Species dependence of A3 adenosine receptor pharmacology and function

Abstract

Efforts to fully understand pharmacological differences between G protein-coupled receptor (GPCR) species homologues are generally not pursued in detail during the drug development process. To date, many GPCRs that have been successfully targeted are relatively well-conserved across species in amino acid sequence and display minimal variability of biological effects. However, the A₃ adenosine receptor (AR), an exciting drug target for a multitude of diseases associated with tissue injury, ischemia, and inflammation, displays as little as 70% sequence identity among mammalian species (e.g., rodent vs. primate) commonly used in drug development. Consequently, the pharmacological properties of synthetic A₃AR ligands vary widely, not only in binding affinity, selectivity, and signaling efficacy, but to the extent that some function as agonists in some species and antagonists in others. Numerous heterocyclic antagonists that have nM affinity at the human A₃AR are inactive or weakly active at the rat and mouse A₃ARs. Positive allosteric modulators, including the imidazo [4,5-c]quinolin-4-amine derivative LUF6000, are only active at human and some larger animal species that have been evaluated (rabbit and dog), but not rodents. A₃AR agonists evoke systemic degranulation of rodent, but not human mast cells. The rat A₃AR undergoes desensitization faster than the human A₃AR, but the human homologue can be completely re-sensitized and recycled back to the cell surface. Thus, comprehensive pharmacological evaluation and awareness of potential A₃AR species differences are critical in studies to further understand the basic biological functions of this unique AR subtype. Recombinant A₃ARs from eight different species have been pharmacologically characterized thus far. In this review, we describe in detail current knowledge of species differences in genetic identity, G protein-coupling, receptor regulation, and both orthosteric and allosteric A₃AR pharmacology.

Keywords: Adenosine receptor; Antagonist; G protein-coupled receptor; Nucleoside; Species differences.

Fig. 1

Chemical structures of representative A₃AR agonists and allosteric modulators, including those that are commercially available (see text)

Fig. 2

Chemical structures of representative A₃AR antagonists, including those that are commercially available (see text). The four 4'-truncated nucleosides (bottom row, right) can display residual low efficacy agonism, depending on the model

Fig. 3

Sequence alignment of thirteen A₃AR species homologues (Species identifiers (https://www.ncbi.nlm.nih.gov/): NP_000668.1, Homo sapiens (human); NP_001289704.2, Pan troglodytes (chimpanzee); NP_001075527.1, Oryctolagus cuniculus (rabbit); NP_001003178.1, Canis lupus familiaris (dog); XP_006182940.1, Camelus ferus (camel); NP_001289720.1, Callithrix jacchus (marmoset); XP_006935064.2, Felis catus (cat); XP_012998623.1, Cavia porcellus (guinea pig); XP_035293911.1, Cricetulus griseus (hamster); XP_021514447.1, Meriones unguiculatus (Mongolian gerbil); mouse (source: Fisher et al. [84]); NP_001289680.1, Rattus novegicus (Norwegian rat); NP_989482.2, Gallus gallus (chicken). TMs are indicated in cyan, and extracellular loops in yellow highlight. The definition of helical regions and residues (bold, human and mouse sequences) corresponding to those in contact (distance ≤ 4Å) with partial agonist ligand MRS7591 (Figure 2) are according to Tosh et al. [82]. Ala mutation of red residues in the human A₃AR sequence diminished agonist binding or activation [85]: L3.32, T3.36, F168 (EL2), M5.38, W6.48, L6.51, N6.55, L7.35, I7.39, S7.42, H7.43. Note: The green highlighted L in the mouse sequence is the correct residue [82]. On GPCRdb (https://gpcrdb.org/) this residue is incorrectly shown as a valine (V301^8.61). Note that residue numbers shown in this alignment may differ from other reports