Serotonin transporter: Recent progress of in silico ligand prediction methods and structural biology towards structure-guided in silico design of therapeutic agents

Serotonin transporter (SERT) is a membrane transporter which terminates neurotransmission of serotonin through its reuptake. This transporter as well as its substrate have long drawn attention as a key mediator and drug target in a variety of diseases including mental disorders. Accordingly, its structural basis has been studied by X-ray crystallography to gain insights into a design of ligand with high affinity and high specificity over closely related transporters. Recent progress in structural biology including single particle cryo-EM have made big strides also in determination of the structures of human SERT in complex with its ligands. Moreover, rapid progress in machine learning such as deep learning accelerates computer-assisted drug design. Here, we would like to summarize recent progresses in our understanding of SERT using these two rapidly growing technologies, limitations, and future perspectives

Acetaminophen improves tardive akathisia induced by dopamine D₂ receptor antagonists

Tardive akathisia is a movement disorder characterized by internal restlessness with an uncontrollable urge to move, leading to repetitive movements. It is a common side effect of long-term treatment with dopamine D₂ receptor antagonists. In the present study, we analyzed the FDA Adverse Event Reporting System and IBM MarketScan Research Database to find a drug that can be used concomitantly with dopamine D₂ receptor antagonists and still reduce the risk of akathisia. Acetaminophen was determined to be the most effective akathisia-suppressing drug. In an experimental validation of the hypothesis, chronic treatment of rats with haloperidol caused akathisia symptoms, including increased stereotyped behavior and locomotor activity, and decreased immobility time. Acute treatment with acetaminophen significantly attenuated haloperidol-induced akathisia. In the ventral striata of these rats, acetaminophen prevented haloperidol-induced decrease in the number of c-Fos⁺ preproenkephalin⁺ neurons. These results suggest that acetaminophen is effective in suppressing tardive akathisia by activating indirect-pathway medium spiny neurons

Early Detection of Adverse Drug Reaction Signals by Association Rule Mining Using Large-Scale Administrative Claims Data

INTRODUCTION: Adverse drug reactions (ADRs) are a leading cause of mortality worldwide and should be detected promptly to reduce health risks to patients. A data-mining approach using large-scale medical records might be a useful method for the early detection of ADRs. Many studies have analyzed medical records to detect ADRs; however, most of them have focused on a narrow range of ADRs, limiting their usefulness. OBJECTIVE: This study aimed to identify methods for the early detection of a wide range of ADR signals. METHODS: First, to evaluate the performance in signal detection of ADRs by data-mining, we attempted to create a gold standard based on clinical evidence. Second, association rule mining (ARM) was applied to patient symptoms and medications registered in claims data, followed by evaluating ADR signal detection performance. RESULTS: We created a new gold standard consisting of 92 positive and 88 negative controls. In the assessment of ARM using claims data, the areas under the receiver-operating characteristic curve and the precision-recall curve were 0.80 and 0.83, respectively. If the detection criteria were defined as lift > 1, conviction > 1, and p-value < 0.05, ARM could identify 156 signals, of which 90 were true positive controls (sensitivity: 0.98, specificity: 0.25). Evaluation of the capability of ARM with short periods of data revealed that ARM could detect a greater number of positive controls than the conventional analysis method. CONCLUSIONS: ARM of claims data may be effective in the early detection of a wide range of ADR signals

Raphe AMPA receptors and nicotinic acetylcholine receptors mediate ketamine-induced serotonin release in the rat prefrontal cortex.

Several lines of evidence indicate that ketamine has a rapid antidepressant-like effect in rodents and humans, but underlying mechanisms are unclear. In the present study, we investigated the effect of ketamine on serotonin (5-HT) release in the rat prefrontal cortex by in vivo microdialysis. A subcutaneous administration of ketamine (5 and 25 mg/kg) significantly increased the prefrontal 5-HT level in a dose-dependent manner, which was attenuated by local injection of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) antagonists into the dorsal raphe nucleus (DRN). Direct stimulation of AMPARs in the DRN significantly increased prefrontal 5-HT level, while intra-DRN injection of ketamine (36.5 nmol) had no effect. Furthermore, intra-DRN injection of an α 4 β 2-nicotinic acetylcholine receptor (nAChR) antagonist, dihydro-β-erythroidine (10 nmol), significantly attenuated the subcutaneous ketamine-induced increase in prefrontal 5-HT levels. These results suggest that AMPARs and α 4 β 2-nAChRs in the DRN play a key role in the ketamine-induced 5-HT release in the prefrontal cortex

Pathophysiological Role of TRPM2 in Age-Related Cognitive Impairment in Mice

Aging causes various functional changes, including cognitive impairment and inflammatory responses in the brain. Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable channel expressed abundantly in immune cells, exacerbates inflammatory responses. Previously, we reported that TRPM2 on resident microglia plays a critical role in exacerbating inflammation, white matter injury, and cognitive impairment during chronic cerebral hypoperfusion; however, the physiological or pathophysiological role of TRPM2 during age-associated inflammatory responses remains unclear. Therefore, we examined the effects of TRPM2 deletion in young (2–3 months) and older (12–24 months) mice. Compared with young wild-type (WT) mice, middle-aged (12–16 months) WT mice showed working and cognitive memory dysfunction and aged (20–24 months) WT mice exhibited impaired spatial memory. However, these characteristics were not seen in TRPM2 knockout (TRPM2-KO) mice. Consistent with the finding of cognitive impairment, aged WT mice exhibited white matter injury and hippocampal damage and an increase in the number of Iba1-positive cells and amounts of pro-inflammatory cytokines in the brain; these characteristics were not seen in TRPM2-KO mice. These findings suggest that TRPM2 plays a critical role in exacerbating inflammatory responses and cognitive dysfunction during aging

Neurotropin inhibits neuronal activity through potentiation of sustained Kv currents in primary cultured DRG neurons

Neurotropin (NTP) is a Japanese analgesic agent for treating neuropathic pain; however, its method of action remains unclear. This study examined the effects of NTP on the activity of small dorsal root ganglion (DRG) neurons using whole-cell patch clamp recordings. After 3 days of treatment, NTP decreased current injection-induced firing activity of cultured DRG neurons by raising the current threshold for action potential generation. Additionally, NTP increased the sustained component of voltage-gated potassium (Kv) channel currents without affecting other K⁺ currents. These results suggest that NTP inhibits the firing activity of DRG neurons through augmentation of sustained Kv current

Prediction of pharmacological activities from chemical structures with graph convolutional neural networks

化合物の薬理作用を予測する技術を開発 --薬理作用ビッグデータを用いて--. 京都大学プレスリリース. 2021-01-13.Many therapeutic drugs are compounds that can be represented by simple chemical structures, which contain important determinants of affinity at the site of action. Recently, graph convolutional neural network (GCN) models have exhibited excellent results in classifying the activity of such compounds. For models that make quantitative predictions of activity, more complex information has been utilized, such as the three-dimensional structures of compounds and the amino acid sequences of their respective target proteins. As another approach, we hypothesized that if sufficient experimental data were available and there were enough nodes in hidden layers, a simple compound representation would quantitatively predict activity with satisfactory accuracy. In this study, we report that GCN models constructed solely from the two-dimensional structural information of compounds demonstrated a high degree of activity predictability against 127 diverse targets from the ChEMBL database. Using the information entropy as a metric, we also show that the structural diversity had less effect on the prediction performance. Finally, we report that virtual screening using the constructed model identified a new serotonin transporter inhibitor with activity comparable to that of a marketed drug in vitro and exhibited antidepressant effects in behavioural studies

TRPM2 exacerbates central nervous system inflammation in experimental autoimmune encephalomyelitis by increasing production of CXCL2 chemokines

多発性硬化症の新たな病態増悪機構を解明 --TRPM2を介したケモカイン産生が神経炎症の増悪に至る好中球の浸潤を引き起こす--. 京都大学プレスリリース. 2018-09-13.Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS) characterized by demyelination and axonal injury. Current therapies that mainly target lymphocytes do not fully meet clinical need due to the risk of severe side effects and lack of efficacy against progressive MS. Evidence suggests that MS is associated with CNS inflammation, although the underlying molecular mechanism is poorly understood. Transient receptor potential melastatin 2 (TRPM2), a Ca²⁺-permeable nonselective cation channel, is expressed at high levels in the brain and by immune cells, including monocyte lineage cells. Here, we show that TRPM2 plays a pathological role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Knockout or pharmacological inhibition of TRPM2 inhibited progression of EAE, and TRPM2-knockout (TRPM2-KO) mice showed lower activation of Iba1-immunopositive monocyte lineage cells and neutrophil infiltration of the CNS than wild-type (WT) mice. Moreover, CXCL2 production in TRPM2-KO mice was significantly reduced at Day 14 although the severity of EAE was the same as that in WT mice at that time point. In addition, we used bone marrow chimeric mice to show that TRPM2 expressed by CNS-infiltrating macrophages contributes to progression of EAE. Since CXCL2 induces migration of neutrophils, these results indicate that reduced expression of CXCL2 in the CNS suppresses neutrophil infiltration and slows progression of EAE in TRPM2-KO mice. Together, the results suggest that TRPM2 plays an important role in progression of EAE pathology and shed light on its putative role as a therapeutic target for MS. SIGNIFICANCE STATEMENT: Current therapies for multiple sclerosis (MS), which mainly target lymphocytes, carry the risk of severe side effects and lack efficacy against the progressive form of the disease. Here, we found that the transient receptor potential melastatin 2 (TRPM2) channel, abundantly expressed in CNS-infiltrating macrophages, plays a crucial role in development of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. EAE progression was suppressed by knockout or pharmacological inhibition of TRPM2; this was attributed to a reduction in CXCL2 chemokine production by CNS-infiltrating macrophages in TRPM2-knockout mice, resulting in suppression of neutrophil infiltration into the CNS. These results reveal an important role of TRPM2 in the pathogenesis of EAE and shed light on its potential as a therapeutic target

The Role of Dorsal Raphe Serotonin Neurons in the Balance between Reward and Aversion

Background: Reward processing is fundamental for animals to survive and reproduce. Many studies have shown the importance of dorsal raphe nucleus (DRN) serotonin (5-HT) neurons in this process, but the strongly correlative link between the activity of DRN 5-HT neurons and rewarding/aversive potency is under debate. Our primary objective was to reveal this link using two different strategies to transduce DRN 5-HT neurons. Methods: For transduction of 5-HT neurons in wildtype mice, adeno-associated virus (AAV) bearing the mouse tryptophan hydroxylase 2 (TPH2) gene promoter was used. For transduction in Tph2-tTA transgenic mice, AAVs bearing the tTA-dependent TetO enhancer were used. To manipulate the activity of 5-HT neurons, optogenetic actuators (CheRiff, eArchT) were expressed by AAVs. For measurement of rewarding/aversive potency, we performed a nose-poke self-stimulation test and conditioned place preference (CPP) test. Results: We found that stimulation of DRN 5-HT neurons and their projections to the ventral tegmental area (VTA) increased the number of nose-pokes in self-stimulation test and CPP scores in both targeting methods. Concomitantly, CPP scores were decreased by inhibition of DRN 5-HT neurons and their projections to VTA. Conclusion: Our findings indicate that the activity of DRN 5-HT neurons projecting to the VTA is a key modulator of balance between reward and aversion

Increased Expression of Proliferating Cell Nuclear Antigen in Rejecting Rat Lung Allografts

The aim of this study was to investigate the expression of proliferating cell nuclear antigen (PCNA) as an index of cell proliferation in the Brown Norway (BN) to Lewis (LEW) rat lung allograft model.Following transplantation of BN left lungs into LEW recipients, counts of PCNA-positive cells in the perivascular cellular infiltrate and bronchus-associated lymphoid tissue (BALT) were compared with the histological grade of rejection. Lungs were excised on postoperative days 3 and 5. LEW-to-LEW donor-recipient transplantation was performed as a control. Routinely processed, paraffinembedded sections were selected and stained with PCNA. The PCNA index (% of nuclei positive for PCNA) in the BALT was significantly higher in allograft (19.1%, p < 0.05) compared with isograft (4.2%) at 3 days following transplantation. Similarly, the PCNA index was also greater in the perivascular cellular infiltrates of rejecting lungs (23.9% at 3 days, 31.6% at 5 days). These findings indicate that the cells stimulated by the rejection reaction could be increase the expression of PCNA, and the increasing severity of rejection was paralleled by an increase in the number of PCNA-positive cells. In conclusion, PCNA may be a useful marker of acute cellular rejection in lung allografts