KPU-300, a Novel Benzophenone–Diketopiperazine–Type Anti-Microtubule Agent with a 2-Pyridyl Structure, Is a Potent Radiosensitizer That Synchronizes the Cell Cycle in Early M Phase

Abstract

KPU-300 is a novel colchicine-type anti-microtubule agent derived from plinabulin (NPI-2358). We characterized the effects of KPU-300 on cell cycle kinetics and radiosensitization using HeLa cells expressing the fluorescent ubiquitination-based cell cycle indicator (Fucci). Cells treated with 30 nM KPU-300 for 24 h were efficiently synchronized in M phase and contained clearly detectable abnormal Fucci fluorescence. Two-dimensional flow-cytometric analysis revealed a fraction of cells distinct from the normal Fucci fluorescence pattern. Most of these cells were positive for an M phase marker, the phosphorylated form of histone H3. Cells growing in spheroids responded similarly to the drug, and the inner quiescent fraction also responded after recruitment to the growth fraction. When such drug-treated cells were irradiated in monolayer, a remarkable radiosensitization was observed. To determine whether this radiosensitization was truly due to the synchronization in M phase, we compared the radiosensitivity of cells synchronized by KPU-300 treatment and cells in early M phase isolated by a combined method that took advantage of shake-off and the properties of the Fucci system. Following normalization against the surviving fraction of cells treated with KPU-300 alone, the surviving fractions of cells irradiated in early M phase coincided. Taken together with potential vascular disrupting function in vivo, we propose a novel radiosensitizing strategy using KPU-300.

Fluctuation in radioresponse of HeLa cells during the cell cycle evaluated based on micronucleus frequency

“…The loose attachment of cells to dishes during mitosis allows M-phase cells to be selectively detected by the shake-off method [1][2][3] , with quite a high purity (> 90%), which has been used in a wide range of studies 16,17 . However, sustainability of the synchronicity was not fully investigated.…”

Radiosensitization of PC3 Prostate Cancer Cells by 5-Thiocyanato-2′-deoxyuridine

“…The radioprotectant effect of hypoxia (healthy, oxygenated cells surrounding the tumor are more sensitive to ionizing radiation than those targeted by RT) and other radio-resistance factors can be reduced or even eliminated by introducing radiosensitizers, i.e., substances that sensitize cells to radiation [3][4][5]. Various radiosensitization strategies are also being developed, e.g., Okuyama et al reported that HeLa cells could be synchronized in early M-phase after treatment with a novel anti-microtubule agent, KPU-300, consequently leading to significant synergistic decrease of surviving fraction after the subsequent radiation exposure [6]. Due to the fact that the number of radiosensitizers employed in clinics is surprisingly small, it seems rational to look for and test new potentially radiosensitizing molecules.…”

Synthesis of monoalkylidene diketopiperazines and application to the synthesis of barettin

“…While the mono-alkylidene natural product 3 has been prepared several times, 7 medicinal chemistry and the structure-activity relationships were advanced using bis-alkylidene derivatives. 8 This structural modification not only improved stability and enhanced the tubulin-stabilizing bioactivity but also greatly simplified the synthesis and enabled rapid generation of many unsaturated non-natural amino acid derivatives for evaluation. The construction of bis-alkylidene DKP structures is now well established and can be easily accomplished through iterative aldol condensations using bisacetoxyglycine anhydride (5) and aromatic aldehydes (see Fig.…”