5 research outputs found

    Targeting the p53/MDM2 protein-protein interaction

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    The p53/MDM2 protein-protein interaction is the most widely characterised proteinprotein interaction to date. As of 2014, there are over 20 compounds that have been shown to the p53-MDM2 protein-protein interaction, however many compounds have not progressed into clinical trials due to their high hydrophobicity. Herein we describe the synthesis, molecular modelling, physical characterisation and biological testing of novel inhibitors of the p53/MDM2 protein-protein interaction based on the natural product chlorofusin. The first focus is a combinatorial library generated in the Searcey laboratory of known p53/MDM2 protein-protein interaction inhibitors with the desire to generate novel analogues and study their interactions with the protein through NMR spectroscopy and molecular modelling. These compounds were tested by in a fluorescence polarisation assay and also in cell lines overexpressing MDM2 as well as p53-null cells as a comparator. This generated two novel compounds shown to have activity selectively for the p53/MDM2 protein-protein interaction. The second chapter focuses on simplified substitutions of the azaphilone (the chromophore portion of chlorofusin, a natural product inhibitor of the p53-MDM2 proteinprotein interaction): initially with simple fused bicyclic carboxylic acids and later using click chemistry substitutions. Interestingly, in vitro studies showed that the click analogues retained activity or activity improved when the peptide portion was removed and hence further studies of the click amino acid analogues were generated. This library generated one analogue that was active in vitro as well as selectively in MDM2-overexpressing cell lines. The third chapter focusses on the azaphilone chromophore present in the natural product chlorofusin. The Sonogashira precursor used to generate azaphilone analogues was synthesised using a methodology adopted by Porco et al and subsequent analogues were generated using a novel double-Sonogashira approach followed by functionalisation published by Boger et al. Once the azaphilone was synthesised, metholodogies were trialled in order to condense the azaphilone with the chlorofusin peptide in order to create analogues containing both the peptide and small molecule portions of chlorofusin. In addition, molecular modelling was attempted to generate novel binding analogues

    Allosteric regulation of MDM2 protein

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    The diverse functions of the MDM2 oncoprotein in growth control and tumourigenesis are managed through coordinated regulation of its discrete domains induced by both extrinsic and intrinsic stimuli. A picture of MDM2 is immerging where structurally discrete but interdependent functional domains are linked through changes in conformation. However compelling insights into how this process is carried out have been hindered by inadequate information on the structure and conformation of the full-length protein. The data presented indicates that the C-terminal RING domain of MDM2, primarily responsible of the E3 ubiquitin ligase activity of the protein, has other intriguing functions. The binding of ATP within the RING domain, triggers conformational changes of MDM2 and its main interaction partner – p53. This in effect promotes efficient binding of the p53 tumour suppressor to specific DNA promoter sequences. Moreover, results presented in this thesis demonstrate a novel role for the RING domain of MDM2 in determining the conformation and activity of its N-terminal hydrophobic cleft, the key target of anticancer drugs designed to activate the function of p53 tumour suppressor protein. Specific modulations within the RING domain, affecting Zinc coordination are synonymous with increased binding affinity of the hydrophobic pocket to the transactivation domain of p53 resulting in a gain of MDM2 transrepressor function thus leading to a decrease in p53-dependant gene expression. ThermoFluor measurements and size exclusion chromatography show that changes in the RING motif lack an effect on the overall integrity of the MDM2 protein. The intrinsic fluorescence measurements manifest that these changes generate long range conformational transitions that are transmitted through the core/central acidic domain of MDM2 resulting in allosteric regulation of the N-terminal hydrophobic pocket. Such RING generated conformational changes result in the relaxation of the hydrophobic pocket. Additionally, it is shown that the cooperation between the RING and the hydrophobic cleft in MDM2 has implications in the efficiency of binding of anticancer drugs such as Nutlin by MDM2. Cooperation between the RING and hydrophobic domain of MDM2 to regulate function demonstrates an allosteric relationship and highlights the need to study MDM2 in a native conformation. In essence the presented data demonstrates that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed towards its hydrophobic pocket

    Development of New Synthetic Approaches to Biologically-Active Peptides

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    Synthetic peptides are widely used in drug research due to their high selectivity and efficaciousness, as well as in chemical biology as tools for modulating protein-protein interactions. While there are a large number of naturally occurring peptides, which can be used for the development of new pharmaceuticals, there is also an increasing interest in rationally designed synthesised and optimised peptides. In the field of chemical biology peptides are used to understand complex biological processes, where gaining knowledge and understanding can lead to improved human health and the development of novel drug targeting strategies. Since the synthesis of the first peptide back in 1882, there have been major changes to the peptide synthesis protocol, leading to robust Fmoc-solid phase peptide synthesis that is used in most laboratories today. However, the peptide purification protocols have been lacking behind, meaning peptide purification is still a major bottleneck in fast, efficient, environmentally friendly peptide synthesis. In this thesis, a new peptide synthesis and purification method has been developed and optimised making use of an acrylamide-scavenging approach. Different strategies to introduce an N-terminal acrylamide cap have been explored, including the use of acryloyl chloride and acrylic anhydride, where the latter was suited for automated SPPS. It was found that replacement of piperidine by DABCO for Fmoc-deprotections was equally efficient and suitable for automated SPPS. The scavenging of acrylamide-tagged deletions was performed making use of a thiol-resin, of which the rate was determined by LCMS in peptide model systems. Upon optimisation of acrylamide-tagging and Fmoc-deprotection conditions, the new purification method was compatible with both manual and automated SPPS, requiring only minor adjustments from standard synthesis protocols. This allowed for the synthesis and purification of the diabetes peptide exenatide (39 amino acids), without making use of preparative-HPLC. An attempt was made to extend this methodology to the synthesis of cyclic thioether peptides; however, this did not provide conclusive results. An alternative approach to cyclising peptides was explored, making use of disulfide crosslinking with hexafluorobenzene in biologically active p53 peptides. Residues of the biologically active pDI peptide that had been shown to be tolerant to substitution were replaced with cysteine (analogues), including D-cysteine, homocysteine and penicillamine. It was found that replacement of L-cysteine by any of the analogues resulted in altered binding affinity and selectivity. Finally, the use of fluorine as a reporter for 19F NMR analysis of biological molecules was investigated. Fluorinated prolines were used to explore protein-protein interactions and dynamics, specifically to study the peptidyl-prolyl isomerase cyclophilin D (CypD). First, short substrates were synthesised, including a section of Bcl2 (8 amino acids) and F0F1 ATPase subunit B (9 amino acids), which formed a proof of principle. Significant line broadening was observed in the 19F NMR spectrum with increased protein concentration, indicating substrate binding. These results were more apparent for the Subunit B substrate, indicating it is a better binder to CypD than the Bcl2 peptide. The synthesis of another CypD substrate, a part of the poly proline motif in p53, was optimised for the incorporation of 4S-fluoroprolines. A very significant finding was that each proline residue had a distinct chemical shift. The native cis-trans ratios of each peptide was determined, after which some changes in the cis-trans ratios were observed in the presence of CypD. For some of the fluorinated proline residues line broadening was observed upon incubation with CypD, indicating the occurrence of protein-protein interactions

    IN SILICO METHODS FOR DRUG DESIGN AND DISCOVERY

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    Computer-aided drug design (CADD) methodologies are playing an ever-increasing role in drug discovery that are critical in the cost-effective identification of promising drug candidates. These computational methods are relevant in limiting the use of animal models in pharmacological research, for aiding the rational design of novel and safe drug candidates, and for repositioning marketed drugs, supporting medicinal chemists and pharmacologists during the drug discovery trajectory.Within this field of research, we launched a Research Topic in Frontiers in Chemistry in March 2019 entitled “In silico Methods for Drug Design and Discovery,” which involved two sections of the journal: Medicinal and Pharmaceutical Chemistry and Theoretical and Computational Chemistry. For the reasons mentioned, this Research Topic attracted the attention of scientists and received a large number of submitted manuscripts. Among them 27 Original Research articles, five Review articles, and two Perspective articles have been published within the Research Topic. The Original Research articles cover most of the topics in CADD, reporting advanced in silico methods in drug discovery, while the Review articles offer a point of view of some computer-driven techniques applied to drug research. Finally, the Perspective articles provide a vision of specific computational approaches with an outlook in the modern era of CADD

    Antioxidant and DPPH-Scavenging Activities of Compounds and Ethanolic Extract of the Leaf and Twigs of Caesalpinia bonduc L. Roxb.

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    Antioxidant effects of ethanolic extract of Caesalpinia bonduc and its isolated bioactive compounds were evaluated in vitro. The compounds included two new cassanediterpenes, 1α,7α-diacetoxy-5α,6β-dihydroxyl-cass-14(15)-epoxy-16,12-olide (1)and 12α-ethoxyl-1α,14β-diacetoxy-2α,5α-dihydroxyl cass-13(15)-en-16,12-olide(2); and others, bonducellin (3), 7,4’-dihydroxy-3,11-dehydrohomoisoflavanone (4), daucosterol (5), luteolin (6), quercetin-3-methyl ether (7) and kaempferol-3-O-α-L-rhamnopyranosyl-(1Ç2)-β-D-xylopyranoside (8). The antioxidant properties of the extract and compounds were assessed by the measurement of the total phenolic content, ascorbic acid content, total antioxidant capacity and 1-1-diphenyl-2-picryl hydrazyl (DPPH) and hydrogen peroxide radicals scavenging activities.Compounds 3, 6, 7 and ethanolic extract had DPPH scavenging activities with IC50 values of 186, 75, 17 and 102 μg/ml respectively when compared to vitamin C with 15 μg/ml. On the other hand, no significant results were obtained for hydrogen peroxide radical. In addition, compound 7 has the highest phenolic content of 0.81±0.01 mg/ml of gallic acid equivalent while compound 8 showed the highest total antioxidant capacity with 254.31±3.54 and 199.82±2.78 μg/ml gallic and ascorbic acid equivalent respectively. Compound 4 and ethanolic extract showed a high ascorbic acid content of 2.26±0.01 and 6.78±0.03 mg/ml respectively.The results obtained showed the antioxidant activity of the ethanolic extract of C. bonduc and deduced that this activity was mediated by its isolated bioactive compounds
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