JAIN, MOHIT,LAXMILAL (2017) Development of Targeted Therapeutics for the Treatment of Glioblastoma. Doctoral thesis, Durham University.
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Author-imposed embargo until 31 July 2020.
Glioblastoma is the most aggressive cancer of the brain. Despite recent advances in cancer biology and multimodality therapies, such as surgery, radiotherapy and chemotherapy, the outcome of patients with high grade glioma remains fatal. The major drawback of current glioma chemotherapeutics is their inability to cross the blood brain barrier, lack of tumour specificity agents and their consequent side effects. Matrix metalloprotease (MMP) activity is central to cancer development, angiogenesis and invasion. They are highly active in the tumour environment and absent or inactive in normal tissues, therefore they represent viable targets for cancer drug discovery. A better understanding of the role of MMPs in human gliomas could potentially have diagnostic, prognostic and therapeutic implications. This study aims to assess the expression of specific MMPs in preclinical human glioma models and clinical glioma samples; evaluate in silico docking to rationalise substrate binding preferences of homologous MMPs; rationally design MMP-subtype-selective tumour activated prodrugs; and determine the feasibility of targeting MMP-selective anticancer prodrugs conjugated to graphene oxide as a local drug delivery approach for glioblastoma.
This study found significant overexpression of MMP-10 in glioma relative to histologically normal brain tissues. Strong correlation was observed between MMP-10 protein and gene expression of glioma cell lines relative to low expression in a normal brain cell line. MMP-10 activity, as measured by fluorogenic substrate cleavage assay, also demonstrated a strong correlation between MMP-10 activity and gene expression levels.
Following demonstration of selective overexpression of MMP-10 in glioma, a reiterative in silico proteolytic docking coupled in vitro biochemical assessment was utilised to rationalise functional similarity and differentiate substrate binding selectivity of homologous MMPs. The binding modes of MMP-substrates within the active site of closely related MMPs were able to accurately predict the cleavage subsites by specific MMPs, as confirmed by in vitro cleavage assay. The success of computational and experimental methodology provided a robust tool for identifying MMP-subtype differences and subsequent development of MMP-10 selective peptide prodrugs.
MMP-subtype selective and MMP-10 selective prodrugs were designed by rational exploitation of MMP-docked complexes of substrates. Peptide residues were modified to achieve selectivity for MMP-2 and MMP-10 (over MMP-3 and MMP-9) demonstrating predicted cleavage at distinct subsites. This selectivity was further exploited to attain MMP-10 selectivity, over MMP-2, MMP-3 and MMP-9. The rationally designed peptide prodrugs were synthesised and were shown to be preferentially cleaved by MMPs at predicted subsites and demonstrated no activation by engineered-out MMPs, as predicted. Compared to MJ02 (MMP-2 and MMP-10 selective doxorubicin prodrug), MJ04 (MMP-10 selective doxorubicin prodrug) demonstrated selective metabolism by glioma cell lines to release chemotherapeutic agents. This therapeutic approach against glioma cell lines depended upon the involvement of MMPs, confirmed using pharmacological inhibition. MJ04 demonstrated negligible activity in the presence of an MMP-10 selective inhibitor, suggesting MMP-10 selective activation of the prodrug in glioma cells relative to normal glial cells.
Following successful development of MMP-10 selective prodrugs, the feasibility of targeting glioma tumour with local delivery of chemotherapeutics from functionalised graphene-oxide tethered prodrug implants, was assessed as a therapeutic strategy to circumvent the blood brain barrier. Graphene oxide conjugated prodrug was synthesised which is shown to be preferentially cleaved in MMP expressing glioma cell lines relative to normal glial cells. This study demonstrates that MMP-10 is overexpressed in glioblastoma and can be used to metabolise anticancer prodrugs that can be activated selectively by local tumour environment.
|Item Type:||Thesis (Doctoral)|
|Award:||Doctor of Philosophy|
|Keywords:||Matrix metalloproteinases, computer-aided drug design, prodrugs, glioblastoma, targeted therapy, in silico|
|Faculty and Department:||Faculty of Social Sciences and Health > Medicine and Health, School of|
|Copyright:||Copyright of this thesis is held by the author|
|Deposited On:||01 Aug 2017 15:21|