Hashwin V. S. Ganesh

sym-Triazines for directed multitarget modulation of cholinesterases and amyloid-β in Alzheimer's disease.

Alzheimers disease (AD) is a complex neurodegenerative disorder marked by numerous causative factors of disease progression, termed pathologies. We report here the synthesis of a small library of novel sym-triazine compounds designed for targeted modulation of multiple pathologies related to AD, specifically human acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and Aβ aggregation. Rational targeting of AChE was achieved by the incorporation of acetylcholine substrate analogues into a sym-triazine core in either a mono-, di-, or trisubstituted regime. A subset of these derivatives demonstrated improved activity compared to several commercially available cholinesterase inhibitors. High AChE/BuChE selectivity was characteristic of all derivatives, and AChE steady-state kinetics indicated a mixed-type inhibition mechanism. Further integration of multiple hydrophobic phenyl units allowed for improved β-sheet intercalation into amyloid aggregates. Several highly effective structures exhibited fibril inhibition greater than the previously reported β-sheet-disrupting penta-peptide, iAβ5p, evaluated by thioflavin T fluorescence spectroscopy and transmission electron microscopy. Highly effective sym-triazines were shown to be well tolerated by differentiated human neuronal cells, as demonstrated by the absence of adverse effects on cellular viability at a wide range of concentrations. Parallel targeting of multiple pathologies using sym-triazines is presented here as an effective strategy to address the complex, multifactorial nature of AD progression.

Quantum Dot Based Fluorometric Detection of Cancer TF-Antigen

Cancer is a major global health challenge that would benefit from advances in screening methods for early detection that are rapid and low cost. TF-antigen is a tumor-associated antigen displayed on cell surface proteins of a high percentage of human carcinomas. Here we present a fluorometric bioassay for TF-antigen (galactose-β-(1→3)-N-acetyl-d-galactosamine) that utilizes quantum dot (QD) technology coupled with magnetic beads for rapid detection of TF-antigen at high sensitivity (10(-7) M range). In the competitive bioassay, 4-aminophenyl β-d-galactopyranoside (4-APG) conjugated to QDs competes with TF-antigen for binding sites on peanut agglutinin (PNA) that is immobilized on magnetic beads. The bioassay is specific and ultrasensitive in the environment of complex protein mixtures, demonstrating its potential applicability for the screening of clinical samples.

Biological activity of sym-triazines with acetylcholine-like substitutions as multitarget modulators of Alzheimer's disease.

The bioactivities of two novel compounds (TAE-1 and TAE-2) that contain a sym-triazine scaffold with acetylcholine-like substitutions are examined as promising candidate agents against Alzheimers disease. Inhibition of amyloid-β fibril formation in the presence of Aβ1-42, evaluated by Thioflavin T fluorescence, demonstrated comparable or improved activity to a previously reported pentapeptide-based fibrillogenesis inhibitor, iAβ5p. Destabilization of Aβ1-42 assemblies by TAE-1 and TAE-2 was confirmed by scanning electron microscopy imaging. sym-Triazine inhibition of acetylcholinesterase (AChE) activity was observed in cytosol extracted from differentiated human SH-SY5Y neuronal cells and also using human erythrocyte AChE. The sym-triazine derivatives were well tolerated by these cells and promoted beneficial effects on human neurons, upregulating expression of synaptophysin, a synaptic marker protein, and MAP2, a neuronal differentiation marker.

Epigallocatechin Gallate-Modified Graphite Paste Electrode for Simultaneous Detection of Redox-Active Biomolecules

In this study, simultaneous electrochemical detection of ascorbic acid (AA), dopamine (DA), and uric acid (UA) was performed using a modified graphite paste electrode (MGPE) with epigallocatechin gallate (EGCG) and green tea (GT) powder. It was shown that the anodic peak current increased in comparison with that of the graphite paste electrode (GPE) in the cyclic voltammograms. The optimal pH for simultaneous determination of a quaternary mixture of AA–DA–UA was determined to be pH 2. The anodic peak potentials for a mixture containing AA–DA–UA were well separated from each other. The catalytic peak currents obtained at the surface of the MGPE/EGCG were linearly dependent on the AA, DA, and UA concentrations up to 23, 14, and 14 µM, respectively. The detection limits for AA, DA, and UA were 190, 90, and 70 nM, respectively. The analytical performance of this sensor has been evaluated for simultaneous detection of AA, DA, and UA in real samples. Finally, a modified electrode was prepared using GT and used for simultaneous determination of AA, DA, and UA. Based on the results, MPGE/GT showed two oxidation peaks at 0.43 and 0.6 V for DA and UA, respectively, without any oxidation peak for AA. The calibration curves at the surface of MGPE/GT were linear up to 14 µM with a detection limit of 0.18 and 0.33 µM for DA and UA, respectively. MGPEs provide a promising platform for the future development of sensors for multiplexed electrochemical detection of clinically important analytes.

Carbon ceramic microelectrodes modified with buckyballs for simultaneous determination of redox-active biomolecules

In this report, simultaneous electrochemical determination of ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp) was achieved using buckyball-modified carbon ceramic microelectrodes (CCMEs). A concentration-dependent increase in anodic peak current signals was observed in comparison with those obtained at bare CCMEs. The optimal pH for simultaneous determination of a quaternary mixture of AA–DA–UA–Trp was determined to be pH 4. The peak separations for the mixture containing AA–DA–UA–Trp were well-defined at the scan rate of 50 mV s−1. The catalytic peak current obtained was linearly dependent on the AA, DA, UA and Trp concentrations in the range of 6.0–600, 6.0–600, 6.0–600 and 4.0–440 μM, respectively. The detection limits for AA, DA, UA and Trp were also determined to be 1.64, 0.82, 0.36 and 1.22 μM, respectively. The analytical performance of this sensor has also been challenged for simultaneous electrochemical detection of AA, DA, UA and Trp in real samples.

NOVEL METHODS TO UNDERSTAND AMYLOID-β AGGREGATION KINETICS IN THE PRESENCE OF SMALL MOLECULES AND METALS

Background:Parkinson’s disease (PD), like other neurological disorders, has been recognized to have a genetic component. Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene are recognized as genetic risk factors for familial PD, and may also represent causal factors in the more common sporadic form of the disease. The central role of LRRK2 in genetic and idiopathic PD has led to significant interest in further characterizing LRRK2 at both the genetic and protein levels. Methods: We developed an ultrasensitive digital immunoassay utilizing single molecule array (Simoa) technology for the purpose of measuring LRRK2 in human cerebrospinal fluid (CSF), peripheral blood mononuclear cells (PBMC) as well as rat and human brain lysate. Limit of detection (LOD) was determined from recombinant protein calibration curves. Sensitivity and dilutional linearity was performed using human CSF and brain lysate to evaluate assay specificity, and spike recovery of recombinant LRRK2 protein was evaluated for measurement accuracy in sample matrix. Cross reactivity was also tested between LRRK2 and LRRK1 recombinant proteins. Using this digital ELISA, LRRK2 levels in PBMC and brain lysates were determined and compared with the results from Singulex Erenna platform. Results:The Simoa assay measured recombinant LRRK2 with a LOD of 0.2pg/mL. Average dilutional linearity was 111% for normal human CSF spiked with LRRK2 recombinant protein. Endogenous LRRK2 parallelism averaged 113% for rat brain lysates. Analyte spike recovery for human CSF ranged from 80% to 116% with a mean of 96%. No cross reactivity was observed between LRRK2 and LRRK1 recombinant proteins. The measured LRRK2 levels correlated between this digital ELISA and Erenna assay for both PBMCs and rat brain lysates. Conclusions:The ultrasensitive digital Simoa assay has potential to accurately quantify LRRK2 in PBMC and brain lysates. Data needs to be replicated with a larger data set including samples from patients diagnosed PD.