Unlocking G-Quadruplexes as Antiviral Targets.

Guanine-rich DNA and RNA sequences can fold into noncanonical nucleic acid structures called G-quadruplexes (G4s). Since the discovery that these structures may act as scaffolds for the binding of specific ligands, G4s aroused the attention of a growing number of scientists. The versatile roles of G4 structures in viral replication, transcription, and translation suggest direct applications in therapy or diagnostics. G4-interacting molecules (proteins or small molecules) may also affect the balance between latent and lytic phases, and increasing evidence reveals that G4s are implicated in generally suppressing viral processes, such as replication, transcription, translation, or reverse transcription. In this review, we focus on the discovery of G4s in viruses and the role of G4 ligands in the antiviral drug discovery process. After assessing the role of viral G4s, we argue that host G4s participate in immune modulation, viral tumorigenesis, cellular pathways involved in virus maturation, and DNA integration of viral genomes, which can be potentially employed for antiviral therapeutics. Furthermore, we scrutinize the impediments and shortcomings in the process of studying G4 ligands and drug discovery. Finally, some unanswered questions regarding viral G4s are highlighted for prospective future projects. SIGNIFICANCE STATEMENT: G-quadruplexes (G4s) are noncanonical nucleic acid structures that have gained increasing recognition during the last few decades. First identified as relevant targets in oncology, their importance in virology is now increasingly clear. A number of G-quadruplex ligands are known: viral transcription and replication are the main targets of these ligands. Both viral and cellular G4s may be targeted; this review embraces the different aspects of G-quadruplexes in both host and viral contexts.

Refining our methodologies for assessing quadruplex DNA ligands; selectivity or an illusion of selectivity?

Regulation of transcription and replication by the tetrad patterns of DNA has drawn the attention of many scientists. In this perspective article, we discuss some disparaged parameters in the study of G-quadruplex structures (G4-tetrads). Besides, the implication of "destabilization as a side-effect" by these ligands on quadruplexes is explained. The lack of strict control of in vitro cell-free experiments in terms of ionic concentration, pH, epigenetic modifications, (macro)molecular crowding, and solvent effects is evident in many previous studies. The role of these factors in ligands binding and their possible effects in G-quadruplex structures are also represented.

Phlorotannins as HIV Vpu inhibitors, an in silico virtual screening study of marine natural products.

The importance of new effective treatment methodologies for human immunodeficiency virus (HIV) is undeniable for the medical society. Viral protein U (Vpu), one of the disparaged accessory proteins of HIV, is responsible for the dissemination of viral particles, and HIV mutants lacking Vpu protein have remarkably reduced pathogenicity. Here, we explored the marine natural products to find the leading structures which can potentially inhibit the activity of Vpu in silico. To fulfill this goal, we set up a virtual screening based on molecular docking to evaluate the binding capacity of different marine products to Vpu. For validation, we used molecular dynamics simulation and monitored the root mean square deviation value and binding interactions. The results were intriguing when we realized that the hit compounds (phlorotannins) had previously been identified as reverse transcriptase and HIV protease inhibitors. This research inaugurates a new road to combat HIV by multifaceted mode of action of these marine natural products without putting the normal cells in jeopardy (with their safe toxicological profile).

Synthetic biology in various cellular and molecular fields: applications, limitations, and perspective.

Synthetic biology breakthroughs have facilitated genetic circuit engineering to program cells through novel biological functions, dynamic gene expressions, as well as logic controls. SynBio can also participate in the rapid development of new treatments required for the human lifestyle. Moreover, these technologies are applied in the development of innovative therapeutic, diagnostic, as well as discovery-related methods within a wide range of cellular and molecular applications. In the present review study, SynBio applications in various cellular and molecular fields such as novel strategies for cancer therapy, biosensing, metabolic engineering, protein engineering, and tissue engineering were highlighted and summarized. The major safety and regulatory concerns about synthetic biology will be the environmental release, legal concerns, and risks of the engineered organisms. The final sections focused on limitations to SynBio.

Association of FSHR missense mutations with female infertility, in silico investigation of their molecular significance and exploration of possible treatments using virtual screening and molecular dynamics.

This study investigated the association of A419T (rs121909661) and T449I (rs28928870) with infertility among Iranian women and possible treatments by agonizing the mutated receptor. 151 women were genotyped at A419T and T449I sites. Homology modeling, pharmacophore modeling, virtual screening, docking and molecular dynamics (MD) were performed. A419T and T449I indicated a significant and a weak association with infertility among Iranian women (P = 0.005 and P = 0.03, respectively). Significant differences found among three genotypes of A419T with FSH (P = 0.01) and LH (P < 0.0001). G-allele carriers of A419T had susceptibility to display higher FSH and LH serum levels. In silico results revealed the most potent agonists among 3041 similar compounds and MD supported this finding. Altogether, genotyping of A419T and T449I as potential markers might be helpful in prognosis and treatment of infertility. Also, a new series of potent FSHR agonists were identified for future drug development and treatment of infertility related to FSHR dysfunction.

Design, synthesis, biological evaluation, and molecular dynamics of novel cholinesterase inhibitors as anti-Alzheimer's agents.

A series of novel chroman-4-one derivatives were designed and synthesized successfully with good to excellent yield (3a-l). In addition, the obtained products were evaluated for their cholinesterase (ChE) inhibitory activities. The results show that among the various synthesized compounds, analogs bearing the piperidinyl ethoxy side chain with 4-hydroxybenzylidene on the 3-positions of chroman-4-one (3l) showed the most potent activity with respect to acetylcholinesterase (anti-AChE activity; IC50 = 1.18 µM). In addition, the structure-activity relationship was studied and the results revealed that the electron-donating groups on the aryl ring of the 3-benzylidene fragment (3k, 3l) resulted in the designed compounds to be more potent ChE inhibitors in comparison with those having electron-withdrawing groups (3h). In this category, the strongest ChE inhibition was found for the compound containing piperidine as cyclic amine, and a hydroxyl group (for AChE, compound 3l) and fluoro group (for butyrylcholinesterase (BuChE, compound 3i) on the para-position of the aryl ring of the benzylidene group. The molecular docking and dynamics studies of the most potent compounds (3i and 3l against BuChE and AChE, respectively) demonstrated remarkable interactions with the binding pockets of the ChE enzymes and confirmed the results obtained through in vitro experiments.

Unexplored power of CRISPR-Cas9 in neuroscience, a multi-OMICs review.

The neuroscience and neurobiology of gene editing to enhance learning and memory is of paramount interest to the scientific community. The advancements of CRISPR system have created avenues to treat neurological disorders by means of versatile modalities varying from expression to suppression of genes and proteins. Neurodegenerative disorders have also been attributed to non-canonical DNA secondary structures by affecting neuron activity through controlling gene expression, nucleosome shape, transcription, translation, replication, and recombination. Changing DNA regulatory elements which could contribute to the fate and function of neurons are thoroughly discussed in this review. This study presents the ability of CRISPR system to boost learning power and memory, treat or cure genetically-based neurological disorders, and alleviate psychiatric diseases by altering the activity and the irritability of the neurons at the synaptic cleft through DNA manipulation, and also, epigenetic modifications using Cas9. We explore and examine how each different OMIC techniques can come useful when altering DNA sequences. Such insight into the underlying relationship between OMICs and cellular behaviors leads us to better neurological and psychiatric therapeutics by intelligently designing and utilizing the CRISPR/Cas9 technology.

Reproductive Toxicity of Nanoparticles: A Comprehensive Review.

The unique characteristics of nanoparticles (NPs) have captivated scientists in various fields of research. However, their safety profile has not been fully scrutinized. In this regard, the effects of NPs on the reproductive system of animals and humankind have been a matter of concern. In this article, we will review the potential reproductive toxicity of various types of NPs, including carbon nanomaterials, dendrimers, quantum dots, silica, gold, and magnetic nanoparticles, reported in the literature. We also mention some notable cases where NPs have elicited beneficial effects on the reproductive system. This review provides extensive insight into the effects of various NPs on sperm and ovum and the outcomes of their passage through blood-testis and placental barriers and accumulation in the reproductive organs.

Antileishmanial effects and drugability characteristics of a heterocyclic copper complex: An in silico, in vitro and molecular study.

Leishmaniasis caused by the protozoan Leishmania presents a severe illness, principally in tropical and subtropical areas. Antileishmanial metal complexes, like Glucantime®ï¸ with proven activity, are routinely studied to probe their potency. We investigated the effects of a Cu (II) homoleptic complex coordinated by two dimethyl-bipyridine ligands against Leishmania major stages in silico and in vitro. The affinity of this heterocyclic Cu (II) complex (CuDMBP) towards a parasitic metacaspase was studied by molecular docking. Key pharmacokinetic and pharmacodynamic properties of the complex were predicted using three web-based tools. CuDMBP was tested for in vitro antileishmanial activities using MTT assay, model murine macrophages, flow cytometry, and quantitative real-time polymerase chain reaction (qPCR). Molecular docking confirmed the tendency between the target macromolecule and the complex. ADMET evaluations highlighted CuDMBP's key pharmacological features, including P-glycoprotein-associated GI absorption and lack of trans-BBB permeability. MTT showed significant inhibitory effects against promastigotes. CuDMBP significantly increased the level of cellular IL-12 expression (p < 0.05), while the upregulation observed in the expression of iNOS was considered not significant (p > 0.05). It decreased the expression of IL-10 significantly (p < 0.05). Findings demonstrated that CuDMBP deserves to be introduced as a leishmanicidal candidate provided further studies are carried out.

A Review on Phosphodiesterase-5 Inhibitors as a Topical Therapy for Erectile Dysfunction.

INTRODUCTION: Due to the prevalence of erectile dysfunction and impotence among men in recent years, several pharmacotherapies have been considered for such problems. Systemic drug therapies in the treatment of erectile dysfunction have significant issues, including drug interactions and contraindications in a wide range of diseases, which makes researchers seek to design drugs and dosage forms with fewer side effects, interactions, and contraindications with maintained efficacy. OBJECTIVES: 5-Phosphodiesterase inhibitors (5-PDEIs or PDE5Is), previously used systemically to treat erectile malfunction, are now appropriate candidates for topical application with considerable potency and fewer complications. METHODS: We sought to investigate the recent findings on the current subject in order to provide a comprehensive overview of the issue using an extensive literature search to pinpoint the latest scientific reports on this subject. RESULTS: In the present review, the function of 5-Phosphodiesterase inhibitors as topical formulations was evaluated with details including formulation type, adsorption, and comparative efficacy in all recent studies as an acceptable alternative therapy to systemic drugs. CONCLUSIONS: Due to the fact that the influential factors in erectile dysfunction interact with many diseases and delinquent treatments, the use of topical therapeutic agents can be promising in mild to moderate cases. The utilization of 5-PDEIs through novel topical and transdermal drug delivery techniques plays a vital role in improving this effectiveness. Hamzehnejadi M, Tavakoli MR, Abiri A, et al. A Review on Phosphodiesterase-5 Inhibitors as a Topical Therapy for Erectile Dysfunction. Sex Med Rev 2022;10:369-384.

In silico analysis and in planta production of recombinant ccl21/IL1ß protein and characterization of its in vitro anti-tumor and immunogenic activity.

CCL21 has an essential role in anti-tumor immune activity. Epitopes of IL1ß have adjuvant activity without causing inflammatory responses. CCR7 and its ligands play a vital role in the immune balance; specifically, in transport of T lymphocytes and antigen-presenting cells such as dendritic cells to the lymph nodes. This study aimed to produce epitopes of CCL21 and IL1ß as a recombinant protein and characterize its in vitro anti-tumor and immunogenic activity. A codon-optimized ccl21/IL1ß gene was designed and synthesized from human genes. Stability and binding affinity of CCL21/IL1ß protein and CCR7 receptor were examined through in silico analyses. The construct was introduced into N. tabacum to produce this recombinant protein and the structure and function of CCL21/IL1ß were examined. Purified protein from transgenic leaves generated a strong signal in SDS PAGE and western blotting assays. FTIR measurement and MALDI-TOF/TOF mass spectrography showed that ccl21/IL-1ß was correctly expressed in tobacco plants. Potential activity of purified CCL21/IL1ß in stimulating the proliferation and migration of MCF7 cancer cell line was investigated using the wound healing method. The results demonstrated a decrease in survival rate and metastasization of cancer cells in the presence of CCL21/IL1ß, and IC50 of CCL21 on MCF7 cells was less than that of non-recombinant protein. Agarose assay on PBMCsCCR7+ showed that CCL21/IL1ß has biological activity and there is a distinguishable difference between chemokinetic (CCL21) and chemotactic (FBS) movements. Overall, the results suggest that CCL21/IL1ß could be considered an effective adjuvant in future in vivo and clinical tests.

Exploring the cancer-testis antigen BORIS to design a novel multi-epitope vaccine against breast cancer based on immunoinformatics approaches.

Recently, cancer immunotherapy has gained lots of attention to replace the current chemoradiation approaches and multi-epitope cancer vaccines are manifesting as the next generation of cancer immunotherapy. Therefore, in this study, we used multiple immunoinformatics approaches along with other computational approaches to design a novel multi-epitope vaccine against breast cancer. The most immunogenic regions of the BORIS cancer-testis antigen were selected according to the binding affinity to MHC-I and II molecules as well as containing multiple cytotoxic T lymphocyte (CTL) epitopes by multiple immunoinformatics servers. The selected regions were linked together by GPGPG linker. Also, a T helper epitope (PADRE) and the TLR-4/MD-2 agonist (L7/L12 ribosomal protein from mycobacterium) were incorporated by A(EAAAK)3A linker to form the final vaccine construct. Then, its physicochemical properties, cleavage sites, TAP transport efficiency, B cell epitopes, IFN-γ inducing epitopes and population coverage were predicted. The final vaccine construct was reverse translated, codon-optimized and inserted into pcDNA3.1 to form the DNA vaccine. The final vaccine construct was a stable, immunogenic and non-allergenic protein that contained numerous CTL epitopes, IFN-γ inducing epitopes and several linear and conformational B cell epitopes. Also, the final vaccine construct formed stable and significant interactions with TLR-4/MD-2 complex according to molecular docking and dynamics simulations. Moreover, its world population coverage for HLA-I and HLA-II were about 93% and 96%, respectively. Taking together, these preliminary results can be used as an appropriate platform for further experimental investigations. Communicated by Ramaswamy H. Sarma.

A review on function and side effects of systemic corticosteroids used in high-grade COVID-19 to prevent cytokine storms.

In December 2019, a cluster of pneumonia caused by a novel coronavirus (2019-nCoV), officially known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in Wuhan, Hubei province, China. Cytokine storm is an uncontrolled systemic inflammatory response resulting from the release of large amounts of pro-inflammatory cytokines and chemokines that occurs at phase 3 of viral infection. Such emergence led to the development of many clinical trials to discover efficient drugs and therapeutic protocols to fight with this single-stranded RNA virus. Corticosteroids suppress inflammation of the lungs during the cytokine storm, weaken immune responses, and inhibit the elimination of pathogen. For this reason, in COVID-19 corticosteroid therapy, systemic inhibition of inflammation is observed with a wide range of side effects. The present review discusses the effectiveness of the corticosteroid application in COVID-19 infection and the related side effects of these agents. In summary, a number of corticosteroids, including and especially methylprednisolone and dexamethasone, have demonstrated remarkable efficacy, particularly for COVID-19 patients who underwent mechanical ventilation.

Discovery of new TLR7 agonists by a combination of statistical learning-based QSAR, virtual screening, and molecular dynamics.

Search for new antiviral medications has surged in the past two years due to the COVID-19 crisis. Toll-like receptor 7 (TLR7) is among one of the most important TLR proteins of innate immunity that is responsible for broad antiviral response and immune system control. TLR7 agonists, as both vaccine adjuvants and immune response modulators, are among the top drug candidates for not only our contemporary viral pandemic but also other diseases. The agonists of TLR7 have been utilized as vaccine adjuvants and antiviral agents. In this study, we hybridized a statistical learning-based QSAR model with molecular docking and molecular dynamics simulation to extract new antiviral drugs by drug repurposing of the DrugBank database. First, we manually curated a dataset consisting of TLR7 agonists. The molecular descriptors of these compounds were extracted, and feature engineering was done to restrict the number of features to 45. We applied a statistically inspired modification of the partial least squares (SIMPLS) method to build our QSAR model. In the next stage, the DrugBank database was virtually screened structurally using molecular docking, and the top compounds for the guanosine binding site of TLR were identified. The result of molecular docking was again screened by the ligand-based approach of QSAR to eliminate compounds that do not display strong EC50 values by the previously trained model. We then subjected the final results to molecular dynamics simulation and compared our compounds with imiquimod (an FDA-approved TLR7 agonist) and compound 1 (the most active compound against TLR7 in vitro, EC50 = 0.2 nM). Our results evidently demonstrate that cephalosporins and nucleotide analogues (especially acyclic nucleotide analogues such as adefovir and cidofovir) are computationally potent agonists of TLR7. We finally reviewed some publications about cephalosporins that, just like pieces of a puzzle, completed our conclusion.

In silico and in vitro inhibitory potential of an organometallic Cu (II) complex on Leishmania major stages.

Leishmaniosis results in a serious complication, principally in the tropical and subtropical areas. Metalcored complexes, like meglumine antimoniate (MA) have proven antileishmanial activity. Similarly, in this research, we investigated the effects of Cu (II) dimethoxy bipyridine (CuDMOBP) against Leishmania major stages in silico and in vitro. Molecular docking analysis was carried out on the complex and a protozoan metacaspase. The complex's antipromastigote and its cytotoxicity towards macrophages were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method to calculate relative Inhibitory Concentration 50% (IC50), Cytotoxic Concentration 50% (CC50), and Selectivity Index (SI). Expression of TNF-α and IL-10 in intracellular amastigotes and induction of apoptosis was also investigated using quantitative real-time PCR. The complex interacted effectively with four amino acid residues including lysine (Lys171), histidine (His193), arginine (Arg44 and Arg243) of the targeted metacaspase. This indicates a potential affinity between the target macromolecule and the complex. MTT results showed significant in vitro inhibitory effects against promastigotes. Reduction in cellular expression of IL-10 and TNF-α was also significant, p<0.05 and p<0.005, respectively. CuDMOBP showed powerful in vitro anti-leishmanial activity and could be introduced as a new leishmanicidal candidate.

The art of suicidal molecular seduction for targeting drug resistance.

The development of drug resistance is one of the most significant challenges of the current century in the pharmaceutical industry. Superinfections, cancer chemoresistance, and resistance observed in many non-infectious diseases are nullifying the efforts and monetary supplies, put in the advent of new drug molecules. Millions of people die because of this drug resistance developed gradually through extensive use of the drugs. Inherently, some drugs are less prone to become ineffective by drug resistance than others. Covalent inhibitors bind to their targets via a biologically permanent bound with their cognate receptor and therefore display more potent inhibiting characteristics. Suicide inhibitors or mechanism-based inhibitors are one of the covalent inhibitors, which require a pre-activation step by their targeting enzyme. This step accrues their selectivity and specificity with respect to other covalent inhibitors. After that pre-activation step, they produce an analogue of the transition state of the catalytic enzyme, which is practically incapable of dissociating from the enzyme. Suicide inhibitors, due to their high intrinsic affinity toward the related enzyme, are resistant to many mechanisms involved in the development of drug resistance and can be regarded as one of the enemies of this scientific hurdle. These inhibitors compete even with monoclonal antibodies in terms of their cost-effectiveness and efficacy.

Exploring the out of sight antigens of SARS-CoV-2 to design a candidate multi-epitope vaccine by utilizing immunoinformatics approaches.

SARS-CoV-2 causes a severe respiratory disease called COVID-19. Currently, global health is facing its devastating outbreak. However, there is no vaccine available against this virus up to now. In this study, a novel multi-epitope vaccine against SARS-CoV-2 was designed to provoke both innate and adaptive immune responses. The immunodominant regions of six non-structural proteins (nsp7, nsp8, nsp9, nsp10, nsp12 and nsp14) of SARS-CoV-2 were selected by multiple immunoinformatic tools to provoke T cell immune response. Also, immunodominant fragment of the functional region of SARS-CoV-2 spike (400-510 residues) protein was selected for inducing neutralizing antibodies production. The selected regions' sequences were connected to each other by furin-sensitive linker (RVRR). Moreover, the functional region of ß-defensin as a well-known agonist for the TLR-4/MD complex was added at the N-terminus of the vaccine using (EAAAK)3 linker. Also, a CD4 + T-helper epitope, PADRE, was used at the C-terminal of the vaccine by GPGPG and A(EAAAK)2A linkers to form the final vaccine construct. The physicochemical properties, allergenicity, antigenicity, functionality and population coverage of the final vaccine construct were analyzed. The final vaccine construct was an immunogenic, non-allergen and unfunctional protein which contained multiple CD8 + and CD4 + overlapping epitopes, IFN-γ inducing epitopes, linear and conformational B cell epitopes. It could form stable and significant interactions with TLR-4/MD according to molecular docking and dynamics simulations. Global population coverage of the vaccine for HLA-I and II were estimated 96.2% and 97.1%, respectively. At last, the final vaccine construct was reverse translated to design the DNA vaccine. Although the designed vaccine exhibited high efficacy in silico, further experimental validation is necessary.

Where ferroptosis inhibitors and paraquat detoxification mechanisms intersect, exploring possible treatment strategies.

Paraquat (PQ) is a fast-acting and effective herbicide that is used throughout the world to eliminate weeds. Over the past years, PQ was considered one of the most popular poisoning substances for suicide, and PQ poisoning accounts for about one-third of suicides around the world. Poisoning with PQ may cause multiorgan failure, pulmonary fibrosis, and ultimately death. Exposure to PQ results in the accumulation of PQ in the lungs, causing severe damage and, eventually, fibrosis. Until now, no effective antidote has been found to treat poisoning with PQ. In general, the toxicity of PQ is due to the formation of high energy oxygen free radicals and the peroxidation of unsaturated lipids in the cell. Ferroptosis is the result of the loss of glutathione peroxidase 4 (GPX4) activity that transforms iron-dependent lipid hydroperoxides to lipid alcohols, which are inert in the biological environment. Impaired iron metabolism and lipid peroxidation are increasingly known as the driving agents of ferroptosis. The contribution of ferroptosis to the development of cell death during poisoning with PQ has not yet been addressed. There is growing evidence about the relationship between PQ poisoning and ferroptosis. This raises the possibility of using ferroptosis inhibitors for the treatment of PQ poisoning. In this hypothesis-driven review article, we elaborated how ferroptosis inhibitors might circumvent the toxicity induced by PQ and may be potentially useful for the treatment of PQ toxicity.

Targets and mechanisms of sulforaphane derivatives obtained from cruciferous plants with special focus on breast cancer - contradictory effects and future perspectives.

Breast cancer is the most common type of cancer among women. Therefore, discovery of new and effective drugs with fewer side effects is necessary to treat it. Sulforaphane (SFN) is an organosulfur compound obtained from cruciferous plants, such as broccoli and mustard, and it has the potential to treat breast cancer. Hence, it is vital to find out how SFN targets certain genes and cellular pathways in treating breast cancer. In this review, molecular targets and cellular pathways of SFN are described. Studies have shown SFN inhibits cell proliferation, causes apoptosis, stops cell cycle and has anti-oxidant activities. Increasing reactive oxygen species (ROS) produces oxidative stress, activates inflammatory transcription factors, and these result in inflammation leading to cancer. Increasing anti-oxidant potential of cells and discovering new targets to reduce ROS creation reduces oxidative stress and it eventually reduces cancer risks. In short, SFN effectively affects histone deacetylases involved in chromatin remodeling, gene expression, and Nrf2 anti-oxidant signaling. This review points to the potential of SFN to treat breast cancer as well as the importance of other new cruciferous compounds, derived from and isolated from mustard, to target Keap1 and Akt, two key regulators of cellular homeostasis.

Phenoxyethyl Piperidine/Morpholine Derivatives as PAS and CAS Inhibitors of Cholinesterases: Insights for Future Drug Design.

Acetylcholinesterase (AChE) catalyzes the conversion of Aß peptide to its aggregated form and the peripheral anionic site (PAS) of AChE is mainly involved in this phenomenon. Also catalytic active site (CAS) of donepezil stimulates the break-down of acetylcholine (ACh) and depletion of ACh in cholinergic synapses are well established in brains of patients with AD. In this study, a set of compounds bearing phenoxyethyl amines were synthesized and their inhibitory activity toward electric eel AChE (eeAChE) and equine butyrylcholinesterase (eqBuChE) were evaluated. Molecular dynamics (MD) was employed to record the binding interactions of best compounds against human cholinesterases (hAChE and hBuChE) as well as donepezil as reference drug. In vitro results revealed that compound 5c is capable of inhibiting eeAChE activity at IC50 of 0.50 µM while no inhibitory activity was found for eqBuChE for up to 100 µM concentrations. Compound 5c, also due to its facile synthesis, small structure and high selectivity for eeAChE would be very interesting candidate in forthcoming studies. The main interacting parts of compound 5c and compound 7c (most potent eeAChE and eqBuChE inhibitors respectively) with receptors which confer selectivity for AChE and BuChE inhibition were identified, discussed, and compared with donepezil's interactions. Also during MD simulation it was discovered for the first time that binding of substrates like donepezil to dual CAS and PAS or solely CAS region might have a suppressive impact on 4-α-helical bundles near the tryptophan amphiphilic tetramerization (WAT) domain of AChE and residues which are far away from AChE active site. The results proposed that residues involved in donepezil interactions (Trp86 and Phe295) which are located in CAS and mid-gorge are the mediator of conformational changes in whole protein structure.