RESEARCH

SINGLE AMINO ACID/METABOLITE SELF-ASSEMBLIES AND ITS IMPLICATIONS IN ETIOLOGY OF METABOLIC DISORDERS AND THEIR ASSOCIATION WITH AMYLOID

There are many diseases that are caused by the accumulation of single amino acids and other metabolites since the enzyme which cause the breakdown of these metabolites are dysfunctional due to specific gene mutations. Such diseases are termed “Inborn errors of metabolisms (IEMs)”. Our aim is to understand the molecular mechanism of these IEMs and their association with amyloid diseases in greater detail. Hence, we are interested to study the self-assembly of amino acids and other metabolites extensively to decipher the nature of these aggregates and investigate their amyloidogenic properties. In this direction, our research team for the very first time reported the self-assembly of non-aromatic amino acids Cysteine (Cys) and Methoinine (Met) to amyloid-like structures (ACS Chemical Neuroscience, 2018, 10, 1230-1239). Incidentally, this research was also the first report wherein the self-assembling properties of non-aromatic amino acids to amyloid-like aggregates were demonstrated. The Cys and Met assemblies were characterized in detail by various microscopy and spectroscopy techniques. The nature of the interactions which lead to the self-assembly of Cys and Met were also studied by various solid-state characterization techniques like ATR-FTIR, solid-state NMR, XRD and TGA analysis. The amyloid nature of assemblies was assessed by co-incubating Cys and Met structures with amyloid binding dyes such as ThT and CR. Cytotoxicity assays on human neuroblastoma, IMR-32 and COS-7 kidney cells suggested that both Cys and Met structures were cytotoxic. These results hence indicate that the pathogenesis of diseases like Cystinuria and Hypermethionemia might be related to the toxic structure formation by Cys and Met and suggests a common etiology between amyloid-associated diseases and IEMs. Further, our group also reported unusual aggregates formed by the self-assembly of Proline (Pro), Hydroxyproline (Hyp) and Lysine (Lys) by various microscopic and spectroscopic techniques and also illustrated their cytotoxic nature through MTT assays (ACS Chemical Neuroscience, 2021, 12, 3237-49). We are also currently pursuing research on the self-assembly of branched-chain amino acids (BCAA) and assessing its implication in Maple syrup urine disease (MSUD) in collaborative work with Prof. Ehud Gazit (Tel Aviv University), In future, we want to extend our studies to other non-aromatic single amino acids like Serine, Threonine, Glutamine, Histidine since our preliminary investigation suggest these amino acids also do have a propensity to self assemble. Further, our group is also interested in studying the self-assembling behavior of metabolites formed in various catabolic or anabolic pathways. In this direction our group is currently pursuing research on metabolites formed in catabolic pathways such as the Ornithine cycle and Krebs cycle.
Further, we also want to investigate the association of single amino acid/metabolite assembly with amyloid-related diseases like Alzheimer’s and Parkinson’s. For these studies, we will pursue cross-seeding experiments of single amino acid assemblies with full- length Aβ42 and its reductionist model diphenylalanine (FF). In this context, we recently reported a chemical perspective on the mechanism of action of anti-amyloid compounds by assessing their interaction with FF (ACS Chemical Neuroscience, 2021, 12, 2851-64). In the future, we want to investigate if the metabolite assemblies have prognostic or antagonistic action. We also want to assess if common drugs can be repurposed for the treatment of both amyloid-related diseases like Alzheimer’s or Parkinson’s and IEMs like Phenylketonria, Cystinuria and MSUD to name a few. We are currently also investigating the effect of the interaction of D-amino acids with L-amino acids with the aim to use D-amino acids as therapeutic remedies.

CONTROLLED SELF-ASSEMBLY OF MODIFIED SINGLE AMINO ACIDS

There is ever increasing demand to find new scaffold for the design of novel micro/nanoarchitectures which can be of potential interest for diverse applications in biology and material science. In this direction, we are interested in assessing the self-assembly of modified single amino acids under controlled conditions. The project aims to find a very simple and facile route for the synthesis of novel scaffolds for self-assembly. In this context, our team reported self-assembly of modified single amino acids i.e. charged aliphatic and aromatic amino acids which are functionalized with different protecting groups such as 9-fluorenylmethoxycarbonyl (Fmoc), tert-Butyloxycarbonyl (Boc), N-carboxybenzyl (Cbz) and tert-Butyl (tBu) to distinct structures (New Journal of Chemistry 2022, DOI: 10.1039/D1NJ05172E). Further, we also studied self-assembled structures formed by Fmoc modified L-amino acids namely Alanine, Leucine, Isoleucine, Valine, Proline, and Glycine to distinct morphologies under controlled conditions of concentration and temperature. Surprisingly, the self-assembled structures formed by different Fmoc amino acids were very unique, and distinct morphologies like different shapes of flowers, fibers, and rod-like structures could be assessed (ChemRxiv Preprint DOI:10.33774/chemrxiv-2021-2930x; Paper under review).Similarly we have studied controlled structural changes in the self-assemblies of N-(9-Fluorenylmethoxycarbonyl)-O-tert-butyl-L-threonine (Fmoc-Thr(tbu)-OH) (FTU) to well defined unique morphologies and explained the structure formation through computational modelling (ChemRxiv Preprint DOI:10.26434/chemrxiv.14255477; under review). In the future, we want to study different single amino acids modified with protecting groups other than -Fmoc like –Boc, -Benzyl, -Trityl and propose the mechanism of structure formation in detail through various kinetic and thermodynamic studies. We could also observe hollow spheres formed by modified amino acids under specific conditions in the studies done so far. We want to study the morphologies of these spheres in detail to determine their hollow nature and investigate their potential as drug delivery tools in the future.

ASSESSING AGGREGATION PROPERTIES OF HETEROCYCLIC COMPOUNDS FOR THEIR APPLICATION AS A DYE AND IN SENSING

The main aim of this project is to synthesize heterocyclic compounds and assess its self-assembling properties and investigate the effect of self-assembly on the photo-physical characteristics of compounds and their subsequent applications as dyes and sensors. Our studies suggest there is a direct correlation between aggregation properties and the photo-physical characteristics of compounds. Some dyes reveal enhanced fluorescence on the aggregation as in aggregation-induced emission dyes while some others reveal better fluorescence on disaggregation. Hence, we want to pursue future studies on finding the molecular mechanism behind the photo-physical properties of the dye from its aggregation perspective. In this context, we reported the self-assembly and photo-physical properties of a new pyridothiazole based aggregation-induced-emission enhancement (AIEE) luminogen 4-(5-methoxy-thiazolo[4,5-b]pyridin-2-yl)benzoic acid (PTC1) and its application for the sensitive detection and monitoring of amyloid fibrillation (ACS Appl. Bio Mater. 2019, 2, 4442–4455). Further, we also reported the self-assembly of an acyl-thiourea based sensor, N-{(6-methoxy-pyridine-2-yl) carbamothioyl}benzamide (NG1), to panchromatic fluorescent fibers and its dual-sensing properties for the sequential detection of Cu2+ ions and lactic acid (Soft Matter, 2021, 17, 4304-4316). In another work, we reported the synthesis and characterization of the structures formed by self-assembly of 4-chloro-2(3H)-benzothiazolone (CBT) into panchromatic fibers and their application in cellular imaging (New J. Chem. 2021, 45, 17211-21). In a very interesting research work which is under review, we have studied crystal structure and self-assembly of 4-(5-methoxythiazolo[4,5-b]pyridin-2-yl)-N,N-dimethylaniline (TPA) to fluorescent panchromatic lotus flower-like architectures and its implications in sensing dichromate ions(ChemRxiv Preprint DOI:10.26434/chemrxiv.14114210). Besides, we have also designed isothiazoloanthrone based 7-Amino-6h-anthra[9,1-cd][1,2]thiazol-6-one (AAT) Excited-State Intramolecular Proton Transfer (ESIPT) dye which reveal color changing properties and can be potentially used for the differential detection of normal and cancer cell through its anticancer activity (ChemRxiv Preprint DOI:10.33774/chemrxiv-2021-2930x; under review).

In our future research endeavours we want to design indole-based conjugates as combinatorial therapeutic tools for the treatment of breast cancer. Our preliminary investigation suggests these indole derivatives possess aggregation-induced emission enhancement properties and they assemble hollow, sphere like, aggregates. Since indoles have known anticancer properties they can also be used for selectively targeting cancer cells and possibly the cell death of cancer cells could also be imaged. Further, we are also interested in designing new AIEE-ACQ conjugates which can potentially be used for sensing. For this project we will conjugate pyridothiazole based AIEE scaffold synthesized in our lab and conjugate it with different Aggregation caused quenching (ACQ) fluorophores like Pyrene, Naphthalene, Anthracene and Phenanthrene. We also wish to conjugate heterocyclic compounds with cell penetrating peptides to enhance their bioactivity. Further conjugation of heterocyclic compounds with aptamers and biomarkers can remarkably enhance their utility as pharmacological products.

NANOPARTICLE BASED ASSAYS/ SYNTHESIS OF COMPOUNDS FOR SENSING AND DETECTION

The main goal of these projects will be to design safe and efficient nanoparticle-based products and heterocyclic compounds for sensitive detection of analytes like drugs, biomarkers and environmental pollutants Functionalization of nanoparticles with specific molecules that can recognize and interact with the analyte is a very efficient strategy to design novel assays which are highly sensitive and specific. In this context, we have already efficiently designed a gold nanoparticle-based assay for facile detection of amyloid inhibitors (ChemRxiv DOI 10.26434/chemrxiv.7819661). We are currently also pursuing research for the design of a novel nanoparticle-based assay for the specific and sensitive detection of cancer specific proteases. Further, we have also synthesized novel Azo dyes for sensitive detection of water pollutants like copper (Cu2+), tin (Sn2+), Aluminium (Al3+) and dichromate (Cr2O72-) ions (ChemRxiv Preprints DOI 10.26434/chemrxiv.13708249; DOI 10.26434/chemrxiv.14135894). We want to pursue research on this topic in greater detail in the future and design highly efficient nanoparticles/compounds which could detect analytes at very minute levels.