DSpace Collection:http://theses.ncl.ac.uk/jspui/handle/10443/1022024-02-04T20:30:56Z2024-02-04T20:30:56ZThe investigation of circulating biomarkers and potential mechanisms of resistance in the ATR/CHK1 signalling pathway in response to CHK1 inhibitor therapyHannaway, Nicola Louisehttp://theses.ncl.ac.uk/jspui/handle/10443/57672023-08-18T09:42:06Z2022-01-01T00:00:00ZTitle: The investigation of circulating biomarkers and potential mechanisms of resistance in the ATR/CHK1 signalling pathway in response to CHK1 inhibitor therapy
Authors: Hannaway, Nicola Louise
Abstract: The DNA damage response is a network of cell checkpoints leading to cellular repair and genome integrity when DNA insults have occurred. In cancers, enhanced tumour replication stress and genomic instability along with mutations in oncogenes and tumour suppressor genes can lead to disruption of the DNA damage response. Tumour survival becomes dependant on critical checkpoint controls. Drugs to target intact pathways, such as CHK1 inhibitors are therefore desirable and are being evaluated in clinical trials as prospective anticancer therapies. Cancer treatments have moved to a personalised approach based on tumour profiling and circulating biomarkers, such as circulating cell free DNA to predict patient response to targeted treatments. The mechanisms underpinning sensitivity and resistance to CHK1 inhibitor treatment are still unclear.
This project explores three distinct strands of research to examine both primary and acquired mechanisms of CHK1i resistance. Firstly, investigating gene and protein expression changes in Eμ-Myc mouse models of B-Cell lymphoma. Wild type Eμ-Myc mice are sensitive to CHK1i treatment, however by contrast Eμ-Myc NF-κB c-Rel -/- and RelA T505A lymphomas are CHK1i resistant. Secondly, the generation and characterisation of U2OS osteosarcoma cells with acquired CHK1i resistance. Lastly, investigating if DNA damage response mutations can be isolated from patient cfDNA samples on a CHK1 inhibitor clinical trial.
Results have shown that resistance to CHK1 is complex and that there are multiple resistance mechanisms in place. The Eμ-Myc NF-κB c-Rel -/- mice completely downregulate the DNA damage response and this pattern is shared with some resistant cell lines, but alternative mechanisms developed in other models. DDR mutations can be detected and tracked in cfDNA samples and show a large degree of heterogeneity between patients. This study highlights the importance of the ATR/CHK1 DNA damage checkpoint and how drug resistance mechanisms can vary between diverse tumour mutational profiles.
Description: Ph. D. Thesis.2022-01-01T00:00:00ZExpanding the scope of DNA compatible chemistry for the application within DNA encoded librariesHunter, James Henryhttp://theses.ncl.ac.uk/jspui/handle/10443/54782022-06-30T09:32:48Z2021-01-01T00:00:00ZTitle: Expanding the scope of DNA compatible chemistry for the application within DNA encoded libraries
Authors: Hunter, James Henry
Abstract: The discovery of potential lead like molecules is a crucial phase for any drug discovery
program. Current methods to identify lead molecules are often resource intensive, often
requiring millions of compounds to be screened in biological assays. Reducing the time and
cost taken to generate, store and screen large compound libraries would have a positive
impact on academia and small pharmaceutical companies. DNA encoded libraries (DELs) aim
to improve upon this by screening an entire library in a single vessel against a target and
utilising the DNA tag to identify potential inhibitors. Current methods of preparing DELs are
limited to chemistry that is compatible with DNA. These chemical methods are often limited
to simple chemical reactions, such as cross-couplings and amide bond formation, which are
used combinatoriality to generate vast libraries. As analogous chemical reactions are used,
current libraries are often populated with compounds with similar physical properties and
have limited structural diversity. The reactions used to generate DELs are often low yielding,
or are limited in substrate scope, further reducing the diversity of potential libraries.
Development of new approaches with DEL synthesis will increase the ability to synthesise
libraries with greater chemical diversity and improved physical property profiles.
Products produced from the encoded transformation paradigm.
A new paradigm termed “encoded transformations” was introduced. This technique involves
encoding a specific chemical transformation of a reactive core molecule instead of coding the
addition of a building block. This would lead to a potential library of compounds with reduced
overall molecular weight and more lead-like physical properties. A common reactive 2-oxobetenamide core was used and several chemical transformations have been successfully
employed. These chemical transformations were developed off-DNA and provide a means to prepare libraries with significant scaffold diversity using DNA compatible chemistry.
Micellar solvents have been shown to improve normal phase organic chemical reactions.
Applying this technique to on-DNA synthesis vastly improved the scope and diversity of the
reactants used. Both amide couplings and Suzuki-Miyaura reactions were successfully
optimised utilising micellar media as a solvent. Optimisation was carried out in both examples
by factorial experimental design (FED), which revealed second order relationships between
variables tested that would not likely be discovered using conventional techniques.
Products produced from novel micellar promoted Suzuki-Miyaura and amide reactions.
These novel reactions were used to synthesise a prototype 6x6 library, which was PCR
amplified and sequenced proving that reactions in micellar media do not cause DNA damage.
The reverse amide coupling was then used to create a 99,405-member library, including
compounds designed to specifically target the SARs-CoV-2 main protease (Mpro) binding site,
using an array of both covalent and non-covalent inhibitors.
Description: PhD Thesis2021-01-01T00:00:00ZPreclinical testing of a targeted TRAIL therapeutic for bone sarcomaGamie, Zakareya Esame Khalilhttp://theses.ncl.ac.uk/jspui/handle/10443/54462022-06-15T08:51:24Z2020-01-01T00:00:00ZTitle: Preclinical testing of a targeted TRAIL therapeutic for bone sarcoma
Authors: Gamie, Zakareya Esame Khalil
Abstract: Background: TNF-related apoptosis-inducing ligand (TRAIL) can induce cell death in
cancer cells after binding to its TRAIL receptors [TRAILR, Death Receptor 4 (DR4) and
Death Receptor 5 (DR5)] while sparing non-malignant cells. The application of TRAIL
provides an approach that can potentially overcome drug resistance and toxicity
associated with high doses of conventional therapies. It could be administered alone or
in combination with conventional therapies and, therefore, may offer a promising new
approach to bone sarcoma treatment. Enhancing the cytotoxic effect of TRAIL involves
targeting a tumour associated antigen (TAA). Here, the aim was to characterise bone
sarcoma cells for TRAILR expression and to assess the effectiveness, both in vitro and
in vivo of a novel TRAIL construct, neural/glial antigen 2 (NG2) targeted TRAIL
(ScFvNG2-Fc-scTRAIL).
Methods: Bone sarcoma cell lines were characterised for TRAILR and NG2 expression
on RNA and protein level. Together with non-malignant cell lines, they were exposed
to the novel TRAIL therapeutic (ScFvNG2-Fc-scTRAIL) in vitro and then tested in vivo in
a newly developed xenograft model of dedifferentiated chondrosarcoma.
Results: Surface DR5 was expressed in all cell lines examined (very high: HT1080,
MG63; moderate: SW153, U2OS, TC71). NG2 was also expressed (very high: SW1353,
MG63; moderate: U2OS, HT1080). ScFvNG2-Fc-scTRAIL demonstrated enhanced
cytotoxicity in DR5- and NG2-expressing cell lines (MG63>HT1080>U2OS), which
increased with doxorubicin and was also found in vivo when engrafting a luciferase
expressing HT1080 cell line in a dedifferentiated chondrosarcoma mouse model.
Conclusion: I demonstrate that a novel targeted TRAIL therapeutic, ScFvNG2-FcscTRAIL, has a selective and significant cytotoxic effect on cell lines expressing both cell
surface DR5 and NG2, and these cytotoxic effects can be enhanced further with
doxorubicin. Such combinations could minimise the risk of treatment failure due to
drug resistance, a common problem of single agent approaches. Furthermore, these
findings provide a framework for the clinical development of ScFvNG2-Fc-scTRAIL and
could potentially be used in the neoadjuvant setting, which would be a shift from the
usual convention of prioritising excision of the sarcoma.
Description: PhD Thesis2020-01-01T00:00:00ZNovel, Differentiated Antibody Drug Conjugate Warheads from Analysis of the NCI Screening DatabaseBrough, Danielhttp://theses.ncl.ac.uk/jspui/handle/10443/52742022-02-17T13:45:15Z2020-01-01T00:00:00ZTitle: Novel, Differentiated Antibody Drug Conjugate Warheads from Analysis of the NCI Screening Database
Authors: Brough, Daniel
Abstract: Antibody-drug conjugates (ADCs) combine the selective nature of targeted therapies with
potent cytotoxic warheads utilising linker technology to deliver selectively the warhead to the
target. Proof of concept has already been observed with five licensed ADCs on the market,
giving confidence to the use of ADCs in cancer treatment, with multiple ADCs in clinical
testing. Many of these ADCs consist of cytotoxic payloads derived from complex natural
products, consequently leading to synthetically long and complicated routes.
To address this problem, novel warheads have been identified through screening the NCI
database, that are structurally simpler drugs with differing cellular activity profiles. This may
also address resistance issues. Out of the seven that were identified, three series have been
explored; the nitroacridines (113), the quinolones (106) and thiosemicarbazones (174).
Nitroacridines inherently target hypoxic cells and are known to intercalate with DNA.
Quinolones are known tubulin binders and compounds synthesised have been evaluated in
biological studies. Thiosemicarbazones are metal chelators, sequestering iron and copper.
They inhibit ribonucleotide reductase, inactivating the enzyme leading to cell death.
Work has been carried out to optimise and resynthesize these parent warheads as well as
analogues. The warheads have been coupled to non-cleavable and cleavable dipeptide linkers
(examples shown below) and subsequent conjugation to an antibody, which have been
assessed in cell assays.
Description: Ph. D. Thesis.2020-01-01T00:00:00Z