Post-Translational Modifications in Tau and Their Roles in Alzheimer's Pathology.

Microtubule-Associated Protein Tau (also known as tau) has been shown to accumulate into paired helical filaments and neurofibrillary tangles, which are known hallmarks of Alzheimer's disease (AD) pathology. Decades of research have shown that tau protein undergoes extensive post-translational modifications (PTMs), which can alter the protein's structure, function, and dynamics and impact the various properties such as solubility, aggregation, localization, and homeostasis. There is a vast amount of information describing the impact and role of different PTMs in AD pathology and neuroprotection. However, the complex interplay between these PTMs remains elusive. Therefore, in this review, we aim to comprehend the key post-translational modifications occurring in tau and summarize potential connections to clarify their impact on the physiology and pathophysiology of tau. Further, we describe how different computational modeling methods have helped in understanding the impact of PTMs on the structure and functions of the tau protein. Finally, we highlight the tau PTM-related therapeutics strategies that are explored for the development of AD therapy.

Small Molecules N-Phenylbenzofuran-2-carboxamide and N-Phenylbenzo[b]thiophene-2-carboxamide Promote Beta-Amyloid (Aß42) Aggregation and Mitigate Neurotoxicity.

This study reports the unusual ability of small molecules N-phenylbenzofuran-2-carboxamide (7a) and N-phenylbenzo[b]thiophene-2-carboxamide (7b) to promote and accelerate Aß42 aggregation. In the in vitro aggregation kinetic assays, 7a was able to demonstrate rapid increases in Aß42 fibrillogenesis ranging from 1.5- to 4.7-fold when tested at 1, 5, 10, and 25 µM compared to Aß42-alone control. Similarly, compound 7b also exhibited 2.9- to 4.3-fold increases in Aß42 fibrillogenesis at the concentration range tested. Electron microscopy studies at 1, 5, 10, and 25 µM also demonstrate the ability of compounds 7a and 7b to promote and accelerate Aß42 aggregation with the formation of long, elongated fibril structures. Both 7a and 7b were not toxic to HT22 hippocampal neuronal cells and strikingly were able to prevent Aß42-induced cytotoxicity in HT22 hippocampal neuronal cells (cell viability ∼74%) compared to the Aß42-treated group (cell viability ∼20%). Fluorescence imaging studies using BioTracker 490 green, Hoeschst 33342, and the amyloid binding dye ProteoStat further demonstrate the ability of 7a and 7b to promote Aß42 fibrillogenesis and prevent Aß42-induced cytotoxicity to HT22 hippocampal neuronal cells. Computational modeling studies suggest that both 7a and 7b can interact with the Aß42 oligomer and pentamers and have the potential to modulate the self-assembly pathways. The 8-anilino-1-naphthalenesulfonic acid (ANS) dye binding assay also demonstrates the ability of 7a and 7b to expose the hydrophobic surface of Aß42 to the solvent surface that promotes self-assembly and rapid fibrillogenesis. These studies demonstrate the unique ability of small molecules 7a and 7b to alter the self-assembly and misfolding pathways of Aß42 by promoting the formation of nontoxic aggregates. These findings have direct implications in the discovery and development of novel small-molecule-based chemical and pharmacological tools to study the Aß42 aggregation mechanisms, and in the design of novel antiamyloid therapies to treat Alzheimer's disease.

Differential Effects of Endocannabinoids on Amyloid-Beta Aggregation and Toxicity.

The regulation and metabolism of the endocannabinoid system has received extensive attention for their potential neuroprotective effect in neurodegenerative diseases such as Alzheimer's disease (AD), which is characterized by amyloid ß (Aß) -induced cell toxicity, inflammation, and oxidative stress. Using in vitro techniques and two cell lines, the mouse hippocampus-derived HT22 cells and Chinese hamster ovary (CHO) cells expressing human cannabinoid receptor type 1 (CB1), we investigated the ability of endocannabinoids to inhibit Aß aggregation and protect cells against Aß toxicity. The present study provides evidence that endocannabinoids N-arachidonoyl ethanol amide (AEA), noladin and O-arachidonoyl ethanolamine (OAE) inhibit Aß42 aggregation. They were able to provide protection against Aß42 induced cytotoxicity via receptor-mediated and non-receptor-mediated mechanisms in CB1-CHO and HT22 cells, respectively. The aggregation kinetic experiments demonstrate the anti-Aß aggregation activity of some endocannabinoids (AEA, noladin). These data demonstrate the potential role and application of endocannabinoids in AD pathology and treatment.

Peptide-Modified Gemini Surfactants as Delivery System Building Blocks with Dual Functionalities for Glaucoma Treatment: Gene Carriers and Amyloid-Beta (Aß) Self-Aggregation Inhibitors.

Retinal ganglion cell (RGC) neurodegeneration in glaucoma has potential links with amyloid-ß (Aß) deposition. Targeting the Aß pathway was shown to reduce RGC apoptosis and protect RGCs from degeneration. We report exploratory studies on the amyloid Aß40 aggregation inhibition properties of four cell adhesion peptide (CAP)-gemini surfactants that are intended as building blocks for gene carrier nanoparticles for glaucoma treatment. The CAP-gemini surfactants (18-7N(p1-4)-18) were evaluated as potential Aß40 peptide aggregation inhibitors by a fluorescence kinetic assay and for their binding interactions with Aß40 dimers by molecular docking studies. In vitro Aß40 peptide aggregation inhibition studies showed that the 18-7N(p3)-18 and 18-7N(p1)-18 ligands inhibit Aß40 peptide aggregation and prevent the formation of higher order structures. CDOCKER energies and CDOCKER interaction energies demonstrated that the CAP-gemini surfactants formed more stable complexes in the Aß40 dimer assembly and underwent both polar and nonpolar interactions compared to CAP peptides alone. Also, 18-7N(p3)-18 showed a significantly lower CDOCKER energy compared to that of the unmodified gemini surfactant 18-7NH-18 (p < 0.0001) and 18-7N(p4)-18 (p < 0.001), 18-7N(p1)-18, and 18-7N(p2)-18. Similarly, 18-7N(p3)-18 showed a significantly lower CDOCKER interaction energy compared to that of 18-7NH-18, 18-7N(p4)-18 (p < 0.0001), and 18-7N(p2)-18 (p < 0.001), while 18-7N(p3)-18 and 18-7N(p1)-18 showed similar CDOCKER interaction energies. These studies suggest that a combination of both hydrophobic and electrostatic interactions contributes to the anti-Aß40 aggregation activity of CAP-gemini surfactants. CAP-gemini surfactants showed 10-fold better Aß40 peptide aggregation inhibition compared to previously reported values and could provide a new opportunity for glaucoma treatment as dual-functional gene carriers.

Apigenin directly interacts with and inhibits topoisomerase 1 to upregulate CD26/DPP4 on colorectal carcinoma cells.

Introduction: CD26/dipeptidyl peptidase IV (DPP4) is a cell-surface glycoprotein present on most epithelial cells that modulates the local response to external signals. We have previously shown that the dietary flavone apigenin (4',5,7-trihydroxyflavone) upregulates cell-surface CD26/DPP4 on human colorectal carcinoma (CRC) cells and regulates its activities. We observed a unique synergistic interaction with the CRC chemotherapeutic agent irinotecan, which through its metabolite SN38 elevates CD26 at doses that are sub-cytotoxic. As SN38 interacts with topoisomerase 1 (Topo1) we evaluated whether apigenin influences Topo1 activity. Methods: We used a radioimmunoassay to selectively measure CD26 at the cell surface of HT-29 cells following various treatments. Topoisomerase 1 mRNA expression was measured by q-RT-PCR and protein abundance by western blot analysis. Direct inhibition of topoisomerase activity was measured using an assay of DNA supercoil relaxation with recombinant human Topo1. The role of Topo1 in the effect of apigenin was shown both pharmacologically and by siRNA silencing of Topo1. Molecular docking analysis was done with SBD computational software using the CDOCKER algorithm. Results: The interplay between apigenin and irinotecan was not observed when apigenin was combined with other chemotherapeutic drugs including the topoisomerase 2 inhibitors doxorubicin or etoposide. There was no enhancement of irinotecan action if apigenin was replaced with its hydroxylated metabolite luteolin (3',4',5,7-tetrahydroxyflavone) or emodin (6-methyl-1,3,8-trihydroxyanthraquinone), which is an inhibitor of the principal kinase target of apigenin, casein kinase 2 (CK2). Apigenin did not alter Topo1 mRNA expression, but siRNA knockdown of functional Topo1 eliminated the effect of apigenin and itself increased CD26 levels. Apigenin inhibited Topo1 activity in intact HT-29 cells and showed comparable inhibition of purified recombinant human Topo1 enzyme activity to that of SN-38, the active metabolite of irinotecan. Apigenin fits into the complex of Topo1 with DNA to directly inhibit Topo1 enzyme activity. Discussion: We conclude that apigenin has a unique fit into the Topo1-DNA functional complex that leads to direct inhibition of Topo1 activity, and suggest that this is the basis for the exceptional interaction with the CRC drug irinotecan. A combined action of these two agents may therefore exert a role to limit local signals that facilitate tumour progression.

Strategies in the design and development of (TAR) DNA-binding protein 43 (TDP-43) binding ligands.

The human transactive responsive (TAR) DNA-binding protein 43 (TDP-43) is involved in a number of physiological processes in the body. Its primary function involves RNA regulation. The TDP-43 protein is also involved in many diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD) and even cancers. These TDP-43 mediated diseases are collectively called as TDP-43 proteinopathies. Intense research in the last decade has increased our understanding on TDP-43 structure and function in biology. The three-dimensional structures of TDP-43 domains such as N-terminal domain (NTD), RNA-recognition motif-1 (RRM1), RNA-recognition motif-2 (RRM2) and the C-terminal domain (CTD) or low-complexity domain (LCD) have been solved. These structures have yielded insights into novel binding sites and pockets at various TDP-43 domains, which can be targeted by designing a diverse library of ligands including small molecules, peptides and oligonucleotides as molecular tools to (i) study TDP-43 function, (ii) develop novel diagnostic agents and (iii) discover disease-modifying therapies to treat TDP-43 proteinopathies. This review provides a summary on recent progress in the development of TDP-43 binding ligands and uses the solved structures of various TDP-43 domains to investigate putative ligand binding regions that can be exploited to discover novel molecular probes to modulate TDP-43 structure and function.

Modeling the Inhibition Kinetics of Curcumin, Orange G, and Resveratrol with Amyloid-ß Peptide.

The ß-amyloid (Aß) protein aggregation into toxic forms is one of the major factors in the Alzheimer's disease (AD) pathology. Screening compound libraries as inhibitors of Aß-aggregation is a common strategy to discover novel molecules as potential therapeutics in AD. In this regard, thioflavin T (ThT)-based fluorescence spectroscopy is a widely used in vitro method to identify inhibitors of Aß aggregation. However, conventional data processing of the ThT assay experimental results generally provides only qualitative output and lacks inhibitor-specific quantitative data, which can offer a number of advantages such as identification of critical inhibitor-specific parameters required to design superior inhibitors and reduce the need to conduct extensive in vitro kinetic studies. Therefore, we carried out mathematical modeling based on logistic equation and power law (PL) model to correlate the experimental results obtained from the ThT-based Aß40 aggregation kinetics for small-molecule inhibitors curcumin, orange G, and resveratrol and quantitatively fit the data in a logistic equation. This approach provides important inhibitor-specific parameters such as lag time λ, inflection point τ, maximum slope v m, and apparent rate constant k app, which are particularly useful in comparing the effectiveness of potential Aß40 aggregation inhibitors and can be applied in drug discovery campaigns to compare and contrast Aß40 inhibition data for large compound libraries.

Drug Repurposing: Dipeptidyl Peptidase IV (DPP4) Inhibitors as Potential Agents to Treat SARS-CoV-2 (2019-nCoV) Infection.

The current outbreak of severe acute respiratory distress syndrome (SARS) or nCOVID-19 pandemic, caused by the coronavirus-2 (CoV-2), continues to wreak havoc globally. As novel vaccines are being discovered and developed, small molecule drugs still constitute a viable treatment option for SARS-CoV-2 infections due to their advantages such as superior patient compliance for oral therapies, reduced manufacturing costs and ease of large scale distribution due to better stability and storage profiles. Discovering new drugs for SARS-CoV-2 infections is a time consuming and expensive proposition. In this regard, drug repurposing is an appealing approach which can provide rapid access to therapeutics with proven record of safety and efficacy. We investigated the drug repurposing potential of a library of dipeptidyl peptidase 4 (DPP4) inhibitors which are currently marketed for type-2 diabetes as treatment option for SARS-CoV-2 infections. These computational studies led to the identification of three marketed DPP4 inhibitors; gemigliptin, linagliptin and evogliptin as potential inhibitors of SARS-CoV-2 Mpro viral cysteine protease. In addition, our computational modeling shows that these drugs have the potential to inhibit other viral cysteine proteases from the beta coronavirus family, including the SAR-CoV Mpro and MERS-CoV CLpro suggesting their potential to be repurposed as broad-spectrum antiviral agents.

Therapeutic opportunities in cancer therapy: targeting the p53-MDM2/MDMX interactions.

Ubiquitination is a key enzymatic post-translational modification that influences p53 stability and function. p53 protein regulates the expression of MDM2 (mouse double-minute 2 protein) E3 ligase and MDMX (double-minute 4 protein), through proteasome-based degradation. Exploration of targeting the ubiquitination pathway offers a potentially promising strategy for precision therapy in a variety of cancers. The p53-MDM2-MDMX pathway provides multiple molecular targets for small molecule screening as potential therapies for wild-type p53. As a result of its effect on molecular carcinogenesis, a personalized therapeutic approach based on the wild-type and mutant p53 protein is desirable. We highlighted the implications of p53 mutations in cancer, p53 ubiquitination mechanistic details, targeting p53-MDM2/MDMX interactions, significant discoveries related to MDM2 inhibitor drug development, MDM2 and MDMX dual target inhibitors, and clinical trials with p53-MDM2/MDMX-targeted drugs. We also investigated potential therapeutic repurposing of selective estrogen receptor modulators (SERMs) in targeting p53-MDM2/MDMX interactions. Molecular docking studies of SERMs were performed utilizing the solved structures of the p53/MDM2/MDMX proteins. These studies identified ormeloxifene as a potential dual inhibitor of p53/MDM2/MDMX interaction, suggesting that repurposing SERMs for dual targeting of p53/MDM2 and p53/MDMX interactions is an attractive strategy for targeting wild-type p53 tumors and warrants further preclinical research.

Histone Demethylase KDM5B as a Therapeutic Target for Cancer Therapy.

Lysine-specific demethylase 5B (KDM5B/PLU1/JARID1B) is found to be overexpressed in numerous malignancies, including breast, lung, skin, liver, and prostate cancer. Identification of molecules targeting the KDM5B enzyme could be a potential lead in cancer research. Although many KDM5B inhibitors with promising outcomes have been developed so far, its further application in clinical practice is limited due to toxicity and lack of target specificity. Here, we summarize the significance of targeting KDM5B in anticancer therapy and report the molecular docking studies of some known anti-viral agents, decitabine, entecavir, abacavir, penciclovir, and 3-deazaneplanocin A in the catalytic domain JmjC of KDM5B. These studies show the repurposing potential of identified anti-viral agents in cancer therapy.

PANX1 in inflammation heats up: New mechanistic insights with implications for injury and infection.

A new study by Yang and colleagues has revealed that TNF-alpha regulates PANX1 levels through an NF-kB-dependent mechanism in human endothelial cells. PANX1 modulates Ca2+ influx contributing to IL-1beta production independent of purinergic signaling. These novel findings expand our understanding of TNF-alpha-mediated upregulation of IL-1beta with implications for responses to tissue injury and infection.

Corrigendum to "Role of UHRF1 in malignancy and its function as a therapeutic target for molecular docking towards the SRA domain" [Int. J. Biochem. Cell Biol. 114 (September 2019) 105558].

Cancer pathogenesis has been attributed to the minor and major disruptions in the cell cycle, with a key role being played by several of the recently discovered epigenetic factors. Lately, UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1), an epigenetic regulator has been shown to be evidently over expressed in numerous malignancies through an in-depth review of literature. Molecular docking studies have found that existing drugs such as propranolol, naphthazarin and thymoquinone have favourable interactions with specific domains of UHRF1. However, these findings would need large scale clinical trials to confirm their potency and safety during chemotherapy. UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1), an epigenetic factor, plays a crucial role as an important checkpoint in the cell machinery. Basic science continues to unravel multiple facets of this five domain protein which includes a detailed elucidation of its roles and mechanisms of interaction with various enzymes during DNA replication. The gene has recently begun to be also termed as the "Universal Oncogene" in response to the results of research conducted in heterogenous populations and in over 17 cancers displaying heightened mRNA and protein expression in breast, liver, lung, head and neck cancers and many more. This gene could therefore, be a potential biomarker for diagnosis and for the prediction of the prognosis and survival of the diseased. A scientifically established solution in the form of targeted treatment must follow such a discovery and therefore, several natural and synthetic compounds such as thymoquinone and the well-known antihypertensive, propranolol have been docked and reported to have favourable interactions with the SRA (Set and Ring Associated) domain of UHRF1 in this review. This comprehensive review is thus, a brief synopsis of details regarding the structure and heightened levels of UHRF1 in several malignancies. Furthermore, pharmacogenomic research revolving around this oncogene is a potential sphere for clinical studies to be conducted in much larger and heterogenous populations to not only validate these therapeutic docking results but to also to bring personalised medicine to the bedside for the benefit of the patients.

Role of UHRF1 in malignancy and its function as a therapeutic target for molecular docking towards the SRA domain.

The figure describes the location of UHRF1 (Ubiquitin-like with containing PHD and RING Finger domains 1) gene, mRNA and protein synthesis in the tumor cell and its structural domains with a focus on the docking of Naphthazarin on the SRA domain of UHRF1, resulting in reduction of tumor size.

Repurposing nitrocatechols: 5-Nitro-α-cyanocarboxamide derivatives of caffeic acid and caffeic acid phenethyl ester effectively inhibit aggregation of tau-derived hexapeptide AcPHF6.

Polyphenols like caffeic acid and its phenethyl ester have been associated with potent anti-aggregating activity. Accordingly, we screened a library of polyphenols and synthetic derivatives thereof for their capacity to inhibit tau-aggregation using a thioflavin T-based fluorescence method. Our results show that the nitrocatechol scaffold is required for a significant anti-aggregating activity, which is enhanced by introducing bulky substituents at the side chain. A remarkable increase in activity was observed for α-cyanocarboxamide derivatives 26-27. Molecular docking studies showed that the amide bond provides superior conformational stability in the steric zipper assembly of tau, which drives the increase in activity. We also found that derivatives 24-27 were potent chelators of copper(II) - a property of pharmacological significance in abnormal protein aggregation. These small molecules can provide promising leads to develop new drugs for tauopathies and AD. These findings open a new window on the repurposing of nitrocatechols beyond their established role as catechol-O-methyltransferase inhibitors.

Interactions of Selective Serotonin Reuptake Inhibitors with ß-Amyloid.

Treating Alzheimer's disease (AD) is a major challenge at the moment with no new drugs available to cure this devastating neurodegenerative disorder. In this regard, drug repurposing, which aims to determine novel therapeutic usage for drugs already approved by the regulatory agencies, is a pragmatic approach to discover novel treatment strategies. Selective serotonin reuptake inhibitors (SSRIs) are a known class of United States Food and Drug Administration approved drugs used in the treatment of depression. We investigated the ability of SSRIs fluvoxamine, fluoxetine, paroxetine, sertraline, and escitalopram on Aß42 aggregation and fibrillogenesis. Remarkably, the aggregation kinetic experiments carried out demonstrate the anti-Aß42 aggregation activity of SSRIs fluoxetine, paroxetine, and sertraline at all the tested concentrations (1, 10, 50, and 100 µM). Both fluoxetine and paroxetine were identified as the most promising SSRIs, showing 74.8 and 76% inhibition of Aß42 aggregation at 100 µM. The transmission electron microscopy experiments and dot-blot study also demonstrate the ability of fluoxetine and paroxetine to prevent Aß42 aggregation and fibrillogenesis, providing further evidence. Investigating the binding interactions of fluoxetine and paroxetine in the Aß42 oligomer and fibril models derived from the solid-state NMR structure suggests that these SSRIs interact at a region close to the N-terminal (Lys16-Glu22) in the S-shaped cross-ß-strand assembly and reduce Aß42 fibrillogenesis. On the basis of this study, a pharmacophore model is proposed which shows that the minimum structural requirements to design novel Aß42 aggregation inhibitors include the presence of one ionizable group, one hydrophobic group, two aromatic rings, and two hydrogen bond donor groups. These studies demonstrate that SSRIs have the potential to prevent Aß42 aggregation by direct binding and could be beneficial to AD patients on SSRIs.

Interactions of polyunsaturated fatty acids with amyloid peptides Aß40 and Aß42.

Polyunsaturated fatty acids (PUFAs) are reported to exert beneficial effects in Alzheimer's disease. Some PUFAs are known to reduce amyloid-beta (Aß) toxicity by promoting its degradation and clearance. Studies on the direct interactions of PUFAs with Aß peptides are limited and contradictory. In this study, we report the interactions of fatty acids docosahexaenoic acid (DHA), eicosatetraenoic acid (EPA), α-linolenic acid (ALA), arachidonic acid (ARA), linoleic acid (LNA) and oleic acid (OA) with Aß peptides by carrying out fluorescence based aggregation kinetic experiments, transmission electron microscopy and molecular docking studies. Our investigations demonstrate that all the fatty acids tested exhibit anti-aggregation properties by preventing both Aß40 and Aß42 fibrillogenesis (∼16-84% inhibition). OA and DHA were identified as excellent inhibitors of Aß40 or Aß42 fibrillogenesis respectively (∼84% and 81% inhibition at 25 µM). Molecular docking studies conducted, using the dimer and oligomer models of Aß40 peptide, suggest that these fatty acids interact in the aggregation prone Phe19-Ala21 and the ß-turn region (Asp23-Lys28) whereas a similar study with Aß42 dimer and oligomer models, indicate that the fatty acids were oriented in a hydrophobic region (Gln15, Leu16, Leu17 and Leu34). These results, suggest that DHA, EPA, ALA, ARA, LNA and OA are capable of directly interacting with both Aß40 and Aß42 peptides. These studies will have implications in developing potential therapeutics for Alzheimer's disease.

Tau Derived Hexapeptide AcPHF6 Promotes Beta-Amyloid (Aß) Fibrillogenesis.

We studied the interactions of a tau derived hexapeptide AcPHF6 with ß-amyloid peptides Aß40 and Aß42 which reveals its unusual ability to promote Aß fibrillogenesis. The results demonstrate that the N-acetylated and C-amidated AcPHF6 hexapeptide can cause significant acceleration in Aß40 and Aß42 fibril growth. Aggregation kinetic studies at pH 7.4 show that at 25 µM, AcPHF6 hexapeptide was able to cause ∼2.3-fold increase in Aß40 fibrillogenesis dramatically changing the aggregation kinetics. In addition, AcPHF6 peptide was able to reduce cellular toxicity mediated by Aß40 and Aß42 in hippocampal neuronal cell line (HT22). Computational studies suggest that the AcPHF6 peptide can act as an anchor and provides a hydrophobic surface for Aß monomer to bind and undergo rapid fibrillogenesis to form less toxic fibrils and alter the aggregation kinetics. At the molecular level we propose a "dock-and-pack" mechanism where the AcPHF6 hexapeptide aggregates can stabilize the ß-hairpin and promote rapid Aß self-assembly and growth to form less toxic oligomers or fibrils. Our results have direct implications in designing novel peptide/peptidomimetics as novel pharmacological tools to study protein aggregation and potentially prevent Aß-mediated toxicity in Alzheimer's disease.

Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity.

The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer's disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC50=33.0µM; 2, IC50=8.5µM) compared to tacrine (CYP1A2 IC50=1.5µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species.

Droplet Microfluidic System with On-Demand Trapping and Releasing of Droplet for Drug Screening Applications.

96-Well plate has been the traditional method used for screening drug compounds libraries for potential bioactivity. Although this method has been proven successful in testing dose-response analysis, the microliter consumption of expensive reagents and hours of reaction and analysis time call for innovative methods for improvements. This work demonstrates a droplet microfluidic platform that has the potential to significantly reduce the reagent consumption and shorten the reaction and analysis time by utilizing nanoliter-sized droplets as a replacement of wells. This platform is evaluated by applying it to screen drug compounds that inhibit the tau-peptide aggregation, a phenomena related to Alzheimer's disease. In this platform, sample reagents are first dispersed into nanolitre-sized droplets by an immiscible carrier oil and then these droplets are trapped on-demand in the downstream of the microfluidic device. The relative decrease in fluorescence through drug inhibition is characterized using an inverted epifluorescence microscope. Finally, the trapped droplets are released on-demand after each test by manipulating the applied pressures to the channel network which allows continuous processing. The testing results agree well with that obtained from 96-well plates with much lower sample consumption (∼200 times lower than 96-well plate) and reduced reaction time due to increased surface volume ratio (2.5 min vs 2 h).

2,4-Disubstituted quinazolines as amyloid-ß aggregation inhibitors with dual cholinesterase inhibition and antioxidant properties: Development and structure-activity relationship (SAR) studies.

A library of fifty-seven 2,4-disubstituted quinazoline derivatives were designed, synthesized and evaluated as a novel class of multi-targeting agents to treat Alzheimer's disease (AD). The biological assay results demonstrate the ability of several quinazoline derivatives to inhibit both acetyl and butyrylcholinesterase (AChE and BuChE) enzymes (IC50 range = 1.6-30.5 µM), prevent beta-amyloid (Aß) aggregation (IC50 range 270 nM-16.7 µM) and exhibit antioxidant properties (34-63.4% inhibition at 50 µM). Compound 9 (N2-(1-benzylpiperidin-4-yl)-N4-(3,4-dimethoxybenzyl)quinazoline-2,4-diamine) was identified as a dual inhibitor of cholinesterases (AChE IC50 = 2.1 µM; BuChE IC50 = 8.3 µM) and exhibited good inhibition of Aß aggregation (Aß40 IC50 = 2.3 µM). Compound 15b (4-(benzylamino)quinazolin-2-ol) was the most potent Aß aggregation inhibitor (Aß40 IC50 = 270 nM) and was ∼4 and 1.4-fold more potent compared to the reference agents curcumin and resveratrol. These comprehensive structure activity-relationship (SAR) studies demonstrate the application of a 2,4-disubstituted quinazoline ring as a suitable template to develop multi-targeting agents to treat AD.