William C. Wetsel

BACKGROUND: Negative clinical trials foster therapeutic nihilism and public skepticism, especially if they follow optimistic preclinical results. One way to increase the likelihood of successful clinical trials is to enhance the preclinical testing of new treatments. Recently, multicenter preclinical testing networks have gained attention as one tool to enhance rigor and reduce the likelihood of erroneously recommending a new candidate treatment for advancement to clinical trial testing. METHODS: The SPAN (Stroke Preclinical Assessment Network) consists of 6 research laboratories performing the experiments and 1 coordinating center at which treatments are prepared in a blinded manner, subjects are randomized, and statistical analysis is performed. We conducted a rigorous trial that included both sexes, multiple comorbidities, randomization, concealment of treatment status, blinded outcome assessments, prespecified sample sizes, and comprehensive data tracking. Five promising treatments were evaluated: BPN-27332, a lipoxygenase inhibitor; uric acid, a potent natural scavenger of free radicals; tatCN19o, a peptide inhibitor of activated CaMKII (Ca ²⁺ /CaM–dependent protein kinase II); GSK2256294, an inhibitor of sEH (soluble epoxide hydrolase); and NVX-058, an oxygen-carrying compound. A behavioral battery was selected as the end point for the primary analysis. The primary analysis used the modified intention-to-treat population. RESULTS: In total, 1768 rodents (882 females and 886 males) were enrolled: 441 young healthy mice, 444 aging mice, 450 mice with obesity-induced hyperglycemia and metabolic syndrome, and 433 spontaneously hypertensive rats. Data missing due to death before 30 days of follow-up were imputed from the same treatment group, animal model, sex, and stage. Although none of the candidates met the prespecified efficacy boundary, GSK2256294 showed an odds ratio for benefit of 1.28 (95% CI, 0.99–1.65), which was significant at P <0.05. CONCLUSIONS: The second SPAN study reaffirms the importance and feasibility of rigorous, multilaboratory preclinical testing networks to potentially reduce false positives in the development pipeline.

Schizophrenia is a severe mental illness whose current treatments primarily target dopamine and serotonin receptors. These drugs often cause side effects and vary in effectiveness across patients. The trace amine-associated receptor 1 (TAAR1), which modulates monoamine signaling, has emerged as a promising alternative target. To discover previously unexplored TAAR1 ligands, we computationally dock 65 million molecules against the active state of TAAR1 and experimentally test 55 of those highly ranked. Fourteen TAAR1 agonists emerge with potencies ranging from mid-nanomolar to micromolar. This high functional selectivity may reflect the compact conformation adopted by the activated TAAR1 orthosteric site. While this is favorable for agonist prioritization, simulations suggest that it can be over-optimized for initial hit rates at the expense of subsequent affinity maturation. Hit optimization yields nanomolar agonists whose docking-predicted poses are confirmed by cryo-EM. Three agonists have high brain exposure and potencies, with one compound showing potency comparable to the investigational drug ulotaront and two compounds exhibiting even greater potency, at levels sufficient for behavioral studies. All three potently normalize amphetamine-induced pre-pulse inhibition in mice, a model for schizophrenia, without catalepsy, a common side effect of traditional antipsychotics.

Polypharmacological molecules are attractive for complex illnesses. Here, we explored large library docking for joint activity against target pairs. Retrospectively, as libraries grew, so too did the number of likely dual-activity molecules. In prospective docking of a 900-million molecule library against three target pairs (α_2A/SERT, MOR/SERT, and α_2A/MOR), we sought analgesic compounds. Both the α_2A/SERT and SERT/MOR campaigns led to dual binders with low μM to high nM activities with high hit rates; tetrahydropyridines from the α_2A/SERT campaign were also active against 5-HT_2A. However, even though cryo-EM structures confirmed the docking-predicted poses, optimization struggled to improve potency. Still, in mouse behavioral assays, the most potent α_2A/SERT compound ('z7149) was effective against pain without inducing conditioned place preference, and the molecule had potent antidepression and anxiolytic drug-like behavior, consistent with its SERT/5-HT_2A activities. This study reveals both advantages and challenges of docking for polypharmacology.

Clinically, no human diseases are currently diagnosed as being directly driven by β- arrestins. Nonetheless, there is growing interest in developing β-arrestin–biased drugs, due to the key regulatory role of β-arrestins in modulating the cellular signaling of most G protein–coupled receptors (GPCRs). In cell-based studies of rhodopsin-family GPCRs, mutations in a conserved proline–hydrophobic (ProH) motif within the second intracellular loop (ICL2) have been shown to alter β-arrestin signaling bias. The clinical relevance of such mutations in humans is unknown. However, if naturally occurring single nucleotide polymorphisms (SNPs) affecting this ProH motif exist across the GPCR family, then cross-referencing these genetic variants with large-scale population sequencing and epidemiologic data could reveal potential roles for β-arrestin signaling in human health. In this report, we identify SNPs in human GPCRs that correspond to ProH substitutions, and we show that, in the neurotensin receptor NTSR1, these variants can indeed shift signaling bias. We also estimate a lower-bound population frequency for such SNPs, suggesting that although rare for any given receptor, their cumulative prevalence across the GPCR superfamily may be large enough to impact phenotypic variation. Together with emerging data from biased ligands, our findings support the idea that genetic variations in β-arrestin signaling could represent a meaningful source of therapeutic relevance. Significance statement: Mutations altering G protein-coupled receptor (GPCR) signaling can affect health. We have identified a specific amino acid determinant in intracellular loop (ICL2) of human rhodopsin family GPCRs that influences β-arrestin signaling. Although the medical consequences of this determinant remain unclear, we show that single nucleotide polymorphisms (SNPs) affecting the determinant routinely occur. By studying neurotensin receptor NTSR1, we confirm that the SNP alters β-arrestin signaling bias. While individually rare per receptor, these SNPs may collectively contribute to β-arrestin-related phenotypic changes in the human population. Our findings, combined with research on biased drugs, support the idea that β-arrestin signaling might serve as a useful therapeutic target, opening new possibilities for precision medicine.

One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving the identification of high-specificity spatial proteomes. The resulting native proximity proteomes are enriched for human genes dysregulated in the brain of autistic individuals, and reveal proximity interactions between proteins from high-confidence risk genes with those of lower-confidence that may provide new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology.

L-DOPA is the mainstay of treatment for Parkinson’s disease (PD). However, over time this drug can produce dyskinesia. A useful acute PD model for screening novel compounds for anti-parkinsonian and L-DOPA-induced dyskinesia (LID) are dopamine-depleted dopamine-transporter KO (DDD) mice. Treatment with α-methyl-para-tyrosine rapidly depletes their brain stores of DA and renders them akinetic. During sensitization in the open field (OF), their locomotion declines as vertical activities increase and upon encountering a wall they stand on one leg or tail and engage in climbing behavior termed “three-paw dyskinesia”. We have hypothesized that L-DOPA induces a stereotypic activation of locomotion in DDD mice, where they are unable to alter the course of their locomotion, and upon encountering walls engage in “three-paw dyskinesia” as reflected in vertical counts or beam-breaks. The purpose of our studies was to identify a valid index of LID in DDD mice that met three criteria: (a) sensitization with repeated L-DOPA administration, (b) insensitivity to a change in the test context, and (c) stimulatory or inhibitory responses to dopamine D1 receptor agonists (5 mg/kg SKF81297; 5 and 10 mg/kg MLM55-38, a novel compound) and amantadine (45 mg/kg), respectively. Responses were compared between the OF and a circular maze (CM) that did not hinder locomotion. We found vertical counts and climbing were specific for testing in the OF, while oral stereotypies were sensitized to L-DOPA in both the OF and CM and responded to D1R agonists and amantadine. Hence, in DDD mice oral stereotypies should be used as an index of LID in screening compounds for PD.

There is now evidence from multiple Phase II clinical trials that psychedelic drugs can exert long-lasting anxiolytic, anti-depressant, and anti-drug abuse (nicotine and ethanol) effects in patients. Despite these benefits, the hallucinogenic actions of these drugs at the serotonin 2A receptor (5-HT2AR) limit their clinical use in diverse settings. Activation of the 5-HT2AR can stimulate both G protein and β-arrestin (βArr) -mediated signaling. Lisuride is a G protein biased agonist at the 5-HT2AR and, unlike the structurally-related lysergic acid diethylamide (LSD), the drug does not typically produce hallucinations in normal subjects at routine doses. Here, we examined behavioral responses to lisuride, in wild-type (WT), βArr1-knockout (KO), and βArr2-KO mice. In the open field, lisuride reduced locomotor and rearing activities, but produced a U-shaped function for stereotypies in both βArr lines of mice. Locomotion was decreased overall in βArr1-KOs and βArr2-KOs relative to wild-type controls. Incidences of head twitches and retrograde walking to lisuride were low in all genotypes. Grooming was decreased in βArr1 mice, but was increased then decreased in βArr2 animals with lisuride. Serotonin syndrome-associated responses were present at all lisuride doses in WTs, but they were reduced especially in βArr2-KO mice. Prepulse inhibition (PPI) was unaffected in βArr2 mice, whereas 0.5 mg/kg lisuride disrupted PPI in βArr1 animals. The 5-HT2AR antagonist MDL100907 failed to restore PPI in βArr1 mice, whereas the dopamine D2/D3 antagonist raclopride normalized PPI in WTs but not in βArr1-KOs. Clozapine, SCH23390, and GR127935 restored PPI in both βArr1 genotypes. Using vesicular monoamine transporter 2 mice, lisuride reduced immobility times in tail suspension and promoted a preference for sucrose that lasted up to 2 days. Together, it appears βArr1 and βArr2 play minor roles in lisuride’s actions on many behaviors, while this drug exerts anti-depressant drug-like responses without hallucinogenic-like activities.