NOVEL AKT PATHWAY INHIBITORS: AN INTEGRATED APPROACH BETWEEN THEORY AND EXPERIMENT
The AKT kinase signaling pathway regulates growth and survival of cells, and it is linked to the progression of numerous types of human cancers. Several studies have shown that deactivation of Akt can promote inhibition of cancer cell growth. The objective of this study was to investigate the efficacy of novel AKT pathway inhibitors in terms of their ability to deactivate AKT and inhibit human tumor cell growth. These compounds were identified using computer-assisted drug design (CADD) methods. Pharmacophore modeling was used to screen hundreds of small molecule compounds as potential AKT pathway inhibitors. Five commercially available putative AKT pathway inhibitors were identified that had the necessary pharmacophoric features. Three compounds, labeled B, C and D were modeled after solenopsin, an alkaloid from the venom of fire ants and the remaining two, compound A and E were designed after a known Akt inhibitor. Cultured tumorigenic ras-transformed cells (WBras1) and human lung carcinoma cells (H2009) were treated with one of the test compounds at varying non-cytotoxic concentrations, or with the vehicle (DMSO). Akt phosphorylation levels at the Ser473 and Thr308 site were monitored. Among all the cells treated, compound B significantly decreased. phosphorylation at varying time points in both tumorigenic cell types at low micromolar concentrations. Downstream Akt effectors were assessed for alternations in pathway regulatory activity. The results indicated that compounds B and C effectively downregulated Akt pathway activation through reduced phosphorylation of Akt and downstream effectors in the Akt pathway. To determine whether any of the CADD compounds directly inhibit Akt, an in-vitro Akt kinase kit assay was utilized. The study revealed that none of the CADD compounds inhibited Akt phosphorylation at the ATP activation site. In this dissertation, it has been revealed that only compounds B and C decreased cancer cell growth in human lung carcinoma cells. The outcome of this investigation confirmed that by inhibiting activation of Akt, inhibition of cancer cell growth can be achieved. Compound B was the most effective of the 5 compounds in inhibiting AKT phosphorylation. Compound B was also the most effective in inhibiting H2009 cell growth. In addition, we studied and showed that an extract of an anticancer herbal mix (labeled as BAH), comprised of plantain (Musca paradisiaca), yam (Dioscorea rotundata) and fish (Atlantic herring), significantly decreased Akt phosphorylation, blocked cancer cell growth and inhibited the activity of Akt in the presence of ATP. Taken together, this study highlights the utility of CADD in identifying promising anti-cancer agents and for the first time unveils novel solenopsin derivatives that hamper the activation of Akt, including alteration of Akt signaling in the pathway. The inhibitory effect in vitro of a proprietary anticancer herbal extract for the first time in human lung cancer cells is also demonstrated.