Phd in PHARMACEUTICAL SCIENCES

Thesis title: From Hypoxia to Human Carbonic Anhydrases: design, synthesis, and evaluation of novel pyran-2-one based derivatives as anti-tumour and anti-inflammatory agents

Tumorigenesis is a dynamic process that drives the transformation of a cell into a cancerous one by altering or escaping the mechanisms that control normal cell biology. Tumour development is characterised by uncontrolled cell growth and proliferation leading to a massive amount of oxygen required. The adaptive response to oxygen deprivation, or hypoxia, is the stabilisation of the hypoxia-inducible factor (HIF) resulting in the transcription of HIF target genes such as CAIX and XII (Carbonic Anhydrase isoforms IX and XII). Carbonic Anhydrases (CAs) are ubiquitous metalloenzymes which catalyse the reversible hydration of carbon dioxide to bicarbonate ions, a reaction involved in a plethora of fundamental cell processes. Dysregulation of these enzymes is associated with the development of several pathological conditions. Particularly, the overexpression of the tumour-related isoforms IX and XII in hypoxic tumours has highlighted their role as promising targets for the treatment of cancer. For this reason, different Carbonic Anhydrase Inhibitors (CAIs) have been reported so far. In my PhD project, based on the molecular simplification of the coumarin scaffold that has been widely exploited for selective CA IX and XII inhibitor development, a series of pyran-2-one derivatives have been designed, synthesised, and evaluated as promising molecules for the selective inhibition of the tumour-related isoforms IX and XII. The novel compounds have been endowed with an amide or ester linker, bearing different (un)substituted aromatic or (hetero)(cyclo)aliphatic residues. Moreover, an additional variation by binding a triazole ring substituted with the previously mentioned residues to the linker has been evaluated. The design of the different libraries was aimed at expanding the chemical space exploitable for inhibitory activity. Furthermore, the different physic-chemical properties of the substituents allow the evaluation of the different interactions occurring between the compounds and the active site of the enzyme. All the compounds exhibited a selective inhibitory activity against the tumour-related human Carbonic Anhydrase isoforms IX and XII. In particular, the derivatives endowed with the amide linker showed to be the most potent and selective inhibitors among the series with inhibition values in the low nanomolar range. For this reason, the possible binding mode of these compounds to the active site of the hCA IX was investigated by molecular docking and molecular dynamics (MD) simulations. Moreover, the compounds exhibiting the best results in the hCA inhibition assay were further investigated against breast adenocarcinoma cell line (MCF7) in mimic hypoxic conditions, evaluating their ability to synergize with doxorubicin. Collectively, the obtained results underline the potential of the pyran-2-one core for the development of selective hCA IX and XII inhibitors thus being a promising scaffold for the development of anti-tumour agents. Furthermore, the pro-tumour effects of inflammatory cells (i.e., angiogenesis, DNA damage, extracellular matrix remodelling, and host defence avoidance) have prompted the development of novel CAI-CORM hybrids based on the pyran-2-one scaffold (CAI) and the dicobalt hexacarbonyl (DCH) metal core (CORM). CO-releasing molecules (CORMs) are systemic carriers able to deliver and release carbon monoxide (CO) to the targeted site in a controlled manner. The bioactivities of this gasotransmitter vary from anti-inflammatory to antiapoptotic effects. Moreover, the CO dose-dependent stabilisation of the hypoxia-inducible factor (HIF) may be translated into therapeutical applications. For this reason, the novel CAI-CORMs could enhance the therapeutic effect of the single entities resulting in a putative synergic anti-tumoral and anti-inflammatory effect. Through a spectrophotometric-based assay, aimed at exploring the influence of CAI fragments on CO-releasing profiles, the CO-releasing rate was assessed showing that the compounds are effective CORM, being able to ensure a fast and significative CO release over time. During my abroad experience, in the research group of Professor Sir Peter Ratcliffe at the University of Oxford, I collaborated on a project aimed at determining the tissue specificity, precisely of the clear cell renal cell carcinoma (ccRCC), driven by the von-Hippel Lindau tumour suppressor gene (VHL in human, Vhl in mice) deletion. However, studying tumorigenesis driven by VHL loss requires a system in which gene loss can be precisely induced and monitored in real-time in vivo. For this reason, the project depended on a novel lineage-marking mouse model of Vhl-inactivation able to report gene loss accurately in vivo. My work aimed to study the earliest events occurring in the kidney after Vhl-loss based on the initial hypothesis that the tissue-specific tumorigenicity of VHL results from VHL-inactivated cells facing opposite cell fates in different tissues. The comparison between Control and KO mice has enabled the investigation of how many Vhl-null persist in different parts of the kidney and whether they proliferate and evolve to a tumorigenic fate.