rats). Ligand concentration-effect relationships correlated with their kinetically derived affinity. Two ligands that dissociated slowly (53 and 130 min) produced prolonged suppression, whereas only transient suppression was observed with a more rapidly dissociating ligand (16 min). Investigating the structure-activity relationship indicated exceptionally low values of k 1 , approaching 100,000-fold less than the diffusion-limited rate. Retrospective interpretation of medicinal chemistry indicates optimizing specific elements of chemical structure overcame kinetic barriers in the association pathway, for example, constraint of the pendant aromatic orthogonal to the ligand core. Collectively, these findings demonstrate receptor binding kinetics provide new dimensions for understanding and potentially advancing the pharmacology of CRF 1 receptor antagonists.

Graphical Abstract Highlights d The gut peptide PYY 3-36 reduces food intake but causes severe GI intolerability d Like in humans, shorter-acting PYY analogs cause emesis in obese rhesus macaques d mAb-cycPYY is a stable, NPY2R-selective analog with a slow infusion profile d This results in a markedly improved efficacy/emesis therapeutic window in rhesus SUMMARYThe gut hormone PYY 3-36 reduces food intake in humans and exhibits at least additive efficacy in combination with GLP-1. However, the utility of PYY analogs as anti-obesity agents has been severely limited by emesis and rapid proteolysis, a profile similarly observed with native PYY 3-36 in obese rhesus macaques. Here, we found that antibody conjugation of a cyclized PYY 3-36 analog achieved high NPY2R selectivity, unprecedented in vivo stability, and gradual infusion-like exposure. These properties permitted profound reduction of food intake when administered to macaques for 23 days without a single emetic event in any animal. Co-administration with the GLP-1 receptor agonist liraglutide for an additional 5 days further reduced food intake with only one animal experiencing a single bout of emesis. This antibody-conjugated PYY analog therefore may enable the long-sought potential of GLP-1/PYY-based combination treatment to achieve robust, well-tolerated weight reduction in obese patients.

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