Microtubules, composed of αβ-tubulin heterodimers, have remained popular anticancer targets for decades. Six known binding sites on tubulin dimers have been identified thus far, with five sites on β-tubulin and only one site on α-tubulin, hinting that compounds binding to α-tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our x-ray crystallography study reveals that, in addition to binding to the vinblastine site, cevipabulin also binds to a new site on α-tubulin. We find that cevipabulin at this site pushes the αT5 loop outward, making the nonexchangeable GTP exchangeable, which reduces the stability of tubulin, leading to its destabilization and degradation. Our results confirm the existence of a new agent binding site on α-tubulin and shed light on the development of tubulin degraders as a new generation of antimicrotubule drugs targeting this novel site.

A series of pyrrolo [2,3-d]pyrimidine derivatives were prepared and optimized for cytotoxic activities against FLT3-ITD mutant cancer cells. Among them, compound 9u possessed nanomolar FLT3 inhibitory activities and subnanomolar inhibitory activities against MV4-11 and Molm-13 cells. It also showed excellent inhibitory activities in FLT3-ITD-D835V and FLT3-ITD-F691L cells which were resistant to quizartinib. Furthermore, 9u exhibited over 40-fold selectivity toward FLT3 relative to c-Kit kinase, which might reduce myelosuppression toxicity. Cellular assays demonstrated that 9u inhibited phosphorylated FLT3 and downstream signaling factors and also induced cell cycle arrest in the G 0 /G 1 stage and apoptosis in MV4-11 and Molm-13 cells. Oral administration of 9u at 10 mg/kg could achieve rapid tumor extinction in the MV4-11 xenograft model and significantly inhibit the tumor growth in the MOLM-13 xenograft model with a tumor growth inhibitory rate of 96% without obvious toxicity. Additionally, 9u demonstrated high bioavailability (F = 59.5%) and suitable eliminated half-life time (T 1/2 = 2.06 h), suggesting that 9u may be a potent candidate for treating acute myelogenous leukemia.

Purpose: This study was to perform preclinical evaluation of a novel class I and IIb HDAC-selective inhibitor, purinostat mesylate, for the treatment of Ph þ B-cell acute lymphoblastic leukemia (B-ALL).Experimental Design: Biochemical assays were used to test enzymatic activity inhibition of purinostat mesylate. Ph þ leukemic cell lines and patient cells were used to evaluate purinostat mesylate activity in vitro. BL-2 secondary transplantation Ph þ B-ALL mouse model was used to validate its efficacy, mechanism, and pharmacokinetics properties in vivo. BCR-ABL(T315I)-induced primary B-ALL mouse model and PDX mouse model derived from relapsed Ph þ B-ALL patient post TKI treatment were used to determine the antitumor effect of purinostat mesylate for refractory or relapsed Ph þ B-ALL. Long-term toxicity and hERG blockade assays were used to safety evaluation of purinostat mesylate. Results: Purinostat mesylate, a class I and IIb HDAC highly selective inhibitor, exhibited robust antitumor activity in hematologic cancers. Purinostat mesylate at low nanomolar concentration induced apoptosis, and downregulated BCR-ABL and c-MYC expression in Ph þ leukemia cell lines and primary Ph þ B-ALL cells from relapsed patients. Purinostat mesylate efficiently attenuated Ph þ B-ALL progression and significantly prolonged the survival both in BL-2 secondary transplantation model with clinical patient symptoms of Ph þ B-ALL, BCR-ABL(T315I)-induced primary B-ALL mouse model, and PDX model derived from patients with relapsed Ph þ B-ALL post TKI treatment. In addition, purinostat mesylate possesses favorable pharmacokinetics and low toxicity properties.Conclusions: Purinostat mesylate provides a new therapeutic strategy for patients with Ph þ B-ALL, including those who relapse after TKI treatment.

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