Ann M. Decker

GPR17, an orphan G protein-coupled receptor, negatively regulates oligodendrocyte differentiation and myelination. Inhibiting GPR17 through selective antagonism is a promising strategy to promote endogenous remyelination in disorders like multiple sclerosis. Through rational structural modification of Pranlukast, a nonselective CysLT1 antagonist, we identified novel, selective small molecule GPR17 antagonist leads 39 and 44. These compounds exhibit GPR17 antagonist potencies (IC50) of ∼400−500 nM, with >5000-fold reduced potency at CysLT1 and minimal off-target activity at closely related CysLT2, P2Y1, P2Y2, P2Y4, and P2Y6 receptors. Structure-based modeling using the cryo-EM structure of GPR17 and the crystal structure of CysLT1 provided insights into the observed potency and selectivity. Treatment of isolated rat OPCs with early selective GPR17 antagonist 22 (10 μM) increased MBP expression and Olig2+ cell expansion, underscoring GPR17’s role in promoting OPC differentiation and myelination. Compounds 39 and 44 represent promising leads for further optimization into potent, drug-like GPR17 antagonists.

The development of effective pharmacotherapies for methamphetamine (METH) use disorder remains a critical challenge, prompting interest in strategies to modulate the endocannabinoid system because of its involvement in reward regulation. We hereby report the identification and characterization of a negative allosteric modulator (NAM) of the CB1 receptor, RTICBM-303 (11), which displayed good potencies in several CB1 in vitro assays, concentration dependently reduced the Emax of the agonist CP55,940, and increased [3H]CP55,940 binding. Compound 11, bearing a 4-fluorosulfonyl substituent, demonstrated considerably improved metabolic stability in rat liver microsomes (T1/2 > 255 min), favorable brain penetration, but limited aqueous solubility. Finally, 11 (10 mg/kg, i.p.) significantly and selectively reduced both cue-induced and drug-primed reinstatement of METH-seeking behavior in rats, without altering operant responding for food. These findings support 11 for further preclinical development and highlight the broader therapeutic potential of CB1 NAMs for treating stimulant use disorders.

Cannabinoid receptor type 1 (CB₁) negative allosteric modulators have emerged as an alternate approach to CB₁ orthosteric antagonists/inverse agonists for cocaine addiction treatment. This study explores aryl-alkyl squaramides as CB₁ allosteric modulators, featuring RTICBM-262 (3) with good in vitro potencies in CB₁ calcium mobilization, [³⁵S]GTPγS binding, and cAMP assays. Molecular modeling studies suggest 3 bound in a similar pocket as Org27569, forming π-stacking with key residues H154^2.41 and W241^4.50, and the potential C98-C107 disulfide bond had limited impact on its binding or receptor activation. ADME and in vivo pharmacokinetic studies suggest that 3 had reasonable metabolic stability, brain penetration, and selectivity against a panel of ∼ 50 targets but poor solubility and high protein binding. At 5.6 mg/kg (i.p.), 3 significantly attenuated both cocaine-seeking behavior specific to cue-induced reinstatement and cocaine-induced behavioral sensitization without altering locomotor activity. These results support squaramides as promising candidates for further investigation for cocaine addiction treatment.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) is increasing globally. MASLD is characterized by clinically significant liver steatosis, and a subset of patients progress to more severe metabolic-disorder-associated steatohepatitis (MASH) with liver inflammation and fibrosis. Cannabinoid receptor 1 (CB1) antagonism is a proven therapeutic strategy for the treatment of the phenotypes that underlie MASLD, though work on early centrally penetrant compounds largely ceased following adverse psychiatric indications in humans. We present here preclinical testing of a CB1 neutral antagonist, N-[1-[8-(2-Chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]-4-phenylpiperidin-4l]methanesulfonamide (RTI-348), with minimal brain exposure when administered to mice. In a diet-induced model of MASLD-induced MASH, administration of RTI-348 decreased the total body and liver weight gain. Animals treated with RTI-348 showed reduced steatosis. Furthermore, they produced lower plasma alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH), biomarkers associated with liver damage. Mice maintained on the MASH diet had elevated expression of genes associated with profibrogenesis, immune response, and extracellular matrix remodeling, and treatment with RTI-348 mitigated these diet-induced changes in gene expression. Using an intracranial electrical self-stimulation model, we also demonstrated that RTI-348 does not produce an anhedonia response, as seen with the first-generation CB1 inverse agonist rimonabant. Altogether, the results herein point to RTI-348 as a promising neutral antagonist for MASH.

The development of synthetic agonists for the orphan receptor GPR88 has recently attracted significant interest, given the promise of GPR88 as a novel drug target for psychiatric and neurodegenerative disorders. Examination of structure-activity relationships of two known agonist scaffolds 2-PCCA and 2-AMPP, as well as the recently resolved cryo-EM structure of 2-PCCA-bound GPR88, led to the design of a new scaffold based on the "reversed amide" strategy of 2-AMPP. A series of novel (4-substituted-phenyl)acetamides were synthesized and assessed in cAMP accumulation assays as GPR88 agonists, which led to the discovery of several compounds with better or comparable potencies to 2-AMPP. Computational docking studies suggest that these novel GPR88 agonists bind to the same allosteric site of GPR88 that 2-PCCA occupies. Collectively, our findings provide structural insight and SAR requirement at the allosteric site of GPR88 and a new scaffold for further development of GPR88 allosteric agonists.

Quality of life is often reduced in patients with sleep-wake disorders. Insomnia is commonly treated with benzodiazepines, despite their well-known side effects. Pellotine (1), a Lophophora alkaloid, has been reported to have short-acting sleep-inducing properties in humans. In this study, we set out to evaluate various in vitro and in vivo properties of 1. We demonstrate that 1 undergoes slow metabolism; e.g. in mouse liver microsomes 65% remained, and in human liver microsomes virtually no metabolism was observed after 4 h. In mouse liver microsomes, two phase I metabolites were identified: 7-desmethylpellotine and pellotine-N-oxide. In mice, the two diastereomers of pellotine-O-glucuronide were additionally identified as phase II metabolites. Furthermore, we demonstrated by DESI-MSI that 1 readily enters the central nervous system of rodents. Furthermore, radioligand-displacement assays showed that 1 is selective for the serotonergic system and in particular the serotonin (5-HT)_1D, 5-HT₆, and 5-HT₇ receptors, where it binds with affinities in the nanomolar range (117, 170, and 394 nM, respectively). Additionally, 1 was functionally characterized at 5-HT₆ and 5-HT₇, where it was found to be an agonist at the former (EC₅₀ = 94 nM, E_max = 32%) and an inverse agonist at the latter (EC₅₀ = 291 nM, E_max = -98.6). Finally, we demonstrated that 1 dose-dependently decreases locomotion in mice, inhibits REM sleep, and promotes sleep fragmentation. Thus, we suggest that pellotine itself, and not an active metabolite, is responsible for the hypnotic effects and that these effects are possibly mediated through modulation of serotonergic receptors.

ID 54516 Poster Board 341 We optimized a high-throughput fluorescent-based assay to assess transporter activity by measuring uptake of the substrate, ASP⁺ - 4-(4-(dimethylamino)styryl)-N-methylpyridinium. ASP⁺ is the fluorescent derivative of the neurotoxin 1-methyl-4-phenylpyridinium MPP⁺, both of which have been used to study various transporter properties. The ASP⁺ microplate method offers an easily teachable platform for microscale adaption to conduct high-throughput screens in 96-well plates. Our current work focuses on organic cation transporters (OCTs), proteins expressed in the brain and throughout the body with vital roles in normal physiology and drug responses. OCTs have a low-affinity and high-capacity to transport substrates across the cell membrane, and can quickly take up a broad range of compounds that include pharmacological drugs, endogenous neurotransmitters like dopamine, norepinephrine, and serotonin, and experimental substrates like ASP⁺. OCT research is limited by the lack of selective ligands for each of the three OCT subtypes (OCT1, 2, and 3). Saturation binding of ASP⁺ was measured in human embryonic kidney (HEK) cells that overexpress human OCT3 to determine the K_D for ASP⁺ in this model. Validation competition analysis was conducted to measure the displacement of ASP⁺ uptake and reduced fluorescence by known OCT3 inhibitors, corticosterone and normetanephrine. A primary screen testing nearly 1500 compounds (at 10 μM) and a subsequent confirmatory screen testing 100 compounds (at 10 μM and 1 μM) were conducted to identify hit compounds that were selected based on their ability to inhibit maximum binding of ASP⁺ at < 75%, similarly to blockade measured by normetanephrine. Screening parameters such as z-factor, % coefficient of variation, and the signal-to-background ratio were within acceptable ranges for this fluorescent ASP⁺ uptake microplate method, confirming its utility for high throughput screens. Twenty-seven compounds had confirmed activity at human OCT3 and were used in further competition analysis studies to determine their IC₅₀ values and identify trends in structure-activity-relationships. Experiments are ongoing to also evaluate the selectivity of lead compounds by testing their inhibition of fluorescent ASP⁺ uptake in cell lines that express human OCT1 or OCT2 for comparison to data obtained in the human OCT3 cell line. Identifying novel experimental selective inhibitors will propel the study of broadly expressed OCTs and expand the utility of the ASP⁺ microplate assay as a rapid and versatile approach to studying ligand interactions and the transport function of OCTs. This work was supported by a pilot, NIDA-funded grant (U18DA052527-01 awarded to LD and BB) and an FSOP internal research award (RFS).