Synthesis, molecular docking and biochemical analysis of aminoalkylated naphthalene-based chalcones as acetylcholinesterase inhibitors

Twelve novel chalcones were synthesized using 2-alkyloxy-naphthaldehydes and Mannich bases of 4-hydroxyacetophenone. The chalcones were characterized using FTIR, 1D and 2D NMR and HRMS spectroscopy. Comparative docking analysis was carried out to screen their affinity towards the AChE enzyme (PDB 1EVE). All chalcones showed lower binding energy (-13.06 to -10.43 kcal/mol) against AChE better than donepezil (-10.52 kcal/mol). All chalcones were potent inhibitors towards AChE, with IC50 values ranging between 0.11 and 5.34 nM better than donepezil (IC50 33.4 nM) and selectivity indexes (0.66–23.83), despite the fact that chalcones 10 and 13 were inactive. The structure activity relationship indicated that introducing diethyl amine in ring A of the chalcone skeleton and the propargyl moiety at ring B was a?rmed to be a prospective drug against AChE. The multifunctional properties of chalcone 15 were all advantages that demonstrate an excellent candidate for the development of an effective drug against AChE

2-Benzyloxynaphthalene aminoalkylated chalcone designed as acetylcholinesterase inhibitor: Structural characterisation, in vitro biological activity and molecular docking studies

The design of an acetylcholinesterase inhibitor with multifunctional properties became the perspective for the development of an effective drug against Alzheimer's disease. Towards this target, 1-{4-hydroxy-3-[(piperidin-1-yl)methyl]phenyl}ethan-1-one (chalcone 3) was prepared and studied as an acetylcholinesterase inhibitor. The novel chalcone 3 was synthesised via Claisen-Schmidt condensation reaction with 84% yield and characterized using 1D and 2D NMR spectroscopy. The in vitro bioactivity studies of chalcone 3 demonstrated excellent inhibitory activity against AChE (IC50 1.0 nM) showing 33-fold better inhibition than donepezil, biometal chelating ability and moderate antioxidant activity. Chalcone 3 with these fascinating multifunctional proprieties can be a good candidate for the development of AD treatments. A molecular modelling investigation revealed that chalcone 3 showed dual binding inhibition of AChE enzyme. XRD shows short intra- and inter-molecular interactions with two chalcone 3 molecules per cell. Interesting Hirshfeld Surface Analysis (HSA) was conducted showing explicit agreement with the XRD analysis

Current status and potential applications of underexplored prokaryotes

Thousands of prokaryotic genera have been published, but methodological bias in the study of prokaryotes is noted. Prokaryotes that are relatively easy to isolate have been well-studied from multiple aspects. Massive quantities of experimental findings and knowledge generated from the well-known prokaryotic strains are inundating scientific publications. However, researchers may neglect or pay little attention to the uncommon prokaryotes and hard-to-cultivate microorganisms. In this review, we provide a systematic update on the discovery of underexplored culturable and unculturable prokaryotes and discuss the insights accumulated from various research efforts. Examining these neglected prokaryotes may elucidate their novelties and functions and pave the way for their industrial applications. In addition, we hope that this review will prompt the scientific community to reconsider these untapped pragmatic resources

Novel thiophene Chalcones-Coumarin as acetylcholinesterase inhibitors: Design, synthesis, biological evaluation, molecular docking, ADMET prediction and molecular dynamics simulation

A series of around eight novel chalcone based coumarin derivatives (23a-h) was designed, subjected to in-silico ADMET prediction, synthesized, characterized by IR, NMR, Mass analytical techniques and evaluated as acetylcholinesterase (AChE) inhibitor for the treatment of Alzheimer's disease (AD). The results of predicted ADMET study demonstrated the drug-likeness properties of the titled compounds with developmental challenges in lipophilicity and solubility parameters. The in vitro assessment of the synthesized compounds revealed that all of them showed significant activity (IC50 ranging from 0.42 to 1.296 µM) towards AChE compared to the standard drug, galantamine (IC50 = 1.142 ± 0.027 µM). Among these, compound 23e displayed the most potent inhibitory activity with IC50 value of 0.42 ± 0.019 µM. Cytotoxicity of all compounds was tested on normal human hepatic (THLE-2) cell lines at three different concentrations using the MTT assay, in which none of the compound showed significant toxicity at the highest concentration of 1000 µg/ml compared to the control group. Based on the docking study against AChE, the most active derivative 23e was orientated towards the active site and occupied both catalytic anionic site (CAS) and peripheral anionic site (PAS) of the target enzyme. In-silico studies revealed tested showed better inhibition activity of AChE compared to Butyrylcholinesterase (BuChE). Molecular dynamics simulation explored the stability and dynamic behavior of 23e- AChE complex

Genetic analysis and molecular basis of G6PD deficiency among malaria patients in Thailand: implications for safe use of 8-aminoquinolines

Background: It was hypothesized that glucose-6-phosphate dehydrogenase (G6PD) deficiency confers a protective effect against malaria infection, however, safety concerns have been raised regarding haemolytic toxicity caused by radical cure with 8-aminoquinolines in G6PD-deficient individuals. Malaria elimination and control are also complicated by the high prevalence of G6PD deficiency in malaria-endemic areas. Hence, accurate identification of G6PD deficiency is required to identify those who are eligible for malaria treatment using 8-aminoquinolines. Methods: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency. Results: Based on phenotypic testing, the prevalence of G6PD deficiency (T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals. Conclusions: With varying degrees of enzyme deficiency, G6PD genotyping can be used as a complement to phenotypic screening to identify those who are eligible for 8-aminoquinolines. The information gained from this study could be useful for management and treatment of malaria, as well as for the prevention of unanticipated reactions to certain medications and foods in the studied population

Characterization of sodium channel mutations in the dengue vector mosquitoes Aedes aegypti and Aedes albopictus within the context of ongoing Wolbachia releases in Kuala Lumpur, Malaysia

Specific sodium channel gene mutations confer target site resistance to pyrethroid insecticides in mosquitoes and other insects. In Aedes mosquito species, multiple mutations that contribute to resistance vary in their importance around the world. Here, we characterize voltage sensitive sodium channel (Vssc) mutations in populations of Aedesaegypti from Kuala Lumpur, Malaysia, and look at their persistence in populations affected by ongoing Wolbachia releases (a dengue control measure). We also describe a Vssc mutation in Aedesalbopictus (F1534L) found for the first time in Malaysia. We show that there are three predominant Vssc haplotypes in Aedesaegypti in this region, which all persist with regular backcrossing, thereby maintaining the original genetic composition of the populations. We identify changes in genotype frequency in closed populations of Ae. aegypti maintained for multiple generations in laboratory culture, suggesting different fitness costs associated with the genotypes, some of which may be associated with the sex of the mosquito. Following population replacement of Ae. aegypti by Wolbachia in the target area, however, we find that the Vssc mutations have persisted at pre-release levels. Mosquitoes in two genotype classes demonstrate a type I pyrethroid resistance advantage over wildtype mosquitoes when exposed to 0.25% permethrin. This resistance advantage is even more pronounced with a type II pyrethroid, deltamethrin (0.03%). The results point to the importance of these mutations in pyrethroid resistance in mosquito populations and the need for regular backcrossing with male mosquitoes from the field to maintain similarity of genetic background and population integrity during Wolbachia releases

Drug discovery - yesterday and tomorrow: the common approaches in drug design and cancer

The process of drug discovery has undergone radical changes and development over years. Traditionally, the drugs were discovered by employing chemistry and pharmacology - based cautious approach. When natural products were the most important source of drugs or drug precursors, but the conventional randomized drug research phenomenon was no longer effective at that time due to many negatives of these approaches like: high expenses of discovering new drugs, time - consuming and reduced success guarantee. Thus, with the development of the era, the concept of “Rational Drug Design” has enabled drug target identification and validation to be more specific. In addition, several novel technologies and approaches have been introducing economics, proteomics and other omics areas such as 3D QSAR, pharmacophore modeling and other, which playing a promising role in accelerating the pace of drug discovery process. Their view of the current research focuses on the importance of drug discovery in modern times and shows how old methods have been replaced and summarized. Some of examples of molecules are identified in addition to computational approaches used to discover it, specifically in the field of anticancer drug design

Biochemical and cellular studies of PI3 kinase inhibition

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that catalyzes the biosynthesis of PI(3)P, PI(3,4)P₂ and PI(3,4,5)P₃ – second messengers that trigger a wide range of downstream signaling cascades involved in cell survival, growth, adhesion and proliferation. The class I PI3K proteins have been the subject of much study due to their association with disease. They are heterodimeric, composed of a regulatory subunit (p85) complexed with either one of four different isoforms of the catalytic subunit (p110α, p110β, p110γ and p110δ). Cancer has been a particular focus of regarding the roles of each isoform. The PI3KCA gene encoding the p110α-isoform has been found to be frequently mutated in cancers such as breast, prostate, colon, liver and brain. Thus, PI3K inhibitors showing selectivity towards the p110α isoform have been identified as targets for treatment of cancer. Other isoforms have also been identified as participating in the progression of certain cancers. This project is divided into two parts; the first part of the study is aimed at elucidating molecular mechanisms governing isoform selective inhibition of PI3K while in the second part of the thesis, the concept of using prodrugs as a means of targeting tissue selectivity is examined. There has been a growing number of class I PI3K inhibitors described to date with some showing selectivity to different PI3K isoforms. However the basis of selectivity of these inhibitors is still ambiguous. Previous studies have shown that specific regions within the catalytic subunit contain non-conserved residues which are involved in isoform selectivity. In order to rationally develop isoform selective inhibitors, the nature of the binding interactions between the enzyme and the inhibitors need to be understood. The approach taken in this thesis has been to use site-directed mutagenesis, to interrogate the role of key residues postulated to be responsible for the observed isoform selectivity of certain inhibitors. We have produced a series of in vitro mutants at position 770 and 859 (p110α-numbering) using two baculovirus expression systems. It was found that the Bac-to-bac baculovirus expression system provides an efficient method to produce in vitro PI3Kα mutant enzymes with enzyme activity comparable to the wild type enzyme. These mutant enzymes were assessed against PI3Kα selective inhibitor PIK-75, pan-PI3K inhibitor ZSTK-474 and some analogues in order to identify the role of the non-conserved residues, Arginine 770 and Glutamine 859, in determining inhibitor potency and selectivity. It was shown that changes to Arginine 770 and Glutamine 859 of p110α isoform did not influence isoform selectivity of the inhibitors. These results were used to compare some conflicting models of PIK-75 binding and were found to contradict some of the theoretical models and support one specific model proposed. Subsequent studies involving mutagenesis at other residues further confirmed this model. In Chapter 4, a similar examination of non-conserved residues in the binding of well characterised pan-PI3K inhibitor, GDC-0941 and an analogue compound 19 (CNIO-19) has been presented. Despite crystallographic evidence of isoform-specific binding interactions, these mutations had little effect on ligand binding. These results suggest that GDC-0941 can bind to PI3K isoforms with high flexibility and therefore that GDC-0941 non-selectivity is a function of redundancy in multiple possible binding orientations in different PI3K isoforms. In vitro mutagenesis using a panel of p110α mutant enzymes showed that mutation of the non-conserved residues did not affect GDC-0941 binding due to another mode of binding being utilised. CNIO-19 has an isosteric structure to GDC-0941 but an altered selectivity profile, with a markedly reduced potency for p110α. The comparison could provide a reference point for validating hypotheses relating to the binding mechanism. Again with this compound, the influence of mutations in p110α had only modest effects upon potency, albeit in the case of R770A and R777A the changes in potency were in opposite directions. It is likely that an extended series of isoform mutants and analogues would be required to adequately assess the role of binding site residues in dictating selectivity. The second part of this thesis presents a different aspect of this project which was the development of a study model to assess drug candidates for cancer therapeutics. The archetypal PI3K inhibitor, LY294002 and a pyridinyl analogue were modified by introduction of a tetrazole substituent at the 6-position resulting in two potent PI3Kα inhibitors, showing a 50-fold increase in potency relative to LY294002 itself. However neither compound showed activity in inhibiting MCF-7 breast cancer cell proliferation, which was proposed to be due to poor cellular permeability. Therefore a prodrug strategy was adopted to enhance cellular uptake whereby a pivaloyl ester group was added to the molecule. Both inhibitors possessed anti-proliferative activity against the MCF-7 cancer cell line when delivered using the prodrug strategy. The susceptibility of the compounds to a model esterase and the MCF-7 cell lysate were examined, which suggested that intracellular cleavage to the active compounds could occur but was possibly incomplete in the in vitro experiments. The results in total suggest this is a potentially promising avenue to pursue in the development of anti-cancer agents with reduced side effects. Overall, this thesis presents our current understanding of the biochemistry and pharmacology of several known PI3K inhibitors. We found that in vitro site-directed mutagenesis of PI3Kα enzymes is a powerful tool to assess the roles of non-conserved amino acids within the enzyme catalytic pocket especially in identifying selective inhibitor:enzyme interactions. The outcome from this study will be a facilitation of future design and development of novel isoform selective PI3K inhibitors based on these characteristics: 1) improved potency, 2) isoform selectivity, 3) cellular permeability and 4) enhanced anti-proliferative activity

Biochemical and cellular studies of PI3 kinase inhibition

Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that catalyzes the biosynthesis of PI(3)P, PI(3,4)P₂ and PI(3,4,5)P₃ – second messengers that trigger a wide range of downstream signaling cascades involved in cell survival, growth, adhesion and proliferation. The class I PI3K proteins have been the subject of much study due to their association with disease. They are heterodimeric, composed of a regulatory subunit (p85) complexed with either one of four different isoforms of the catalytic subunit (p110α, p110β, p110γ and p110δ). Cancer has been a particular focus of regarding the roles of each isoform. The PI3KCA gene encoding the p110α-isoform has been found to be frequently mutated in cancers such as breast, prostate, colon, liver and brain. Thus, PI3K inhibitors showing selectivity towards the p110α isoform have been identified as targets for treatment of cancer. Other isoforms have also been identified as participating in the progression of certain cancers. This project is divided into two parts; the first part of the study is aimed at elucidating molecular mechanisms governing isoform selective inhibition of PI3K while in the second part of the thesis, the concept of using prodrugs as a means of targeting tissue selectivity is examined. There has been a growing number of class I PI3K inhibitors described to date with some showing selectivity to different PI3K isoforms. However the basis of selectivity of these inhibitors is still ambiguous. Previous studies have shown that specific regions within the catalytic subunit contain non-conserved residues which are involved in isoform selectivity. In order to rationally develop isoform selective inhibitors, the nature of the binding interactions between the enzyme and the inhibitors need to be understood. The approach taken in this thesis has been to use site-directed mutagenesis, to interrogate the role of key residues postulated to be responsible for the observed isoform selectivity of certain inhibitors. We have produced a series of in vitro mutants at position 770 and 859 (p110α-numbering) using two baculovirus expression systems. It was found that the Bac-to-bac baculovirus expression system provides an efficient method to produce in vitro PI3Kα mutant enzymes with enzyme activity comparable to the wild type enzyme. These mutant enzymes were assessed against PI3Kα selective inhibitor PIK-75, pan-PI3K inhibitor ZSTK-474 and some analogues in order to identify the role of the non-conserved residues, Arginine 770 and Glutamine 859, in determining inhibitor potency and selectivity. It was shown that changes to Arginine 770 and Glutamine 859 of p110α isoform did not influence isoform selectivity of the inhibitors. These results were used to compare some conflicting models of PIK-75 binding and were found to contradict some of the theoretical models and support one specific model proposed. Subsequent studies involving mutagenesis at other residues further confirmed this model. In Chapter 4, a similar examination of non-conserved residues in the binding of well characterised pan-PI3K inhibitor, GDC-0941 and an analogue compound 19 (CNIO-19) has been presented. Despite crystallographic evidence of isoform-specific binding interactions, these mutations had little effect on ligand binding. These results suggest that GDC-0941 can bind to PI3K isoforms with high flexibility and therefore that GDC-0941 non-selectivity is a function of redundancy in multiple possible binding orientations in different PI3K isoforms. In vitro mutagenesis using a panel of p110α mutant enzymes showed that mutation of the non-conserved residues did not affect GDC-0941 binding due to another mode of binding being utilised. CNIO-19 has an isosteric structure to GDC-0941 but an altered selectivity profile, with a markedly reduced potency for p110α. The comparison could provide a reference point for validating hypotheses relating to the binding mechanism. Again with this compound, the influence of mutations in p110α had only modest effects upon potency, albeit in the case of R770A and R777A the changes in potency were in opposite directions. It is likely that an extended series of isoform mutants and analogues would be required to adequately assess the role of binding site residues in dictating selectivity. The second part of this thesis presents a different aspect of this project which was the development of a study model to assess drug candidates for cancer therapeutics. The archetypal PI3K inhibitor, LY294002 and a pyridinyl analogue were modified by introduction of a tetrazole substituent at the 6-position resulting in two potent PI3Kα inhibitors, showing a 50-fold increase in potency relative to LY294002 itself. However neither compound showed activity in inhibiting MCF-7 breast cancer cell proliferation, which was proposed to be due to poor cellular permeability. Therefore a prodrug strategy was adopted to enhance cellular uptake whereby a pivaloyl ester group was added to the molecule. Both inhibitors possessed anti-proliferative activity against the MCF-7 cancer cell line when delivered using the prodrug strategy. The susceptibility of the compounds to a model esterase and the MCF-7 cell lysate were examined, which suggested that intracellular cleavage to the active compounds could occur but was possibly incomplete in the in vitro experiments. The results in total suggest this is a potentially promising avenue to pursue in the development of anti-cancer agents with reduced side effects. Overall, this thesis presents our current understanding of the biochemistry and pharmacology of several known PI3K inhibitors. We found that in vitro site-directed mutagenesis of PI3Kα enzymes is a powerful tool to assess the roles of non-conserved amino acids within the enzyme catalytic pocket especially in identifying selective inhibitor:enzyme interactions. The outcome from this study will be a facilitation of future design and development of novel isoform selective PI3K inhibitors based on these characteristics: 1) improved potency, 2) isoform selectivity, 3) cellular permeability and 4) enhanced anti-proliferative activity