Plinabulin (NPI-2358) (NPI-2358) is a vascular disrupting agent (VDA) against tubulin-depolymerizing tumor cells ( IC50: 9.8-18 nM). Plinabulin selectively targets and binds to the colchicine-binding site of tubulin, thereby interrupting equilibrium of microtubule dynamics. This disrupts mitotic spindle assembly leading to cell cycle arrest at M phase and blockage of cell division.

NPI-2358 induces a reduction in tumor perfusion in a time- and dose-dependent manner. In mice carrying human plasmacytoma xenografts, NPI-2358 (7.5 mg/kg) significantly inhibits tumor growth. Compared to its efficacy in C3H tumors, NPI-2358 demonstrates superior activity against KHT sarcomas, and its anticancer effectiveness is enhanced when combined with radiation therapy.

In human umbilical vein endothelial cells, NPI-2358 (20 nM) rapidly induces the depolymerization of microtubule proteins and penetrates the monolayer of cells. It arrests MM cells in the early phases of mitosis and induces cell death. NPI-2358 binds to the colchicine binding site of microtubule proteins, effectively inhibiting human tumor cell lines that overexpress Pgp, or reducing the catalytic activity of nuclear Topo II (IC50: 9.8-18 nM). Moreover, it inhibits microtubule formation and the migration of endothelial and MM cells, leading to dysfunction in the tumor vasculature. NPI-2358 induces mitotic arrest or death in MM cells, but this effect can be deactivated by blocking the JNK pathway.

The adherent cells are plated in 96-well flat-bottomed plates and allowed to attach for 24 hours at 37 °C. HL-60 and HL-60/MX2 cells are plated in 96-well plates on the day of NPI-2358 addition. Serially diluted NPI-2358 is added to cells at concentrations ranging from 2 pM to 20 μM. Cells treated with a final concentration of 0.25% (v/v) DMSO serves as the vehicle control. Cell viability is assessed 48 hours later by measuring the reduction of resazurin with a fluorimeter. The IC50 value is calculated.(Only for Reference)