Hatfield, Amelia
(2024)
Investigating the Mechanism of Action of Antitumour Benzoprims.
PhD thesis, University of Nottingham.
Abstract
KRAS is one of the most frequently mutated oncogenes, with activating mutations occurring in ~ 20% of all human cancers, including >90% of pancreatic cancer, ~40% of colorectal cancer (CRC) and ~30% of non-small cell lung cancer (NSCLC). Until recently, KRAS inhibitors have not been successful clinically despite years of effort, mainly due to RAS proteins not appearing to possess suitable binding pockets to which small molecule inhibitors can bind. However, in 2021, the first KRASG12C inhibitor was approved for clinical use - Sotorasib®.
Experimental anti-tumour Benzoprims, analogues of the anti- malarial drug pyrimethamine, were found previously to possess anti-tumour activity against metastatic melanoma cells, mainly by inhibition of dihydrofolate reductase (DHFR), a validated target in cancer therapy. However, methylbenzoprim (MBP) and dichlorobenzoprim (DCB) were also found to elicit selective, potent activity in CRC and NSCLC cell lines possessing mutant KRAS, indicating an additional mechanism of action, independent of anti-DHFR activity. Therefore, the additional mechanism(s) of action of benzoprims MBP, DCB and further analogues SM1235, SM1243, SM1244 and SM1245 involving KRAS were investigated.
Benzoprims were found to be potent inhibitors of CRC cell growth and were found to be more selective for KRASG13D mutant cells compared to K-, N and HRASG12V cells, as well as cells possessing wild-type RAS, apart from SM1245 which displayed no selectivity. SM1235 was found to be the most potent compound overall and possessed a 130-fold higher potency in KRASG13D cells compared to wild-type RAS cells. All benzoprims, except SM1245, exerted net cytostatic effects, indicative of perturbation of cell signalling pathways. All benzoprims were found to decrease phosphorylation of AKT, ERK1/2, GSK3α, HSP27, JNK, MEK1, MKK3, MKK6, P38, P53, P70S6K, RSK1 and RSK2, all of which are proteins within the mitogen activated protein kinase (MAPK) pathway, downstream to RAS. Proteomic analysis of SM1235 did not reveal RAS as a direct target.
This study has investigated the mechanisms of action of benzoprims and highlighted proteins and pathways which are independent of both DHFR and RAS inhibition. Due to this, benzoprims activity which is independent of their anti-DHFR activity is unlikely to be a consequence of direct RAS inhibition or even inhibition of a single protein. Instead, benzoprims appear to elicit their anti-tumour effects through mediation of several biological processes and pathways, including the MAPK pathway, DNA synthesis and metabolism.
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