Computational Chemistry in Structure-Based Solute Carrier Transporter Drug Design: Recent Advances and Future Perspectives.

Solute carrier transporters (SLCs) are a class of important transmembrane proteins that are involved in the transportation of diverse solute ions and small molecules into cells. There are approximately 450 SLCs within the human body, and more than a quarter of them are emerging as attractive therapeutic targets for multiple complex diseases, e.g., depression, cancer, and diabetes. However, only 44 unique transporters (∼9.8% of the SLC superfamily) with 3D structures and specific binding sites have been reported. To design innovative and effective drugs targeting diverse SLCs, there are a number of obstacles that need to be overcome. However, computational chemistry, including physics-based molecular modeling and machine learning- and deep learning-based artificial intelligence (AI), provides an alternative and complementary way to the classical drug discovery approach. Here, we present a comprehensive overview on recent advances and existing challenges of the computational techniques in structure-based drug design of SLCs from three main aspects: (i) characterizing multiple conformations of the proteins during the functional process of transportation, (ii) identifying druggability sites especially the cryptic allosteric ones on the transporters for substrates and drugs binding, and (iii) discovering diverse small molecules or synthetic protein binders targeting the binding sites. This work is expected to provide guidelines for a deep understanding of the structure and function of the SLC superfamily to facilitate rational design of novel modulators of the transporters with the aid of state-of-the-art computational chemistry technologies including artificial intelligence.

Follistatin-like 1 (FSTL1) interacts with Wnt ligands and Frizzled receptors to enhance Wnt/ß-catenin signaling in obstructed kidneys in vivo.

Follistatin (FS)-like 1 (FSTL1) is a member of the FS-SPARC (secreted protein, acidic and rich in cysteine) family of secreted and extracellular matrix proteins. The functions of FSTL1 have been studied in heart and lung injury as well as in wound healing; however, the role of FSTL1 in the kidney is largely unknown. Here, we show using single-cell RNA-Seq that Fstl1 was enriched in stromal cells in obstructed mouse kidneys. In addition, immunofluorescence demonstrated that FSTL1 expression was induced in fibroblasts during kidney fibrogenesis in mice and human patients. We demonstrate that FSTL1 overexpression increased renal fibrosis and activated the Wnt/ß-catenin signaling pathway, known to promote kidney fibrosis, but not the transforming growth factor ß (TGF-ß), Notch, Hedgehog, or Yes-associated protein (YAP) signaling pathways in obstructed mouse kidneys, whereas inhibition of FSTL1 lowered Wnt/ß-catenin signaling. Importantly, we show that FSTL1 interacted with Wnt ligands and the Frizzled (FZD) receptors but not the coreceptor lipoprotein receptor-related protein 6 (LRP6). Specifically, we found FSTL1 interacted with Wnt3a through its extracellular calcium-binding (EC) domain and von Willebrand factor type C-like (VWC) domain, and with FZD4 through its EC domain. Furthermore, we show that FSTL1 increased the association of Wnt3a with FZD4 and promoted Wnt/ß-catenin signaling and fibrogenesis. The EC domain interacting with both Wnt3a and FZD4 also enhanced Wnt3a signaling. Therefore, we conclude that FSTL1 is a novel extracellular enhancer of the Wnt/ß-catenin pathway.

Subtype-selective mechanisms of negative allosteric modulators binding to group I metabotropic glutamate receptors.

Group I metabotropic glutamate receptors (mGlu1 and mGlu5) are promising targets for multiple psychiatric and neurodegenerative disorders. Understanding the subtype selectivity of mGlu1 and mGlu5 allosteric sites is essential for the rational design of novel modulators with single- or dual-target mechanism of action. In this study, starting from the deposited mGlu1 and mGlu5 crystal structures, we utilized computational modeling approaches integrating docking, molecular dynamics simulation, and efficient post-trajectory analysis to reveal the subtype-selective mechanism of mGlu1 and mGlu5 to 10 diverse drug scaffolds representing known negative allosteric modulators (NAMs) in the literature. The results of modeling identified six pairs of non-conserved residues and four pairs of conserved ones as critical features to distinguish the selective NAMs binding to the corresponding receptors. In addition, nine pairs of residues are beneficial to the development of novel dual-target NAMs of group I metabotropic glutamate receptors. Furthermore, the binding modes of a reported dual-target NAM (VU0467558) in mGlu1 and mGlu5 were predicted to verify the identified residues that play key roles in the receptor selectivity and the dual-target binding. The results of this study can guide rational structure-based design of novel NAMs, and the approach can be generally applicable to characterize the features of selectivity for other G-protein-coupled receptors.

The binding mode of vilazodone in the human serotonin transporter elucidated by ligand docking and molecular dynamics simulations.

Vilazodone is a novel antidepressant used for the treatment of major depressive disorder (MDD) with a primary action mechanism of inhibiting the human serotonin reuptake transporter (hSERT) and acting as a 5-HT1A receptor partial agonist. The interaction between vilazodone and the 5-HT1A receptor has been reported, however, the binding mode of vilazodone in the hSERT remains elusive. In the current study, to elucidate the molecular mechanism of vilazodone binding in the hSERT, the drug and its five analogs were docked into the hSERT crystal structure as initial conformations and were sampled by 400 ns molecular dynamics (MD) simulations. Through the analysis of the profiles of protein-ligand binding free energies, interaction fingerprints, and conformational rearrangements, the binding mode of vilazodone in the hSERT was revealed. As a result, unlike the classical antidepressants located in the S1 site of the hSERT, vilazodone adopted a linear pose in the binding pocket. Its arylpiperazine fragment occupies the central site (S1) and interacts with Y95, D98, I172, Y176, F335, F341, S438, and T439, while the indole fragment extends to the allosteric site (S2) via interacting with the ionic switch (R104/E403) between the two sites. The new insights obtained are not only helpful in understanding the binding mode of vilazodone in the hSERT, but also provide valuable guidance to the discovery of novel antidepressant drugs.

Prediction of the binding mode and resistance profile for a dual-target pyrrolyl diketo acid scaffold against HIV-1 integrase and reverse-transcriptase-associated ribonuclease H.

The rapid emergence of drug-resistant variants is one of the most common causes of highly active antiretroviral therapeutic (HAART) failure in patients infected with HIV-1. Compared with the existing HAART, the recently developed pyrrolyl diketo acid scaffold targeting both HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) is an efficient approach to counteract the failure of anti-HIV treatment due to drug resistance. However, the binding mode and potential resistance profile of these inhibitors with important mechanistic principles remain poorly understood. To address this issue, an integrated computational method was employed to investigate the binding mode of inhibitor JMC6F with HIV-1 IN and RNase H. By using per-residue binding free energy decomposition analysis, the following residues: Asp64, Thr66, Leu68, Asp116, Tyr143, Gln148 and Glu152 in IN, Asp443, Glu478, Trp536, Lys541 and Asp549 in RNase H were identified as key residues for JMC6F binding. And then computational alanine scanning was carried to further verify the key residues. Moreover, the resistance profile of the currently known major mutations in HIV-1 IN and 2 mutations in RNase H against JMC6F was predicted by in silico mutagenesis studies. The results demonstrated that only three mutations in HIV-1 IN (Y143C, Q148R and N155H) and two mutations in HIV-1 RNase H (Y501R and Y501W) resulted in a reduction of JMC6F potency, thus indicating their potential role in providing resistance to JMC6F. These data provided important insights into the binding mode and resistance profile of the inhibitors with a pyrrolyl diketo acid scaffold in HIV-1 IN and RNase H, which would be helpful for the development of more effective dual HIV-1 IN and RNase H inhibitors.

Computational characterization of the selective inhibition of human norepinephrine and serotonin transporters by an escitalopram scaffold.

Human norepinephrine and serotonin transporters (hNET and hSERT) are closely related monoamine transporters (MATs) that regulate neurotransmitter signaling in neurons and are primary targets for a wide range of therapeutic drugs used in the treatment of mood disorders. The subtle modifications of an escitalopram scaffold exhibit distinct selective inhibition profiles of hNET and hSERT. However, the structural details of escitalopram scaffold binding to hSERT and (or) hNET are poorly understood and still remain a great challenge. In this work, on the basis of more recently solved X-ray crystallographic structure of hSERT in complex with escitalopram, 3 µs long all-atom MD simulations and binding free energy calculations via MM/GB(PB)SA, thermodynamic integration (TI) and MM/3D-RISM methods were performed to reproduce experimental free energies. And both MM/GBSA and TI have a high correlation coefficient (R2 = 0.95 and 0.96, respectively) between the relative binding free energies of the calculated and experimental values. Furthermore, MM/GBSA per-residue energy decomposition, molecular interaction fingerprints and thermodynamics-structure relationship analysis were employed to investigate and characterize the selectivity of the escitalopram scaffold with three modifications (escitalopram, ligand10 and talopram) to hNET and hSERT. As a result, 4 warm spots (A73, Y151, A477 and I481) in hNET and 4 warm spots (A96, A173, T439 and L443) in hSERT were thus discovered to exert a pronounced effect on the selective inhibition of hNET and hSERT by the studied ligands. These simulation results would provide great insight into the design of inhibitors with the desired selectivity to hNET and hSERT, thus further promoting the research of more efficacious antidepressants.

Computational identification of the binding mechanism of a triple reuptake inhibitor amitifadine for the treatment of major depressive disorder.

Amitifadine, the only drug ever clinically tested in Phase 3 for treating depression, is a triple reuptake inhibitor (TRI) that simultaneously interacts with human monoamine transporters (MATs) including hSERT, hNET and hDAT. This novel multi-target strategy improves drug efficacy and reduces the toxic side effects of drugs. However, the binding modes accounting for amitifadine's polypharmacological mode of action are still elusive, and extensive exploration of the amitifadine-target interactions between amitifadine and MATs is urgently needed. In this study, a total of 0.63 µs molecular dynamics (MD) simulations with an explicit solvent as well as endpoint binding free energy (BFE) calculation were carried out. MD simulation results identified a shared binding mode involving eleven key residues at the S1 site of MATs for the binding of amitifadine, and the results of the BFE calculations were in good agreement with experimental reports. Moreover, by analyzing the per-residue energy contribution variation at the S1 site of three MATs and additional cross-mutagenesis simulations, the variation in the inhibition ratio of amitifadine between hSERT and two other MATs was discovered to mainly come from non-conserved residues (Y95, I172 and T439 in hNET and Y95, I172, A169 and T439 in hDAT). As the rational inhibition ratio of multi-target drugs among various therapeutic targets was found to be the key to their safety and tolerance, the findings of this study may further facilitate the rational design of more potent but less toxic multi-target antidepressant drugs.

Differentiating physicochemical properties between NDRIs and sNRIs clinically important for the treatment of ADHD.

BACKGROUND: Drugs available for treating attention-deficit hyperactivity disorder (ADHD) are mainly selective norepinephrine (sNRIs) and dual norepinephrine-dopamine (NDRIs) reuptake inhibitors. The major problem of sNRIs lines in their delayed onset of action and partial- or non-responses, which makes NDRIs distinguished in drug efficacy. Understanding of the differential binding modes of these 2 types of drugs to their corresponding targets can give great insights into the discovery of privileged drug-like scaffolds with improved efficacy. So far, no such study has been carried out. METHODS: A combinatorial computational strategy, integrating homology modeling, molecular docking, molecular dynamics (MD) and binding free energy calculation, was employed to analyze the binding modes of 8 clinically important ADHD drugs in their targets. RESULTS: Binding modes of 2 types of ADHD drugs (sNRIs and NDRIs) in their targets was identified for the first time by MD simulation, and 15 hot spot residues were discovered as crucial for NDRIs' binding in hNET and hDAT. Comparing to sNRIs, a clear reduction in the hydrophobic property of NDRIs' one functional group was observed, and the depth of drugs' aromatic ring stretched into the pocket of both targets was further identified as key contributors to drugs' selectivity. CONCLUSIONS: The hydrophobic property of NDRI ADHD drugs' one functional group contributes to their selectivity when bind hNET and hDAT. GENERAL SIGNIFICANCE: These results provide insights into NDRI ADHD drugs' binding mechanisms, which could be utilized as structural blueprints for assessing and discovering more efficacious drugs for ADHD therapy.

Revealing vilazodone's binding mechanism underlying its partial agonism to the 5-HT1A receptor in the treatment of major depressive disorder.

It has been estimated that major depressive disorder (MDD) will become the second largest global burden among all diseases by 2030. Various types of drugs, including selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), and serotonin receptor partial agonist/reuptake inhibitors (SPARIs), have been approved and become the primary or first-line medications prescribed for MDD. SPARI was expected to demonstrate more enhanced drug efficacy and a rapid onset of action as compared to SSRI and SNRI. As one of the most famous SPARIs, vilazodone was approved by the FDA for the treatment of MDD. Because of the great clinical importance of vilazodone, its binding mechanism underlying its partial agonism to the 5-HT1A receptor (5-HT1AR) could provide valuable information to SPARIs' drug-like properties. However, this mechanism has not been reported to date; consequently, the rational design of new efficacious SPARI-based MDD drugs is severely hampered. To explore the molecular mechanism of vilazodone, an integrated computational strategy was adopted in this study to reveal its binding mechanism and prospective structural feature at the agonist binding site of 5-HT1AR. As a result, 22 residues of this receptor were identified as hotspots, consistently favoring the binding of vilazodone and its analogues, and a common binding mechanism underlying their partial agonism to 5-HT1AR was, therefore, discovered. Moreover, three main interaction features between vilazodone and 5-HT1AR have been revealed and schematically summarized. In summary, this newly identified binding mechanism will provide valuable information for medicinal chemists working in the field of rational design of novel SPARIs for MDD treatment.

Discovery of novel 3-benzylquinazolin-4(3H)-ones as potent vasodilative agents.

In the present study, a series of 3-benzylquinazolin-4(3H)-ones were synthesized and characterized. Their vasodilative effects were evaluated by wire myograph on isolated rat mesenteric arterial ring induced contraction with 60mM KCl. The SAR of target compounds was discussed preliminarily. Among these compounds, 2a and 2c displayed potent vasodilatation action and could compete significantly the rat mesenteric arterial rings induced contraction with phenylephrine. Compounds 2a and 2c were further tested for their antihypertensive effects in SHR by oral administration. The results indicated that 2a and 2c could reduce significantly both diastolic and systolic blood pressure. Moreover, 2c displayed antihypertensive effect in a dose dependent manner, and could maintain the effects for 6h at a dosage of 4.0mg/kg. These findings suggest that the title compounds are novel vasodilative agents, representing a novel series of promising antihypertensive agents.

Targeting PI4KA sensitizes refractory leukemia to chemotherapy by modulating the ERK/AMPK/OXPHOS axis.

Background: The emergence of chemoresistance in leukemia markedly impedes chemotherapeutic efficacy and dictates poor prognosis. Recent evidence has revealed that phosphatidylinositol 4 kinase-IIIα (PI4KA) plays a critical role in tumorigenesis. However, the molecular mechanisms of PI4KA-regulated chemoresistance and leukemogenesis remain largely unknown. Methods: Liquid chromatography-mass spectrometry (LC-MS), patient samples and leukemia xenograft mouse models were used to investigate whether PI4KA was an effective target to overcome chemoresistance in leukemia. Enzyme-linked immunosorbent assay (ELISA) and molecular mechanics/generalized born surface area (MM/GBSA) method were employed to identify cepharanthine (CEP) as a novel PI4KA inhibitor. Results: High expression of PI4KA was observed in drug-resistant leukemia cells or in relapsed leukemia patients, which was correlated with poor overall survival. Depletion of PI4KA sensitized drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo by regulating ERK/AMPK/OXPHOS axis. We also identified cepharanthine (CEP) as a novel PI4KA inhibitor, which could undermine the stability of the PI4KA/TTC7/FAM126 complex, enhancing the sensitivity of drug-resistant leukemia cells to chemotherapeutic drugs in vitro and in vivo. Conclusions: Our study underscored the potential of therapeutic targeting of PI4KA to overcome chemoresistance in leukemia. A combination of the PI4KA inhibitor with classic chemotherapeutic agents could represent a novel therapeutic strategy for the treatment of refractory leukemia.

Rational Remodeling of Atypical Scaffolds for the Design of Photoswitchable Cannabinoid Receptor Tools.

Azobenzene-embedded photoswitchable ligands are the widely used chemical tools in photopharmacological studies. Current approaches to azobenzene introduction rely mainly on the isosteric replacement of typical azologable groups. However, atypical scaffolds may offer more opportunities for photoswitch remodeling, which are chemically in an overwhelming majority. Herein, we investigate the rational remodeling of atypical scaffolds for azobenzene introduction, as exemplified in the development of photoswitchable ligands for the cannabinoid receptor 2 (CB2). Based on the analysis of residue-type clusters surrounding the binding pocket, we conclude that among the three representative atypical arms of the CB2 antagonist, AM10257, the adamantyl arm is the most appropriate for azobenzene remodeling. The optimizing spacer length and attachment position revealed AzoLig 9 with excellent thermal bistability, decent photopharmacological switchability between its two configurations, and high subtype selectivity. This structure-guided approach gave new impetus in the extension of new chemical spaces for tool customization for increasingly diversified photo-pharmacological studies and beyond.

Selective Inhibition of HDAC1 by Macrocyclic Polypeptide for the Treatment of Glioblastoma: A Binding Mechanistic Analysis Based on Molecular Dynamics.

Glioblastoma (GBM) is the most common and aggressive intracranial malignant brain tumor, and the abnormal expression of HDAC1 is closely correlated to the progression, recurrence and metastasis of GBM cells, making selective inhibition of HDAC1 a promising strategy for GBM treatments. Among all available selective HDAC1 inhibitors, the macrocyclic peptides have gained great attention due to their remarkable inhibitory selectivity on HDAC1. However, the binding mechanism underlying this selectivity is still elusive, which increases the difficulty of designing and synthesizing the macrocyclic peptide-based anti-GBM drug. Herein, multiple computational approaches were employed to explore the binding behaviors of a typical macrocyclic peptide FK228 in both HDAC1 and HDAC6. Starting from the docking conformations of FK228 in the binding pockets of HDAC1&6, relatively long MD simulation (500 ns) shown that the hydrophobic interaction and hydrogen bonding of E91 and D92 in the Loop2 of HDAC1 with the Cap had a certain traction effect on FK228, and the sub-pocket formed by Loop1 and Loop2 in HDAC1 could better accommodate the Cap group, which had a positive effect on maintaining the active conformation of FK228. While the weakening of the interactions between FK228 and the residues in the Loop2 of HDAC6 during the MD simulation led to the large deflection of FK228 in the binding site, which also resulted in the decrease in the interactions between the Linker region of FK228 and the previously identified key amino acids (H134, F143, H174, and F203). Therefore, the residues located in Loop1 and Loop2 contributed in maintaining the active conformation of FK228, which would provide valuable hints for the discovery and design of novel macrocyclic polypeptide HDAC inhibitors.

Recent Advances and Challenges of the Drugs Acting on Monoamine Transporters.

BACKGROUND: The human Monoamine Transporters (hMATs), primarily including hSERT, hNET and hDAT, are important targets for the treatment of depression and other behavioral disorders with more than the availability of 30 approved drugs. OBJECTIVE: This paper is to review the recent progress in the binding mode and inhibitory mechanism of hMATs inhibitors with the central or allosteric binding sites, for the benefit of future hMATs inhibitor design and discovery. The Structure-Activity Relationship (SAR) and the selectivity for hit/lead compounds to hMATs that are evaluated by in vitro and in vivo experiments will be highlighted. METHODS: PubMed and Web of Science databases were searched for protein-ligand interaction, novel inhibitors design and synthesis studies related to hMATs. RESULTS: Literature data indicate that since the first crystal structure determinations of the homologous bacterial Leucine Transporter (LeuT) complexed with clomipramine, a sizable database of over 100 experimental structures or computational models has been accumulated that now defines a substantial degree of structural variability hMATs-ligands recognition. In the meanwhile, a number of novel hMATs inhibitors have been discovered by medicinal chemistry with significant help from computational models. CONCLUSION: The reported new compounds act on hMATs as well as the structures of the transporters complexed with diverse ligands by either experiment or computational modeling have shed light on the poly-pharmacology, multimodal and allosteric regulation of the drugs to transporters. All of the studies will greatly promote the Structure-Based Drug Design (SBDD) of structurally novel scaffolds with high activity and selectivity for hMATs.

Exploring the Binding Mechanism of Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators in Clinical Trials by Molecular Dynamics Simulations.

Metabotropic glutamate receptor 5 (mGlu5) plays a key role in synaptic information storage and memory, which is a well-known target for a variety of psychiatric and neurodegenerative disorders. In recent years, the increasing efforts have been focused on the design of allosteric modulators, and the negative allosteric modulators (NAMs) are the front-runners. Recently, the architecture of the transmembrane (TM) domain of mGlu5 receptor has been determined by crystallographic experiment. However, it has been not well understood how the pharmacophores of NAMs accommodated into the allosteric binding site. In this study, molecular dynamics (MD) simulations were performed on mGlu5 receptor bound with NAMs in preclinical or clinical development to shed light on this issue. In order to identify the key residues, the binding free energies as well as per-residue contributions for NAMs binding to mGlu5 receptor were calculated. Subsequently, the in silico site-directed mutagenesis of the key residues was performed to verify the accuracy of simulation models. As a result, the shared common features of the studied 5 clinically important NAMs (mavoglurant, dipraglurant, basimglurant, STX107, and fenobam) interacting with 11 residues in allosteric site were obtained. This comprehensive study presented a better understanding of mGlu5 receptor NAMs binding mechanism, which would be further used as a useful framework to assess and discover novel lead scaffolds for NAMs.

What Contributes to Serotonin-Norepinephrine Reuptake Inhibitors' Dual-Targeting Mechanism? The Key Role of Transmembrane Domain 6 in Human Serotonin and Norepinephrine Transporters Revealed by Molecular Dynamics Simulation.

Dual inhibition of serotonin and norepinephrine transporters (hSERT and hNET) gives greatly improved efficacy and tolerability for treating major depressive disorder (MDD) compared with selective reuptake inhibitors. Pioneer studies provided valuable information on structure, function, and pharmacology of drugs targeting both hSERT and hNET (serotonin-norepinephrine reuptake inhibitors, SNRIs), and the differential binding mechanism between SNRIs and selective inhibitors of 5-HT (SSRIs) or NE (sNRIs) to their corresponding targets was expected to be able to facilitate the discovery of a privileged drug-like scaffold with improved efficacy. However, the dual-target mechanism of SNRIs was still elusive, and the binding mode distinguishing SNRIs from SSRIs and sNRIs was also unclear. Herein, an integrated computational strategy was adopted to discover the binding mode shared by all FDA approved SNRIs. The comparative analysis of binding free energy at the per-residue level discovered that residues Phe335, Leu337, Gly338, and Val343 located at the transmembrane domain 6 (TM6) of hSERT (the corresponding residues Phe317, Leu319, Gly320, and Val325 in hNET) were the determinants accounting for SNRIs' dual-acting inhibition, while residues lining TM3 and 8 (Ile172, Ser438, Thr439, and Leu443 in hSERT; Val148, Ser419, Ser420, and Met424 in hNET) contributed less to the binding of SNRIs than that of SSRIs and sNRIs. Based on these results, the distances between an SNRI's centroid and the centroids of its two aromatic rings (measuring the depth of rings stretching into hydrophobic pockets) were discovered as the key to the SNRIs' dual-targeting mechanism. This finding revealed SNRIs' binding mechanism at an atomistic level, which could be further utilized as structural blueprints for the rational design of privileged drug-like scaffolds treating MDD.

Exploring the Inhibitory Mechanism of Approved Selective Norepinephrine Reuptake Inhibitors and Reboxetine Enantiomers by Molecular Dynamics Study.

Selective norepinephrine reuptake inhibitors (sNRIs) provide an effective class of approved antipsychotics, whose inhibitory mechanism could facilitate the discovery of privileged scaffolds with enhanced drug efficacy. However, the crystal structure of human norepinephrine transporter (hNET) has not been determined yet and the inhibitory mechanism of sNRIs remains elusive. In this work, multiple computational methods were integrated to explore the inhibitory mechanism of approved sNRIs (atomoxetine, maprotiline, reboxetine and viloxazine), and 3 lines of evidences were provided to verify the calculation results. Consequently, a binding mode defined by interactions between three chemical moieties in sNRIs and eleven residues in hNET was identified as shared by approved sNRIs. In the meantime, binding modes of reboxetine's enantiomers with hNET were compared. 6 key residues favoring the binding of (S, S)-reboxetine over that of (R, R)-reboxetine were discovered. This is the first study reporting that those 11 residues are the common determinants for the binding of approved sNRIs. The identified binding mode shed light on the inhibitory mechanism of approved sNRIs, which could help identify novel scaffolds with improved drug efficacy.