One- and Two-Photon Excitable Photoswitchable Orthoand Allosteric Ligands of Class A GPCRs

Photoswitchable ligands for two class A G protein-coupled receptors (GPCRs) were designed, synthesized, and characterized. For the serotonin 2A receptor (5-HT2AR), a photoswitchable derivative of the psychedelic N,N-dimethyltryptamine (photo-DMT) was created that binds with similar affinities in its cis- and trans-configurations but produces different levels of receptor activation, acting as an efficacy switch. For the muscarinic acetylcholine receptor 1 (M1R), photoswitchable derivatives of the agonist xanomeline, called xanoswitches, were optimized for two-photon near-infrared light activation, enabling bidirectional modulation of neuronal activity in vivo, and for one-photon amber-light activation, allowing control of calcium oscillations and zebrafish motility. The work also presents the first allosteric photoswitch for class A GPCRs, using the M1 positive allosteric modulator BQCA to create Photo-BQCAs that dynamically modulate cooperativity.

Study at a glance

Abstract

Photopharmacology is a vivid field of research that combines the unsurpassed spatiotemporal resolution of light with the principles of pharmacology. By introduction of synthetic chemical photoswitches into the structures of biologically active compounds, researchers can obtain control over biological processes. This is especially useful in the research of G protein-coupled receptors (GPCRs) where scientific and therapeutic interest is enormous. When traditional pharmacology lacks spatiotemporal resolution or selectivity, photopharmacology can provide the necessary precision, opening up a vast potential of possible applications, ranging from the use as scientific tools to therapeutic applications. In this work photoswitchable ligands for Class A GPCRs were designed, synthesized and extensively characterized with regard to their photophysical properties and their pharmacological profile. The targeted receptors are the serotonin receptor 2A (5-HT2AR) and the muscarinic acetylcholine receptor 1 (M1R). Both receptors have been of great historical relevance and are still subjects of enormous scientific interest as they are implicated in clinical disorders as for example addiction and depression (5-HT2A) or in Alzheimer’s disease (M1R). For the 5-HT2AR, N,N-dimethyltryptamine (DMT), an in nature abundantly occurring psychedelic agent, was rendered photoswitchable by incorporation of its indole-core into a photoswitchable moiety. Together with photoswitchable serotonin derivatives from the Trauner group, they constitute the first photoswitchable ligands for this receptor. Complementary to the photoswitchable serotonin, the presented photo-DMT acts as efficacy switch with sub-micromolar potencies. This means, that photo-DMT is binding with almost identical affinities in its cis- and trans-configuration to the 5-HT2A but with a disparate degree of maximal receptor activation. The muscarinic receptors have been prototypical systems for the development of photopharmacological agents, hence, a number of photoswitches already exists for this receptor family. However, all of those photoswitchable ligands show higher activity in their thermodynamically more stable configuration and their operational wavelengths are of low tissue penetrating capabilities. By azo-extension of the aliphatic side-chain of the clinical promising muscarinic agonist xanomeline (M1R/M4R preferring), the first cison switches at the muscarinic receptors, termed ”xanoswitches”, were developed. These xanoswitches were optimized by chemical modifications with regard to their operational wavelengths. In a first project the xanoswitches were chemically modified to allow photoisomerization by two-photon (2P) absorption processes, using a pulsed near-infrared light (NIR) laser. This was done using single point, bio-isosteric modifications at the azobenzene. With these 2P-xanoswitches, for the first time, bidirectional, reversible photomodulation of neuronal activity in vivo using NIR-light could be achieved. In a second project, xanoswitches were optimized for red-shifted one-photon (1P) excitation. By incorporation of a tetra-ortho-chlorinated azobenzene into the molecular structure, amber-light switchable xanoswitches were obtained. Complementary to the two-photon switches these 1P-xanoswitches are operational with standard LEDs and were shown to allow reversible modulation of intracellular Ca2+ oscillations and optical control over zebrafish motility. Moreover, this work presents the first allosteric photoswitch for class A GPCRs. Using the prototypical M1 positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) a set of Photo-BQCAs was developed exhibiting complementary photopharmacological properties. While BQCisA is more active in its thermodynamically less stable cis-configuration, BQCtrAns exhibits a higher activity in its more stable transconfiguration. Together they serve as proof of principle compounds, showing that cooperativity can be dynamically modulated using photoswitchable ligands and that the desired photopharmacological behavior can be introduced by design. The achievements described in this work can give important new insights into various aspects of GPCR pharmacology by allowing precise control of receptor activation. This might shed new light on the complex signaling pathways that underlie many physiological processes and diseases or might lead to therapeutic application of photoswitchable compounds.