Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin with tubulin degradation effect

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.

Utidelone inhibits growth of colorectal cancer cells through ROS/JNK signaling pathway

Utidelone (UTD1), a novel microtubule stabilizing agent, is an epothilone B analogue which was produced by genetic engineering. UTD1 has exhibited broad antitumor activity in multiple solid tumors. However, its activity and mechanism in colorectal cancer (CRC) remain to be studied. In this study, UTD1 dramatically inhibited CRC cell proliferation (with 0.38 µg/ml, 0.77 µg/ml IC50 in RKO and HCT116, respectively) in vitro. Immunofluorescence staining showed that UTD1 induced the formation of microtubule bundling and asters in RKO cells. Flow cytometry analysis demonstrated that UTD1 induced cell cycle to arrest in G2/M phase, subsequent apoptosis. Significantly, UTD1 exhibited stronger effect on inducing apoptosis than paclitaxel and 5-FU, especially in HCT15 cells which is ABCB1 high-expression. UTD1 exposure cleaved caspase-3 and poly ADP-ribose polymerase (PARP), decreased mitochondrial membrane potential, released cytochrome c, increased the production of active oxygen and activated c-Jun N-terminal kinase (JNK), suggesting ROS/JNK pathway was involved in this process. Moreover, UTD1 inhibited tumor growth and was more effective and safer compared with paclitaxel and 5-FU in RKO xenograft in nude mice. Taken together, our findings first indicate that UDT1 inhibits tumor growth in CRC xenograft model and may be a promising agent for CRC treatment.

Triazoloacridone C-1305 impairs XBP1 splicing by acting as a potential IRE1α endoribonuclease inhibitor

Inositol requiring enzyme 1 alpha (IRE1α) is one of three signaling sensors in the unfolding protein response (UPR) that alleviates endoplasmic reticulum (ER) stress in cells and functions to promote cell survival. During conditions of irrevocable stress, proapoptotic gene expression is induced to promote cell death. One of the three signaling stressors, IRE1α is an serine/threonine-protein kinase/endoribonuclease (RNase) that promotes nonconventional splicing of XBP1 mRNA that is translated to spliced XBP1 (XBP1s), an active prosurvival transcription factor. Interestingly, elevated IRE1α and XBP1s are both associated with poor cancer survival and drug resistance. In this study, we used next-generation sequencing analyses to demonstrate that triazoloacridone C-1305, a microtubule stabilizing agent that also has topoisomerase II inhibitory activity, dramatically decreases XBP1s mRNA levels and protein production during ER stress conditions, suggesting that C-1305 does this by decreasing IRE1α’s endonuclease activity.

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin and provides insights into microtubule dynamic instability

Microtubule, composed of αβ-tubulin heterodimers, remains as one of the most popular anticancer targets for decades. To date, anti-microtubule drugs mainly target β-tubulin to inhibit microtubule dynamic instability (MDI) while agents binding to α-tubulin are less well characterized and also the molecular mechanism of MDI is far from being articulated. Cevipabulin, an oral microtubule-active antitumor clinical candidate, is widely accepted as a microtubule stabilizing agent (MSA) but binds to the microtubule-destabilization vinblastine site on β-tubulin and this unusual phenomenon has so far failed to be explained. Our X-ray crystallography study reveals that, in addition binding to the vinblastine site, cevipabulin also binds to a novel site on α-tubulin (named the seventh site) which located at the region spatially corresponding to the vinblastine site on β-tubulin. Interestingly, cevipabulin exhibits two unique site-dependent functions. Cevipabulin binding to the seventh site promotes tubulin degradation through interaction of the non-exchengeable GTP to reduce tubulin stability. Cevipabulin binding to the vinblastine site enhances longitudinal interactions but inhibits lateral interactions of tubulins, thus inducing tubulin protofilament polymerization (but not microtubule polymerization like MSAs), and then tangling into irregular tubulin aggregates. Importantly, the tubulin-cevipabulin structure is an intermediate between “bent” and “straight” tubulins and the involved bent-to-straight conformation change will be helpful to fully understand the molecular mechanism of tubulin assembly. Our findings confirm cevipabulin is not an MSA and shed light on the development of a new generation of anti-microtubule drugs targeting the novel site on α-tubulin and also provide new insights into MDI.

Comparison of efficacy and safety of second-line palliative chemotherapy with paclitaxel plus raltitrexed and paclitaxel alone in patients with metastatic gastric adenocarcinoma: a randomized phase 2 clinical trial

BackgroundPaclitaxel is a microtubule stabilizing agent, used as standard second line chemotherapy in the treatment of advanced gastric cancer. This study was designed to compare the clinical outcome of paclitaxel plus raltitrexed regimen as second line treatment in MGC patients.MethodsAn open, randomized, multi centers phase II clinical trial. 148 patients were randomly assigned and treated with either RP (raltitrexed 3 mg/m2 day1 and paclitaxel 135 mg/m2 day1, 3week) or P (paclitaxel 135 mg/m2 day1, 3week) as second-line palliative chemotherapy. The primary endpoint is PFS; secondary endpoint is ORR, OS and safety.ResultsProgression free survival has a tendency to be prolonged with RP versus P (2.7month vs. 1.7month, p = 0.148). Overall survival has also a tendency to be prolonged with RP versus P (10.2month vs. 6.1month, p = 0.140). Overall response rate was equal with RP versus P (6.8% vs.4.0%, p = 0.72). DCR in the RP and P group was 56.2% and 36.0% respectively. Grade 3 to 4 treatment-related adverse events occurred in 36.2% (RP) vs. 28.2% (P) of patients. Frequent grade 3 to 4 toxicities for RP vs. P were: neutropenia (11.0% vs. 4.0%), anemia (1.4% vs. 4.0%), thrombocytopenia (1.4% vs. 5.3%), and all grade peripheral neurotoxicity (12.3% vs. 17.3%). All grades found with elevated aminotransferase (27.4% vs. 14.1%). Subgroup analysis shows if the disease combined with ascites or peritoneal involved OS of RP regimen is longer (p = 0.05).ConclusionsSecond-line palliative chemotherapy with paclitaxel plus raltitrexed have tendency to prolong PFS and OS, especially some patients with ascites or peritoneal involved, which needs to be confirmed by larger sample studies.Trial registrationNCT02072317. Registered 26 February 2014

Two Antagonistic Microtubule Targeting Drugs Act Synergistically to Kill Cancer Cells

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for pharmaceutics which could potentiate its therapeutic effect. We screened a chemical library and selected Carba1, a carbazole, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. Catastrophe induction by Carba1 promotes paclitaxel binding to microtubule ends, providing a mechanistic explanation of the observed synergy. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel binding to dynamic microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

Two antagonistic microtubule targeting drugs act synergistically to kill cancer cells

Paclitaxel is a microtubule stabilizing agent and a successful drug for cancer chemotherapy inducing, however, adverse effects. To reduce the effective dose of paclitaxel, we searched for drugs which could potentiate its therapeutic effect. We have screened a chemical library and selected Carba1, a carbazolone, which exerts synergistic cytotoxic effects on tumor cells grown in vitro, when co-administrated with a low dose of paclitaxel. Carba1 targets the colchicine binding-site of tubulin and is a microtubule-destabilizing agent. The Carba1-induced modulation of microtubule dynamics increases the accumulation of fluorescent paclitaxel inside microtubules, providing a mechanistic explanation of the observed synergy between Carba1 and paclitaxel. The synergistic effect of Carba1 with paclitaxel on tumor cell viability was also observed in vivo in xenografted mice. Thus, a new mechanism favoring paclitaxel accumulation in microtubules can be transposed to in vivo mouse cancer treatments, paving the way for new therapeutic strategies combining low doses of microtubule targeting agents with opposite mechanisms of action.

New Zampanolide Mimics: Design, Synthesis, and Antiproliferative Evaluation

Zampanolide is a promising microtubule-stabilizing agent (MSA) with a unique chemical structure. It is superior to the current clinically used MSAs due to the covalent nature of its binding to β-tubulin and high cytotoxic potency toward multidrug-resistant cancer cells. However, its further development as a viable drug candidate is hindered by its limited availability. More importantly, conversion of its chemically fragile side chain into a stabilized bioisostere is envisioned to enable zampanolide to possess more drug-like properties. As part of our ongoing project aiming to develop its mimics with a stable side chain using straightforward synthetic approaches, 2-fluorobenzyl alcohol was designed as a bioisosteric surrogate for the side chain based on its binding conformation as confirmed by the X-ray structure of tubulin complexed with zampanolide. Two new zampanolide mimics with the newly designed side chain have been successfully synthesized through a 25-step chemical transformation for each. Yamaguchi esterification and intramolecular Horner–Wadsworth–Emmons condensation were used as key reactions to construct the lactone core. The chiral centers at C17 and C18 were introduced by the Sharpless asymmetric dihydroxylation. Our WST-1 cell proliferation assay data in both docetaxel-resistant and docetaxel-naive prostate cancer cell lines revealed that compound 6 is the optimal mimic and the newly designed side chain can serve as a bioisostere for the chemically fragile N-acetyl hemiaminal side chain in zampanolide.