Adrenomedullin 2/intermedin is a slow off-rate, long-acting endogenous agonist of the adrenomedullin2 G protein-coupled receptor.

Adrenomedullin 2/intermedin (AM2/IMD), adrenomedullin (AM), and calcitonin gene-related peptide (CGRP) have functions in the cardiovascular, lymphatic, and nervous systems by activating three heterodimeric receptors comprising the class B GPCR CLR and a RAMP1, -2, or -3 modulatory subunit. CGRP and AM prefer the RAMP1 and RAMP2/3 complexes, respectively, whereas AM2/IMD is thought to be relatively nonselective. Accordingly, AM2/IMD exhibits overlapping actions with CGRP and AM, so the rationale for this third agonist for the CLR-RAMP complexes is unclear. Here, we report that AM2/IMD is kinetically selective for CLR-RAMP3, known as the AM2R, and we define the structural basis for its distinct kinetics. In live cell biosensor assays, AM2/IMD-AM2R elicited longer-duration cAMP signaling than the other peptide-receptor combinations. AM2/IMD and AM bound the AM2R with similar equilibrium affinities, but AM2/IMD had a slower off-rate and longer receptor residence time, thus explaining its prolonged signaling capacity. Peptide and receptor chimeras and mutagenesis were used to map the regions responsible for the distinct binding and signaling kinetics to the AM2/IMD mid-region and the RAMP3 extracellular domain (ECD). Molecular dynamics simulations revealed how the former forms stable interactions at the CLR ECD-transmembrane domain interface and how the latter augments the CLR ECD binding pocket to anchor the AM2/IMD C terminus. These strong binding components only combine in the AM2R. Our findings uncover AM2/IMD-AM2R as a cognate pair with unique temporal features, reveal how AM2/IMD and RAMP3 collaborate to shape CLR signaling, and have significant implications for AM2/IMD biology.

When structure-affinity relationships meet structure-kinetics relationships: 3-((Inden-1-yl)amino)-1-isopropyl-cyclopentane-1-carboxamides as CCR2 antagonists.

Chemokine ligand 2 (CCL2) mediates chemotaxis of monocytes to inflammatory sites via interaction with its G protein-coupled receptor CCR2. Preclinical animal models suggest that the CCL2-CCR2 axis has a critical role in the development and maintenance of inflammatory disease states (e.g., multiple sclerosis, atherosclerosis, insulin resistance, restenosis, and neuropathic pain), which can be treated through inhibition of the CCR2 receptor. However, in clinical trials high-affinity inhibitors of CCR2 have often demonstrated a lack of efficacy. We have previously described a new approach for the design of high-affinity CCR2 antagonists, by taking their residence time (RT) on the receptor into account. Here, we report our findings on both structure-affinity relationship (SAR) and structure-kinetic relationship (SKR) studies for a series of 3-((inden-1-yl)amino)-1-isopropyl-cyclopentane-1-carboxamides as CCR2 antagonists. SAR studies showed that this class of compounds tolerates a vast diversity of substituents on the indenyl ring with only small changes in affinity. However, the SKR is affected greatly by minor modifications of the structure. The combination of SAR and SKR in the hit-to-lead process resulted in the discovery of a new high-affinity and long-residence-time CCR2 antagonist (compound 15a, Ki = 2.4 nM; RT = 714 min).

Structure-activity relationships (SAR) and structure-kinetic relationships (SKR) of bicyclic heteroaromatic acetic acids as potent CRTh2 antagonists III: the role of a hydrogen-bond acceptor in long receptor residence times.

The correct positioning and orientation of an hydrogen bond acceptor (HBA) in the tail portion of the biaryl series of CRTh2 antagonists is a requirement for long receptor residence time. The HBA in combination with a small steric substituent in the core section (R(core) ≠ H) gives access to compounds with dissociation half-lives of ⩾ 24h.