C-4 analogs of podophyllotoxin inhibit tubulin polymerization: A generalizable platform for the discovery of antimitotic therapeutics for cancer

Antimitotic agents, such as podophyllotoxin and its derivatives, act through a ubiquitous mechanism of action by targeting the most fundamental mechanisms of cell division, and have demonstrated broad clinical potential as anticancer therapeutics. Given the pharmacological importance of the C-4 hydroxyl on podophyllotoxin, we sought to establish a structure-activity relationship between chemical modification and in vitro potency. We previously reported that increased C-4 sterics of podophyllotoxin esters have minimal effect on cell-free tubulin polymerization, yet decrease in vitro potency against human colon cancer cells. In this study, the antiproliferative activity of 26 novel carbonate, carbamate, and silyl ether podophyllotoxin analogs was evaluated for inhibition of in vitro cell viability, cell cycle arrest, cell-free tubulin polymerization, immunofluorescence imaging, and docking models. In human and murine colorectal cancer (HCT-116, HT-29, CT-26), lung cancer (A549, Calu-1), triple-negative breast cancer (MDA-MB-231 P), liver carcinoma (Hep G2), ovarian adenocarcinoma (SK-OV-3), and embryonic kidney cells (HEK-293), the carbonate analogs exhibited greater antiproliferative activity when compared to their carbamate counterparts, while the silyl ether analogs performed the poorest. Most notably, the t-butyl carbonate exhibited the most potent activity with IC50 at or below single digit nanomolar ranges, while the carbamates exhibited IC50 values greater than 42 nM. Correspondingly, the most potent analogs also exhibited potent cell cycle G2/M arrest by flow cytometry, with t-butyl carbonate causing arrest to the greatest percentage (69.2%), affirming tubulin inhibition as the primary mechanism of action. However, those differences were not reflected in the cell-free biochemical assay, where the best carbonates and carbamates showed similar percentages of tubulin inhibition (85.78% and 85.28% respectively), and no correlation was observed between C-4 substituent and tubulin inhibition. Additionally, in silico modeling revealed a minimal difference in each analog's binding affinity, providing further evidence that C-4 functionalization does not directly alter a compound's ability to bind to tubulin. Collectively, our results suggest that absolute binding to tubulin is not the primary determinant of biological activity for C-4 analogs of podophyllotoxin across a broad panel of cancer cell lines, and that the impact of structural changes at C-4 on biological potency is agnostic to logP and steric bulk. Altogether, the structure-activity relationship described enables future development of podophyllotoxin-based antimitotic agents for cancer.