Recent Advances in Combretastatin A-4 Inspired Inhibitors of Tubulin Polymerization-An Update

Cancer is one of the leading causes of fatality and mortality worldwide. Investigations on developing therapeutic strategies for cancer are supported throughout the world. The massive achievements in molecular sciences involving biochemistry, molecular chemistry, medicine, and pharmacy, and high throughput techniques such as genomics and proteomics have helped to create new potential drug targets for cancer treatment. Microtubules are very attractive targets for cancer therapy because of the crucial roles they play in cell division. In recent years, lots of efforts have been put into the identification of new microtubule-targeting agents (MTAs) in anticancer therapy. Combretastatin A-4 (CA-4) is a natural compound that binds to microtubules’ colchicine binding site and inhibits microtubule polymerization. Due to CA-4’s structural simplicity, many analogs have been synthesized. This article summarizes the new molecule development efforts to reach CA-4 analogs by modifications on its pharmacophore groups, published since 2015.

Beneficiary Effects of Colchicine on Inflammation and Fibrosis in A Mouse Model of Chronic Kidney Disease

Introduction: Low grade inflammation is seen in many chronic illnesses, including chronic kidney disease (CKD). We have recently reported on beneficiary effects of anti-inflammatory treatment in the interleukin (IL-)1 pathway on anemia as well as CKD extent in a mouse model. Colchicine has been shown to have beneficiary effects in several inflammatory conditions through various mechanisms, including inhibition of tubulin polymerization as well as caspase 1 mediated IL1 activation.Methods: CKD was induced by administering an adenine diet to 8-week-old C57BL/6J mice. Mice were treated with colchicine (Col) (30µg/kg) or saline injections for 3 weeks, generating 4 groups: C, C-Col, CKD and CKD-Col.Results: Uremic animals had an increase in inflammation indices in blood (neutrophils), liver and kidneys (p-STAT3, IL-6, SOCS-3). Increased kidney tubulin polymerization and caspase 1 in CKD, as well as kidney Mid88 and IRAK4 (downstream of IL1) were inhibited in CKD-Col. Kidney macrophage infiltration (F4/80 and MAC-2), the percentage of fibrotic area and TGFb mRNA levels were lower in CKD-Col Vs CKD.Conclusions: colchicine improves kidney macrophage infiltration and fibrosis in CKD through inhibition of tubulin polymerization and Caspase 1 activation. Given its reported safety profile for long term anti-inflammatory therapy without increasing infection tendency, it may serve as novel therapeutic approach in CKD.

Angiogenesis, Anti-Tumor, and Anti-Metastatic Activity of Novel α-Substituted Hetero-Aromatic Chalcone Hybrids as Inhibitors of Microtubule Polymerization

A library of new heteroaromatic ring-linked chalcone analogs were designed and synthesized of these, compound 7m with α-CH3 substitution and bearing a benzofuran ring, displaying the most potent activity, with IC50 values of 0.07–0.183 µM against three cancer cells. Its low cytotoxicity toward normal human cells and strong potency on drug-resistant cells revealed the possibility for cancer therapy. It also could moderately inhibit in vitro tubulin polymerization with an IC50 value of 12.23 µM, and the disruption of cellular architecture in MCF-7 cells was observed by an immunofluorescence assay. Cellular-based mechanism studies elucidated that 7m arrested the cell cycle at the G2/M phase and induced apoptosis by regulating the expression levels of caspases and PARP protein. Importantly, the compound 7 m was found to inhibit HUVEC tube formation, migration, and invasion in vitro. In vivo assay showed that 7m could effectively destroy angiogenesis of zebrafish embryos. Furthermore, our data suggested that treatment with 7m significantly reduced MCF-7 cell metastasis and proliferation in vitro and in zebrafish xenograft. Collectively, this work showed that chalcone hybrid 7m deserves further investigation as dual potential tubulin polymerization and angiogenesis inhibitor.

Repurposing of the ALK Inhibitor Crizotinib for Acute Leukemia and Multiple Myeloma Cells

Crizotinib was a first generation of ALK tyrosine kinase inhibitor approved for the treatment of ALK-positive non-small-cell lung carcinoma (NSCLC) patients. COMPARE and cluster analyses of transcriptomic data of the NCI cell line panel indicated that genes with different cellular functions regulated the sensitivity or resistance of cancer cells to crizotinib. Transcription factor binding motif analyses in gene promoters divulged two transcription factors possibly regulating the expression of these genes, i.e., RXRA and GATA1, which are important for leukemia and erythroid development, respectively. COMPARE analyses also implied that cell lines of various cancer types displayed varying degrees of sensitivity to crizotinib. Unexpectedly, leukemia but not lung cancer cells were the most sensitive cells among the different types of NCI cancer cell lines. Re-examining this result in another panel of cell lines indeed revealed that crizotinib exhibited potent cytotoxicity towards acute myeloid leukemia and multiple myeloma cells. P-glycoprotein-overexpressing CEM/ADR5000 leukemia cells were cross-resistant to crizotinib. NCI-H929 multiple myeloma cells were the most sensitive cells. Hence, we evaluated the mode of action of crizotinib on these cells. Although crizotinib is a TKI, it showed highest correlation rates with DNA topoisomerase II inhibitors and tubulin inhibitors. The altered gene expression profiles after crizotinib treatment predicted several networks, where TOP2A and genes related to cell cycle were downregulated. Cell cycle analyses showed that cells incubated with crizotinib for 24 h accumulated in the G2M phase. Crizotinib also increased the number of p-H3(Ser10)-positive NCI-H929 cells illustrating crizotinib’s ability to prevent mitotic exit. However, cells accumulated in the sub-G0G1 fraction with longer incubation periods, indicating apoptosis induction. Additionally, crizotinib disassembled the tubulin network of U2OS cells expressing an α-tubulin-GFP fusion protein, preventing migration of cancer cells. This result was verified by in vitro tubulin polymerization assays. In silico molecular docking also revealed a strong binding affinity of crizotinib to the colchicine and Vinca alkaloid binding sites. Taken together, these results demonstrate that crizotinib destabilized microtubules. Additionally, the decatenation assay showed that crizotinib partwise inhibited the catalytic activity of DNA topoisomerase II. In conclusion, crizotinib exerted kinase-independent cytotoxic effects through the dual inhibition of tubulin polymerization and topoisomerase II and might be used to treat not only NSCLC but also multiple myeloma.

Anti-Aggregative Effect of the Antioxidant DJ-1 on the TPPP/p25-Derived Pathological Associations of Alpha-Synuclein

DJ-1, a multi-functional protein with antioxidant properties, protects dopaminergic neurons against Parkinson’s disease (PD). The oligomerization/assembly of alpha-synuclein (SYN), promoted by Tubulin Polymerization Promoting Protein (TPPP/p25), is fatal in the early stage of PD. The pathological assembly of SYN with TPPP/p25 inhibits their proteolytic degradation. In this work, we identified DJ-1 as a new interactive partner of TPPP/p25, and revealed its influence on the association of TPPP/p25 with SYN. DJ-1 did not affect the TPPP/p25-derived tubulin polymerization; however, it did impede the toxic assembly of TPPP/p25 with SYN. The interaction of DJ-1 with TPPP/p25 was visualized in living human cells by fluorescence confocal microscopy coupled with Bifunctional Fluorescence Complementation (BiFC). While the transfected DJ-1 displayed homogeneous intracellular distribution, the TPPP/p25-DJ-1 complex was aligned along the microtubule network. The anti-aggregative effect of DJ-1 on the pathological TPPP/p25-SYN assemblies was established by the decrease in the intensity of their intracellular fluorescence (BiFC signal) and the increase in the proteolytic degradation of SYN complexed with TPPP/p25 due to the DJ-1-derived disassembly of SYN with TPPP/p25. These data obtained with HeLa and SH-SY5Y cells revealed the protective effect of DJ-1 against toxic SYN assemblies, which assigns a new function to the antioxidant sensor DJ-1.

Synthesis, Computational Analysis, and Antiproliferative Activity of Novel Benzimidazole Acrylonitriles as Tubulin Polymerization Inhibitors: Part 2

We used classical linear and microwave-assisted synthesis methods to prepare novel N-substituted, benzimidazole-derived acrylonitriles with antiproliferative activity against several cancer cells in vitro. The most potent systems showed pronounced activity against all tested hematological cancer cell lines, with favorable selectivity towards normal cells. The selection of lead compounds was also tested in vitro for tubulin polymerization inhibition as a possible mechanism of biological action. A combination of docking and molecular dynamics simulations confirmed the suitability of the employed organic skeleton for the design of antitumor drugs and demonstrated that their biological activity relies on binding to the colchicine binding site in tubulin. In addition, it also underlined that higher tubulin affinities are linked with (i) bulkier alkyl and aryl moieties on the benzimidazole nitrogen and (ii) electron-donating substituents on the phenyl group that allow deeper entrance into the hydrophobic pocket within the tubulin’s β-subunit, consisting of Leu255, Leu248, Met259, Ala354, and Ile378 residues.