Chaudhry, T (2023) Peptide Conjugates for MicroRNA-21 Inhibition in Glioblastoma Multiforme. Doctoral thesis, Liverpool John Moores University.

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Abstract

Glioblastoma multiforme (GBM) is the most common and lethal primary malignant brain tumour in adults (Osuka and Van Meir, 2017), following diagnosis it is estimated that less than 5% of GBM patients survive for more than 5 years (Tamimi and Juweid,2017). The current standard practise for GBM treatment includes surgery followed by a regimen daily radiotherapy and oral chemotherapy, typically Temozolomide (TMZ). However, TMZ has a poor effect on overall patient survival rates and the prognosis following GBM diagnosis remains bleak (Tamimi and Juweid, 2017). For this reason, alternative strategies have been explored for the treatment of GBM. This includes the development of miRNA-21 (miR-21) targeted GBM therapies. The function of miR-21 in relation to the development and progression of solid tumour cancers, including GBM, has been studied extensively (Volinia et al., 2006; Medina, Nolde and Slack, 2010). In relation to GBM, the oncogenic function of miR-21 in GBM was first reported by Kosik and co-workers, they demonstrated that the knockdown of miR-21 in cultured GBM cells triggered the activation of caspases, increasing apoptotic cell death. This highlighted the function of miR-21 as an antiapoptotic factor in GBM (Chan, Krichevsky and Kosik, 2005). Thus, with the view to develop miR-21 targeted cancer therapies, peptides have been reported to bind to pri-miR-21 and pre-miR-21 to inhibit the formation of mature oncogenic miR-21 (Diaz et al., 2014; Bose et al., 2015; Shortridge et al., 2017). This includes PIMP21 (peptide 49) which has been reported to bind pre-miR-21 with a binding affinity (KD) of 0.0127 µM and prevent the Dicer cleavage of pre-miR-21 (IC50 of 0.5 µM), using in-vitro studies (Bose et al., 2015). The peptide was further demonstrated to knockdown miR-21 expression at a cellular level, in MCF-7 cells, by 6-fold with respect to a scrambled control peptide (Bose et al., 2015). This highlighted the potential of peptides in the development of miR-21 targeted anti-cancer therapies. However, the use of peptides as medicine is still limited, one of the reasons for this is the limitation associated with the delivery of therapeutic peptides to their active site in cells and across difficult to penetrate barriers, like the BBB (Kadry, Noorani and Cucullo, 2020). Cell penetrating peptides (CPPs) like peptide 779 (SYPGWSW) have been highlighted to successfully deliver therapeutic cargo across the BBB and to the cytoplasm of GBM cells (Ran et al., 2017). For this reason, in this study the cell penetrating function of peptide 77 was further investigated, this led to the discovery of novel peptides 87 (S(Cha)PGWSW) and 91 (S(Nal)PGWSW) with the increased ability to penetrate GBM, relative to the parent peptide 77. It was further found that the increased cellular uptake of peptide 87 was likely associated with the increase in peptide lipophilicity, which facilitated increased hydrophobic interactions between peptide 87 and the cell membrane of GBM cells. Meanwhile, the increased cellular uptake of peptide 91 was linked to the increased proteolytic stability of the peptide (t½ = 35.54 h), when compared to parent peptide 77 (t½ = 19.25 h). Subsequently, a peptide conjugate was designed to determine the ability of the peptide 77 to deliver therapeutic cargo like the PIMP21 peptide (49) to GBM cells, to knockdown the expression of miR-21 and establish the basis for a potential miR21 targeted GBM therapy. This led to the discovery that peptide conjugate 109 (SYPGWSW-Ahx-Lys-Ahx-ALWPPNLHAWVP) was able to translocate across the BBB and penetrate GBM cells. The peptide conjugate was further demonstrated to knockdown miR-21 expression by 15-fold relative to a scrambled control peptide conjugate. Furthermore, Peptide conjugate 109 (0-100 µM) was highlighted to have no significant effect on GBM cell viability following 24 h of exposure. This was the first example of miR-21 modulation in GBM using therapeutic peptides and highlighted the therapeutic potential of peptide conjugate 109 in the development of a potential GBM targeted anti-cancer therapy.

Item Type:	Thesis (Doctoral)
Uncontrolled Keywords:	Peptides; microRNA; Drug Delivery; Blood brain barrier; Cell penetrating peptides
Subjects:	R Medicine > RC Internal medicine > RC0254 Neoplasms. Tumors. Oncology (including Cancer) R Medicine > RM Therapeutics. Pharmacology
Divisions:	Pharmacy & Biomolecular Sciences
SWORD Depositor:	A Symplectic
Date Deposited:	17 Apr 2023 09:19
Last Modified:	15 Feb 2024 16:17
DOI or ID number:	10.24377/LJMU.t.00019237
Supervisors:	Ross, K and Coxon, C
URI:	https://researchonline.ljmu.ac.uk/id/eprint/19237

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