Microtubules are dynamic assemblies of ab-tubulin heterodimers and have been recognized as highly attractive targets for cancer chemotherapy. A broad range of agents bind to tubulin and interfere with microtubule assembly. Despite having a long history of characterization, colchicine binding site inhibitors (CBSIs) have not yet reached the commercial phase as anti-cancer drugs to date. We determined the structures of tubulin complexed with a set of structurally diverse CBSIs (lexibulin, nocodazole, plinabulin and tivantinib), among which nocodazole and tivantinib are both binary-function inhibitors targeting cancer-related kinases and microtubules simultaneously. High resolution structures revealed the detailed interactions between these ligands and tubulin. Our results showed that the binding modes of the CBSIs were different from previous docking models, highlighting the importance of crystal structure information in structurebased drug design. A real structure-based pharmacophore was proposed to rationalize key common interactions of the CBSIs at the colchicine domain. Our studies provide a solid structural basis for developing new anti-cancer agents for the colchicine binding site. DatabaseThe atomic coordinates and structure factors for tubulin complexed with lexibulin, nocodazole, plinabulin and tivantinib have been deposited in the Protein Data Bank under accession codes 5CA0, 5CA1, 5C8Y and 5CB4, respectively.

Electron microscopy and crystallography studies of α4β7 integrin reveal the mechanism by which this atypical integrin enables rolling adhesion prior to integrin activation.

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.

As a major component of the cytoskeleton, microtubules consist of αβ-tubulin heterodimers and have been recognized as attractive targets for cancer chemotherapy. Microtubule-stabilizing agents (MSAs) promote polymerization of tubulin and stabilize the polymer, preventing depolymerization. The molecular mechanisms by which MSAs stabilize microtubules remain elusive. Here we report a 2.05 Å crystal structure of tubulin complexed with taccalonolide AJ, a newly identified taxane-site MSA. Taccalonolide AJ covalently binds to β-tubulin D226. On AJ binding, the M-loop undergoes a conformational shift to facilitate tubulin polymerization. In this tubulin–AJ complex, the E-site of tubulin is occupied by GTP rather than GDP. Biochemical analyses confirm that AJ inhibits the hydrolysis of the E-site GTP. Thus, we propose that the β-tubulin E-site is locked into a GTP-preferred status by AJ binding. Our results provide experimental evidence for the connection between MSA binding and tubulin nucleotide state, and will help design new MSAs to overcome taxane resistance.

Integrin α X β 2 functions as complement receptor for iC3b and mediates recognition and phagocytosis of pathogens. We used negative-stain EM to examine the α X β 2 interaction with iC3b. EM class averages of α X β 2 in complex with iC3b define the binding sites on both the integrin and iC3b. iC3b contains C3c and thioester domain moieties linked by a long flexible linker. The binding site is on the key ring of the C3c moiety, at the interface between the MG3 and MG4 domains. Similar complexes are seen between α X β 2 and the C3c fragment. α X β 2 binds through the α X αI domain, on the face known to bear the metal ion-dependent adhesion site, at the opposite end of the αI domain from its site of insertion in the β-propeller domain.

Edited by Wolfgang Peti KXO1 (tirbanibulin or KX2-391) is as a non-ATP-competitive inhibitor of SRC proto-oncogene nonreceptor tyrosine kinase (SRC) and is being clinically investigated for the management of various cancers and actinic keratosis. Recently, KXO1 has also been shown to strongly inhibit tubulin. Interestingly, unlike conventional tubulin-targeting drugs, KXO1 has exhibited low toxicity in preclinical and clinical studies, but the reason for this remains elusive, as are the KXO1-binding site and other details of the interaction of KXO1 with tubulin. Here, cellbased experiments revealed that KXO1 induces tubulin depolymerization and G 2 /M phase cell cycle arrest at low nanomolar concentrations, similar to colchicine, used as a positive control. Results from biochemical experiments, including an N,Nethylenebis(iodoacetamide) competition assay, disclosed that KXO1 binds to the colchicine-binding site on ␤-tubulin, further confirmed by the crystal structure of the tubulin-KXO1 complex at 2.5-Å resolution. A high-quality electron density map of the crystallographic data enabled us to unambiguously determine the position and orientation of KXO1 in the colchicinebinding site, revealing the detailed interactions between KXO1 and tubulin. We also found that KXO1 binds reversibly to purified tubulin, induces a totally reversible cellular effect (G 2 /M cell cycle arrest), and possesses no cellular toxicity 5 days after drug washout, explaining KXO1's low toxicity. In summary, we show that KXO1 binds to the colchicine-binding site of tubulin and resolved the crystal structure of the tubulin-KXO1 complex. Importantly, KXO1's reversible binding to tubulin explains its clinically low toxicity, an insight that could guide further clinical applications of KXO1.

Background: How does the multiple sclerosis therapeutic antibody natalizumab bind to ␣4 integrins? Results: Natalizumab binds the ␣4 ␤-propeller domain outside the ligand binding groove for domain 1 of vascular cell adhesion molecule (VCAM) and non-competitively antagonizes binding. Conclusion: Natalizumab may push domain 2 of VCAM into a non-preferred orientation upon integrin binding. Significance: Positioning of species-specific substitutions outside of ligand-binding sites leads to surprising antibody mechanisms of action.

Atherosclerosis plays a key role in the development of cardiovascular diseases, and is often associated with oxidative stress and local inflammation. Thymol, a major polyphenolic compound in thyme, exhibits antioxidant and anti-inflammatory properties. In this study, we measured the in vitro antioxidant activity of thymol, and investigated the effect of thymol on high-fat-diet-induced hyperlipidemia and atherosclerosis. New Zealand white rabbits were fed with regular chow, high-fat and high-cholesterol diet (HC), T3, or T6 (HC with thymol supplementation at 3 mg/kg/d or 6 mg/kg/d, respectively) for 8 weeks. Aortic intimal thickening, serum lipid parameters, multiple inflammatory markers, proinflammatory cytokines, and atherosclerosis-associated indicators were significantly increased in the HC group but decreased upon thymol supplementation. In summary, thymol exhibits antioxidant activity, and may suppress the progression of high-fat-diet-induced hyperlipidemia and atherosclerosis by reducing aortic intimal lipid lesion, lowering serum lipids and oxidative stress, and alleviating inflammation-related responses.