Structural convergence for tubulin binding of CPAP and vinca domain microtubule inhibitors

Microtubule dynamics is regulated by various cellular proteins and perturbed by small molecule compounds. To what extent the mechanism of the former resembles that of the latter is an open question. We report here structures of tubulin bound to the PN2-3 domain of CPAP, a protein controlling the length of the centrioles. We show that an α-helix of the PN2-3 N-terminal region binds and caps the longitudinal surface of the tubulin β subunit. Moreover, a PN2-3 N-terminal stretch lies in a β-tubulin site also targeted by fungal and bacterial peptide-like inhibitors of the vinca domain, sharing a very similar binding mode with these compounds. Therefore, our results identify several characteristic features of cellular partners that bind to this site and highlight a structural convergence of CPAP with small molecules inhibitors of microtubule assembly.

Characterization of Microtubule Destabilizing Drugs: A Quantitative Cell-Based Assay That Bridges the Gap between Tubulin Based- and Cytotoxicity Assays

(1) Background: Microtubule depolymerizing agents (MDAs) are commonly used for cancer treatment. However, the therapeutic use of such microtubule inhibitors is limited by their toxicity and the emergence of resistance. Thus, there is still a sustained effort to develop new MDAs. During the characterization of such agents, mainly through in vitro analyses using purified tubulin and cytotoxicity assays, quantitative comparisons are mandatory. The relationship between the effect of the drugs on purified tubulin and on cell viability are not always direct. (2) Methods: We have recently developed a cell-based assay that quantifies the cellular microtubule content. In this study, we have conducted a systematic comparative analysis of the effect of four well-characterized MDAs on the kinetics of in vitro tubulin assembly, on the cellular microtubule content (using our recently developed assay) and on cell viability. (3) Conclusions: These assays gave complementary results. Additionally, we found that the drugs’ effect on in vitro tubulin polymerization is not completely predictive of their relative cytotoxicity. Their effect on the cellular microtubule content, however, is closely related to their effect on cell viability. In conclusion, the assay we have recently developed can bridge the gap between in vitro tubulin assays and cell viability assays.

Synthesis and Investigations of Building Blocks with Dibenzo[b,f] Oxepine for Use in Photopharmacology

The synthesis of photoswitchable azo-dibenzo[b,f]oxepine derivatives and microtubule inhibitors were described. Subsequently, we examined the reaction of methoxy derivative 3-nitrodibenzo[b,f]oxepine with different aldehydes and in the presence of BF3·OEt2 as a catalyst. Our study provided a very concise method for the construction of the azo-dibenzo[b,f]oxepine skeleton. The analysis of products was run using experimental and theoretical methods. Next, we evaluated the E/Z isomerization of azo-dibenzo[b,f]oxepine derivatives, which could be photochemically controlled using visible-wavelength light.

Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore represent promising drug targets. While αβ-tubulin, the molecular building block of microtubules, is an established drug target in a variety of cancers, we still lack substantial knowledge of the biochemistry of parasite tubulins, which would allow us to exploit the structural divergence between parasite and human tubulins. Indeed, mechanistic insights have been limited by the lack of purified, functional parasite tubulin. In this study, we isolated Plasmodium falciparum tubulin that is assembly-competent and shows specific microtubule dynamics in vitro. We further present mechanistic evidence that two compounds selectively interact with parasite over host microtubules and inhibit Plasmodium microtubule polymerization at substoichiometric compound concentrations. The ability of compounds to selectively disrupt protozoan microtubule growth without affecting human microtubules provides the exciting possibility for the targeted development of novel antimalarials.

Microtubule inhibitors enhance DNA transfection efficiency through autophagy receptor p62/SQSTM1

Ectopic gene expression is an indispensable tool in biology and medicine. However, it is often limited by the low efficiency of DNA transfection. It is known that depletion of p62/SQSTM1 enhances DNA transfection efficiency by preventing the degradation of transfected DNA. Therefore, p62 is a potential target of drugs to increase transfection efficiency. To identify drugs that enhance transfection efficiency, a non-biased high-throughput screening was applied to over 4,000 compounds from the Osaka University compound library, and their p62-dependency was evaluated. The top-scoring drugs were mostly microtubule inhibitors, such as colchicine and vinblastine, and all of them showed positive effects only in the presence of p62. To understand the mechanisms, the time of p62-dependent ubiquitination was examined using polystyrene beads that were introduced into cells as materials that mimicked transfected DNA. The microtubule inhibitors caused a delay in ubiquitination. Furthermore, the level of phosphorylated p62 at S405, which is required for ubiquitination during autophagosome formation, markedly decreased in the drug-treated cells. These results suggest that microtubule inhibitors inhibit p62-dependent autophagosome formation. Our findings provide new insights into the mechanisms of DNA transfection and also provide a solution to increase DNA transfection efficiency.

Discovery, characterization and functional improvement of kumamonamide as a novel plant growth inhibitor that disturbs plant microtubules

The discovery and useful application of natural products can help improve human life. Chemicals that inhibit plant growth are broadly utilized as herbicides to control weeds. As various types of herbicides are required, the identification of compounds with novel modes of action is desirable. In the present study, we discovered a novelN-alkoxypyrrole compound, kumamonamide fromStreptomyces werraensisMK493-CF1 and established a total synthesis procedure. Resulted in the bioactivity assays, we found that kumamonamic acid, a synthetic intermediate of kumamonamide, is a potential plant growth inhibitor. Further, we developed various derivatives of kumamonamic acid, including a kumamonamic acid nonyloxy derivative (KAND), which displayed high herbicidal activity without adverse effects on HeLa cell growth. We also detected that kumamonamic acid derivatives disturb plant microtubules; and additionally, that KAND affected actin filaments and induced cell death. These multifaceted effects differ from those of known microtubule inhibitors, suggesting a novel mode of action of kumamonamic acid, which represents an important lead for the development of new herbicides.

The Frequent Sampling of Wound Scratch Assay Reveals the “Opportunity” Window for Quantitative Evaluation of Cell Motility-Impeding Drugs

Wound healing assay performed with automated microscopy is widely used in drug testing, cancer cell analysis, and similar approaches. It is easy to perform, and the results are reproducible. However, it is usually used as a semi-quantitative approach because of inefficient image segmentation in transmitted light microscopy. Recently, several algorithms for wound healing quantification were suggested, but none of them was tested on a large dataset. In the current study, we develop a pipeline allowing to achieve correct segmentation of the wound edges in >95% of pictures and extended statistical data processing to eliminate errors of cell culture artifacts. Using this tool, we collected data on wound healing dynamics of 10 cell lines with 10 min time resolution. We determine that the overall kinetics of wound healing is non-linear; however, all cell lines demonstrate linear wound closure dynamics in a 6-h window between the fifth and 12th hours after scratching. We next analyzed microtubule-inhibiting drugs’, nocodazole, vinorelbine, and Taxol, action on the kinetics of wound healing in the drug concentration-dependent way. Within this time window, the measurements of velocity of the cell edge allow the detection of statistically significant data when changes did not exceed 10–15%. All cell lines show decrease in the wound healing velocity at millimolar concentrations of microtubule inhibitors. However, dose-dependent response was cell line specific and drug specific. Cell motility was completely inhibited (edge velocity decreased 100%), while in others, it decreased only slightly (not more than 50%). Nanomolar doses (10–100 nM) of microtubule inhibitors in some cases even elevated cell motility. We speculate that anti-microtubule drugs might have specific effects on cell motility not related to the inhibition of the dynamic instability of microtubules.

Apoptotic Blocks in Primary Non-Hodgkin B Cell Lymphomas Identified by BH3 Profiling

To determine causes of apoptotic resistance, we analyzed 124 primary B cell NHL samples using BH3 profiling, a technique that measures the mitochondrial permeabilization upon exposure to synthetic BH3 peptides. Our cohort included samples from chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), high-grade B cell lymphoma with translocations in MYC and BCL2 (HGBL-DH), mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL). While a large number of our samples displayed appropriate responses to apoptosis-inducing peptides, pro-apoptotic functional defects, implicating BAX, BAK, BIM or BID, were seen in 32.4% of high-grade NHLs (12/37) and in 3.4% of low-grade NHLs (3/87, p < 0.0001). The inhibition of single anti-apoptotic proteins induced apoptosis in only a few samples, however, the dual inhibition of BCL2 and MCL1 was effective in 83% of samples, indicating MCL1 was the most common cause of lack of response to the BCL2 inhibitor, venetoclax. We then profiled Toledo and OCI-Ly8 high-grade lymphoma cell lines to determine which drugs could reduce MCL1 expression and potentiate venetoclax responses. Doxorubicin and vincristine decreased levels of MCL1 and increased venetoclax-induced apoptosis (all p < 0.05). Overall, in primary NHLs expressing BCL2 that have no defects in pro-apoptotic signaling, a poor response to venetoclax is primarily due to the presence of MCL1, which may be overcome by combining venetoclax with doxorubicin and vincristine-based chemotherapy or with other anti-microtubule inhibitors.