A novel cardenolide glycoside isolated from Xysmalobium undulatum reduces levels of the Alzheimer’s disease-associated β-amyloid peptides aβ42 in vitro

Elevated levels of the amylo β-proteins (Aβ), particularly Aβ42, are associated with a high risk of Alzheimer’s disease (AD). The Aβ proteins are produced from cellular processing of the amyloid precursor proteins (APPs). To identify natural products that block the formation of Aβ-proteins from APPs, we previously screened a library of plant extracts and identified Xysmalobium undulaum (Apocynaceae) as a potential plant for further research. Here, we provide a report on the isolation and identification of the active principles from the plant species using a bioassay-guided fractionation. Fractions and resulting pure compounds from the purification process of the extract of X. undulatum were screened in vitro against APPs transfected HeLa cell lines. Three compounds, acetylated glycosydated crotoxogenin (1), xysmalogenin-3, β-D-glucopyranoside (2), and crotoxigenin 3-Oglucopyranoside (3), were subsequently isolated and their structures elucidated using NMR and mass spectrometry. Compound 1, a novel cardenolide, and 2 significantly decreased the Aβ42 levels in a dose-dependent manner while compound 3 was inactive. In silico investigations identified the AD’s β-secretase enzyme, BACE1, as a potential target for these compounds with the glycoside moiety being of significance in binding to the enzyme active site. Our study provides the first report of a novel cardenolide and the potential of cardenolides as chemical scaffolds for developing AD treatment drugs.The University of Pretoria Post Graduate Research Support Bursary, South Africa; the Bio-Synergy Research Project; the Bio & Medical Technology Development Program of the Ministry of Science, ICT, and Future Planning through the National Research Foundation, Korea.http://www.mdpi.com/journal/pharmaceuticalspm2022BiochemistryChemistryGeneticsMicrobiology and Plant Patholog

A cytotoxic bis(1,2,3-triazol-5-ylidene)carbazolide gold(III) complex targets DNA by partial intercalation

The syntheses of bis(triazolium)carbazole precursors and their corresponding coinage metal (Au, Ag) complexes are reported. For alkylated triazolium salts, di- or tetranuclear complexes with bridging ligands were isolated, while the bis (aryl) analogue afforded a bis(carbene) AuI -CNC pincer complex suitable for oxidation to the redox-stable [AuIII(CNC) Cl]+ cation. Although the ligand salt and the [AuIII(CNC)Cl]+ complex were both notably cytotoxic toward the breast cancer cell line MDA-MB-231, the AuIII complex was somewhat more selective. Electrophoresis, viscometry, UV-vis, CD and LD spectroscopy suggest the cytotoxic [AuIII(CNC)Cl]+ complex behaves as a partial DNA intercalator. In silico screening indicated that the [AuIII(CNC)Cl]+ complex can target DNA three-way junctions with good specificity, several other regular B-DNA forms, and Z-DNA. Multiple hydrophobic πtype interactions involving T and A bases appear to be important for B-form DNA binding, while phosphate O···Au interactions evidently underpin Z-DNA binding. The CNC ligand effectively stabilizes the AuIII ion, preventing reduction in the presence of glutathione. Both the redox stability and DNA affinity of the hit compound might be key factors underpinning its cytotoxicity in vitro.https://chemistry-europe.onlinelibrary.wiley.com/journal/15213765pm2021ChemistryPhysiolog

The in silico and in vitro analysis of donepezil derivatives for Anopheles acetylcholinesterase inhibition

Current studies on Anopheles anticholinesterase insecticides are focusing on identifying agents with high selectivity towards Anopheles over mammalian targets. Acetylcholinesterase (AChE) from electric eel is often used as the bioequivalent enzyme to study ligands designed for activity and inhibition in human. In this study, previously identified derivatives of a potent AChE, donepezil, that have exhibited low activity on electric eel AChE were assessed for potential AChE-based larvicidal effects on four African malaria vectors; An. funestus, An. arabiensis, An. gambiae and An. coluzzii. This led to the identification of four larvicidal agents with a lead molecule, 1-benzyl-N-(thiazol-2-yl) piperidine-4-carboxamide 2 showing selectivity for An. arabiensis as a larvicidal AChE agent. Differential activities of this molecule on An. arabiensis and electric eel AChE targets were studied through molecular modelling. Homology modelling was used to generate a three-dimensional structure of the An. arabiensis AChE for this binding assay. The conformation of this molecule and corresponding interactions with the AChE catalytic site was markedly different between the two targets. Assessment of the differences between the AChE binding sites from electric eel, human and Anopheles revealed that the electric eel and human AChE proteins were very similar. In contrast, Anopheles AChE had a smaller cysteine residue in place of bulky phenylalanine group at the entrance to the catalytic site, and a smaller aspartic acid residue at the base of the active site gorge, in place of the bulky tyrosine residues. Results from this study suggest that this difference affects the ligand orientation and corresponding interactions at the catalytic site. The lead molecule 2 also formed more favourable interactions with An. arabiensis AChE model than other Anopheles AChE targets, possibly explaining the observed selectivity among other assessed Anopheles species. This study suggests that 1- benzyl-N-(thiazol-2-yl) piperidine-4-carboxamide 2 may be a lead compound for designing novel insecticides against Anopheles vectors with reduced toxic potential on humans.DATA AVAILABILITY STATEMENT : Amino acid sequence data are available UniProt Knowledge Base (Accession numbers: A0A182HKN4, A0A6E8V9T9, and A0A182RZ85). The 3D molecular structure data are available on Protein Data Bank (https:// www.rcsb.org/) with the ID numbers: 5YDI, 5YDH, 1EVE, and 4EY7.The Department of Science and Innovation (DSI)/National Research Foundation (NRF) Research Chairs Initiative Grant.http://www.plosone.orgdm2022Chemistr

Binding pose analysis of hydroxyethylamine based β-secretase inhibitors and application thereof to the design and synthesis of novel indeno[1,2-b]indole based inhibitors

β-Secretase (BACE1) is recognised as a target for the treatment of Alzheimer’s disease, and transition-state isosteres such as hydroxyethylamines have shown promise when incorporated into BACE1 inhibitors. A computational investigation of previously reported carbazole-based hydroxylethylamines with contradictory binding poses was undertaken using molecular dynamic simulations to rationalise the ligands preferred binding preference. Visual inspection of the confirmed binding pocket showed unoccupied space surrounding the carbazole moiety which was probed through the synthesis of seventeen ligands wherein the carbazole ring system was replaced with an indeno[1,2-b]indole ring system. The most active compound, rac-1- [benzyl(methyl)amino]-3-(indeno[1,2-b]indol-5(10H)-yl)propan-2-ol, indicated an inhibition of 91% at 10 µM against β-secretase with a cytotoxicity IC50 value of 10.51 ± 1.11 µM against the SH-SY5Y cell line.This work was supported by the National Research Foundation (NRF) of South Africa (Thuthuka grant number 106959), the University of Pretoria (Research and Development Program) and the Council for Scientific and Industrial Research (CSIR), South Africa.The National Research Foundation (NRF) of South Africa, the University of Pretoria (Research and Development Program) and the Council for Scientific and Industrial Research (CSIR), South Africa.http://www.arkat-usa.orgpm2021ChemistryPharmacologyPhysiolog

Nucleophilic substitution reactions of α-haloketones : a computational study

This dissertation describes the computational modelling of reactions between α-haloketones and various nucleophiles. Nucleophilic substitution reactions of α-haloketones (also known as α- halocarbonyls in literature) are utilised in synthetic laboratories to obtain 1,2-disconnections; which are typically difficult to obtain otherwise. To gain insight into these reactions, DFT modelling was carried out in this project, with further understanding into these reactions being obtained using Quantum Fragment Along Reaction Pathway (QFARP) which is an extension of Interacting Quantum Atoms (IQA). The nucleophilic substitution reaction was modelled between α-bromoacetophenone (α- BrAcPh), to represent α-haloketones, and the common nucleophiles phenolate (PhO–) and acetate (AcO–). QFARP provided insight into the reactions which could not have been obtained with other computational approaches. It was shown that the reaction with AcO– results in greater destabilisation for the α-group of α-BrAcPh as compared to the reaction of PhO–, explaining the difference in activation energies for the reactions. Diatomic- and fragment-interactions provided awareness into the driving force of the reactions and showed how the hydrogens for the α-group of α-BrAcPh provide significant attractive interactions with the nucleophiles during the initial stages of the nucleophilic substitution reaction. Furthermore, reactions modelled between α-BrAcPh and MeO– was done, as experimental literature has reported the presence of two competing reactions: nucleophilic substitution and epoxidation. Modelling showed the two reactions have low activation energies which are comparable with another. Interestingly, the rate determining step for the epoxidation reaction is not the formation of the transition state structure but rather the rotational barrier which is required to allow the leaving group, bromine, to be trans to the carbonyl-O of α-BrAcPh. Previous reports indicated that the presence of an electron donating/withdrawing group on the phenyl ring of α-BrAcPh had a significant influence on the reaction rate and selectivity between the two reactions. These experimental observations correlated well with the modelling results when comparing the potential energy surfaces (PES) of the reactions. Analysis using QFARP was also applied to these reactions to gain a more fundamental understanding of the reactions and how they differ. While QFARP was not able to explain the selectivity with different substituents present, insight into the dominating interactions that are involved in the reactions was recovered.Dissertation (MSc)--University of Pretoria, 2017.ChemistryMScUnrestricte

Combining in silico and in vitro approaches to discover novel inhibitors of Acetylcholinesterase

Alzheimer’s disease (AD) is the most common neurodegenerative disease which is a significant socio-economic problem. The number of patients affected by the disease is increasing at an alarming rate, largely due to expanding population sizes and longer life expectancy. While significant amounts of research into AD have been conducted the cause and pathogenesis of the disease are not well understood, with several hypotheses being noted in literature. To date, four drugs have been approved by the FDA, but these compounds only provide symptomatic relief. This study describes the uses of computer-aided drug discovery (CADD) techniques to identify novel inhibitors of Acetylcholinesterase (AChE), a target for AD. High throughput virtual screening (HTVS) was employed to predict potential inhibitors of AChE – an approach which, due to the associated difficulties of modelling the enzyme has to date not been reported to be successful in literature. Validation of enrichment was performed with the “Directory of Useful Decoys, enhanced” DUD-E dataset, showing that an ensemble of binding pocket conformations is critical when a diverse set of ligands are being screened. HTVS of a library of 20 000 compounds was performed. Cross-validation of the model was conducted by in vitro screening of 720 compounds, which led to 25 hits being identified with IC50 values of less than 50 μM. The majority of these hits belonged to two scaffolds: 1-ethyl-3-methoxy-3-methylpyrrolidine and 1H-pyrrolo[3,2-c]pyridin-6-amine, the latter being found through serendipity. Both scaffolds were noted to be promising compounds for further optimisation. Computational analysis of the active hits were performed to gain a deeper understanding of the binding pose to AChE. As various possible binding poses were suggested from molecular docking, molecular dynamic (MD) simulations were employed to validate the poses. In the case of the most active compounds identified, a critical, stable water bridge formed deep within the pocket. This, in part, explains the lack of activity for subsets of compounds that are not able to form this critical water bridge. The pKa analysis of AChE inhibitors showed a preference for pKa values higher than physiological pH leading to the ligands being cations and allowing the inhibitor to better mimic the substrate of AChE. Implications of using pKa as a guideline to improve potency and selectivity for AChE inhibitors are discussed. Further development of the docking protocol was performed with the use of a popular machine learning approach, Random Forest (RF). The approach is largely based on SIEVE-Score which takes the interaction energies between the ligand and residues in the pocket into account. By employing an ensemble of receptor conformations, significant enrichment over previous studies was obtained. Finally, additional secondary projects are reported which cover the computational analysis of compounds synthesised within the research group which inhibit either AChE or β-secretase (BACE1). Analysis of the interactions that inhibitors make with residues in the binding pocket allowed for an improved understanding of the system for future lead-optimisation studies.Thesis (PhD)--University of Pretoria, 2020.NRFChemistryPhDUnrestricte

A Cytotoxic Bis(1,2,3‐triazol‐5‐ylidene)carbazolide Gold(III) Complex Targets DNA by Partial Intercalation

The syntheses of bis(triazolium)carbazole precursors and their corresponding coinage metal (Au, Ag) complexes are reported. For alkylated triazolium salts, di- or tetranuclear complexes with bridging ligands were isolated, while the bis (aryl) analogue afforded a bis(carbene) AuI -CNC pincer complex suitable for oxidation to the redox-stable [AuIII(CNC) Cl]+ cation. Although the ligand salt and the [AuIII(CNC)Cl]+ complex were both notably cytotoxic toward the breast cancer cell line MDA-MB-231, the AuIII complex was somewhat more selective. Electrophoresis, viscometry, UV-vis, CD and LD spectroscopy suggest the cytotoxic [AuIII(CNC)Cl]+ complex behaves as a partial DNA intercalator. In silico screening indicated that the [AuIII(CNC)Cl]+ complex can target DNA three-way junctions with good specificity, several other regular B-DNA forms, and Z-DNA. Multiple hydrophobic πtype interactions involving T and A bases appear to be important for B-form DNA binding, while phosphate O···Au interactions evidently underpin Z-DNA binding. The CNC ligand effectively stabilizes the AuIII ion, preventing reduction in the presence of glutathione. Both the redox stability and DNA affinity of the hit compound might be key factors underpinning its cytotoxicity in vitro.https://chemistry-europe.onlinelibrary.wiley.com/journal/15213765pm2021ChemistryPhysiolog

Sustaining fermentation in high-gravity ethanol production by feeding yeast to a temperature-profiled multifeed simultaneous saccharification and co-fermentation of wheat straw

Background: Considerable progress is being made in ethanol production from lignocellulosic feedstocks by fermentation, but negative effects of inhibitors on fermenting microorganisms are still challenging. Feeding preadapted cells has shown positive effects by sustaining fermentation in high-gravity simultaneous saccharification and co-fermentation (SSCF). Loss of cell viability has been reported in several SSCF studies on different substrates and seems to be the main reason for the declining ethanol production toward the end of the process. Here, we investigate how the combination of yeast preadaptation and feeding, cell flocculation, and temperature reduction improves the cell viability in SSCF of steam pretreated wheat straw. Results: More than 50% cell viability was lost during the first 24 h of high-gravity SSCF. No beneficial effects of adding selected nutrients were observed in shake flask SSCF. Ethanol concentrations greater than 50 g L−1 led to significant loss of viability and prevented further fermentation in SSCF. The benefits of feeding preadapted yeast cells were marginal at later stages of SSCF. Yeast flocculation did not improve the viability but simplified cell harvest and improved the feasibility of the cell feeding strategy in demo scale. Cultivation at 30 °C instead of 35 °C increased cell survival significantly on solid media containing ethanol and inhibitors. Similarly, in multifeed SSCF, cells maintained the viability and fermentation capacity when the temperature was reduced from 35 to 30 °C during the process, but hydrolysis yields were compromised. By combining the yeast feeding and temperature change, an ethanol concentration of 65 g L−1, equivalent to 70% of the theoretical yield, was obtained in multifeed SSCF on pretreated wheat straw. In demo scale, the process with flocculating yeast and temperature profile resulted in 5% (w/w) ethanol, equivalent to 53% of the theoretical yield. Conclusions: Multifeed SSCF was further developed by means of a flocculating yeast and a temperature-reduction profile. Ethanol toxicity is intensified in the presence of lignocellulosic inhibitors at temperatures that are beneficial to hydrolysis in high-gravity SSCF. The counteracting effects of temperature on cell viability and hydrolysis call for more tolerant microorganisms, enzyme systems with lower temperature optimum, or full optimization of the multifeed strategy with temperature profile.Forestry, Faculty ofNon UBCReviewedFacult