Targeting Class III Phosphoinositide 3-Kinase (Pi3k) for Anticancer Drug Discovery

Phosphoinositide 3-kinases (PI3Ks), which are divided into class I, class II and class III PI3Ks, phosphorylate the 3-hydroxyl group of the inositol ring of phosphatidylinositol (PtdIns) lipid substrates. Among PI3Ks, PIK3C3 is the only class III PI3K, which specifically generates phosphatidylinositol 3-phosphate (PtdIns3P). It in turn recruits proteins containing FYVE or PX domains, thereby initiating various complexes at the membranes of endosomes, phagosomes and autophagosomes. There are at least two distinct complexes reported, both contains PIK3C3, Vps15, Beclin-1 plus either Atg14L/Barkor or UVRAG. Atg14L-containing complex participates in the generation of autophagosomes, whereas the UVRAG-containing complex is found at the endosomes. Recent studies have shown that mammalian PIK3C3 is required for the activation of the mTOR/S6K1 pathway, which regulates protein synthesis in response to nutrient availability. On the basis of the crucial roles of autophagy, endosomal maturation and protein synthesis in cancer maintenance and progression, we hypothesize that class III PI3K (PIK3C3) may be a potential target for the discovery of novel anticancer agents. Currently, most of the drugs are pan-PI3K inhibitors, such as 3-methyladenine, wortmannin, LY294002, which have limited potency and have off-target effects on different lipid and protein kinases. SAR405 is reportedly the most potent selective PIK3C3 inhibitor, which has a binding equilibrium constant KD of 1.5 nM. Recently, we identified compound 93, which has a different chemical structure, exhibits better potency than SAR-405 with a KD value of 0.53 nM. Our preliminary data suggested that compound 93 displays better cytotoxicity than SAR405 and 3-methyladenine (3-MA) in bladder cancer cells. Compound 93 also increases the protein level of SQSTM1 and induces lucent vacuoles at the peri-nuclear region, suggesting that blocking PIK3C3 kinase activity affects vesicle trafficking and successfulness of autophagy. Based on the above rationale, we hypothesize that PIK3C3 is a potential target for the discovery of novel anticancer agents. To address this hypothesis, we proposed three aims as listed below: Aim 1: (i) Target validation of PIK3C3, and (ii) investigate the molecular mechanisms of PIK3C3 inhibitors in cancer cells. Compound 93 is selected as our hit compound and we will examine its anticancer activity in more cancer cell lines. Aim 2: Hit-to-lead discovery of specific PIK3C3 inhibitors. The goal of this stage of the work is to produce more potent and selective compounds, which has favorable PK properties adequate to examine their efficacy in animal models. We will also evaluate the combination of our most potential derivative with the clinical therapeutic drugs (chemotherapeutic or targeted therapeutic agents) in cancer cells. Aim 3: After the achievement of the first two aims, several potential compounds will be obtained and the anticancer mechanism will be clearly studied. We will perform animal studies for the determination of in vivo efficacy of lead compounds. The mechanisms associated with observed signaling cascades in vitro will be confirmed. By executing these aims, we expect to achieve the specific goals and discover potential derivatives for advanced development.