Scientists Have Screened New Serotonin Transporter Inhibitors From Over 20 Billion Molecules

Highlights
•Large-library docking finds conformationally and target-selective SERT inhibitors
•Cryo-EM supports the computationally predicted structure
•The inhibitors are anti-depressant-like and alleviate opioid withdrawal in mice
•Targeting of transporters for structure-based ligand discovery

 

5-hydroxytryptamine transporter (SERT) is a common target for antidepressants as it can “transport” serotonin from synapses.

 

Transporting protein transporters is a process of “swallowing and vomiting”, completed by alternating protein conformations opening outward and inward. Research has shown that the transport mechanism of transporters can be divided into three stages: outward opening, outward closing, and inward opening. Currently, antidepressants target SERT’s outward opening state, with one exception: Ibergine, an alkaloid extracted from plants in central Africa, targets the inward opening state and has unusual antidepressant and substance withdrawal effects.

 

However, ibozantine does not specifically target SERT and has severe cardiac toxicity, which limits its clinical conversion. During a vacation, Gary Rudnick from Yale University, who had been studying SERT, introduced SERT and ibuprofen to Brian Shoichet from the University of California, San Francisco. This made Shoichet, who was originally studying brain receptor docking to help develop depression and pain drugs, very interested. He hoped that ibuprofen could better target SERT and develop new drugs.

 

In 2018, the Shoichet team took over this project. Recently, their latest research results were published in the journal Cell. Through the collaboration of multiple research teams, they discovered two new molecules similar to Ebergitine but with more specific targeting. They have an antidepressant effect about 200 times stronger than fluoxetine in mice and can effectively inhibit opioid addiction and withdrawal symptoms.

 

Thanks to the research results on the conformational determination of the SERT Eberga base complex in an inward open state using cryo-EM, the research team can search for conformational selective inhibitors based on this.

This is the first time the Shoichet team has conducted docking experiments on transporters. They used a super large virtual library containing over 200 million molecules. The first round of docking experiments reduced this number to 49, of which 36 can be synthesized. These molecules are topologically different from each other and also different from known SERT inhibitors, and prioritize targeting SERT inward opening states.

 

The Rudnick team assisted them in testing each of these molecules and found that 13 of them could inhibit SERT. They selected the 5 molecules with the strongest activity and hoped to optimize them, with the goal of improving the affinity of the molecules.

 

The research team used Smallworld and Arthur search engines to screen over 20 billion on-demand customized analog molecules and tested interactions in specific models. In the end, out of the five molecules, two analogs of molecules 2313, 8090, and 8219, stood out. The inhibition constant (Ki) of molecule 2313 on SERT was 0.92μM, indicating that the smaller the value, the stronger the inhibition ability of the inhibitor on the target. The Ki of 8090 and 8219 decreased to 14nM and 3nM.

 

With the help of colleagues, cryo-EM expert Aash Manglik, they found that the binding form of 8090 to SERT at the atomic level is very similar to that simulated by the computer in the molecular screening stage. Of course, it is also similar to the form of inhibition of SERT by Ebergine, but different from the long-term hallucinogenic effect of Ebergine, 8090 is strong and selective, and has no additional impact on hundreds of other receptors and transporters.

 

Shoichet handed over these two molecules to his colleagues Allan Basbaum and William Wetsel of Duke University Medical Center for animal experiments to verify their therapeutic effects on addiction, depression, and anxiety.

 

The researchers used two mouse models, one is VMAT2 heterozygous mice, and the other is learned helplessness (LH) mice (which subjected the mice to inevitable and unpredictable electrical shocks in an experimental chamber, simulating environmental stress).

 

In VMAT2 heterozygous mice, compared with the blank control, 20mg/kg fluoxetine, 30mg/kg ibozantine, 1 or 2mg/kg 8090, and 0.1 or 0.5mg/kg 8219 injections significantly reduced depressive-like reactions (immobility, giving up self-rescue) in tail suspension experiments. 0.5mg/kg 8219 was more effective than the same dose of 8090, and 0.1mg/kg 8219 was roughly equivalent to 20mg/kg fluoxetine, that is to say, the difference in efficacy between fluoxetine and 8219 is approximately 200 times.

 

 

In general, after 8090 and 8219 treatment, LH mice showed a less depressive reaction in the tail suspension test, more sensitivity to foot shock, better escape performance, and reduced escape delay. In the 0 maze test, the treated LH mice entered the open area more quickly and had more desire to explore. In the 8-arm maze test to verify working memory, the treated LH mice had lower working memory error frequency (repeated entry into the same arm/total arm entry times), these all indicate the effectiveness of 8090 and 8219.

 

The only inconsistent result was the sucrose preference experiment, where LH mice treated with 8090 had a restored preference for sucrose, but 8219 did not, and this was not related to total sugar water intake. The intake levels among the groups were similar.

 

Not only that, 8219 also showed the potential to reduce opioid withdrawal symptoms. Researchers compared the efficacy of 8219 with the commonly used opioid addiction withdrawal drug paroxetine. In morphine-addicted mice, naloxone was used to promote withdrawal, and paroxetine inhibited the jumping and tail erection behavior caused by the withdrawal. 8219 not only inhibited jumping, but also inhibited front paw shaking and overall tremor.

 

Therefore, researchers believe that 8219 is equivalent to or even better than paroxetine in alleviating opioid withdrawal symptoms.

 

The research team believes that the powerful antidepressant effects and inhibitory effects of drug addiction withdrawal symptoms exhibited by 8090 and 8219 may bring better therapeutic effects to patients in these two fields in the future. Researchers have now provided the structures of these two molecules to pharmaceutical company Sigma Aldrich, hoping to receive further testing while continuing to search for new, more precise targeted molecules.

 

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Reference

Singh I, Seth A, Billesbølle CB, Braz J, Rodriguiz RM, Roy K, Bekele B, Craik V, Huang XP, Boytsov D, Pogorelov VM, Lak P, O’Donnell H, Sandtner W, Irwin JJ, Roth BL, Basbaum AI, Wetsel WC, Manglik A, Shoichet BK, Rudnick G. Structure-based discovery of conformationally selective inhibitors of the serotonin transporter. Cell. 2023 May 11;186(10):2160-2175.e17. doi: 10.1016/j.cell.2023.04.010. Epub 2023 May 2. PMID: 37137306.