Pharmacy
http://hdl.handle.net/10012/9947
2024-03-28T21:58:45ZArylbenzamide and Arylcarboxamide Derivatives as Modulators of Amyloid-Beta Aggregation
http://hdl.handle.net/10012/20392
Arylbenzamide and Arylcarboxamide Derivatives as Modulators of Amyloid-Beta Aggregation
Zhao, Yusheng
Alzheimer’s disease (AD) is a complex neurodegenerative disease with increasing incidence and prevalence globally. The current AD therapies based on small molecules offer only symptomatic relief and are not curative therapies. The recently launched anti-amyloid monoclonal antibodies hold promise although these are new to the market and their long-term benefits and potential disease-modifying effects are unknown. The global increases in the aging population and increasing life span mandate the need to understand the mechanisms of AD and discover effective and safe therapies. Over the past several decades, few hypotheses have been proposed to explain the pathophysiology of AD, among which the amyloid beta (Aβ) cascade is now considered as one of the initiating factors that drives the progression and other pathological factors of AD. The aggregation of Aβ into oligomers and fibrils together with its downstream signaling pathway are neurotoxic. Thus, small molecule modulators that could reduce the overall toxic burden of Aβ aggregates are thought to be beneficial in treating AD.
In this thesis, a library of 72 small molecule derivatives were designed based on the chemical structure of chalcone and curcumin, two bioactive natural compounds that are able to modulate Aβ aggregation and reduce their neurotoxicity. The derivatives reported in this thesis encompass four different templates, namely, N-benzyl (Chapter 2), N-phenethyl (Chapter 3), N-benzyloxy (Chapter 4), and N-phenyl (Chapter 5) benzamide and carboxamides. These compounds were synthesized by coupling the amine substrates with either acid halides or carboxylic acids to obtain the target compounds in 72-93.6% yields. A number of biophysical and biochemical experiments were carried out to determine the ability of these small molecules to modulate the aggregation properties of Aβ42. The experiments carried out include i) thioflavin T based fluorescence aggregation kinetics experiments; ii) transmission electron microscopy studies; iii) 8-anilino-1-naphthalenesulfonic acid based fluorescence spectroscopy; iv) antioxidant assay by fluorescence spectroscopy; iv) cell viability studies in mouse hippocampal HT22 neuronal cells and Aβ42-induced neurotoxicity assay; v) fluorescence microscopy studies to assess the neurotoxicity using Proteostat dye, and vi) computational modelling studies to determine the interactions of small molecules with Aβ42 aggregates. From this library, 51 aggregation inhibitors were identified (inhibition of Aβ42 ranging from 7-53.1% at 25 µM). These derivatives were able to provide significant neuroprotection from Aβ42-induced cytotoxicity in mouse hippocampal HT22 cells (cell viability ranging from 80.8-96.8% versus 38.7% for Aβ42-treated control). Molecular docking studies indicate that these derivatives were able to interact with the hydrophobic domains of the Aβ42 oligomer and fibril through hydrophobic interactions. In a striking and unusual finding, 8 derivatives were identified as Aβ42 aggregation promotors with the ability to promote the aggregation by 1.2-5.1 folds. Two lead promotors 14b (N-benzylbenzofuran-2-carboxamide) and 14c (N-benzylbenzo[b]thiophene-2-carboxamide) were identified. These two compounds were able to rescue HT22 cells from Aβ42-induced cytotoxicity (cell viability 73.8% and 73.9% for 14b and 14c versus 19.7% for Aβ42-treated control). These two compounds have the ability to increase the surface hydrophobicity of Aβ42 aggregates and promote fibrillogenesis. Molecular docking studies suggested that Aβ42 aggregates might undergo conformational change upon binding and thus transit to much more stable and less toxic/nontoxic fibrils. Further structure-activity relationship study indicated that the hydroxy- and methoxy-disubstituted phenyl moiety was required to possess Aβ42 inhibition activity, where the presence of bicyclic aromatic rings such as benzofuran and benzothiophene, and 4-methoxyphenyl moiety is required for pro-aggregation activity. The results show that these benzamides and carboxamides possessing N-benzyl, N-phenethyl, N-benzyloxy, and N-phenyl templates hold promise in the design and development of novel small molecules as Aβ42 aggregation modulators. Remarkably 14b (N-benzylbenzofuran-2-carboxamide) and 14c (N-benzylbenzo[b]thiophene-2-carboxamide) were able to accelerate Aβ42 aggregation and remodel the aggregation pathway to form less toxic/nontoxic aggregates suggesting their application as novel chemical tools to understand the mechanisms of Aβ42 aggregation cascade.
2024-03-11T00:00:00ZEnhancing the Ministring DNA (msDNA) Purification Using PI-Sce1 Homing Endonuclease/CRISPR-Cas3 Recombinant System
http://hdl.handle.net/10012/20334
Enhancing the Ministring DNA (msDNA) Purification Using PI-Sce1 Homing Endonuclease/CRISPR-Cas3 Recombinant System
Fernando, Merium
In the generation of msDNA the recombinant E. coli cells are transformed by a msDNA generating precursor plasmid, whereupon expression of the Tel protelomerase enzyme, acting on the pal target sequence present in the precursor plasmid, generated linear covalently closed (LCC) msDNA. However, the in vivo recombinant platform to produce msDNA results in a mixture of plasmids including unprocessed precursor plasmid, unwanted LCC bacterial backbone, and their topological isoforms, which interferes with the purification of the target species. For larger scale synthesis, the plasmid extract needs to be pretreated with commercially available restriction enzymes before being purified through chromatographic columns. Meanwhile, at the laboratory scale, msDNA is purified from agarose gels based on their size. These purification processes are time-consuming and inefficient and therefore, there is a need to optimize the process.
To address this issue, we developed two in vivo recombinant systems for digesting the unwanted prokaryotic backbone and unprocessed precursor plasmid. These systems are the PI-SceI homing endonuclease enzyme system and the clustered regularly interspaced short palindromic repeats-Cas3 (CRISPR-Cas3) system. Homing endonucleases are highly specific DNA cleaving enzymes. The homing endonuclease PI-SceI, encoding gene vma from Saccharomyces cerevisiae was successfully integrated into the tel integrated bacterial chromosome via site-specific recombination using conditional replication and integration (CRIM) plasmid. The double integrants, both vma and tel integrated recombinant bacteria, were transformed with msDNA synthesizing precursor plasmids and induced the msDNA synthesis and vma gene overexpression. Even though the double integrants were able to overexpress the homing endonuclease enzyme and digest the precursor plasmid, they were not able to synthesize msDNA. Therefore, the Tel protelomerase enzyme was expressed episomally inside the vma integrated recombinant bacteria. This vma gene is under the control of an inducible PBAD promoter. In the presence of L-arabinose in the media, the Tel protelomerase enzyme was episomally expressed and synthesized msDNA by acting on the precursor plasmid. Subsequently, the overexpressed PI-SceI homing endonuclease enzyme digested the undesired byproducts of msDNA synthesis as expected. Introducing homing endonuclease enzyme recognition sequences into the Tel protelomerase enzyme-expressing plasmid will further improve the purification process. The other recombinant system that was developed is the utilization of the CRISPR-Cas3 system which is naturally present in W3110 E. coli K-12 bacteria. A pre-crRNA targeting the origin of replication (ori) of the msDNA synthesizing precursor plasmid was successfully designed and cloned into the low copy number plasmid. The pre-crRNA expressing gene cassette was placed under the control of the PBAD promoter. Upon overexpression, crRNA was synthesized inside the W3110 E. coli K-12 bacteria. The crRNA bound to the expressed CRISPR-Cas3 protein cascade of the bacteria, guided the effector complex to the target sequence and successfully digested the targeted precursor plasmid. Even though the W3110 tel+ recombinant bacteria synthesized msDNA in a pre-crRNA expressing background, an efficient degradation of the unwanted by-products of msDNA synthesis was not observed. This could be due to the disruption of the CRISPR locus of W3110 tel+ recombinant bacteria. Episomal expression of the CRISPR-Cas genes inside W3110 tel+ recombinant bacteria will enhance the digestion of the non-msDNA species.
2024-02-05T00:00:00ZDevelopment of Modular Polymeric NPs for Drug Delivery Using Amine-Reactive Chemistry
http://hdl.handle.net/10012/20307
Development of Modular Polymeric NPs for Drug Delivery Using Amine-Reactive Chemistry
Wong, Calvin
Cancer remains one of the leading causes of death worldwide and very often requires chemotherapy treatment. Despite advances in chemotherapy treatments, some cancers remain difficult to treat due to tumour type, location, and in some cases, the development of drug resistances. In order to tackle cancer more effectively, researchers have explored and developed novel chemotherapy agents. However, many of these agents suffer from low bioavailability or prohibitively high toxicity to the body. Nanotechnology-based drug delivery systems aim to assist in protection and site-specific delivery of these potential anti-cancer agents, increasing their effectiveness and lowering toxic effects. Polymeric NP delivery systems can encapsulate drugs and be coated with functional groups or moieties to enhance various properties such as targeting.
In this project, poly(lactic-co-glycolic) acid (PLGA ) NPs were synthesized to encapsulate curcumin (CUR) via single emulsion method. CUR, the principal constituent of Curcuma longa, commonly known as turmeric, has been explored for its anti-cancer potential, but is severely limited by its hydrophobicity and sensitivity to light and water. The PLGA NPs were coated with oligomeric chitosan (COS) and RGD peptide (peptide consisting of Arg-Gly-Asp) using amine-reactive chemistry (NHS and EDC). Both COS and RGD had been previously shown to accumulate and target many different types of cancer cells. NPs were characterised based on size distribution, zeta potential, and binding efficiency of RGD peptide. They were also evaluated on encapsulation efficiency, and stability, of CUR within the NPs. OVCAR-3 cancer cells were treated with COS and RGD-coated PLGA NPs loaded with Coumarin-6 dye for fluorescent imaging of cell uptake. They were also treated with CUR-loaded NPs to determine cytotoxicity and effectiveness of delivery.
The NPs exhibited size distribution and zeta potential within expected values, though binding efficiency of RGD was low. CUR-loaded NPs showed significant increase in cytotoxicity over free (unencapsulated) CUR, and void (empty) NPs, suggesting successful delivery of CUR as an anti-cancer agent; the performance of COS and RGD coated NPs over bare PLGA NPs was inconclusive, however. Optimization will be required to improve formulation during the coating steps. Further investigation may be required into alternative binding chemistry, such as click chemistry.
2024-01-26T00:00:00ZProbing the interactions that drive RNA binding and self-association of hnRNPA1 implicated in neurodegeneration
http://hdl.handle.net/10012/20239
Probing the interactions that drive RNA binding and self-association of hnRNPA1 implicated in neurodegeneration
Fatima, Syeda Sakina
The heteronuclear ribonucleoprotein A1 (hnRNPA1 or A1) is associated with the pathology of different diseases, including neurological disorders and cancers. In particular, the aggregation and dysfunction of A1 has been identified as a critical driver for neurodegeneration in Multiple Sclerosis (MS). Structurally, A1 includes a low-complexity domain (LCD) and two RNA-recognition motifs (RRMs), and their interdomain coordination may play a crucial role in A1 aggregation. Previous studies propose that RNA-inhibitors or nucleoside analogs that bind to RRMs can potentially prevent A1 self-association. Therefore molecular-level understanding on the RNA recognition by A1 RRMs remains of scientific interest. Although several crystal structures of RNA-bound RRM complexes have been reported in the literature, there are still open questions about which RRM RNA prefers to bind and why only specific RNA sequences tend to bind A1. This thesis aims at probing the structures, dynamics and nucleotide interactions with A1’s RRMs using a combination of advanced computational methods. Our research to-date has revealed that adenine and guanine in RNAs (or DNAs), and the key residues from the interdomain linker connecting the two RRM domains contribute significantly for RNA binding to A1 RRMs. Further research will seek to address the impact of RNA length on its binding and how RNA specificities vary between the RRMs. Critical residues for RNA-binding have been identified and their molecular-level insights on their nucleotide preferences have been evaluated. As a final addition, the full-length A1 protein for which a crystal structure in the PDB does not exist, is modeled, to analyze the interactions that occur between the RRMs and the LCD domain that could promote A1’s aggregation. Both of A1’s known isoforms, isoform A (320aa) and isoform B (372aa) have been modeled and studied, with and without RNA bound to them. Our data suggests that interplay between the LCD and the RRMs may block exposure of critical RNA-binding residues to the environment when RNA is not already bound to the protein. Taken together, this thesis elaborates on full protein dynamics and nucleotide-protein interactions that may be helpful in designing therapeutics. Nucleotide-based therapies or nucleoside analogs in particular, can be designed based on specific interactions outlined in this thesis.
2024-01-17T00:00:00Z