Sviluppo del saggio enzimatico e caratterizzazione cinetica di nuovi inibitori di Fosfatidilinositolo 4-chinasi IIIβ come agenti antivirali ad ampio spettro.

Phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) is a lipid kinase that belongs to the phosphatidylinositol 4-kinases (PI4Ks) family. This family synthesizes phosphatidylinositol 4-phosphate (PI4P) from phosphatidylinositol (PI). PI4P is involved in signaling and cellular trafficking mainly at the Golgi and trans-Golgi network, to contributes to the definition of the characteristics of plasma membranes, and it activates a variety of ion channels. In mammals four PI4K isoforms are present, classified as type II (PI4KIIα and PI4KIIβ) or type III (PI4KIIIα and PI4KIIIβ) based on their primary sequences and catalytic properties. Type III PI4Ks are hijacked by several ss(+)RNA viruses to remodel cellular membranes and generate PI4P lipid-enriched organelles specialized for viral replication. Given the essential role of PI4KIIIβ in RNA replication of Enteroviruses, this kinase represents a valuable target for antiviral therapy. A major aim in the development of PI4KIIIs inhibitors is to achieve selective inhibition of the α or β isoforms. To date, there is just one known inhibitor- PIK93, which potently inhibits the β isoform, however it also displays detectable activity towards the α isoform, as well as towards PI3-kinases. In addition, only a limited number of methods to detect PI4KIIIβ kinase activity are available, based on laborious substrate precipitation or plate-filter based assay. The aim of my thesis was to develop a novel PI4KIIIβ assay and optimize the experimental conditions, in order to screen for novel potent inhibitors against this kinase. We have investigated different agents active as broad-spectrum antivirals by targeting PI4KIIIβ. These putative inhibitors were analyzed through the novel kinetic assay that we have developed and consequently optimized. The residual activity of PI4KIIIβ kinase was measured by the detection of [γ33P] ATP incorporated into the appropriate lipidic substrate immobilized on the filter by means of liquid scintillation. Moreover, we were able to adapt this assay also to PI4KIIIα and PIK3C kinases that were utilized to analyze the selectivity of the promising inhibitors against PI4KIIIβ. Thereafter, the most potent inhibitors were investigated for the putative (ir)reversible binding to the kinase.