ABSTRACT

The 5-HT 5A receptor (5-HT 5A R), for which no selective agonists and only a few antagonists exist, remains the least understood serotonin (5-HT) receptor. A single commercial antagonist (SB-699551) has been widely used to investigate central nervous system (CNS) 5-HT 5A R function in neurological disorders, including pain. However, because SB-699551 has affinity for many 5-HTRs, lacks inactive property-matched controls, and has assay interference concerns, it has liabilities as a chemical probe. To better illuminate 5-HT 5A R function, we developed a probe set through iterative rounds of molecular docking, pharmacological testing, and optimization. Docking over six million lead-like molecules against a 5-HT 5A R homology model identified five mid-μM ligand starting points with unique scaffolds. Over multiple rounds of structure-based design and testing, a new quinoline scaffold with high affinity and enhanced selectivity for the 5-HT 5A R was developed, leading to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT 5A R with a much more restricted off-target profile and decreased assay liabilities vs. SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678, which was also consistent with recent published 5-HTR structures. Surprisingly, property-matched analogs of UCSF678 that were either inactive across 5-HTRs or retained affinity for UCSF678’s off-targets revealed that 5-HT 5A R engagement is nonessential for alleviating pain in a mouse model, contrary to previous studies using less-selective ligands. Relative to SB-699551, these molecules constitute a well-characterized and more selective probe set with which to study the function of the 5-HT 5A receptor.

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The 5-HT_5A receptor (5-HT_5AR), for which no selective agonists and a few antagonists exist, remains the least understood serotonin receptor. A single commercial antagonist, SB-699551, has been widely used to investigate the 5-HT_5AR function in neurological disorders, including pain, but this molecule has substantial liabilities as a chemical probe. Accordingly, we sought to develop an internally controlled probe set. Docking over 6 million molecules against a 5-HT_5AR homology model identified 5 mid-μM ligands, one of which was optimized to UCSF678, a 42 nM arrestin-biased partial agonist at the 5-HT_5AR with a more restricted off-target profile and decreased assay liabilities versus SB-699551. Site-directed mutagenesis supported the docked pose of UCSF678. Surprisingly, analogs of UCSF678 that lost the 5-HT_5AR activity revealed that 5-HT_5AR engagement is nonessential for alleviating pain, contrary to studies with less-selective ligands. UCSF678 and analogs constitute a selective probe set with which to study the function of the 5-HT_5AR.

The neuromodulator melatonin synchronizes circadian rhythms and related physiological functions through the actions of two G-protein-coupled receptors: MT₁ and MT₂. Circadian release of melatonin at night from the pineal gland activates melatonin receptors in the suprachiasmatic nucleus of the hypothalamus, synchronizing the physiology and behaviour of animals to the light-dark cycle^1-4. The two receptors are established drug targets for aligning circadian phase to this cycle in disorders of sleep^5,6 and depression^1-4,7-9. Despite their importance, few in vivo active MT₁-selective ligands have been reported^2,8,10-12, hampering both the understanding of circadian biology and the development of targeted therapeutics. Here we docked more than 150 million virtual molecules to an MT₁ crystal structure, prioritizing structural fit and chemical novelty. Of these compounds, 38 high-ranking molecules were synthesized and tested, revealing ligands with potencies ranging from 470 picomolar to 6 micromolar. Structure-based optimization led to two selective MT₁ inverse agonists-which were topologically unrelated to previously explored chemotypes-that acted as inverse agonists in a mouse model of circadian re-entrainment. Notably, we found that these MT₁-selective inverse agonists advanced the phase of the mouse circadian clock by 1.3-1.5 h when given at subjective dusk, an agonist-like effect that was eliminated in MT₁- but not in MT₂-knockout mice. This study illustrates the opportunities for modulating melatonin receptor biology through MT₁-selective ligands and for the discovery of previously undescribed, in vivo active chemotypes from structure-based screens of diverse, ultralarge libraries.

Excipients, considered "inactive ingredients," are a major component of formulated drugs and play key roles in their pharmacokinetics. Despite their pervasiveness, whether they are active on any targets has not been systematically explored. We computed the likelihood that approved excipients would bind to molecular targets. Testing in vitro revealed 25 excipient activities, ranging from low-nanomolar to high-micromolar concentration. Another 109 activities were identified by testing against clinical safety targets. In cellular models, five excipients had fingerprints predictive of system-level toxicity. Exposures of seven excipients were investigated, and in certain populations, two of these may reach levels of in vitro target potency, including brain and gut exposure of thimerosal and its major metabolite, which had dopamine D3 receptor dissociation constant K _d values of 320 and 210 nM, respectively. Although most excipients deserve their status as inert, many approved excipients may directly modulate physiologically relevant targets.

The MRGPRX family of receptors (MRGPRX1-4) is a family of mas-related G-protein-coupled receptors that have evolved relatively recently¹. Of these, MRGPRX2 and MRGPRX4 are key physiological and pathological mediators of itch and related mast cell-mediated hypersensitivity reactions^2-5. MRGPRX2 couples to both G_i and G_q in mast cells⁶. Here we describe agonist-stabilized structures of MRGPRX2 coupled to G_i1 and G_q in ternary complexes with the endogenous peptide cortistatin-14 and with a synthetic agonist probe, respectively, and the development of potent antagonist probes for MRGPRX2. We also describe a specific MRGPRX4 agonist and the structure of this agonist in a complex with MRGPRX4 and G_q. Together, these findings should accelerate the structure-guided discovery of therapeutic agents for pain, itch and mast cell-mediated hypersensitivity.

There is considerable interest in screening ultralarge chemical libraries for ligand discovery, both empirically and computationally^1-4. Efforts have focused on readily synthesizable molecules, inevitably leaving many chemotypes unexplored. Here we investigate structure-based docking of a bespoke virtual library of tetrahydropyridines-a scaffold that is poorly sampled by a general billion-molecule virtual library but is well suited to many aminergic G-protein-coupled receptors. Using three inputs, each with diverse available derivatives, a one pot C-H alkenylation, electrocyclization and reduction provides the tetrahydropyridine core with up to six sites of derivatization^5-7. Docking a virtual library of 75 million tetrahydropyridines against a model of the serotonin 5-HT_2A receptor (5-HT_2AR) led to the synthesis and testing of 17 initial molecules. Four of these molecules had low-micromolar activities against either the 5-HT_2A or the 5-HT_2B receptors. Structure-based optimization led to the 5-HT_2AR agonists (R)-69 and (R)-70, with half-maximal effective concentration values of 41 nM and 110 nM, respectively, and unusual signalling kinetics that differ from psychedelic 5-HT_2AR agonists. Cryo-electron microscopy structural analysis confirmed the predicted binding mode to 5-HT_2AR. The favourable physical properties of these new agonists conferred high brain permeability, enabling mouse behavioural assays. Notably, neither had psychedelic activity, in contrast to classic 5-HT_2AR agonists, whereas both had potent antidepressant activity in mouse models and had the same efficacy as antidepressants such as fluoxetine at as low as 1/40th of the dose. Prospects for using bespoke virtual libraries to sample pharmacologically relevant chemical space will be considered.