Cuminaldehyde as a Lipoxygenase Inhibitor: In Vitro and In Silico Validation

The search for lipoxygenase (LOX) inhibitors has been carried out for decades due to its importance in inflammatory diseases. In the present study, it was observed that the methanolic extract of Cuminum cyminum L. inhibited LOX activity. Activity-guided screening of the C. cyminum crude extracts helped the identification and isolation of cuminaldehyde as a 15-LOX inhibitor. The enzyme kinetics analysis suggested cuminaldehyde to be a competitive inhibitor and the IC ₅₀ value derived from LB plots is 1,370 μM. Binding constants of cuminaldehyde on LOX was deduced by isothermal titration calorimetry. The combined thermodynamics and molecular modeling analyses suggested cuminaldehyde as a competitive LOX inhibitor. It is proposed from the present study that the coordinate bond between the Fe²⁺ atom in the active site of the enzyme and the cuminaldehyde may be responsible for the enzyme inhibition. The study suggests that cuminaldehyde may be acting as an anti-inflammatory compound and may be therefore included in the category of leads for developing dual COX-LOX inhibitors as non-steroidal anti-inflammatory drugs (NSAIDs).     

Identification of Putative Vaccine and Drug Targets against the Methicillin-Resistant Staphylococcus aureus by Reverse Vaccinology and Subtractive Genomics Approaches

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and responsible for causing life-threatening infections. The emergence of hypervirulent and multidrug-resistant (MDR) S. aureus strains led to challenging issues in antibiotic therapy. Consequently, the morbidity and mortality rates caused by S. aureus infections have a substantial impact on health concerns. The current worldwide prevalence of MRSA infections highlights the need for long-lasting preventive measures and strategies. Unfortunately, effective measures are limited. In this study, we focus on the identification of vaccine candidates and drug target proteins against the 16 strains of MRSA using reverse vaccinology and subtractive genomics approaches. Using the reverse vaccinology approach, 4 putative antigenic proteins were identified; among these, PrsA and EssA proteins were found to be more promising vaccine candidates. We applied a molecular docking approach of selected 8 drug target proteins with the drug-like molecules, revealing that the ZINC4235426 as potential drug molecule with favorable interactions with the target active site residues of 5 drug target proteins viz., biotin protein ligase, HPr kinase/phosphorylase, thymidylate kinase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine ligase, and pantothenate synthetase. Thus, the identified proteins can be used for further rational drug or vaccine design to identify novel therapeutic agents for the treatment of multidrug-resistant staphylococcal infection.     

In Silico Identification and Validation of Organic Triazole Based Ligands as Potential Inhibitory Drug Compounds of SARS-CoV-2 Main Protease

The SARS-CoV-2 virus is highly contagious to humans and has caused a pandemic of global proportions. Despite worldwide research efforts, efficient targeted therapies against the virus are still lacking. With the ready availability of the macromolecular structures of coronavirus and its known variants, the search for anti-SARS-CoV-2 therapeutics through in silico analysis has become a highly promising field of research. In this study, we investigate the inhibiting potentialities of triazole-based compounds against the SARS-CoV-2 main protease (M^pro). The SARS-CoV-2 main protease (M^pro) is known to play a prominent role in the processing of polyproteins that are translated from the viral RNA. Compounds were pre-screened from 171 candidates (collected from the DrugBank database). The results showed that four candidates (Bemcentinib, Bisoctrizole, PYIITM, and NIPFC) had high binding affinity values and had the potential to interrupt the main protease (M^pro) activities of the SARS-CoV-2 virus. The pharmacokinetic parameters of these candidates were assessed and through molecular dynamic (MD) simulation their stability, interaction, and conformation were analyzed. In summary, this study identified the most suitable compounds for targeting Mpro, and we recommend using these compounds as potential drug molecules against SARS-CoV-2 after follow up studies.     

Virtual Screening for Biomimetic Anti-Cancer Peptides from Cordyceps militaris Putative Pepsinized Peptidome and Validation on Colon Cancer Cell Line

Colorectal cancer is one of the leading causes of cancer-related death in Thailand and many other countries. The standard practice for curing this cancer is surgery with an adjuvant chemotherapy treatment. However, the unfavorable side effects of chemotherapeutic drugs are undeniable. Recently, protein hydrolysates and anticancer peptides have become popular alternative options for colon cancer treatment. Therefore, we aimed to screen and select the anticancer peptide candidates from the in silico pepsin hydrolysate of a Cordyceps militaris (CM) proteome using machine-learning-based prediction servers for anticancer prediction, i.e., AntiCP, iACP, and MLACP. The selected CM-anticancer peptide candidates could be an alternative treatment or co-treatment agent for colorectal cancer, reducing the use of chemotherapeutic drugs. To ensure the anticancer properties, an in vitro assay was performed with “CM-biomimetic peptides” on the non-metastatic colon cancer cell line (HT-29). According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results from peptide candidate treatments at 0–400 µM, the IC₅₀ doses of the CM-biomimetic peptide with no toxic and cancer-cell-penetrating ability, original C. militaris biomimetic peptide (C-ori), against the HT-29 cell line were 114.9 µM at 72 hours. The effects of C-ori compared to the doxorubicin, a conventional chemotherapeutic drug for colon cancer treatment, and the combination effects of both the CM-anticancer peptide and doxorubicin were observed. The results showed that C-ori increased the overall efficiency in the combination treatment with doxorubicin. According to the acridine orange/propidium iodine (AO/PI) staining assay, C-ori can induce apoptosis in HT-29 cells significantly, confirmed by chromatin condensation, membrane blebbing, apoptotic bodies, and late apoptosis which were observed under a fluorescence microscope.     

Repositioning of Ligands That Target the Spike Glycoprotein as Potential Drugs for SARS-CoV-2 in an In Silico Study

The worldwide health emergency of the SARS-CoV-2 pandemic and the absence of a specific treatment for this new coronavirus have led to the use of computational strategies (drug repositioning) to search for treatments. The aim of this work is to identify FDA (Food and Drug Administration)-approved drugs with the potential for binding to the spike structural glycoprotein at the hinge site, receptor binding motif (RBM), and fusion peptide (FP) using molecular docking simulations. Drugs that bind to amino acids are crucial for conformational changes, receptor recognition, and fusion of the viral membrane with the cell membrane. The results revealed some drugs that bind to hinge site amino acids (varenicline, or steroids such as betamethasone while other drugs bind to crucial amino acids in the RBM (naldemedine, atovaquone, cefotetan) or FP (azilsartan, maraviroc, and difluprednate); saquinavir binds both the RBM and the FP. Therefore, these drugs could inhibit spike glycoprotein and prevent viral entry as possible anti-COVID-19 drugs. Several drugs are in clinical studies; by focusing on other pharmacological agents (candesartan, atovaquone, losartan, maviroc and ritonavir) in this work we propose an additional target: the spike glycoprotein. These results can impact the proposed use of treatments that inhibit the first steps of the virus replication cycle.     

Monoamine Oxidase (MAO) as a Potential Target for Anticancer Drug Design and Development

Monoamine oxidases (MAOs) are oxidative enzymes that catalyze the conversion of biogenic amines into their corresponding aldehydes and ketones through oxidative deamination. Owing to the crucial role of MAOs in maintaining functional levels of neurotransmitters, the implications of its distorted activity have been associated with numerous neurological diseases. Recently, an unanticipated role of MAOs in tumor progression and metastasis has been reported. The chemical inhibition of MAOs might be a valuable therapeutic approach for cancer treatment. In this review, we reported computational approaches exploited in the design and development of selective MAO inhibitors accompanied by their biological activities. Additionally, we generated a pharmacophore model for MAO-A active inhibitors to identify the structural motifs to invoke an activity.     

Discovery of New Inhibitors of eEF2K from Traditional Chinese Medicine Based on In Silico Screening and In Vitro Experimental Validation

Eukaryotic elongation factor 2 kinase (eEF2K) is a highly conserved α kinase and is increasingly considered as an attractive therapeutic target for cancer as well as other diseases. However, so far, no selective and potent inhibitors of eEF2K have been identified. In this study, pharmacophore screening, homology modeling, and molecular docking methods were adopted to screen novel inhibitor hits of eEF2K from the traditional Chinese medicine database (TCMD), and then cytotoxicity assay and western blotting were performed to verify the validity of the screen. Resultantly, after two steps of screening, a total of 1077 chemicals were obtained as inhibitor hits for eEF2K from all 23,034 compounds in TCMD. Then, to verify the validity, the top 10 purchasable chemicals were further analyzed. Afterward, Oleuropein and Rhoifolin, two reported antitumor chemicals, were found to have low cytotoxicity but potent inhibitory effects on eEF2K activity. Finally, molecular dynamics simulation, pharmacokinetic and toxicological analyses were conducted to evaluate the property and potential of Oleuropein and Rhoifolin to be drugs. Together, by integrating in silico screening and in vitro biochemical studies, Oleuropein and Rhoifolin were revealed as novel eEF2K inhibitors, which will shed new lights for eEF2K-targeting drug development and anticancer therapy.     

Preliminary Insight of Pyrrolylated-Chalcones as New Anti-Methicillin-Resistant Staphylococcus aureus (Anti-MRSA) Agents

Bacterial infections are regarded as one of the leading causes of fatal morbidity and death in patients infected with diseases. The ability of microorganisms, particularly methicillin-resistant Staphylococcus aureus (MRSA), to develop resistance to current drugs has evoked the need for a continuous search for new drugs with better efficacies. Hence, a series of non-PAINS associated pyrrolylated-chalcones (1–15) were synthesized and evaluated for their potency against MRSA. The hydroxyl-containing compounds (8, 9, and 10) showed the most significant anti-MRSA efficiency, with the MIC and MBC values ranging from 0.08 to 0.70 mg/mL and 0.16 to 1.88 mg/mL, respectively. The time-kill curve and SEM analyses exhibited bacterial cell death within four hours after exposure to 9, suggesting its bactericidal properties. Furthermore, the docking simulation between 9 and penicillin-binding protein 2a (PBP2a, PDB ID: 6Q9N) suggests a relatively similar bonding interaction to the standard drug with a binding affinity score of −7.0 kcal/mol. Moreover, the zebrafish model showed no toxic effects in the normal embryonic development, blood vessel formation, and apoptosis when exposed to up to 40 µM of compound 9. The overall results suggest that the pyrrolylated-chalcones may be considered as a potential inhibitor in the design of new anti-MRSA agents.     

Deep Learning and Structure-Based Virtual Screening for Drug Discovery against NEK7: A Novel Target for the Treatment of Cancer

NIMA-related kinase7 (NEK7) plays a multifunctional role in cell division and NLRP3 inflammasone activation. A typical expression or any mutation in the genetic makeup of NEK7 leads to the development of cancer malignancies and fatal inflammatory disease, i.e., breast cancer, non-small cell lung cancer, gout, rheumatoid arthritis, and liver cirrhosis. Therefore, NEK7 is a promising target for drug development against various cancer malignancies. The combination of drug repurposing and structure-based virtual screening of large libraries of compounds has dramatically improved the development of anticancer drugs. The current study focused on the virtual screening of 1200 benzene sulphonamide derivatives retrieved from the PubChem database by selecting and docking validation of the crystal structure of NEK7 protein (PDB ID: 2WQN). The compounds library was subjected to virtual screening using Auto Dock Vina. The binding energies of screened compounds were compared to standard Dabrafenib. In particular, compound 762 exhibited excellent binding energy of −42.67 kJ/mol, better than Dabrafenib (−33.89 kJ/mol). Selected drug candidates showed a reactive profile that was comparable to standard Dabrafenib. To characterize the stability of protein–ligand complexes, molecular dynamic simulations were performed, providing insight into the molecular interactions. The NEK7–Dabrafenib complex showed stability throughout the simulated trajectory. In addition, binding affinities, pIC50, and ADMET profiles of drug candidates were predicted using deep learning models. Deep learning models predicted the binding affinity of compound 762 best among all derivatives, which supports the findings of virtual screening. These findings suggest that top hits can serve as potential inhibitors of NEK7. Moreover, it is recommended to explore the inhibitory potential of identified hits compounds through in-vitro and in-vivo approaches.     

Discovery of 5-Methylthiazole-Thiazolidinone Conjugates as Potential Anti-Inflammatory Agents: Molecular Target Identification and In Silico Studies

A series of previously synthesized 5-benzyliden-2-(5-methylthiazole-2-ylimino)thiazoli- din-4-one were evaluated for their anti-inflammatory activity on the basis of PASS predictive outcomes. The predictive compounds were found to demonstrate moderate to good anti-inflammatory activity, and some of them displayed better activity than indomethacin used as the reference drug. Structure–activity relationships revealed that the activity of compounds depends not only on the nature of the substituent but also on its position in the benzene ring. The most active compounds were selected to investigate their possible mechanism of action. COX and LOX activity were determined and found that the title compounds were active only to COX-1 enzymes with an inhibitory effect superior to the reference drug naproxen. As for LOX inhibitory activity, the derivatives failed to show remarkable LOX inhibition. Therefore, COX-1 has been identified as the main molecular target for the anti-inflammatory activity of our compounds. The docking study against COX-1 active site revealed that the residue Arg 120 was found to be responsible for activity. In summary, the 5-thiazol-based thiazolidinone derivatives have been identified as a novel class of selective COX-1 inhibitors.     

Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by liposome-delivered photosensitising agents

The uptake of two photosensitising agents (hematoporphyrin and chlorophyll a) by a highly pathogenic bacterium, namely methicillin-resistant Staphylococcus aureus (MRSA), has been studied by using unilamellar liposomes of different size, fluidity and electric charge as carriers. Optimal results are obtained by using hematoporphyrin embedded in fluid cationic vesicles composed by the monocationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), which yield an endocellular concentration of photosensitiser much higher than that obtained by incubation of the cells with the free porphyrin, yet promote a tighter binding and a more efficient photoinactivation of MRSA. Apparently, the photosensitiser is successfully transferred from the liposome to the bacterial cells when the presence of the tetrapyrrolic derivative does not appreciably perturb the native three-dimensional organisation of the lipid vesicle, such as it occurs with hematoporphyrin. On the other hand, chlorophyll, which causes a marked structural alteration of the DOTAP vesicles as shown by electron microscopy and fluorescence anisotropy measurements, does not show any detectable photocytotoxicity toward MRSA, contrary to what observed for the free dye.     

Phytochemical Screening,In Vitro and In Silico Studies of Volatile Compounds from Petroselinum crispum (Mill) Leaves Grown in Saudi Arabia

The herbal plant Petroselinum crispum (P. crispum) (Mill) is commonly available around the world. In this study, the leaves of the herbal plant P. crispum were collected from the central region of Al-Kharj, Saudi Arabia, to explore their in vitro pharmacological activity. Essential oil from the leaves of P. crispum was isolated using the hydrodistillation method. The composition of P. crispum essential oil (PCEO) was determined using Gas chromatography-mass spectrometry (GC-MS). A total of 67 components were identified, representing approximately 96.02% of the total volatile composition. Myristicin was identified as the principal constituent (41.45%). The in vitro biological activity was assessed to evaluate the antioxidant, antimicrobial, and anti-inflammatory potential of PCEO. PCEO showed the highest antimicrobial activity against Candida albicans and Staphylococcus aureus among all the evaluated microbial species. In vitro anti-inflammatory evaluation using albumin and trypsin assays showed the excellent anti-inflammatory potential of PCEO compared to the standard drugs. An in silico study of the primary PCEO compound was conducted using online tools such as PASS, Swiss ADME, and Molecular docking. In silico PASS prediction results supported our in vitro findings. Swiss ADME revealed the drug likeness and safety properties of the major metabolites present in PCEO. Molecular docking results were obtained by studying the interaction of Myristicin with an antifungal (PDB: 1IYL and 3LD6), antibacterial (PDB: 1AJ6 and 1JIJ), antioxidant (PDB: 3NM8 and 1HD2), and anti-inflammatory (3N8Y and 3LN1) receptors supported the in vitro results. Therefore, PCEO or Myristicin might be valuable for developing anti-inflammatory and antimicrobial drugs.     

In Silico Approach for the Evaluation of the Potential Antiviral Activity of Extra Virgin Olive Oil (EVOO) Bioactive Constituents Oleuropein and Oleocanthal on Spike Therapeutic Drug Target of SARS-CoV-2

Since there is an urgent need for novel treatments to combat the current coronavirus disease 2019 (COVID-19) pandemic, in silico molecular docking studies were implemented as an attempt to explore the ability of selected bioactive constituents of extra virgin olive oil (EVOO) to act as potent SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) antiviral compounds, aiming to explore their ability to interact with SARS-CoV-2 Spike key therapeutic target protein. Our results suggest that EVOO constituents display substantial capacity for binding and interfering with Spike (S) protein, both wild-type and mutant, via the receptor-binding domain (RBD) of Spike, or other binding targets such as angiotensin-converting enzyme 2 (ACE2) or the RBD-ACE2 protein complex, inhibiting the interaction of the virus with host cells. This in silico study provides useful insights for the understanding of the mechanism of action of the studied compounds at a molecular level. From the present study, it could be suggested that the studied active phytochemicals could potentially inhibit the Spike protein, contributing thus to the understanding of the role that they can play in future drug designing and the development of anti-COVID-19 therapeutics.     

In Silico Identification of Potential Sites for a Plastic-Degrading Enzyme by a Reverse Screening through the Protein Sequence Space and Molecular Dynamics Simulations

The accumulation of polyethylene terephthalate (PET) seriously harms the environment because of its high resistance to degradation. The recent discovery of the bacteria-secreted biodegradation enzyme, PETase, sheds light on PET recycling; however, the degradation efficiency is far from practical use. Here, in silico alanine scanning mutagenesis (ASM) and site-saturation mutagenesis (SSM) were employed to construct the protein sequence space from binding energy of the PETase–PET interaction to identify the number and position of mutation sites and their appropriate side-chain properties that could improve the PETase–PET interaction. The binding mechanisms of the potential PETase variant were investigated through atomistic molecular dynamics simulations. The results show that up to two mutation sites of PETase are preferable for use in protein engineering to enhance the PETase activity, and the proper side chain property depends on the mutation sites. The predicted variants agree well with prior experimental studies. Particularly, the PETase variants with S238C or Q119F could be a potential candidate for improving PETase. Our combination of in silico ASM and SSM could serve as an alternative protocol for protein engineering because of its simplicity and reliability. In addition, our findings could lead to PETase improvement, offering an important contribution towards a sustainable future.     

Ultrasound Assisted Synthesis and In Silico Modelling of 1,2,4-Triazole Coupled Acetamide Derivatives of 2-(4-Isobutyl phenyl)propanoic acid as Potential Anticancer Agents

The development of an economical method for the synthesis of biologically active compounds was the major goal of this research. In the present study, we have reported the ultrasound-radiation-assisted synthesis of a series of novel N-substituted 1,2,4-triazole-2-thiol derivatives. The target compounds 6a–f were efficiently synthesized in significant yields (75–89%) by coupling 1,2,4-triazole of 2-(4-isobutylphenyl) propanoic acid 1 with different electrophiles using ultrasound radiation under different temperatures. The sonication process accelerated the rate of the reaction as well as yielded all derivatives compared to conventional methods. All derivatives were confirmed by spectroscopic (FTIR, ¹HNMR, ¹³CNMR, HRMS) and physiochemical methods. All derivatives were further screened for their anticancer effects against the HepG2 cell line. Compound 6d containing two electron-donating methyl moieties demonstrated the most significant anti-proliferative activity with an IC₅₀ value of 13.004 µg/mL, while compound 6e showed the lowest potency with an IC₅₀ value of 28.399 µg/mL. The order of anticancer activity was found to be: 6d > 6b > 6f > 6a > 6c > 6e, respectively. The in silico modelling of all derivatives was performed against five different protein targets and the results were consistent with the biological activities. Ligand 6d showed the best binding affinity with the Protein Kinase B (Akt) pocket with the lowest ∆G value of −176.152 kcal/mol. Compound 6d has been identified as a promising candidate for treatment of liver cancer.     

Discovery of Monoacylglycerol Lipase (MAGL) Inhibitors Based on a Pharmacophore-Guided Virtual Screening Study

Monoacylglycerol lipase (MAGL) is an important enzyme of the endocannabinoid system that catalyzes the degradation of the major endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL is associated with pathological conditions such as pain, inflammation and neurodegenerative diseases like Parkinson’s and Alzheimer’s disease. Furthermore, elevated levels of MAGL have been found in aggressive breast, ovarian and melanoma cancer cells. Due to its different potential therapeutic implications, MAGL is considered as a promising target for drug design and the discovery of novel small-molecule MAGL inhibitors is of great interest in the medicinal chemistry field. In this context, we developed a pharmacophore-based virtual screening protocol combined with molecular docking and molecular dynamics simulations, which showed a final hit rate of 50% validating the reliability of the in silico workflow and led to the identification of two promising and structurally different reversible MAGL inhibitors, VS1 and VS2. These ligands represent a valuable starting point for structure-based hit-optimization studies aimed at identifying new potent MAGL inhibitors.     

In Silico Drug Repurposing of FDA-Approved Drugs Highlighting Promacta as a Potential Inhibitor of H7N9 Influenza Virus

Influenza virus infections continue to be a significant and recurrent public health problem. Although vaccine efficacy varies, regular immunisation is the most effective method for suppressing the influenza virus. Antiviral drugs are available for influenza, although two of the four FDA-approved antiviral treatments have resulted in significant drug resistance. Therefore, new treatments are being sought to reduce the burden of flu-related illness. The time-consuming development of treatments for new and re-emerging diseases such as influenza and the high failure rate are increasing concerns. In this context, we used an in silico-based drug repurposing method to repurpose FDA-approved drugs as potential therapies against the H7N9 virus. To find potential inhibitors, a total of 2568 drugs were screened. Promacta, tucatinib, and lurasidone were identified as promising hits in the DrugBank database. According to the calculations of MM-GBSA, tucatinib (−54.11 kcal/mol) and Promacta (−56.20 kcal/mol) occupied the active site of neuraminidase with a higher binding affinity than the standard drug peramivir (−49.09 kcal/mol). Molecular dynamics (MD) simulation studies showed that the C-α atom backbones of the complexes of tucatinib and Promacta neuraminidase were stable throughout the simulation period. According to ADME analysis, the hit compounds have a high gastrointestinal absorption (GI) and do not exhibit properties that allow them to cross the blood–brain barrier (BBB). According to the in silico toxicity prediction, Promacta is not cardiotoxic, while lurasidone and tucatinib show only weak inhibition. Therefore, we propose to test these compounds experimentally against the influenza H7N9 virus. The investigation and validation of these potential H7N9 inhibitors would be beneficial in order to bring these compounds into clinical settings.     

BDNF as a Putative Target for Standardized Extract of Ginkgo biloba-Induced Persistence of Object Recognition Memory

Despite considerable progress on the study of the effect of standardized extract of Gingko biloba (EGb) on memory processes, our understanding of its role in the persistence of long-term memory (LTM) and the molecular mechanism underlying its effect, particularly episodic-like memory, is limited. We here investigated the effects of EGb on the long-term retention of recognition memory and its persistence and BDNF expression levels in the dorsal hippocampal formation (DHF). Adult male Wistar rats (n = 10/group) were handled for 10 min/5 day. On day 6, the animals were treated with vehicle or 0.4 mg/kg diazepam (control groups) or with EGb (250, 500 or 100 mg/kg) 30 min before the training session (TR1), in which the animals were exposed to two sample objects. On day 7, all rats underwent a second training session (TR2) as described in the TR1 but without drug treatment. Object recognition memory (ORM) was evaluated on day 8 (retention test, T1) and day 9 (persistence test, T2). At the end of T1or T2, animals were decapitated, and DHF samples were frozen at −80 °C for analyses of the differential expression of BDNF by Western blotting. EGb-treated groups spent more time exploring the novel object in T2 and showed the highest recognition index (RI) values during the T1 and T2, which was associated with upregulation of BDNF expression in the DHF in a dose-and session-dependent manner. Our data reveal, for the first time, that EGb treatment before acquisition of ORM promotes persistence of LTM by BDNF differential expression.