Yang Xie

Supplementary Figure S1 shows the 1H NMR spectrum of QW-5-70.

Supplementary Figure S3 shows HPLC analysis confirming purity of QW-5-70.

Supplementary Figure S2 shows the HRMS spectrum of QW-5-70.

Drug resistance driven by efflux transporters and altered tubulin dynamics limits the clinical efficacy of taxanes and vincristine in high-risk neuroblastoma and castration-resistant prostate cancer. We developed QW-5-70, a colchicine binding site inhibitor (CBSI), engineered to retain low-nanomolar potency in both parental and drug-resistant cancer cells by showing minimal dependence on P-glycoprotein (P-gp)–mediated efflux. QW-5-70 binds to the colchicine site on tubulin, inhibits tubulin polymerization, disrupts microtubule networks, and induces mitotic arrest. Across a panel of neuroblastoma and prostate cancer lines, QW-5-70 maintained low-nanomolar activity and remained effective in vincristine-resistant BE2C/VCR and paclitaxel-resistant PC-3/TxR cells. Unlike vincristine and paclitaxel, the activity and intracellular accumulation of QW-5-70 were unaffected by pharmacologic or genetic inhibition of P-gp, indicating effective circumvention of efflux-mediated resistance. In vitro, QW-5-70 significantly reduced colony formation and impaired migration in both parental and resistant cancer cells and induced G2–M cell-cycle arrest and mitochondrial apoptosis. In vivo, QW-5-70 significantly suppressed tumor growth in drug-resistant PC-3/TxR and BE2C/VCR xenografts, with modest weight loss and no evident histopathology in the major organs. Combination studies revealed combination index–defined synergistic interactions at selected dose pairs with the ornithine decarboxylase inhibitor α-difluoromethylornithine and enhanced apoptotic and clonogenic suppression when combined with the Aurora A kinase inhibitor MLN8237. Collectively, QW-5-70 is a potent CBSI that circumvents P-gp–associated resistance, triggers mitotic arrest and apoptosis, and achieves significant antitumor activity in multidrug-resistant tumor models with acceptable tolerability, supporting its further preclinical development alone and in combination with other drugs.

Supplementary Figure S4 shows immunofluorescence analysis of microtubule disruption and acetylated α-tubulin loss induced by QW-5-70.

Supplementary Figure S8 shows in vivo effects of QW-5-70 on proliferation, angiogenesis, and apoptosis markers.

Supplementary Figure S5 shows the inhibitory effects of QW-5-70 on colony formation and cell migration.

Supplementary Figure S6 shows ABC transporter expression and the effect of transporter inhibition on drug response.

Supplementary Figure S7 shows cell cycle arrest and apoptosis induced by QW-5-70.

Supplementary Table S4 summarizes IC50 values and resistance indices in parental and resistant cell lines.