Narrow Results

Myocardial infarction (MI) is a significant threat to human health worldwide. Following MI, cardiomyocytes (CMs) undergo pyroptosis, exacerbating the damage caused by infarction. Ginseng may play a role in alleviating CM pyroptosis. However, further exploration is needed regarding its main active ingredients and effects. By employing network pharmacology on the active ingredients of ginseng, MI and pyroptosis, and employing molecular docking between such ingredients and pyroptosis-related proteins, we screened for the main ingredient of ginseng. Through network pharmacology and molecular docking, we identified ginsenoside Rh2, which acts on MI and cell pyroptosis, as the most likely active ingredient that stably binds to pyroptosis-related proteins. We subsequently constructed a neonatal rat CM oxygen–glucose deprivation (OGD) model in vitro and an MI mouse model in vivo. Ginsenoside Rh2 was administered, with losartan used as a positive control. In the in vitro OGD model, ginsenoside Rh2 increased the viability of primary rat CMs and mitigated OGD-induced pyroptosis. In the in vivo MI model, ginsenoside Rh2 reduced CM pyroptosis, decreased infarct size, and subsequently improved cardiac function. Our study provides a novel therapeutic strategy for MI by attenuating CM pyroptosis.

SARS-CoV-2, the new coronavirus variant, has been a worldwide health crisis that may outbreak at any time in the future. Over spans of human history, preparations derived from natural products have always been recognized as a preliminary source of medications. Taking into account the SARS-CoV-2 main protease (Mpro) as the essential element of the viral cycle and as a main target, herein we highlight a computer-aided comprehensive virtual screening for a focused chemical list of 14 laulimalides marine macrolides against SARS-CoV-2 Mpro using a set of integrated modern computational techniques including molecular docking (MDock), molecule dynamic simulations (MDS) and structure–activity relationships (SARs). Based on their remarkable ligand-protein energy scores and relevant binding affinities with SARS-CoV-2 (Mpro) pocket residues, two promising macrolides [laulimalides LA4 (6) and LA18 (13)] are selected as proposed inhibitor compounds. Consequently, they are thermodynamically investigated by deciphering their MD simulations at 100 ns, where they show noticeable stability within the accommodated (Mpro) pockets. Moreover, in-depth SARs studies suggest crucial roles for C-23 substituted side chain and C-20 methoxy as essential pharmacophoric structural features for activity. Further in vitro/vivo examinations of the selected marine macrolides would pave the way towards developing effective antiviral drugs from natural resources.

Mycobacterium tuberculosis (Mtb), the bacterium responsible for tuberculosis (TB), employs mycolic acids to build its cell wall. This robust structure plays a vital role in protecting the bacterium from external threats and contributes to its resistance against antibiotics. Mycobacterial membrane protein Large 3 (MmpL3), a secondary resistance nodulation division transporter, is essential in mycolic acid biosynthesis, transporting mycolic acid precursors into the periplasm using the proton motive force. Due to its role in bacterial cell wall formation, it is a promising target for new tuberculosis treatments. In this study, starting with 85 known MmPL3 compounds, the artificial intelligence (AI)-assisted tool “Design of Druglike Analogues (DeLA-Drug)” was employed to generate about 15,000 novel molecules. These compounds were then subjected to structure-based high-throughput virtual screening and molecular dynamics (MD) simulations to identify potential novel inhibitors of MmpL3. The binding affinity was obtained by docking the above molecules at the SQ109 binding site in MmPL3, followed by pharmacokinetics and toxicity, which were used to reduce the chemical space. Finally, five ligands were subjected to 100 ns MD simulations to investigate the binding energetics of inhibitors to MmpL3. These compounds demonstrated stable binding and favorable drug-like properties, indicating that they could serve as potential novel inhibitors of MmpL3 for Mtb.

Although cancer has been the main reason for death for decades, until now, no safe certified drugs have been discovered to efficiently inhibit its progression. Currently, we aim to employ an in-silico model to predict the action mechanism of phenolic components that we previously demonstrated as inhibitors of colon, prostate and lung cancer. The work was performed by testing pharmacokinetics and pharmacodynamics proprieties of selected molecules. Four compounds: Kaempferol, chrysin, vanillin and p-hydroxybenzoic acid widely obeyed the five rules and the ADME/T criteria with minimum toxicity. Docking prediction recorded an intense affinity between kaempferol-CDK1(−17.72 ± 0.02 kcal/mol), chrysin-CDK1 (−17.61 ± 0.01 kcal/mol) and kaempferol-caspase 8 (−9.30 ± 0.00 kcal/mol), chrysin-caspase 8 (−8.18 ± 0.12 kcal/mol), vanillin-MMP9 (−9.83 ± 0.04 kcal/mol) and p-hydroxybenzoic acid-Ca2 (−8.91 ± 0.01 kcal/mol). Likewise, MMGBSA results demonstrated that these compounds are evidently bound to the targets’ active sites with solid interactions. The passed compounds might target the responsible factors for transcription as inactivators. The molecular mechanism based on kaempferol and vanillin actions was predicted to target CDK1 and MMP9 via cell cycle arrest and apoptosis. All results were confirmed through RMSD analysis suggesting that kaempferol and vanillin are the best ligands to suppress cancer cell proliferation. Thus, these findings allowed us to imagine a potential molecular mechanism of these binaries, phenolic compounds/target proteins to lead further trails on cancer, of which the synergistic activity between phenolic compounds could promote the therapeutic capacity.

Aims: This study aims to prepare and investigate the potential of theobromine derivative-loaded chitosomes as an effective anticancer drug. Background: $N$-Benzyl-2-(3,7-dimethyl-2,6-dioxo-2,3,6,7-tetrahydro-1$H$-purin-1-yl)acetamide (T-1-BA) exhibited promising anticancer potential. Loaded chitosomes, highlighting their suitability as a drug delivery system. The study builds on the significance of targeted drug delivery systems for enhanced anticancer efficacy. Objectives: To prepare T-1-BA-loaded chitosomes (T-1-BA-PC-CS complex) using the thin film hydration technique. To examine the colloidal characteristics, entrapment efficiency, and stability of the T-1-BA-PC-CS complex. To assess the in vitro anticancer efficacy of the T-1-BA-PC-CS complex against Hct116 and A549 cancer cell lines comparing the free T-1-BA, blank chitosomes, and the standard EGFR inhibitor, Lapatinib. Methods: The T-1-BA-PC-CS complex was prepared through the thin film hydration technique. Colloidal characteristics, including particle size, PDI, and zeta potential, were examined. In vitro anticancer efficacy was assessed through IC${}_{50}$ values, comparative analyses, SI calculations, cell cycle analysis, flow cytometry, and wound healing assays. Results: The T-1-BA-PC-CS complex demonstrated reduced IC${}_{50}$ values against Hct116 and A549 cell lines, indicating enhanced cytotoxicity compared to free T-1-BA and blank chitosomes. Comparative analyses highlighted superior anticancer activity. SI values indicated preferential cytotoxic effects on cancer cell lines. Flow cytometry analysis revealed cell cycle alterations and increased apoptosis. Wound healing assays showed a significant impact on migration and wound healing in A549 cells. Conclusion: The findings suggest that the T-1-BA-PC-CS complex holds great promise as an anticancer therapeutic by efficiently inducing favorable alterations in cell cycle phases and apoptosis, demonstrating its potential for further development in cancer treatment.

A series of novel hybrid bipyrimidine derivatives were designed, synthesized and evaluated for their anticancer activity against the breast cancer cell line MCF-7 and the lung cancer cell line A549. The structures of all the synthesized molecules were confirmed using various spectroscopic techniques. Molecular docking studies were conducted using Maestro software from Schrödinger, targeting the crystal structure of the ErbB4 kinase (PDB ID: 3BBT). Lapatinib and gefitinib were used as reference drugs to compare binding affinities with the ErbB family of receptor tyrosine kinases. Several of the synthesized bipyrimidine derivatives demonstrated promising activity, with IC${}_{50}$ values comparable to the standard drug doxorubicin and gefitinib. Notably, compound PP-10 exhibited equipotent effects when compared to the reference drugs in both breast and lung cancer cell lines. Additionally, compounds PP-07 and PP-09 showed significant potency against the MCF-7 cell line, while compounds PP-02, PP-09, PP-11 and PP-14 were active against the A549 cell line. These novel hybrid bipyrimidine derivatives served as potential lead molecules in the development of novel drug-like molecules for the treatment of breast and lung cancer.

Background: Monoamine oxidase-B (MAO-B) is crucial in breaking down biogenic and dietary amines, with its dysregulation linked to Parkinson’s disease. Objective: This study aims to explore the MAO-B inhibitory potential of 34 hydrazine-linked thiazole derivatives through computational techniques. Methods: Molecular docking against MAO-B (2BYB) assessed binding affinity, complemented by MMGBSA for binding energy evaluation. Pharmacophore modeling and QSAR analysis identified structural features essential for inhibition, while molecular dynamics (MD) simulations validated the stability of ligand-MAO-B complexes. Results: Compound T18 exhibited outstanding binding affinity, while T9 showed the lowest. In vitro studies revealed that T2 had the weakest inhibitory potential, while T19 demonstrated the highest. Pharmacophore modeling identified the six-feature hypothesis ADHRRR_1 as the key structural feature for optimal inhibition. QSAR analysis provided insight into the importance of pharmacophoric features, including the electron-deficient 3-pyridine at N1 of hydrazine, which acts as an electron-withdrawing group that enhances interactions with the enzyme active site, making pyridine a valuable pharmacophore. Additionally, the electronegative fluorinated phenyl ring at C4 of thiazole enhances blood-brain barrier (BBB) permeability and selectivity for MAO-B. Notably, the methyl group on T19 acted as a hydrophobic feature, increasing electron density and boosting inhibitory activity. MD simulations confirmed the stability of the T18/2BYB and T19/2BYB complexes. Furthermore, T19 displayed superior CNS activity and BBB permeability, confirming the methyl group as crucial for enhanced action. Conclusion: These findings underscore the potential of hydrazine-linked thiazole derivatives as potent MAO-B inhibitors. Future in vivo studies are essential to explore their therapeutic benefits further.

Penicillin resistance is a commonly present and controversial matter due to the misuse by people for various reasons. However, few studies have examined the bioactivity of 5- and 6- membered rings. In this study, we aimed to synthesize a new compound containing a 5-membered ring following a short and low-cost method and combined it with oxazepine ring via Schiff bases to produce a bicyclic molecule (Lactozepine). In vitro examinations were conducted to assess the bioactivity of the prepared compound, including anti-bacterial, anti-fungal and antioxidant activities, which showed a wide zone of inhibition of lactozepine against Streptococcus pneumoniae but no inhibition was shown against Klebsiella pneumoniae and Staphylococcus aureus except at a high concentration similar to the result of the anti-fungal assessment. Furthermore, lactozepine showed significant antioxidant activity against free radical formation. The molecular modeling and docking assessment showed the ability of lactozepine to bind to bacterial proteins and inhibit their growth with the lowest free energy for the greatest and strongest binding affinity with the PDB crystal structures 1VQQ, 2WAE, 1PYY and 1IYS ranging from −6.5 and −7.9 kcal/mol. Moreover, the molecular dynamics (MD) simulation showed that RMSF for the assessed protein’s amino acids remained consistent and tightly bound to lactozepine in the dynamic state. The novel compound lactozepine, with $\delta$-lactam rings attached to oxazepine showed bioactivity promising for in vivo studies in the future.

Background: Cancer remains a significant global health challenge, with ongoing research focused on identifying effective therapeutic targets. Human agmatinase (AGMAT), a binuclear manganese enzyme, has been associated with cancer progression through its role in polyamine production and its involvement in the NO/MAPK/PI3K signaling pathway in lung tissue. Objective: This study aimed to identify potential inhibitors of AGMAT from a library of FDA-approved drugs using computational methods, intending to find novel therapeutic agents for cancer treatment. Methods: The three-dimensional structure of AGMAT was determined using homology modeling, with Pseudomonas guanidinobutyrase (GbuA) serving as the template due to its 74% sequence similarity to AGMAT. The model was validated using PROCHECK, Verify3D, ProSA and ERRAT software. High-throughput virtual screening was conducted on a chemical library of approximately 7922 FDA-approved drugs to identify potential AGMAT inhibitors. Molecular docking simulations assessed the binding affinities of these drugs. Results: The virtual screening identified ten lead FDA-approved molecules: Nemifitidum, Examorelin, Pralmorelin hydrochloride, Nonathymulin, Ebiratide, Lypressin, Histrelin, Agripressin, Cetrorelix and Ornipressin that exhibited significant binding affinities to AGMAT. Conclusion: The identified lead compounds represent promising candidates for further investigation as potential cancer therapeutics targeting AGMAT. These findings provide a basis for future experimental validation and the development of new drugs toward cancer treatments.

Hepatocellular carcinoma, presenting a significant health challenge, requires innovative approaches for treatment and prevention. Therefore, this study aimed to comprehensively characterize the potential of compounds from Achillea arabica on the mTOR, Akt and ERK signaling pathways using in silico methods. The drug likeness and ADME/T properties of the compounds were investigated using online tools. Molecular docking and molecular dynamics simulations were used to examine the key interactions of the selected compounds with the mTOR, Akt and ERK target proteins along with the known inhibitors. Three phenolic compounds demonstrated a strong binding affinity as the top candidates. Compounds quercetin, luteolin and apigenin exhibit potential as nontoxic alternatives for hepatocellular carcinoma treatment, typically used as synthetic chemical inhibitors.

The ongoing COVID-19 pandemic has spurred international efforts to discover effective treatments against SARS-CoV-2. The essential oil (EO) of Algerian Origanum vulgare, which was extracted by steam distillation from aerial portions of the plant, contained a variety of bioactive substances that we were able to identify using a combination of gas chromatography–mass spectrometry and infrared spectroscopy (IR). The objective of this work is to ascertain this oil’s organoleptic and physicochemical characteristics. The drug interactions with important proteins involved in SARS-CoV-2 replication were evaluated using molecular docking studies. Certain compounds exhibited a strong affinity for binding, suggesting that they may have the ability to suppress viral activity. The stability and effectiveness of these interactions were validated by molecular dynamics simulations wherein carvacrol complexes demonstrated enhanced structural stability decreased flexibility compactness and decreased solvent exposure in contrast to other ligands. Both carvacrol and thymol are bioavailable and well absorbed according to ADMET analysis however there is a chance of skin irritation and liver toxicity. Carvacrol in particular showed minimal toxicity risks such as blockage of hERG or AMES low CNS permeability and slight long-term toxicity. These findings imply that Origanum vulgare essential oil might be a valuable natural resource for creating more effective COVID-19 treatments.

Malaria, caused by Plasmodium parasites, remains life-threatening, with Plasmodium falciparum responsible for severe cases. The parasite’s invasion of Red Blood Cells (RBCs) is critical for infection. The interaction between Reticulocyte Binding Protein Homologue 5 (RH5), Basigin, monoclonal antibodies and Cysteine-Rich Protective Antigen (CyRPA) with glycan on RBCs is essential for this process. This study aims to identify small molecules inhibiting the RH5-CyRPA-RIPr invasion complex. A library of 2299 compounds from FDA-approved drugs and African natural products in the ZINC database was screened using molecular docking. Binding sites for Basigin, monoclonal antibodies and glycans on RH5 and CyRPA were targeted, with binding affinity cutoffs of −8.5 kcal/mol for monoclonal antibodies and −7.0 kcal/mol for CyRPA and Basigin. Compounds were assessed for pharmacokinetics, solubility, drug-likeness, lead-likeness and biological activity using the PASS online server. Molecular dynamics simulations and free energy calculations evaluated stability and binding efficiency. Nine hits for RH5 and three for CyRPA showed favorable stability and anti-protozoal activity (up to 0.65). African natural products exhibited similarities with drugs like Nequinate and Cyanidin, supporting further research. Promising small molecules were identified as potential inhibitors of P. falciparum invasion, laying a foundation for experimental validation and drug development.

In pre-antibiotic times, various highly contagious diseases like cholera, smallpox and tuberculosis were widespread worldwide. Penicillin discovery in the late 1920s was a groundbreaking moment in medical history, saving countless lives. However, over the next few decades, microbes developed antibiotic resistance, leading to a global public health threat known as antimicrobial resistance (AMR). Pseudomonas aeruginosa is a major contributor to hospital-acquired infections, affecting millions of patients and causing numerous deaths annually. Several non-$\beta$-lactam antibiotics combat these infections effectively, while their effect on P. aeruginosa quorum sensing (QS) has been insufficiently explored. We have undertaken comprehensive research to understand the effect of non-$\beta$-lactam antibiotics on various targets of P. aeruginosa. Using molecular simulations, we scrutinize these antibiotics” dynamic behavior and stability. Based on toxicity, binding energy and binding site, platensimycin and sulfasalazine were identified as promising candidates against various targets of P. aeruginosa. The binding energies for sulfasalazine and platensimycin with LasA were found to be −8.1 and −8.6 kcal/mol, respectively. Both of these leading antibiotics were interacting at the active sites of all tested proteins (LasA, LasI and PqsR). The examination of molecular dynamics confirmed the stable complex formation of the lead non-$\beta$-lactam antibiotics with all selected target proteins under normal physiological conditions. These findings emphasize the potential efficacy of platensimycin and sulfasalazine. They could potentially be repurposed for targeting the QS of P. aeruginosa.

Panax ginseng exerts good neuroprotective activity at the cell and animal level, but the specific bioactive compounds and action mechanism are needed to be investigated, verified, and confirmed. In this work, affinity ultrafiltration (AUF), UPLC-QTOF-MS, and molecular docking were integrated into one strategy to screen, identify, and evaluate the bioactive compounds in ginseng at the molecular level. Three biological macromolecules (AChE, MAO-B, and NMDA receptor) were selected as the target protein for AUF-MS screening for the first time, and 16 potential neuroactive compounds were found with suitable binding degree. Then, the bioactivity of ginseng and its components were evaluated by AChE-inhibitory test and DPPH assay, and the data indicate that ginseng extract and the screened compounds have good neuroactivity. The interaction between the three targets and the screened compounds was further analyzed by molecular docking, and the results were consistent with a few discrepancies in comparison with the AUF results. Finally, according to the corresponding relation between component-target-pathway, the action mechanism of ginseng elucidated that ginseng exerts a therapeutic effect on AD through multiple relations of components, targets, and pathways, which is in good accordance with the TCM theory.

Saponins from the roots of Platycodon grandiflorum, an edible medicinal plant, have shown a wide range of beneficial effects on various biological processes. In this study, an animal model was established by a single intraperitoneal injection of cisplatin (20mg/kg) for evaluating the protective effects of saponins from the roots of P. grandiflorum (PGS, 15mg/kg and 30mg/kg) in mice. The results indicated that PGS treatment for 10 days restored the destroyed intestinal mucosal oxidative system, and the loosened junctions of small intestinal villi was significantly improved. In addition, a significant mitigation of apoptotic effects deteriorated by cisplatin exposure in small intestinal villi was observed by immunohischemical staining. Also, western blot showed that PGS could effectively prevent endoplasmic reticulum (ER) stress-induced apoptosis caused by cisplatin in mice by restoring the activity of PERK (an ER kinase)-eIF2$\alpha$-ATF4 signal transduction pathway. Furthermore, molecular docking results of main saponins in PGS suggested a better binding ability with target proteins. In summary, the present work revealed the underlying protective mechanisms of PGS on intestinal injury induced by cisplatin in mice.

Ginsenoside Rg5 (G-Rg5) is a rare ginsenoside isolated from ginseng (Panax ginseng C.A. Meyer), and this compound is increasingly known for its potent pharmacological activities. This study aimed to provide a comprehensive review of the main activities and mechanisms of G-Rg5 by adopting network pharmacological analysis combined with a summary of published articles. The 100 target genes of G-Rg5 were searched through available database, subjected to protein–protein interaction (PPI) network generation and then core screening. The results showed that G-Rg5 has promising anticancer and neuroprotective effects. By summarizing these two pharmacological activities, we found that G-Rg5 exerts its therapeutic effects mainly through PI3K/AKT, MAPK signaling pathways, and the regulation of apoptosis and cell cycle. And these results were corroborated by KEGG analysis. Likewise, molecular docking of the related proteins was performed, and the binding energies were all less than $-$7.0kJ/mol, indicating that these proteins had excellent binding capacity with G-Rg5. The network pharmacology results revealed many potential G-Rg5 mechanisms, which need to be further explored. We expect that the network pharmacology approach and molecular docking techniques can help us gain a deeper understanding of the therapeutic mechanisms of different ginsenosides and even the ginseng plant, for further developing their therapeutic potential as well as clinical applications.

Rosa roxburghii Tratt is a traditional Chinese plant that has been used to treat different inflammatory diseases. The purpose of this study was to investigate the mechanism of action of Rosa roxburghii Tratt extract (RRTE) against ulcerative colitis (UC) using network pharmacology and experimental validation. HPLC-Q/Orbitrap MS was used to rapidly identify the substances contained in RRTE after extracting the active components from the fruit. Then, network pharmacology combined with molecular docking was used to explore the critical target and potential mechanism of RRTE against UC using the active ingredients in RRTE as the research object. Data are presented in a visual manner. Finally, the pharmacological effects of RRTE in alleviating UC were further verified using a DSS-induced UC model of NCM460. The results showed that 25 components in RRTE were identified. A total of 250 targets of the active components and 5376 targets associated with UC were collected. Furthermore, a systematic analysis of the Protein–Protein Interaction (PPI) networks suggests that epidermal growth factor receptor (EGFR), phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), and serine/threonine kinase 1 (AKT1) are critical targets for RRTE in the treatment of UC. A comprehensive regulatory network analysis showed that RRTE alleviated UC through the EGFR-mediated PI3K/Akt pathway, and molecular docking showed that active components could strongly bind to EGFR, PIK3R1, and AKT1. In addition, RRTE alleviated dextran sulfate sodium salt (DSS)-induced cell injury and significantly decreased the protein expression levels of EGFR, PIK3R1, and p-AKT in NCM460 cells in vitro. Furthermore, RRTE significantly regulated the expression of the apoptosis-related proteins Apoptotic protease-activating factor 1 (Apaf1), cleaved caspase-3, B-cell lymphoma-2 (Bcl2), and Bcl2 associated X protein (Bax). In conclusion, the components of RRTE are complex, and RRTE can relieve UC through the EGFR-mediated PI3K/Akt pathway.

Metabolic syndrome (MetS) represents a considerable clinical and public health burden worldwide. Mangiferin (MF), a flavonoid compound present in diverse species such as mango (Mangifera indica L.), papaya (Pseudocydonia sinensis (Thouin) C. K. Schneid.), zhimu (Anemarrhena asphodeloides Bunge), and honeybush tea (Cyclopia genistoides), boasts a broad array of pharmacological effects. It holds promising uses in nutritionally and functionally targeted foods, particularly concerning MetS treatment. It is therefore pivotal to systematically investigate MF’s therapeutic mechanism for MetS and its applications in food and pharmaceutical sectors. This review, with the aid of a network pharmacology approach complemented by this experimental studies, unravels possible mechanisms underlying MF’s MetS treatment. Network pharmacology results suggest that MF treats MetS effectively through promoting insulin secretion, targeting obesity and inflammation, alleviating insulin resistance (IR), and mainly operating via the phosphatidylinositol 3 kinase (PI3K)/Akt, nuclear factor kappa-B (NF-$\kappa$B), microtubule-associated protein kinase (MAPK), and oxidative stress signaling pathways while repairing damaged insulin signaling. These insights provide a comprehensive framework to understand MF’s potential mechanisms in treating MetS. These, however, warrant further experimental validation. Moreover, molecular docking techniques confirmed the plausibility of the predicted outcomes. Hereafter, these findings might form the theoretical bedrock for prospective research into MF’s therapeutic potential in MetS therapy.

Molecular docking and molecular dynamics (MD) are powerful tools used to investigate protein-ligand interactions. Molecular docking programs predict the binding pose and affinity of a protein-ligand complex, while MD can be used to incorporate flexibility into docking calculations and gain further information on the kinetics and stability of the protein-ligand bond. This review covers state-of-the-art methods of using molecular docking and MD to explore protein-ligand interactions, with emphasis on application to drug discovery. We also call for further research on combining common molecular docking and MD methods.

With the aid of the automatic molecular docking, the affinity of CYP2C9 and CYP2D6 for imrecoxib was studied by InsightII/Affinity program. The results indicate that CYP2C9–imrecoxib complex has higher stability and stronger affinity because CYP2C9 has more favorable interaction energy (-62.72 kcal/mol) and higher Ludi score (610) with imrecoxib than CYP2D6 (-50.22 kcal/mol and 551) and this is consistent with the results of the kinetic experiments by Li et al. By analyzing the theoretical results combined with the experimental ones, we suggest that the affinity difference is caused by the difference of the structure between CYP2C9 and CYP2D6, and the most important residues for enzyme–substrate complexes, such as Phe476, Asn204, Phe100, Leu366 and Arg108 of CYP2C9 and Phe120, Glu216, and Phe483 of CYP2D6 were also identified.