Abstract
Apelin peptide analogues displaying bias towards G protein signalling pathways have beneficial cardiovascular actions compared with the native peptide in humans in vivo. Our aim was to determine whether small molecule agonists could retain G protein bias. We have identified a biased small molecule, CMF-019, and characterised it in vitro and in vivo.In competition radioligand binding experiments in heart homogenates, CMF-019 bound to the human, rat and mouse apelin receptor with high affinity (pKi=8.58±0.04, 8.49±0.04 and 8.71±0.06 respectively). In cell-based functional assays, whereas, CMF-019 showed similar potency for the Gαi pathway to the endogenous agonist [Pyr1]apelin-13 (pD2=10.00±0.13 vs 9.34±0.15), in β-arrestin and internalisation assays it was less potent (pD2=6.65±0.15 vs 8.65±0.10 and pD2=6.16±0.21 vs 9.28±0.10 respectively). Analysis of these data demonstrated a bias of ∼400 for the Gαi over the β-arrestin pathway and ∼6000 over receptor internalisation. CMF-019 was tested for in vivo activity using intravenous injections into anaesthetised male Sprague–Dawley rats fitted with a pressure-volume catheter in the left ventricle. CMF-019 caused a significant increase in cardiac contractility of 606±112mmHg/s (p<0.001) at 500nmol. CMF-019 is the first biased small molecule identified at the apelin receptor and increases cardiac contractility in vivo. We have demonstrated that Gαi over β-arrestin/internalisation bias can be retained in a non-peptide analogue and predict that such bias will have the therapeutic benefit following chronic use. CMF-019 is suitable as a tool compound and provides the basis for design of biased agonists with improved pharmacokinetics for treatment of cardiovascular conditions such as pulmonary arterial hypertension.
Highlights
The apelin receptor is a class A G protein-coupled receptor first cloned in 1993 [1] and was deorphanised in 1998 when its cognate ligand, apelin, was identified from bovine stomach extracts [2]
Binding of CMF-019 was predicted through molecular modelling to occur to a region in the apelin receptor that, by comparison with other species, is highly conserved and is crucial for interaction with the SHK amino acid sequence of apelin-13
Acute apelin infusion is beneficial in pulmonary arterial hypertension (PAH) and heart failure (HF) and it is predicted that apelin agonists would display efficacy in treating these conditions
Summary
The apelin receptor is a class A G protein-coupled receptor first cloned in 1993 [1] and was deorphanised in 1998 when its cognate ligand, apelin, was identified from bovine stomach extracts [2]. ⇑ Corresponding author at: Experimental Medicine and Immunotherapeutics, Level 6 Centre for Clinical investigation, Box 110 Addenbrooke’s Hospital, Cambridge CB2 0QQ, UK These properties of apelin signalling have made it of particular interest as a potential therapeutic for a number of diseases such as pulmonary arterial hypertension (PAH) and heart failure (HF) [13]. Previous work has shown that infusion of apelin is beneficial in a number of disease models of PAH [22] and HF [8,9,10,23,24,25], but long term therapeutic efficacy is limited by a short halflife of a few minutes. Upon activation of the receptor, bound apelin is rapidly internalised through the b-arrestin pathway with the rate of recycling back to the plasma membrane highly dependent on the ligand [26,27]
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