AI-derived therapeutic development of a serotonin receptor–targeting drug for the treatment of opioid use disorder

Significance

The premise of these studies was to develop and validate a therapeutic for opioid use disorder. Using an AI-based platform aimed at polypharmacy-focused drug discovery, two compounds were synthesized and tested, GATC-021 and GATC-1021. We found that GATC-1021, which targets the 5-HT_2A and 5-HT₆ receptors,_, was highly selective in attenuating fentanyl self-administration in male and female rats, with retained effectiveness following repeated dosing. Following GATC-1021 treatment, an increase in the percentage of thin dendritic spines in the hippocampus and changes in gene expression pathways involved in neuroplasticity in the prefrontal cortex were evidenced. Taken together, these findings provide a foundation to support the further clinical development of GATC-1021 as a potential treatment for individuals suffering from opioid use disorder.

Abstract

The opioid epidemic has led to a devastating loss of life nationwide. Of those dependent on opioids, many individuals desire to quit or reduce use, but their efforts are often unsuccessful given the powerful reinforcing properties associated with opioid drugs, including fentanyl. Here, we developed a therapeutic drug based on an AI-based platform, which was rationally designed to identify markers of dysregulation from human drug user postmortem brain tissue. Two top candidate compounds, GATC-021 and GATC-1021, were synthesized and validated with in vitro screening for target specificity. Thereafter, the drug candidates were examined for their effectiveness in modulating opioid reinforcement and intake. GATC-1021 emerged as the most efficacious compound as it significantly decreased fentanyl intake in both male and female rats, without any notable side effects. GATC-1021 was further found to modulate gene expression patterns in addiction-relevant brain regions following fentanyl-induced alterations. These findings validate GATC’s AI-based platform with its polypharmacy-focused drug development approach and further support the clinical potential of GATC-1021 as a promising therapeutic for those suffering from opioid use disorder.

Dysregulated opioid use has led to devastating consequences for the lives of many individuals, affecting millions worldwide (1). In 2017, the opioid epidemic was declared a national emergency in the United States (2). It has been estimated that around three million individuals nationwide currently suffer from opioid use disorder (OUD) (3). Alarmingly, the vast majority of recent deaths have been attributed to synthetic opioids such as fentanyl. Current treatment options for OUD include methadone, buprenorphine, and naloxone (1). However, these medications are significantly limited by suboptimal safety margins, inadequate long-term efficacy, and/or treatment adherence challenges. Thus, the significant morbidity, mortality, and economic consequences imposed by opioid misuse represent a pressing global concern that calls for novel and innovative treatment strategies.

Fentanyl is a synthetic analgesic that exhibits high potency and lipophilic properties, leading to relatively quick pharmacokinetic actions in the brain and thereby inferring fast analgesic properties, high potential for respiratory depression, and high addiction liability (3, 4). Fentanyl exhibits agonist properties with direct binding to the μ-, κ-, and δ-opioid receptors, leading to downstream intracellular changes in β arrestin and G_i-protein signaling cascades (4–7). Recent in vitro evidence also suggests that fentanyl may act on the α1 adrenoceptor subtypes, dopamine D1 and D4 receptors, and serotonin 5-HT_1A and 5-HT_2A receptors, in addition to blocking catecholamine uptake through actions on the vesicular monoamine transporter 1 (5, 8–10). Interestingly, with regard to serotonergic signaling, fentanyl has been shown to modulate activity of a recently identified μ-opioid/5-HT_1A receptor heterodimer at the plasma membrane in vitro, in which fentanyl may increase the downstream intracellular 5-HT_1A signaling pathways MAPK and Erk1/2 (11). Moreover, long-term treatment with fentanyl can induce persistent changes in receptor and related circuit function, for instance as evidenced at the μ-opioid receptor with tolerance and increased desensitization (12). The development of clinical symptoms related to chronic opioid abuse, including dependence and tolerance, is thought to involve opioid receptors within the mesolimbic reward system (13). Specifically, μ opioid receptors agonists, such as fentanyl, increase dopamine release from ventral tegmental area (VTA) neurons projecting into the nucleus accumbens (NAc), which is thought to underlie the reinforcing properties of the drug (14), whereas tolerance and withdrawal-mediated effects are thought to involve inhibitory signaling within and into the VTA (15–17). Importantly, 5-HT receptors are also localized within addiction-associated brain regions, including the VTA, NAc, prefrontal cortex (PFC), and hippocampus (18, 19). Therefore, it is of particular interest to target pathways involved in the molecular changes associated with opioid’s effects as an approach for therapeutic development.

Artificial or augmented intelligence (AI) therapeutic discovery strategies have rapidly emerged across the healthcare market and currently represent a source of innovation within various fields, including neurology, oncology, and cardiology. Here, we employed the GATC Multi-omics Advanced Technology (MAT) platform, which was developed using a complex integration of large genomic, transcriptomic, proteomic, and metabolomic datasets, that were generated from postmortem human brain tissue in patients dependent on opioids. From the data, 20 high probability causative biomarkers were identified and subsequently delivered to the GATC MAT platform to analyze the systems biology and predict the treatment markers and mechanisms. This biomarker signature converged on therapeutic modulation of ZFP36/TTP, 5-HT_2A, and 5-HT₆. Using the GATC AI platform, we identified a dual-target profile involving 5-HT_2A/5-HT₆ agonist paired with a sulbutiamine-like CNS penetration motif. Reinforcement learning-guided optimization reproducibly converged on a bipartite disulfide-indole scaffold, and systematic iteration of the indole/tryptamine pharmacophore generated >80 candidate compounds predicted to modulate pathways implicated in OUD. Two molecules, GATC-1021 and GATC-021, exhibited the highest in-silico dual serotonergic affinity and CNS drug-likeness and were advanced to in vitro receptor binding assays, with GATC-021 also supported by prior classification as a nonhallucinogenic psychedelic analogue [e.g., compound 14 in Cameron et al. (20)]. We thus examined the effectiveness of GATC-021 and GATC-1021 to modulate synaptic targets predicted to mitigate the long-term effects of opioid use with in vitro assays, followed by preclinical studies assessing the effectiveness in mitigating fentanyl intake with intravenous self-administration, the ‘gold-standard’ rodent model of drug taking behavior with high translational relevance to human patterns of drug use. Taken together, our studies validate the GATC MAT platform for the identification of targets for therapeutic development and further provide evidence that AI-directed methods can serve as promising tools for therapeutic design and biological targeting with OUD.

Results

In Vitro Assessment of Binding Specificity.

We first validated the targeting of GATC-021 and GATC-1021 to serotonergic signaling, as predicted by AI-derived target analysis, and GATC-021 was synthesized and examined for its effectiveness in acting on various serotonin receptors (Fig. 1A and SI Appendix, Figs. S1 and S3). In cell-based assays, we found that the greatest efficacy of GATC-021 was on the 5HT_2A and 5HT₆ receptors, with negligible effects on 5HT_2B and 5HT_7D (Fig. 1 B and C). As a control, we also examined TrkB and found no detectable signaling. In examining the effects of GATC-021 on generalized behavior, an inhibitory effect emerged at the highest dose of 70 mg/kg, with a statistically significant decrease in locomotor with distance traveled (Fig. 1D) and in freezing time, time mobile, and center time (SI Appendix, Fig. S4), suggesting off-target behavioral effects.

Fig. 1.

GATC-021 and GATC-1021 exhibit agonist activity on serotonergic receptors. (A) Chemical structure of GATC-021. (B) GATC-021 analysis in the bioluminescence resonance energy transfer (BRET) assay shows activation of serotonin receptors HTR2A and HTR6, with minimal actions on HTR2B, HTR7D, and TrkB. (C) Percent efficacy from functional assay for GATC-021 on each receptor subtype. (D) Following injection of GATC-021, locomotor behavior was assessed in the open field in rats (n = 14). A significant reduction in locomotion was found with 70 mg/kg GATC-021 treatment, indicating potential off-target effects. **P < 0.05. (E) GATC-1021 chemical structure. (F) BRET assay for GATC-1021 shows activation of serotonin receptors HTR2A and HTR6, with negligible activity at the HTR2B, HTR7D, and TrkB receptors. (G) Percent efficacy from functional assay for GATC-1021 on each receptor subtype. (H) No differences in locomotor behavior were found across doses up to 70 mg/kg for GATC-1021 (n = 8). Each dot represents one subject. Data are expressed as mean ± SEM.

GATC-1021 was next synthesized and examined for its binding specificity (Fig. 1E and SI Appendix, Fig. S2). We found that GATC-1021 exhibited agonist action selectivity for the 5HT_2A receptor, with lesser agonist efficacy on the 5HT₆ receptor (Fig. 1 F and G), demonstrating an enhanced selectivity profile when compared to GATC-021. Negligible effects were found for 5HT_2B, 5HT_7D, and TrkB receptors. In examining locomotor behavior, we found no general behavioral effects of GATC-1021 across the doses of 25, 40, and 70 mg/kg compared to vehicle control in all movement analyses (Fig. 1H and SI Appendix, Fig. S4). These findings indicate that GATC-1021 may be a preferred compound given the lack of off-target side effects relative to locomotor behaviors across a range of doses.

Effects of GATC-021 and GATC-1021 on Fentanyl Intake.

Male and female rats were first surgerized to implant indwelling jugular catheters and then were provided access to self-administer intravenous fentanyl across 9 sessions at a dose of 2.5 μg/kg/infusion. All subjects achieved a stable level of responding prior to administration of the GATC compounds (SI Appendix, Fig. S5). Given that no effects of sex were found with administration of GATC compounds, data have been compiled for analysis, and separated sex-specific data are located in the supplementary file (SI Appendix, Figs. S6 and S7).

GATC-021 was first examined to determine whether it would alter fentanyl intake following an acute dose (1 d of treatment) or extended daily dosing period (5 d of treatment). Acute administration of GATC-021 resulted in a reduction in fentanyl intake at the 50 mg/kg dose (Fig. 2A). Next, given variability in baseline responding due to individual differences, data were normalized to the baseline mean of the prior 3 d to examine the percent change from baseline with each dose, resulting in both the 25 and 50 mg/kg GATC-021 doses leading reductions in fentanyl infusions (Fig. 2B). After five days of repeated injections with GATC-021 and fentanyl self-administration, the effects of the compound showed no significant differences when examining the total number of infusions (Fig. 2F). However, this lack of an effect may have been obscured by individual differences in responding or in off-target behavioral actions, since the percent change from baseline provided evidence of a persistent effect in reducing fentanyl intake at both the 25 and 50 mg/kg doses (Fig. 2G). The effects of GATC-021 on self-administration behavior were analyzed by sex, but no statistically significant sex differences were found at any dose of GATC-021 (SI Appendix, Fig. S6).

Fig. 2.

GATC-1021 preferentially decreases fentanyl self-administration with both acute and repeated dosing paradigms. (A–E) The effects of acute dosing were examined for both GATC compounds. (A) GATC-021 with sulbutiamine induced a significant decrease in the number of fentanyl infusions at the dose of 50 mg/kg. **P < 0.01. (n = 12 to 13/group) (B) Data were transformed to percent change from baseline, given individual variability in responding. GATC-021 at both the 25 and 50 mg/kg doses was found to reduce responding in consideration of baseline variability. *P < 0.05, ***P < 0.001. (C) The effects of sulbutiamine alone were examined and compared to the control saline self-administration condition. Subjects self-administering fentanyl, either with or without sulbutiamine injection, exhibited similar responding, which was significantly greater than that found for saline. ***P < 0.001, ****P < 0.0001. (n = 12 to 13/group) (D) GATC-1021 with sulbutiamine induced a significant decrease in the number of infusions earned for all doses from 25 to 70 mg/kg. The 40 mg/kg GATC-1021 dose was examined in the absence of sulbutiamine [40(-sulb)], and this dosing paradigm was found to be equally effective as the dose with sulbutiamine. ****P < 0.0001. (n = 12 to 13/group) (E) In examining the percent change from baseline, all of the GATC-1021 dose groups exhibited a substantial decrease in responding for fentanyl infusions. ****P < 0.0001. (F–J) The effects of repeated dosing were examined for both GATC compounds, and data are shown for the final, fifth day. (F) GATC-021 did not alter the total number of fentanyl infusions following repeated administration. (G) When examining based on percent change from baseline, the 25 and 50 mg/kg treatment groups exhibited a slight reduction in the percent change. *P < 0.05, ***P < 0.001. (H) After five days of injection, subjects self-administering fentanyl, either with or without sulbutiamine, maintained significantly greater responding than that found with saline. However, the repeated dosing of sulbutiamine led to a decrease in the number of fentanyl infusions earned relative to fentanyl group without sulbutiamine. **P < 0.01, ****P < 0.0001 vs saline. ^P < 0.05 vs fentanyl without sulbutiamine. (I) Repeated dosing of GATC-1021 substantially reduced the number of fentanyl infusions earned across all doses examined. *P < 0.05, ****P < 0.0001. (J) In examining the percent change from baseline, all GATC-1021 doses demonstrated a significant reduction in fentanyl intake, with the 40 mg/kg without sulbutiamine group showing the greatest mean percent reduction in fentanyl infusions. **P < 0.01, ****P < 0.0001. Each dot represents one subject. Data are shown as mean ± SEM.

In consideration of the potential effects of individual differences, we next conducted a control study to examine whether the administration of sulbutiamine, which was administered to increase thiamine levels, was independently altering self-administration behavior. Subjects were examined with fentanyl self-administration, either with or without sulbutiamine, which was compared to control levels of saline self-administration (no fentanyl). On the first day of injection, both of the fentanyl self-administering groups exhibited significantly higher levels of the number of infusions earned, independent of sulbutiamine injection, compared to the saline group (Fig. 2C). On the fifth day of injections, similar effects were found with a higher level of responding for fentanyl compared to saline (Fig. 2H). However, the post hoc also revealed a significant reduction in the fentanyl group injected with, versus the group without, sulbutiamine. These findings suggest that repeated, but not acute, administration of sulbutiamine alone may induce an independent, modest reduction in fentanyl self-administration behavior.

GATC-1021 was then examined for its effectiveness in attenuating fentanyl intake. Given the promising safety profile from our initial analyses, a broader dose range was used that included 0, 25, 40, and 70 mg/kg, all with coadministration of sulbutiamine. Further, given the potential effects of sulbutiamine alone with repeated dosing, we also included a treatment group with the moderate dose and no sulbutiamine [denoted as 40(−sulb)]. On the first day of administration, all doses of GATC-1021 were found to be highly effective in attenuating the responding for fentanyl infusions (Fig. 2D). When analyzed as the percent change from baseline (e.g., mean of the 3 d prior to drug treatment) to account for potential individual differences in responding, the significant reduction in fentanyl intake was maintained across all doses, with a mean level of reduced responding greater than 60% (Fig. 2E). On the fifth day of administration, similar effectiveness was found in the actions of GATC-1021, with all groups exhibiting reduced responding compared to the vehicle control (Fig. 2I). This pronounced effect of GATC-1021 in decreasing fentanyl intake was further evidenced when responding was assessed relative to baseline levels for each subject (Fig. 2J). Of note, the effects of GATC-1021 on self-administration behavior were analyzed by sex, but no statistically significant sex differences were found at any dose of GATC-1021 (SI Appendix, Fig. S7). Taken together, these data provide compelling evidence that validate GATC-1021 as being an effective treatment to reduce fentanyl intake, with a broad dose range and similar effectiveness either in the presence or absence of sulbutiamine for fentanyl reduction.

Assessment of Potential Adverse Outcomes.

To examine for any potential adverse effects of the GATC compounds on organ systems, liver samples were pathologically examined following the fentanyl self-administration with GATC dosing studies. For GATC-021, no significant differences were found with any of the histological markers in the liver when compared to the control condition (SI Appendix, Fig. S8A). For GATC-1021, no significant group differences were found for the histological indications in both the brain and liver, with exception of the cytoplasmic vacuoles in the liver, in which reduced vacuoles were found in the 40(-sulb) group compared to the 25 to 70 mg/kg GATC doses (SI Appendix, Fig. S8 B, S8 C). These findings demonstrate that while limited pathological indications were affected with administration of the GATC compounds, removal of sulbutiamine from GATC-1021 dosing may result in a better safety profile.

In consideration of the above findings, GATC-1021 was thereafter focused on as the top drug candidate for further validation. Since GATC-1021 was shown to target the 5HT_2A and 5HT₆ receptors, the next set of studies sought to determine whether GATC-1021 would induce a hallucinogenic-associated response. In rodents, this is classically determined by a head twitch behavior following injection of a hallucinogen, such as DOI (21). While acute administration of DOI resulted in a significant number of head twitch responses, little to no effect was found for GATC-1021 across doses (Fig. 3A). Interestingly, repeated dosing with DOI across five days led to a reduction in the number of head twitches, which is indicative of tolerance, but GATC-1021 still did not induce any significant head twitch responses across doses (Fig. 3B). These findings demonstrate that while GATC-1021 acts on serotonin receptors, there is no evidence that this agonist induces hallucinogenic-like responses.

Fig. 3.

GATC-1021 crosses the blood–brain barrier and alters dendritic spine plasticity, without behavioral effects found with 5HT_2A targeting psychedelics. (A and B) GATC-1021 did not induce the head twitch response compared to the hallucinogen DOI with acute dosing (A) or on the fifth day of dosing (B). ***P < 0.001, ****P < 0.0001 vs DOI. (n = 4/group) (C–G) Pharmacokinetic analysis in the blood and brain following injection with GATC-1021 at time points 15-, 45-, and 75-min. (n = 8 to 10/group) (C) Plasma levels of GATC-1021 peaked at 15 min postinjection and declined at subsequent time points. Sulbutiamine pretreatment did not significantly alter these levels. *P < 0.05, ***P < 0.001, ****p < 0.0001 vs 0 mg/kg. (D) Plasma thiamine levels were elevated in all groups receiving GATC-1021 with sulbutiamine, compared to subjects not receiving sulbutiamine. ^#P < 0.05 all groups vs 40 mg/kg(-sulb). (E) Brain concentrations of GATC-1021 were detected for the 25, 40, and 70 mg/kg groups. Higher levels of GATC-1021 were found at 15 min for 70 mg/kg (^@P < 0.05 vs 0 mg/kg) and 40 mg/kg (-sulb) (****P < 0.001 vs 0 mg/kg), and at 45 min for 40 mg/kg(-sulb) (****P < 0.0001 vs 0 mg/kg). (F) Brain thiamine levels were similar across all dose groups. (G) At 15-min postinjection, brain thiamine pyrophosphate (TPP) levels were significantly higher in the 40 mg/kg(-sulb) group compared to the 0, 25, 40, and 70 mg/kg GATC-1021 groups coadministered sulbutiamine. The TPP in the 40 mg/kg without sulbutiamine group remained elevated at 45 min postinjection compared to the 25 and 40 mg/kg groups with sulbutiamine. ^#P < 0.05 40 mg/kg(-sulb) vs all other groups. ^P < 0.05 40 mg/kg(-sulb) vs 25 and 40 mg/kg. (H) Representative photomicrographic images of dendritic spines in the hippocampus across groups. Red arrows denote examples of thin spines. (Scale bar, 50 μm.) (I) Dendritic spine density (spines/μm) was quantified for each subtype (thin, stubby, mushroom, filopodia, branched). Increased thin spines and decreased stubby spines were found in the GATC-1021 40 mg/kg (-sulb) group compared to the saline or fentanyl self-administration groups. **P < 0.01 compared to saline. ^P < 0.05, ^^^P < 0.001 comparing to fentanyl. (n = 6/group) Each dot represents one subject. Data are shown as mean ± SEM.

Absorption of GATC-1021 into the Blood and Brain.

To validate the ability of GATC-1021 to cross the blood–brain barrier, a pharmacokinetic study was next conducted. Following GATC-1021 injections, blood and brain samples were collected and examined at time points 15-, 45-, and 75-min. All GATC-1021 doses from 25 to 70 mg/kg were found to exhibit high levels in the plasma at the 15 min time point and then declined thereafter (Fig. 3C). As expected, increased thiamine levels were found in all groups receiving the synthetic derivative of thiamine, sulbutiamine, compared to lower endogenous levels in the absence of sulbutiamine (Fig. 3D). Increased blood thiamine levels were found at the 15-, 45-, and 75-min time points for all GATC-1021 doses, as compared to dosing without sulbutiamine. Importantly, in the brain, the highest level of GATC-1021 was found in the absence of sulbutiamine with the 40 mg/kg dose (Fig. 3E). Specifically, while GATC-1021 was detected in the brain at all time points above baseline levels for the 25, 40, and 70 mg/kg doses, a significant increase in brain GATC-1021 was noted at time point 15 min for 70 and 40(-sulb), and at time point 45 min for 40(-sulb). Given the behavioral effects in reducing fentanyl intake at all GATC-1021 doses, this provides evidence of a wide range of therapeutic efficacy for GATC-1021 in the brain. Interestingly, brain thiamine was found at similar levels independent of sulbutiamine injection (Fig. 3F), indicating that peripheral injection with sulbutiamine did not alter brain levels. Moreover, when examining thiamine pyrophosphate (TPP), the active form of thiamine in the brain, increased levels of GATC-1021 were found with the 40(-sulb) dose, but not any of the doses paired with sulbutiamine (Fig. 3G). These findings suggest that administration of sulbutiamine did not result in an increase in the active form of thiamine in the brain, but rather, appeared to reduce the absorption of GATC-1021 into the brain tissue with the formulation and dose used in this study. However, additional studies are warranted to determine whether absorption and/or metabolism would differ across specific brain regions with varying dosing paradigms and in the presence/absence of fentanyl.

Effects of GATC-1021 on Dendritic Spine Plasticity.

Dendritic spines exhibit plasticity throughout the lifespan, and as such, changes in spine density have been proposed to underlie the normal adaptive plasticity that is critical to sustain hippocampal learning and memory function (22). Interestingly, serotonin 5HT_2A receptor agonists alter dendritic spine morphology, and this induced neuroplasticity has been argued to be an underlying factor mediating beneficial cognitive effects found with psychedelic administration (23–27). Further, the 5HT₆ receptor has also been shown to modulate neuronal developmental processes (28). Given that an acute injection of experimenter-administered fentanyl has been shown to alter synaptic plasticity in the hippocampus (29), we first examined hippocampal brain sections for changes in spine morphology across the following subtypes: thin, stubby, mushroom, filopodia, and branched. Fentanyl self-administering rats treated with GATC-1021 (40 mg/kg without sulbutiamine) exhibited changes in spine morphology (Fig. 3 H, 3I). Specifically, the fentanyl group treated with GATC-1021 had a greater percentage of thin spines compared to the saline and fentanyl groups. GATC-1021 treatment with fentanyl also led to a reduction in the percentage of stubby spines compared to the fentanyl only group. In a separate cohort of subjects in which we validated the effects of GATC-1021 to attenuate fentanyl intake, we examined the PFC (SI Appendix, Fig. S9), but no differences were found in dendritic spine density in this region at the time point examined. In the literature, 5-HT2A agonists have exhibited drug-specific and time-course differences on dendritic spine plasticity in the PFC. For instance, DOI was found to exert an increase in spine plasticity within a discrete time point scale, in which increased density was detected at 30 min, but not at 15, 45, and 60 min, postinjection (23). This is in contrast to psilocybin, for which an increase in spine density was found at 24 h and sustained up to 34 d (27). Thus, conclusions drawn based on the lack of difference in the PFC are limited by the time point of analysis in the current studies (e.g., ~75 min post-GATC-1021 injection), as differences may have been imposed either before or after the time of examination. Thus, based on the hippocampal data, these findings provide a potential functional mechanism through which GATC-1021 can alter dendritic spine plasticity, potentially leading to subsequent effects on behavior.

GATC-1021 Alters the Transcriptomic Profile Across Addiction-Related Brain Regions.

We next explored gene expression pathways in fentanyl self-administering subjects treated with GATC-1021 (40 mg/kg without sulbutiamine), comparing to those self-administering fentanyl or saline, by performing differential expression analysis (DEA) of whole-transcriptome profiled PFC, VTA, and NAc tissue samples (30). Sex was included as a covariate in the DEA model to account for baseline male and female differences in expression, but no treatment-by-sex interaction terms were found to be significant in the present analysis. The PFC, VTA, and NAc were selected for total RNA sequencing and analysis given their well-documented roles in various OUD-related processes (1). Aiming to first characterize the transcriptomic changes associated with OUD, we performed DEA between the fentanyl (comparison) and saline (reference) self-administration groups at the adjusted P-value < 0.10 false detection rate (FDR) threshold across the PFC, NAc, and VTA (Fig. 4 A–C, respectively). The fentanyl vs saline self-administration comparison revealed a higher number of differentially expressed genes (DEGs) in the VTA compared to the other brain regions (298 DEGs in the VTA compared to the 45 DEGs in the PFC and 33 DEGs in the NAc) (Fig. 4J). The downregulated DEGs in the VTA with fentanyl are consistent with prior reports, such as the classic plasticity gene Grin2a and immediate-early gene Egr4 (31), and were recapitulated by the current DEA (SI Appendix, Fig. S10). Treatment with GATC-1021 with fentanyl self-administration reduced the number of overall RNA transcripts changed in the VTA compared to the control (Fig. 4F). In the PFC (Fig. 4D) and NAc (Fig. 4E), GATC-1021 induced upregulation and downregulation of a larger set of genes, compared to that found comparing fentanyl to saline self-administration. These findings suggest that GATC-1021 may attenuate fentanyl self-administration by engaging cortico-striatal networks in the PFC and NAc, rather than the VTA. Finally, fentanyl with GATC-1021 did not result in many transcript changes comparing to saline alone in the PFC, NAc, and VTA (Fig. 4 G–I, respectively), suggesting GATC-1021’s actions were selective by being predominantly modulatory for subjects using fentanyl. We next performed gene ontology (GO) analysis for each brain region (SI Appendix, Fig. S10), selecting candidate genes from neuroplasticity-associated terms across biological process, molecular function, and cellular component ontologies. Bdnf gene expression was subsequently examined across treatment groups in the PFC (Fig. 4K), NAc (Fig. 4L), and VTA (Fig. 4M). In the PFC, the post hoc analysis revealed that Bdnf mRNA was significantly increased in the fentanyl group treated with GATC-1021, comparing to both saline and fentanyl only. Given the relevance of this finding, we then conducted a validation study with a separate cohort of subjects (SI Appendix, Fig. S7) and employed RT-qPCR analysis of the PFC; we found a similar effect with increased expression of Bdnf mRNA in the fentanyl group treated with GATC-1021 (SI Appendix, Fig. S9). Next, for RNAseq, Camk2a gene expression was also examined across treatment groups in the PFC (Fig. 4N), NAc (Fig. 4O), and VTA (Fig. 4P). In the VTA, Camk2a was associated with “regulation of synapse organization” and “dendrite development.” In the PFC, the post hoc analysis indicated that the fentanyl group treated with GATC-1021 exhibited increased levels of Camk2a mRNA compared to the saline and fentanyl GATC-1021 groups. GO analysis of the DEGs in the GATC-1021 vs fentanyl comparison implicated the involvement of multiple synaptic plasticity pathways in each brain region (SI Appendix, Fig. S10), and the top 50 genes altered with each comparison to fentanyl self-administration were reported and organized based on sex, brain region, and group (SI Appendix, Figs. S11 and S12). Together, these findings reveal the effects of GATC-1021 in mediating gene expression changes in the brain, with changes in synaptic plasticity mechanisms induced across brain regions.

Fig. 4.

GATC-1021 treatment modifies fentanyl-induced transcriptional changes in key reward-related brain regions. (A–I) Volcano plots showing log₂ fold changes versus -log₁₀ FDR for differential gene expression in the prefrontal cortex (PFC), nucleus accumbens (NAc), and ventral tegmental area (VTA) (n = 12-13/group). Significantly upregulated (green) and downregulated (magenta) genes are highlighted (FDR < 0.10). Gene expression changes comparing the fentanyl vs saline groups for the PFC (A), NAc (B), and VTA (C). Gene expression changes comparing fentanyl with GATC-1021 40 mg/kg (-sulb) vs fentanyl in the PFC (D), NAc (E), and VTA (F). Gene expression changes in the VTA comparing fentanyl with GATC-1021 40 mg/kg (-sulb) vs saline in the PFC (G), NAc (H), and VTA (I). (J) Venn diagrams illustrate the overlap of differentially expressed genes between fentanyl self-administration and GATC-1021 treatment across PFC, NAc, and VTA (Left to Right). Shared genes suggest a potential modulatory effect of GATC-1021 on fentanyl-induced transcriptional changes. (K–M) Bdnf gene expression was significantly increased in the PFC of the fentanyl group treated with GATC-1021 [40 mg/kg (–sulb)] compared to the saline and fentanyl groups (K). No differences in Bdnf expression were found in the NAc (L) or VTA (M). *P < 0.05 vs saline. ^^P < 0.01 vs fentanyl. (N–P) Camk2a gene expression was significantly increased in the PFC of the fentanyl group treated with GATC-1021 [40 mg/kg (–sulb)] compared to the saline and fentanyl groups (N). No differences in Camk2a expression were found in the NAc (O) or VTA (P). *P < 0.05 vs saline. ^^P < 0.01 vs fentanyl.

Discussion

Our top drug candidates, GATC-021 and GATC-1021, were rationally designed to target the 5HT_2A and 5HT₆ receptors based on our AI platform predictions. The AI platform also predicted that increased thiamine levels with sulbutiamine would enhance the actions of the GATC compounds. We found that GATC-1021 was highly efficacious in reducing fentanyl intake in both male and female rats, with retained effectiveness following repeated dosing. Interestingly, enhanced brain absorption and safety profile were found in the absence of sulbutiamine for GATC-1021 with the formulations examined. Moreover, an increase in the percentage of thin dendritic spines in the hippocampus and changes in gene expression pathways involved in neuroplasticity in the prefrontal cortex were evidenced following GATC-1021 treatment. Taken together, these findings provide evidence to support the further clinical development of GATC-1021 as a treatment for OUD.

Targeting the Serotonin System for OUD.

A renewed focus on the therapeutic potential of psychedelics has emerged with studies in humans showing long-term benefits in treating patients with psychiatric disorders (32–35). Psychedelic and hallucinogenic effects have largely been attributed to action on multiple serotonin receptors (33). The 5HT_2A receptor specifically has been shown to modulate dendritic spine plasticity, which is theorized to, at least in part, underlie some of the main beneficial effects related to its use as a therapeutic (23, 25, 26). The formation and maturation of spines is a normal, dynamic process based on various inputs that drive synaptic processing. Given the action of GATC-1021 on 5HT_2A receptors, we examined whether changes in dendritic spine morphology may have occurred with the five days of treatment during access to fentanyl self-administration. An upregulation in the percentage of thin dendritic spines, with a corresponding reduction in stubby dendritic spines, was observed in the hippocampus following GATC-1021 treatment in fentanyl self-administering rats. Thin spines are characterized by a small head with a narrow neck connecting to the dendritic shaft, whereas stubby spines have a short bulbous structure with no distinction of a neck or head. Of note, small, thin spines have been proposed to be main contributors for learning processes associated with long-term potentiation and memory function given their efficiency in transmitting Ca²⁺ signals (22, 36, 37). Importantly, while GATC-1021 acts on 5HT_2A receptors, it did not induce an increase in head twitch behavioral responses, which are thought to be indicative of hallucinogenic-type effects in rodent models. Further evidence of the actions of GATC-1021 in mitigating synaptic plasticity is also supported by the transcriptional changes observed with sequencing data across the VTA, NAc, and PFC, in addition to the specific effects in the PFC with Bdnf and Camk2a, both of which are important proteins involved in altering synaptic plasticity and various aspects of cognitive processes relevant to drug use and abuse (38–40).

Considerations for Sex Differences.

At baseline, females exhibited a higher level of fentanyl intake than males. These findings are in accordance with prior reports from the field, which suggest estradiol levels may regulate drug self-administration (41, 42). Given this higher level of drug consumption, it was important to examine the effects of GATC-021 and GATC-1021 in both male and female subjects across doses and to normalize based on level of baseline responding. While we did not find any significant differences of sex for either drug compound, GATC-1021 was found to be the most efficacious in both females and males for reducing fentanyl intake across a larger dose range, thereby supporting therapeutic development for both sexes.

Acute Versus Longer-Term Dosing Paradigms.

Differential effectiveness of GATC-021 was found based on the dose and duration of testing. GATC-021 demonstrated limited efficacy in reducing fentanyl intake across time, with the 50 mg/kg dose no longer demonstrating effectiveness in reducing the number of fentanyl infusions unless normalized to baseline levels of responding. This suggests that GATC-021 may exhibit tolerance, which is commonly observed with psychedelics (33), similar to that found for DOI and the head twitch response in the current studies. In contrast, GATC-1021 remained highly effective in reducing fentanyl intake after repeated dosing, and the GATC-1021 25 to 40 mg/kg doses were further shown to have no adverse effects on brain and liver tissues across multiple pathological indicators. Thus, these findings further reinforce the enhanced therapeutic potential of GATC-1021.

Conclusions.

The current findings demonstrate that therapeutic targeting with a polypharmacy serotonin-directed approach leads to a reduction in indications of opioid use disorder in a preclinical rodent model. These findings provide a foundation to support the further clinical development of GATC-1021 as a potential treatment for individuals suffering from OUD. Moreover, these benefits may extend to other psychiatric disorders since 5-HT_2A receptor agonists have been proposed to potentially ameliorate the negative and cognitive symptoms of schizophrenia and other disorders (25, 43).

Materials and Methods

Detailed methods are located in the Supplementary File. Further information about the AI pipeline can be found at: https://gatchealth.com

Preparation of GATC-021 and GATC-1021.

3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indol-9-ol hydrochloride (GATC-021) and 8-fluoro-9-methoxy-3-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole (GATC-1021) were synthesized and verified via High Resolution Mass Spectrometry (HRMS) and NMR (NMR) Spectroscopy (SI Appendix, Figs. S1 and S2).

In-Vitro GPCR Biosensor Assays.

GATC compounds were assessed on 5 targets (5-HTR2A, 5-HTR6, 5- HTR7D, 5- HTR2B, and TrKB) using GPCR and Kinase biosensor assays (Eurofins DiscoverX, USA).

In Vivo Testing.

Adult male and female Wistar rats were purchased from Charles River. All procedures were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine.

Open field locomotor test.

To examine the effects of the GATC compounds on general behavior, subjects were injected and then placed in a large square arena to freely explore the open field, which was video recorded and scored by Any-maze video tracking software.

Fentanyl self-administration.

Male and female subjects were implanted with intravenous jugular catheters and assessed across 1 h daily sessions for fentanyl self-administration. After stabilized responding was achieved across 10 sessions, subjects were pretreated with sulbutiamine, and 20 min thereafter, the GATC compounds were injected 15 min prior to each self-administration session, which occurred across 5 consecutive sessions with a between-subject design. GATC-021 doses included 0, 25, and 50 mg/kg i.p., GATC-1021 doses included 0, 25, 40, and 70 mg/kg i.p., and sulbutiamine was administered at a dose of 50 mg/kg s.c. Control groups included 1) intravenous saline self-administration, 2) intravenous fentanyl self-administration, 3) intravenous fentanyl self-administration with sulbutiamine injection, and 4) intravenous fentanyl self-administration with GATC-1021 (40 mg/kg), no sulbutiamine injection.

Head twitch assessment.

Subjects were pretreated with sulbutiamine or vehicle, and 20 min thereafter, the GATC-1021 and were tested 15 min thereafter. As a comparison, subjects were injected with DOI (1 mg/kg) and then examined 15 min thereafter. Testing was conducted in the open field chamber for 40 min and video recorded.

Pharmacokinetic (PK) Analysis.

Subjects were analyzed at three distinct time points: 15, 45, and 75 min postinjection of GATC-1021. The sulbutiamine was administered subcutaneously 20 min prior to each injection of GATC-1021 i.p., except for the last group, which did not receive sulbutiamine [40(–sulb)]. Both plasma and brain tissue were obtained for analysis.

RNAseq and Golgi Staining.

On the final day of treatment, brains were removed immediately after the self-administration session. For RNA sequencing, brain tissue was microdissected from the PFC, VTA, and NAc, placed in RNAlater, and stored at −80 °C. Total RNA was extracted and quantified, and only samples with RNA integrity number (RIN) ≥ 7.0 (range 7.2 to 9.1) were used for library construction. Libraries were generated using the QIAseq UPXome RNA Library Kit (QIAGEN) and were sequenced on an Illumina NovaSeq 6000 (paired-end 2 × 150 bp). Transcript-level reads were summarized to gene-level counts for all analyses, and differential expression was modeled with sex included as a covariate. For Golgi staining, brain tissue containing the hippocampus or prefrontal cortex was processed using the FD Rapid GolgiStain™ Kit (FD NeuroTechnologies). Coronal sections were prepared, and dendritic spine density and morphology were quantified in pyramidal neurons using Neurolucida 360. Data were averaged per animal, with the animal treated as the unit of analysis.

Tissue Histology.

Liver and brain tissue (cerebellum) were removed on the final experimental day at the same time as above, and fixed in 4% paraformaldehyde, followed by storage in 30% sucrose. Histological analysis was performed by Reveal Biosciences.

Supplementary Material

Appendix 01 (PDF)

Data, Materials, and Software Availability

Sequencing data are available in OSF (https://doi.org/10.17605/OSF.IO/MFEQY) (30). All other study data are included in the article and/or SI Appendix.