Garces, Aimie E.
(2019)
Biological Evaluation of Synergistic Drug Combinations, Design and Synthesis of Potent and Selective PI3K Inhibitors and the Design of Bifunctional Ligands Incorporating Pictilisib & Ceritinib for the Treatment of EML4-ALK Positive Lung Cancer.
PhD thesis, University of Nottingham.
Abstract
Lung cancer remains the leading cause of cancer death in the United Kingdom. Non-small cell lung cancer makes up the majority of all lung cancers, approximately 80-85%. Of these non-small cell lung cancers, 3-7% possess chromosomal rearrangements of anaplastic lymphoma kinase (ALK). These lead to constitutively active ALK fusion proteins such as the fusion of the echinoderm microtubule-associated protein-like 4 (EML4) and ALK genes to give the EML4-ALK fusion gene. This activates downstream signals of the Ras/Raf/MEK/ERK1/2, JAK/STAT and PI3K/AKT/mTOR pathways.
Chapter 1 gives an introduction into lung cancer, and, in particular, EML4-ALK positive lung cancer. The chapter explores the current FDA approved inhibitors (crizotinib, ceritinib, alectinib, and brigatinib) for the treatment of EML4-ALK positive lung cancer, and discusses that, although efficacious, resistance rapidly emerges. The mechanisms of resistance are discussed, where it is suggested that the use of up-front combination therapies is a strategy for overcoming resistance, where the benefit is the synergistic response of the combined therapy vs the response of the single agent. In addition, combination therapies targeting one or more signalling nodes suppress the survival and emergence of resistant tumour cells.
Chapter 2 goes on to explore 1:1 molar ratio combinations with ceritinib, one of the approved ALK inhibitors. Combinations were explored using an MTT cell viability assay against an isogenic pair of human lung carcinoma cell lines, A549 and A549 EML4-ALK+. Combinations include P2Y2, VEGFR, dual mTOR/PI3K, AKT, PI3K, and JAK inhibitors. It was seen that the pan PI3K inhibitor, pictilisib, exhibited excellent synergy with ceritinib, thus this combination was evaluated further. The combination of ceritinib and pictilisib was investigated using immunoblot analysis, where it was found that the combination perturbs the JAK/STAT pathway. Additionally, cell cycle analysis was used to examine the effects of pictilisib, ceritinib and their combination on the cell cycle, where it was found that the combination induced G1 arrest. Clonogenic assays were performed on the combination, and it was seen that after 24 h exposure, there was no significant reduction in colonies. However, after 72 h exposure a significant reduction in colony formation was observed. An annexin-V FITC/PI apoptosis assay showed that after 72 h exposure neither ceritinib, pictilisib, nor their combination induce apoptosis, which is indicative of cytostasis.
Chapter 3 brings the focus towards PI3K inhibitors, due to the success of the pan PI3K inhibitor pictilisib in combination with ceritinib. The chapter provides an in-depth introduction to PI3K, and a review of the literature around the design and synthesis of selective inhibitors to the four Class I isoforms of PI3K (α, β, δ, and γ). Following this introduction, an array of PI3Kδ inhibitors with a thienopyrimidine core were designed and synthesised. The chemical synthesis of the series, the structure activity relationships and the physicochemical properties are discussed in detail. The highlight of the series is compound 3, a highly potent and highly selective PI3Kδ inhibitor, which was tested in combination with ceritinib by MTT cell viability assay. A further two series of benzothiazole containing inhibitors were designed and synthesised, where their chemical synthesis, structure activity relationships, and physicochemical properties are discussed in detail. The highlight of these series’ is compound 28, a potent and selective PI3Kγ inhibitor, which was tested in combination with ceritinib by MTT cell viability assay. Additionally, a PI3Kα inhibitor, alpelisib, was evaluated in combination with ceritinib by MTT cell viability assay. The combinations of the selective α, δ, and γ PI3K inhibitors did not exhibit the same level of synergy as observed with the pan PI3K inhibitor pictilisib, and thus, the combination with pictilisib was selected to be incorporated into a bifunctional ligand.
Chapter 4 discusses the design of bifunctional inhibitors. The chapter begins with a brief introduction to bifunctional ligands, linked together via a linker, although all examples in the current literature are of covalent linkers. With anti-cancer therapies, including ceritinib and pictilisib, exhibiting severe side effects, a reduction of dose is highly desirable. One strategy for lowering dosage is through drug delivery to the site of action via controlled release i.e. in the form of a prodrug. In this chapter, the combination therapy of ceritinib and pictilisib were brought together into a bifunctional ligand, where the inhibitors are linked via a pH dependent cleavable linker, introducing a tunable method for controlled release, since cancer cells can have a range of pH states (5.7 - 7.8), mainly due to hypoxia. The design of these prodrugs began with model systems of ceritinib and pictilisib linked together with novel linkers . The synthesis and kinetic studies of these systems are discussed in detail. Additionally, a mechanism of release is proposed. Following this, synthesis progressed to linking pictilisib and ceritinb to give AEG-118, which was shown to simultaneously release the active pay-loads, ceritinib and pictilisb, in a pH dependant manner.
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