Distinctive patterns of G protein coupling induced by the structurally similar 5-HT

Antipsychotic activity of certain 5-HT2A receptor (5-HT2AR) antagonists has been attributed to inverse agonism. 5-HT2AR hallucinogenic agonists exhibit biased agonism toward Gi/o proteins. Ketanserin and altanserin are 2 structurally similar orthosteric drugs used as 5-HT2AR therapeutic and radiotracers tools. This study assessed the pharmacological properties of ketanserin and altanserin, previously described as neutral 5-HT2AR antagonists, using postmortem human prefrontal cortex. Receptor radioligand binding experiments and functional [35S]GTPγS (sulfur 35 labeled guanosine-5'-O-[gamma-thio]-triphosphate) binding assays followed by immunocapture with anti-Gαq/11 and anti-Gαi1 protein antibodies were performed. Molecular dynamics simulations were used to ascertain binding modes to the 5-HT2AR. [3H]Ketanserin binding to 5-HT2ARs was decreased by guanylyl-5'-imido-diphosphate, a GTP analog. Guanylyl-5'-imido-diphosphate transformed the biphasic inhibition of [3H]ketanserin binding by the agonist (±)DOI into a monophasic low-affinity curve. Ketanserin stimulated [35S]GTPγS binding to Gαq/11 but not Gαi1 proteins. Conversely, altanserin inhibited [35S]GTPγS binding to Gαi1 but not Gαq/11 proteins. These effects were blocked by the neutral antagonist MDL-11,939. Altanserin-induced inhibition was abolished by the Gαi/o alkylating agent N-ethylmaleimide. Similar findings were observed in wild type but disappeared in 5-HT2AR knockout mice. Ketanserin and altanserin showed distinct interactions within residues S2.61, S5.46, and N6.55 in the 5-HT2AR binding pocket. Therefore, ketanserin and altanserin elicit biased signaling with different patterns of G protein modulation upon interacting with 5-HT2ARs in brain. Ketanserin behaves as partial agonist on Gq/11, whereas altanserin acts as inverse agonist on Gi1 proteins. These findings should influence the design of antipsychotic drugs targeting this receptor and when used as antagonists and radiotracers for the 5-HT2AR assessment. SIGNIFICANCE STATEMENT: Ketanserin and altanserin are categorized as neutral 5-HT2A receptor antagonists. This study shows differential patterns of 5-HT2A receptor coupling to Gαq/11 and Gαi1 proteins between ketanserin and altanserin and demonstrates biased signaling and inverse agonism in human brain.