Chemical dissection of the cell cycle: probes for cell biology and anti-cancer drug development

Cancer cell proliferation relies on the ability of cancer cells to grow, transition through the cell cycle, and divide. To identify novel chemical probes for dissecting the mechanisms governing cell cycle progression and cell division, and for developing new anti-cancer therapeutics, we developed and performed a novel cancer cell-based high-throughput chemical screen for cell cycle modulators. This approach identified novel G1, S, G2, and M-phase specific inhibitors with drug-like properties and diverse chemotypes likely targeting a broad array of processes. We further characterized the M-phase inhibitors and highlight the most potent M-phase inhibitor MI-181, which targets tubulin, inhibits tubulin polymerization, activates the spindle assembly checkpoint, arrests cells in mitosis, and triggers a fast apoptotic cell death. Importantly, MI-181 has broad anti-cancer activity, especially against BRAF^V600E melanomas.The cell cycle is a set of coordinated events that culminate in the formation of two cells from one mother cell. It''s composed of four major phases; G1 (growth phase 1), S (DNA synthesis phase), G2 (growth phase 2) and M (mitosis), which function to integrate environment sensing signaling pathways with cell growth and proliferation.¹ Cancer cells often deregulate the cell cycle and undergo unscheduled cell divisions, therefore inhibition of the cell cycle represents an opportunity for therapeutic intervention in treating proliferative diseases like cancer.² Most anti-cancer drugs perturb the proliferation cycle of tumor cells by inhibiting/damaging cell cycle events, which activate checkpoints, arrest cells and induce apoptosis.³ For example, inhibitors targeting DNA replication (5-fluorouracil) and cell division (microtubule-stabilizing paclitaxel) have been used successfully for treating a broad array of cancers including breast and colorectal cancers.² Nevertheless, due to toxicity issues, drugs targeting the cell division machinery like mitotic kinases (AurKA/B and Plk1) and kinesins (Kif11 and CENP-E) have been developed.³ However, these drugs have shown limited efficacy in vivo.⁴ Thus, there is a critical need to identify novel drug-like molecules that inhibit cancer cell cycle progression, which can be developed into novel cancer therapies.Genome wide studies aimed at depleting the expression of human genes and characterizing their contribution to cell cycle progression have generated a wealth of information regarding the enzymatic machinery required for proliferation.⁵ These enzymes have become the focus of targeted screening campaigns aimed at finding inhibitors to their activities. For example, an in vitro chemical screen targeting Plk1 identified the small molecule BI2536.⁶ BI2536 was not only used to define novel roles for Plk1 during cell division, it was further developed into an anti-cancer drug whose efficacy is being evaluated in clinical trials.⁷ Therefore, beyond their therapeutic potential, inhibitors can be used as molecular probes for dissecting the function of enzymes critical for cell cycle progression in an acute and temporal manner. However, there are no inhibitors to the majority of the cell cycle machinery and the discovery and characterization of such inhibitors would aid our ability to understand the mechanisms regulating cell division.Although molecularly targeted screens have grown in popularity, they rely on the previous identification and validation of specific cancer targets with druggable activities/interactions.⁸ As an alternative, unbiased high-throughput chemical screens have tried to identify inhibitors to a single cell cycle phase,^{9, 10, 11, 12, 13, 14, 15} which limited their ability to identify novel anti-proliferative agents to other phases of the cell cycle. Nonetheless, G2-phase, M-phase, and cytokinesis screens successfully identified inhibitors to Kif11, Plk1, RhoA, and microtubules.^{9, 10, 11, 12, 13, 14, 15} These inhibitors aided the functional characterization of these proteins and were instrumental for developing drugs with therapeutic potential. However, these screens were conducted with a limited number of compounds (100–38 000) or cell extract fractions, with several screens using the same library of 16 320 compounds, thus limiting compound diversity, chemical coverage, and opportunities for novel discoveries. Most screens also lacked chemical analyses to understand the physiochemical properties of bioactive compounds and their cellular targets. In addition, previous screens have not analyzed the four phases of the cell cycle as a biological system. Thus, there is a critical need to develop new screening strategies to discover novel anti-cancer drugs.This, prompted us to establish an integrated high-throughput screening cell-based strategy for identifying small molecule cell cycle modulators, for use in dissecting the mechanisms of cancer cell division, and for developing novel cancer therapies. We report the development of this novel cell-based screening platform, the discovery of cell cycle phase specific inhibitors, the chemical analyses of these inhibitors, the cell culture characterization of cell division inhibitors, and the detailed examination of MI-181, which has potent anti-cancer activity, especially against melanomas.