X‑ray Crystal Structure-Guided Discovery of Novel Indole Analogues as Colchicine-Binding Site Tubulin Inhibitors with Immune-Potentiating and Antitumor Effects against Melanoma

A series of novel indole analogues were discovered as
colchicine-binding
site inhibitors of tubulin. Among them, 3a exhibited
the highest antiproliferative activity (average IC50 =
4.5 nM), better than colchicine (IC50 = 65.3 nM). The crystal
structure of 3a in complex with tubulin was solved by
X-ray crystallography, which explained the improved binding affinity
of 3a to tubulin and thus its higher anticancer activity
(IC50 = 4.5 nM) than the lead compound 12b (IC50 = 32.5 nM). In vivo, 3a (5 mg/kg) displayed significant antitumor efficacy against B16-F10
melanoma with a TGI of 62.96% and enhanced the antitumor efficacy
of a small-molecule PD-1/PD-L1 inhibitor NP19 (TGI = 77.85%). Moreover, 3a potentiated the antitumor immunity of NP19 by activating
the tumor immune microenvironment, as demonstrated by the increased
tumor-infiltrating lymphocytes (TIL). Collectively, this work shows
a successful example of crystal structure-guided discovery of a novel
tubulin inhibitor 3a as a potential anticancer and immune-potentiating
agent.