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A method is described for the affinity purification of antibodies using protein samples that have been electrophoretically transferred to diazotized paper. Using differentiated neuroblastoma cells as the protein sample and a heterogeneous anti-microtubule protein serum, antibodies were isolated that specifically bound only to tubulin on blots and that stained microtubule networks in cells. The general utility of this method for various types of applications is discussed.

Two monoclonal antibodies (16 D3 and 24 E3) were used to map tubulin domains in human spermatozoa by indirect immunofluorescence. Their specificity to tubulin in these cells was established by Western blotting. Whereas 16 D3 uniformly stained the principal piece of the flagellum, the staining provided by 24 E3 decreased along the tail to become very weak 30 micron further away from the midpiece. This latter antibody also reacted with the proximal centriole as well as the midpiece, but not all spermatozoa stained identically at this level indicating heterogeneity within the population of sperm cells from a given donor. 16 D3 reacted weakly with the head, and the staining was interrupted after a bright spot in the neck. The study of a pathological case (the short tail spermatozoon) with an abnormal arrangement of dense fibers was consistent with a correlation between the distribution of the epitope defined by 24 E3 and that of peri-axenomal structures. The existence of tubulin domains interacting with these structures is postulated.

A new class of simple synthetic antimitotic compounds based on 2-aroylindoles was discovered. (5-Methoxy-1H-2-indolyl)-phenylmethanone (1) as well as analogous 3-fluorophenyl- (36) and 3-methoxyphenyl (3) derivatives displayed high cytotoxicity of IC(50) = 20 to 75 nM against the human HeLa/KB cervical, SK-OV-3 ovarian, and U373 astrocytoma carcinoma cell lines. The inhibition of proliferation correlated with the arrest in the G2/M phase of the cell cycle. In in vitro assays with tubulin isolated from bovine brain, in general antiproliferative activity correlated with inhibition of tubulin polymerization. Thus, the antimitotic activity of 2-aroylindoles is explained by interference with the mitotic spindle apparatus and destabilization of microtubules. In contrast to colchicine, vincristine, nocodazole, or taxol, 1 did not significantly affect the GTPase activity of beta-tubulin. Interestingly, selected compounds inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. In xenograft experiments, 1 was highly active after oral administration at 200 mg/kg against the human amelanocytic melanoma MEXF 989 in athymic nude mice. We conclude, that 2-aroylindoles constitute an interesting new class of antitubulin agents with the potential to be clinically developed for cancer treatment.

Hypomyelination with atrophy of the basal ganglia and cerebellum is a recently described tubulinopathy caused by a mutation in the tubulin beta 4a isoform, expressed in oligodendrocytes. The taiep rat is the only spontaneous tubulin beta 4a mutant available for the study of this pathology. We aimed to identify the effects of the tubulin mutation on freshly collected, unstained samples of the central white matter of taiep rats using second harmonic generation microscopy. Cytoskeletal differences between the central white matter of taiep rats and control animals were found. Nonlinear emissions from the processes and somata of oligodendrocytes in tubulin beta 4a mutant rats were consistently detected, in the shape of elongated structures and cell-like bodies, which were never detected in the controls. This signal represents the second harmonic trademark of the disease. The tissue was also fluorescently labeled and analyzed to corroborate the origin of the nonlinear signal. Besides enabling the description of structural and molecular aspects of H-ABC, our data open the door to the diagnostic use of nonlinear optics in the study of neurodegenerative diseases, with the additional advantage of a label-free approach that preserves tissue morphology and vitality., (© 2022. The Author(s).)

Microtubules are composed of αβ-tubulin heterodimers, and drugs that interfere with microtubule dynamics are used widely in cancer chemotherapy. Small synthetic molecules with an indole nucleus as a core structure have been identified as microtubule inhibitors and recognized as anticancer agents. However, structural information for the interactions between indole derivatives and tubulin is sparse. Here, we present the 2.55 Å crystal structure of tubulin in complex with the indole derivative D64131. We compare the binding modes of D64131, colchicine, and five other indole derivatives to tubulin. These results reveal the interactions between the indole derivatives and tubulin, explain previous results of structure-activity-relationship (SAR) studies and, thus, provide insights into the development of new indole derivatives targeting the colchicine binding site., (© 2019 Federation of European Biochemical Societies.)

Nowadays, hybrid molecule with dual targets activity or effect is regarded as an effective strategy for combating the drug resistance development in cancer therapy. Herein, novel of bifunctional conjugates targeting tubulin and MMPs inhibitors were synthesized. Among them, 15j exhibited robust anticancer activity in vitro and in vivo, with IC 50 values of 0.154-0.296 μM against four human cancer cells and a 74.7 % (@20 mg/kg) tumor growth inhibition in vivo without obvious systemic toxicity. Mechanistic studies indicated that 15j exerted inhibitory effects on both tubulin polymerization, MMP-2 and MMP-9 activity. Moreover, 15j remarkably inhibited cell proliferation, migration and invasion, and accordingly disrupted the NF-κB signaling transduction. Furthermore, 15j effectively initiated mitochondria-dependent apoptotic pathway by causing mitochondrial dysfunction, promoting the accumulation of reactive oxygen species, and inducing DNA damage. Collectively, these results demonstrated that 15j, as a tubulin/MMPs dual-targeting inhibitor, has exhibited significant potential for the lung cancer therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Masson SAS. All rights reserved.)

The Objective Reduction (OR) theory suggested by Sir Roger Penrose presented a novel perspective on the measurement problem of quantum mechanics and consciousness. Subsequently, based on the assertion of anesthesiologist Dr. Hameroff, the theory proposed that the phenomenon of OR could also manifest in microtubules within neurons. This would, serve as the trigger for consciousness, thereby forming the basis of Orchestrated OR (Orch OR). The peculiarity of this theory lies in the claim that tubulin and its higher neuronal information structures are not simply additive, rather, they are formed in a topological manner. Specifically, higher information structure of tubulin, helical pathways are presumed to form an intersecting pattern in both left-handed and right-handed directions, following a repeating Fibonacci series (3, 5, 8, 13, …), and are interconnected with each other. There have been attempts to examine these unique characteristics. However, experimenting upon these quantum characteristics in the microtubule appears to be plagued by certain limitations. Therefore, this study proposed a surface code model to implement this biological model on a quantum computer, focusing on its quantum properties. To the best of our knowledge, this is the first study to attempt this. The study emphasizes that interpreting asymmetric Fibonacci helical pathways as logical qubits can stabilize surface code. In addition, we analyzed the conditions required for experimenting with this model based on the development of current quantum computer. Although the experimental feasibility of this study is dependents on future quantum computer development, it provides significant insights into Orch OR research by offering a novel perspective., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Novel 1,2,3-triazole hybrids bearing various substituents have been synthesized as potential anticancer agents. Ligand-based approach has been adopted to design these compounds relying on the hybridization of 1,2,3-triazole with α,β-unsaturated carbonyl, 5- and 6-membered heterocyclic scaffolds. All synthesized members were investigated for their cytotoxic potency against nine types comprising 60 panels of human cancerous cells by the US National Cancer Institute: Development Therapeutic Program (US_NCI_DTP). Among the tested members, 4b, 4e, and 4h showed prominent cytotoxic effects (> 80 % growth inhibition: GI) on a wide panel of tested cancer cell lines, mainly melanoma and colorectal cancer redeeming their selection for five dose testing. Presenting low nanomolar GI 50 concentrations, two representative potent anticancer compounds 4b and 4e were subjected to cytotoxicity testing on colon normal cell (FHC) to investigate their safety window and they showed less toxicity to normal cells at the concentration required to produce anticancer effect. Furthermore, 4b and 4e were exposed to additional mechanistic studies in colorectal cancer cell HCT-116 suggesting multifaceted mechanisms of action. A study into the effects of cytotoxic chemicals 4b and 4e on cell cycle progression regulation showed triggered the arrest of cell cycles during the G1 and S phases. Moreover, 4b and 4e caused cell death mainly through apoptosis the thing that has been reinforced by the elevated Bax: Bcl2 ratio, as well as concentrations of caspases 3 and 9 within HCT-116. Further, both compounds showed prominent inhibition profiles against tubulin polymerization as well as EGFR catalytic activity reaching down to low-digit micromolar and sub-micromolar concentrations, respectively, as compared to positive reference controls. Compounds' impacts on gene expression of cancer-associated and EGFR-downstream signaling markers including TNFα, IL-6, and mTOR, were explored in HCT-116 highlighted significant downregulations versus the untreated cells. Docking studies demonstrated the specific fit of 4b and 4e into EGFR and the colchicine binding site of tubulin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025. Published by Elsevier Inc.)

Combination therapies play a pivotal role in cancer treatment due to the intricate nature of the disease. Tubulin, a protein crucial for cellular functions, is a prime target in tumor therapy as it regulates microtubule dynamics. Combining tubulin inhibitors with other different inhibitors as dual targeting inhibitors has shown synergistic anti-tumor effects, amplifying therapeutic outcomes. Despite clinical approval of several tubulin inhibitors, their efficacy is hampered by drug resistance and toxic side effects. Dual targeting inhibitors of tubulin and other cancer-related pathways have emerged as vital components in cancer therapy, with promising prospects in both market availability and ongoing clinical trials. The rational design of hybrid inhibitors targeting both pathways presents an innovative approach to combatting cancer. However, despite the potent anti-tumor activity exhibited by several compounds, research on their anti-angiogenic potential remains limited. This review emphasizes the significance of tubulin based dual-target inhibitors, elucidating their mechanisms of action. Recent advances in exploring therapeutic efficacy, toxicity profiles, and challenges such as MDR are discussed. By presenting the research progress of tubulin based dual-target inhibitors as potential anticancer agents, this study delivers valuable insights for the development of more efficient drugs for cancer therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

Microtubule-targeting agents (MTAs) are considered as one of the most successful chemotherapy drugs for lung adenocarcinoma (LUAD). However, the clinical application of MTAs is often significantly plagued by multidrug resistance (MDR). To overcome this limitation in the quest of more effective MTAs for tumor therapy, a series of novel diaryl-substituted nitrogenous fused heterocycles were designed, synthesized and evaluated. Through four rounds of structure-activity relationship studies, the benzoimidazole derivative 37 was identified as a potent cytotoxic agent against both paclitaxel-sensitive and -resistant A549 (A549/T) cells, effectively overcoming multidrug resistance of A549/T cells against various MTAs. Mechanistic investigations revealed that 37 could disrupt microtubule assembly and induce cell cycle arrest at the G2/M phase, and hence trigger the cell apoptosis. Furthermore, 37 was found to be a poor substrate for P-glycoprotein (P-gp), a major contributor to multidrug resistance, and could reduce the level of P-gp in resistant cells, thereby effectively overcoming P-gp-mediated multidrug resistance. Notably, 37 exhibited higher liver microsomal stability and better water solubility than those of the reference combretastatin A-4 (CA-4). In vivo studies using an A549/T xenograft model demonstrated that 37 significantly inhibited tumor growth without obvious toxicity, outperforming the positive controls CA-4 and paclitaxel. As a novel tubulin polymerization inhibitor, compound 37 is marked by potent anticancer activity and remarkable anti-MDR properties. These salient features, coupled with the low toxicity of 37, would render it quite promising as a lead for further drug development towards clinical treatment of multidrug-resistant LUAD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

We designed, synthesized, and evaluated the antitumor activity of a series of novel quinazoline-4-(6-methoxytetrahydroquinoline) analogues. Among the tested compounds, 4a4 exhibited the most potent antiproliferative activities across four human cancer cell lines with half-maximal inhibitory concentration (IC 50 ) values ranging from 0.4 to 2.7 nM, more potent than the lead compound. The 2.71 Å resolution co-crystal structure of 4a4 with tubulin (PDB code: 8YER) confirmed its critical binding at the colchicine site. Moreover, 4a4 inhibited the polymerization of tubulin, colony formation, and tumor cell migration, while inducing G2/M phase arrest and apoptosis. In vivo, 4a4 significantly delayed primary tumor growth in the SKOV3 xenograft model without obvious side effect. Our research enhances the structure-activity relationships (SARs) understanding of the quinazoline-4-tetrahydroquinoline scaffold and provides new insights for potential structural optimization and the development of novel colchicine binding site inhibitors (CBSIs)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Cancer treatment always a huge challenge amidst the resistance and relapse caused by the various treatments. Inhibitors targeting mitosis have been considered as promising therapeutic drugs in clinic, of which tubulins play an important role. Selenium (Se) as an essential microelement in humans and animals, playing a crucial role in the formation of anti-oxidase (glutathione peroxidase) and selenoprotein, also attracted broad attention in cancer therapy. Because the introduction of Se atom could change the length and angle of chemical bond and alter their functional properties, regulating selenized chemotherapeutics has become one of the hot spots. However, little attention has been paid to studying the combination of Se and tubulin inhibitors. Herein, we review the latest research results of selenized tubulin inhibitors in cancer therapy, including its mechanisms, categories and biological activities, providing a theoretical basis for different selenized microtubules inhibitors therapies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Neurons are equipped with microtubules of different stability with stable and dynamic domains often coexisting on the same microtubule. While dynamic microtubules undergo random transitions between disassembly and assembly, stable ones persist long enough to serve as platforms for tubulin-modifying enzymes (known as writers) that attach molecular components to the α- or β-tubulin subunits. The combination of these posttranslational modifications (PTMs) results in a "tubulin code," dictating the behavior of selected proteins (known as readers), some of which were shown to be crucial for neuronal function. Recent research has further highlighted that disturbances in tubulin PTMs can lead to neurodegeneration, sparking an emerging field of investigation with numerous questions such as whether and how tubulin PTMs can affect neurotransmission and synaptic plasticity and whether restoring balanced tubulin PTM levels could effectively prevent or mitigate neurodegenerative disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Recent development of immune checkpoint inhibitors has revolutionized cancer immunotherapy. Although these drugs show dramatic effects on a subset of cancer patients, many other tumors are non-responsive and the pathological mechanism of the resistance is largely unknown. To identify genes underlying anti-PD-1 immunotherapy resistance using a systematic approach, we performed an in vivo genome wide CRISPR screening in lung cancer cells. We integrated our results with multi-omics clinical data and performed both in vitro and in vivo assays to evaluate the role of the top candidate in regulating cytotoxic T cell killing. We identified TUBB3 as a potential target to overcome the resistance and enhance the efficacy of anti-PD-1 immunotherapy. TUBB3 expression is upregulated in lung cancer patients, and its higher expression correlates with poorer patients' survival. We found that TUBB3 expression was significantly elevated in the non-responders compared to responders in our patient cohort that received immunotherapies. Importantly, the results of our preclinical experiments showed that inhibition of TUBB3 with a small molecule inhibitor synergized with anti-PD-1 treatment and enhanced tumor cell killing by cytotoxic T cells. Consistently, anti-PD-1 resistant cells showed significantly higher expression of TUBB3; however, TUBB3 inhibition rendered the resistant cells more susceptible to T cell killing. Mechanistic studies revealed that blocking TUBB3 suppressed the expression of PD-L1 through the EMT-related SNAI1 gene. Our results provide a rationale for a novel combination therapy consisting of the TUBB3 inhibition and anti-PD-1 immunotherapy for lung cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Inhibitors of tubulin polymerization represent a promising therapeutic approach for the treatment of solid tumors. Molecules that bind to the colchicine site are of interest as they can function with a dual mechanism of action as both potent antiproliferative agents and tumor-selective vascular disrupting agents (VDAs). One such example is a 2-aryl-3-aroyl-indole molecule (OXi8006) from our laboratory that demonstrates potent inhibition of tubulin polymerization and strong antiproliferative activity (cytotoxicity) against a variety of human cancer cell lines. A water-soluble prodrug OXi8007, synthesized from OXi8006, demonstrates in vivo disruption of tumor-associated microvessels in several tumor types (mouse models). The molecular framework of OXi8006 inspired a series of fourteen new 2-aryl-3-aroyl-indole analogues that incorporated various functional group modifications on both the indole core and the aroyl ring. Electron withdrawing and donating groups at the mono-substituted 3' position and the di-substituted 3',5' positions were all accommodated while maintaining inhibition of tubulin polymerization (IC 50  < 5 μM), with several analogues demonstrating activity comparable to OXi8006 and the benchmark natural product combretastatin A-4 (CA4). Preliminary structure-activity relationship (SAR) studies were further enhanced by molecular docking to predict possible colchicine site interactions. Two analogues (KGP366 and KGP369) previously synthesized in our laboratory were re-synthesized using a somewhat modified route to increase synthetic efficiency and were subsequently converted to their corresponding water-soluble phosphate prodrug salts to evaluate their efficacy as VDAs. Administration of the prodrug salt (KGP415) of KGP369 caused significant reduction in bioluminescence signal from an orthotopic kidney tumor (RENCA-luc) in BALB/c mice, indicative of VDA activity. Collectively, these new functionalized indole-based analogues have extended SAR knowledge related to the colchicine binding site, and the most biologically active analogues hold promise for continued development as pre-clinical candidates for cancer therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Kevin G. Pinney reports financial support was provided by National Institutes of Health. Kevin G. Pinney reports financial support was provided by Cancer Prevention and Research Institute of Texas. Mary Lynn Trawick reports financial support was provided by National Institute of Health. Mary Lynn Trawick reports financial support was provided by Cancer Prevention and Research Institute of Texas. Ralph P. Mason reports financial support was provided by National Institutes of Health. Kevin G. Pinney has patent pending to Baylor University. Mary Lynn Trawick has patent pending to Baylor University. Zhe Shi has patent pending to Baylor University. Rebecca Vairin has patent pending to Baylor University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Chalcones have the characteristics of simple structure, easy synthesis and potent anti-tumor activity. Herein, a small library of fifty-five novel indole-chalcone derivatives were rationally designed and facilely synthesized. Consequently, their antiproliferative activity was systematically evaluated. Among which, compound 26 exhibited the most potent antiproliferative activity, with IC 50 value of 0.764 μM against MD-MBA-231 cells. Moreover, it displayed a 5-fold selectivity compared with normal human cells. Further investigation revealed that compound 26 bound at the colchicine binding site of tubulin, disrupted their fibrous structure, thereby blocking the progression of the cell cycle and inducing apoptosis. Molecular docking and cellular thermal shift assay (CETSA) experiments further demonstrated that compound 26 could specifically bind to hexokinase 2 (HK2) and inhibit its activity, leading to impaired mitochondrial function and hindered mitochondrial respiration. Based on the quantitative structure-activity relationship study, further structure modifications were performed. Employing biotin probe pull-down assays, we demonstrated that compound 26 exerted its antiproliferative activity through a dual targeting mechanism, which simultaneously disrupted microtubule function and inhibited HK2 activity. Taken together, these results highlighted that compound 26 might be a promising antiproliferative agent for human cancer therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Novel N-substituent diphenylamine derivatives as tubulin inhibitors targeting colchicine-binding site have been designed based on structural simplification and structural fusing strategy. Most designed compounds exhibited the moderate or potent antiproliferative activities against five cancer cell lines. Among them, compound 4k displayed the significant selectivity for osteosarcoma cells MG-63 and U2OS with the IC 50 value of 0.08-0.14 μM. Further investigations verified 4k could inhibit tubulin polymerization by targeting colchicine binding site. Meanwhile, compound 4k not only effectively induced tumor cell cycle arrest at the G2/M phase, but also slightly induced cell apoptosis. These results indicated that N-substituent of diphenylamine derivatives are deserved for further development as tubulin colchicine binding site inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Building on previous research indicating the robust biological effects of coumarins, we focused on exploring imino-coumarin 2-benzazole conjugates. Compounds were tested for antiproliferative activity in vitro, with the most active ones further examined to determine the mechanism of biological action. Five derivatives exhibited significant antiproliferative activity against all tested cancer cells (IC 50 ranging from 0.04 to 8.5 μM), falling within the low micromolar/submicromolar range of inhibitory concentrations. Three compounds had remarkable antiproliferative effects against Capan-1 (IC 50 0.04-0.05 μM) and DND-41 (IC 50 0.06-0.07 μM). Promising compounds were further investigated, confirming their mechanism of action through tubulin polymerization inhibition. Computational docking and molecular dynamics simulations confirmed the high affinity of potent derivatives for the tubulin colchicine site and justified the suitability of the employed skeleton by identifying crucial protein-ligand interactions promoting binding. This insight highlights a strategy for further potency improvements through substituents that can donate hydrogen bonds or bear a positive charge, allowing such ligands to more optimally adapt to the identified anionic binding site environment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Marijana Hranjec, Robert Vianello reports financial support was provided by Croatian Science Foundation. Marijana Hranjec, Robert Vianello reports a relationship with Croatian Science Foundation that includes: funding grants. − If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As the vital the biomacromolecule in eukaryotic cells, tubulin protein is essential for preserving cell shape, facilitating cell division, and cell viability. Tubulin has been approved as promising target for anticancer, and antifungal therapy. However, there are still many gaps in tubulin-targeted fungicidal discovery. To expand the structural diversity of benzimidazoles and achieve the distinct interaction model, a series of novel benzimidazole hydrazone derivatives were therefore synthesized. Antifungal results showed that compound A 9 was the most effective, achieving an EC 50 value of 2.88 μg/mL in vitro against Colletotrichum sublineola. In vivo assay, compound A 9 displayed encouraging efficacy, outperforming the reference agents ferimzone and tetramethylthiuram disulfide. Interestingly, mechanistic studies indicated that, compared with carbendazim, compound A 9 might form stronger interactions with tubulin, exemplified by the presence of multiple hydrogen bonds and π-π interactions, leading to intracellular microtubule aggregation in compound A 9 -treated cells. The significantly different interactions models between A 9 -tubulin and carbendazim-tubulin complexes may endow to produce the low resistance risk. Additionally, compound A 9 possessed low phytotoxicity and satisfactory ADME properties. This study not only provides a structural basis for the development of benzimidazole-based fungicides targeting tubulin but also offers new insights into the use of immunofluorescence assays in tubulin-targeting studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

The cardiac actin cytoskeleton has a dynamic pattern of polymerisation. It is uncertain how far actin destabilisation impacts mitochondrial energetics and biogenesis, cell signal status, and structural entities in cardiomyocytes, particularly in hypoxic conditions. We thus tested the in vitro action of cytochalasin D (Cyt D), an inhibitor of actin polymerisation, in hypoxic ventricular explants to elucidate the role of the actin in mitochondrial energetics and biogenesis, cell signals and actin/tubulin/hsps/MMPs dynamics in hypoxic air-breathing fish hearts. The COX activity increased upon Cyt D exposure, whereas hypoxia lowered COX and SDH activities but increased LDH activity. The ROS increased, and NO decreased by Cyt D. COX and LDH activities, and NO content reversed after Cyt D exposure in hypoxic hearts. Cyt D exposure upregulated actin isoform expression (Actc1 and Actb1) but downregulated tubulin isoform (Tedc1). Hypoxia upregulated actin (Acta1a, Actb1, Actb2, Actc1a) tubulin (Tuba, Tubb5, Tedc1, Tubd1) and hsp (Hspa5, Hspa9, Hspa12a, Hspa14, Hspd1, Hsp90) isoform transcript expression and Cyt D in hypoxic hearts reversed these isoform's expression. Hypoxia upregulated Mmp2 and 9 transcript expressions but downregulated Mfn1, Fis1, Nfkb1, Prkacaa, and Aktip expressions, and Cyt D exposure reversed almost all these markers in hypoxic hearts. The data provide novel evidence for the mechanistic role of actin in integrating mitochondrial energetics and biogenesis, cell signal status and actin/tubulin/Hsp/MMP entity, indicating its critical cardioprotective role in defending against hypoxia. Besides proposing an air-breathing fish heart as a model, the study further brings the therapeutic potential of Cyt D towards hypoxia intervention., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Inc. All rights reserved.)

The boronic acid group plays an important role in drug discovery. Following our discovery of a boronic acid analog of combretastatin A-4 (CA-4), a series of analogs featuring a boronic acid group on the C phenyl ring of CA-4 was synthesized and evaluated for cytotoxicity, as well as for their ability to inhibit tubulin polymerization, inhibit the binding of [ 3 H]colchicine to tubulin and cause depolymerization of cellular microtubules. Modifications on the C ring of CA-4, either eliminating the methoxy group or replacing the C phenyl ring with a pyridine ring, resulted in a reduced potency for inhibiting tubulin polymerization, colchicine binding and cytotoxic activities as compared to CA-4. Replacing the phenol group with a boronic acid group on the C ring of phenstatin led to a slight increase in cytotoxic potency but a decreased potency for inhibition of tubulin assembly and colchicine binding. Moreover, there was a significant decrease in activity by replacing the C phenyl ring with a pyridine ring. Our results indicate the critical importance of the methoxy group on the C ring as well as the importance of the C phenyl ring compared to a pyridine ring, despite the latter providing a nitrogen atom as a hydrogen bond donor/acceptor, which was predicted by molecular modeling to enhance interaction with the target. The decreased activities of our modified CA-4 boronic analogs may be attributed to weakened hydrogen bonding in our docking model based on the crystal structure of colchicine bound to αβ-tubulin. Notably, even though their effectiveness in inhibiting tubulin polymerization and colchicine binding and causing microtubule depolymerization in cells, the majority of these boronic acid analogs exhibited substantial cytotoxicity. This suggests that they may have additional cellular targets that contribute to their cytotoxicity, and this warrants further evaluation of these unique boronic acid compounds as potential anticancer agents., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Tubulin is a critical target for cancer therapy, with colchicine binding site inhibitors (CBSIs) being the most extensively researched. A series of quinazoline derivatives designed to target the colchicine binding site of tubulin were synthesized and evaluated for their biological activities. The antiproliferative effects of these compounds were tested against six human cancer cell lines, and compound Q19 demonstrated potent antiproliferative activity against the HT-29 cell line, with an IC 50 value of 51 nM. Additionally, further investigation revealed that Q19 effectively inhibited microtubule polymerization by binding to the colchicine binding site on tubulin. Furthermore, compound Q19 arrested the HT-29 cell cycle at the G2/M phase, induced apoptosis in these cells, and disrupted angiogenesis. Finally, compound Q19 exhibited potent inhibitory effects on tumor growth in HT-29 xenografted mice while demonstrating minimal toxic side effects and acceptable pharmacokinetic properties. These findings suggested that Q19 hold promise as a potential candidate for colon cancer therapy targeting tubulin., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Yong Yin reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

There is a growing number of reports on the occurrence of benzimidazole resistance-associated single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various helminths of veterinary, and public health concerns. However, a comprehensive analysis of their occurrence, and their contributions to conferring benzimidazole resistance among hookworms has yet to be done. The objectives of this systematic review are to summarize and synthesize peer-reviewed evidence on the occurrence of these resistance-associated mutations in hookworms, document their geographical distribution, and assess their contributions to conferring phenotypic resistance. Three databases were systematically searched using specific keywords. Research that assessed the occurrence of benzimidazole resistance-associated SNPs in hookworms, papers that reported the geographical distribution of these SNPs, and studies that investigated the SNPs' resistance-associated phenotypic effects were included in the review. Research that was not done in hookworms, papers not in the English language, and literature reviews and book chapters were excluded. Critical appraisal checklists were used to determine the risk of bias in the selected papers. Data were extracted from the selected studies and analyzed. PROSPERO Systematic Review Protocol Registration No.: CRD42024510924. A total of 29 studies were included and analyzed. Of these, four were conducted in a laboratory setting, eight described the development and validation of SNP detection methods, and the remaining 17 involved field research. Seven SNP-induced amino acid substitutions at four loci were reported among several hookworm species: Q134H, F167Y, E198A, E198K, E198V, F200Y, and F200L. SNPs have been reported in isolates occurring in the United States, Canada, Brazil, Haiti, Australia, New Zealand, Kenya, Ghana, Mozambique, and Tanzania. Resistance mutations have not been reported in Asia. E198A and F200L were reported in Ancylostoma ceylanicum with laboratory-induced resistance. F167Y and Q134H conferred resistance in A. caninum, as revealed by in vitro investigations and field assessments. There is insufficient peer-reviewed evidence to prove the association between SNP occurrence and resistance. Mutations in the β-tubulin isotype 1 gene confer benzimidazole resistance in A. caninum and A. ceylanicum, but similar evidence is lacking for other human hookworms. Understanding benzimidazole resistance through further research can better inform treatment, prevention, and control strategies., Competing Interests: Declarations. Ethical approval: Ethics clearance is not required due to the nature of the study. Consent for publication: Not applicable. Competing interest: The authors declare no competing interests. Generative AI in the writing process: This research did not utilize any large language models in the writing process. Clinical trial number: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Concerns about the emergence of benzimidazole resistance in soil-transmitted helminths (STH) infections, particularly against Trichuris trichiura, have arisen. Previous studies of veterinary nematodes have linked benzimidazole resistance to single-nucleotide polymorphisms (SNPs) at three specific codons in the beta-tubulin gene, but similar associations in STH have not been consistently observed. In this work, we screened the complete beta-tubulin gene previously linked to benzimidazole resistance in T. trichiura by deep-amplicon sequencing to identify genetic variants and associate levels of diversity with drug response to albendazole. We used 99 DNA samples extracted from T. trichiura pooled eggs, previously semi-purified from human stool samples collected in Manhiça district, Mozambique. We obtained a set of 39 amplicons of the complete gene by subjecting the pooled eggs to long-read PCR and subsequently sequencing them. Of those amplicons, 22 and 17 were obtained from stool samples collected before, and 21 days after albendazole treatment, respectively. We observed genetic variation across the whole gene sequence, in both exons and introns; however, none were associated with the previously proposed resistance-associated SNPs, and none were predicted to significantly affect protein function. No significant differences in genetic diversity were observed between pre- and post-treatment samples. Using publicly available genome-wide data, we also analysed a second beta-tubulin isotype in the T. trichiura genome. We focused on detecting the canonical SNPs and assessing for signatures of genetic selection around this second isotype gene. This analysis did not reveal evidence supporting this second isotype's role in anthelmintic resistance. Despite the limitations of our study, such as a small sample size, particularly paired pre- and post-treatment samples (n = 6), or a restricted geographical area, we found no evidence linking either of the two beta-tubulin genes to benzimidazole resistance in T. trichiura, suggesting that genetic markers of drug resistance likely exist outside the beta-tubulin genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Triple negative breast cancer (TNBC) has long been a challenging disease owing to its high aggressive behaviour, poor prognosis and its limited treatment options. The growing demand of new therapeutics against TNBC enables us to examine the therapeutic efficiency of an emerging class of anticancer compounds, azapodophyllotoxin derivative (HTDQ), a nitrogen analogue of podophyllotoxin, using different biochemical, spectroscopic and computational approaches. The anticancer activities of HTDQ are studied by performing MTT assay in a dose depended manner on Triple negative breast cancer cells using MDA-MB-468 and MDA-MB-231 cell lines with IC 50 value 937 nM and 1.13 µM respectively while demonstrating minimal effect on normal epithelial cells. The efficacy of HTDQ was further tested in 3D tumour spheroids formed by the human TNBC cell line MDA-MB468 and also the murine MMTV positive TNBC cell line 4 T1. The shrinkage that observed in the tumor spheroid clearly indicates that HTDQ remarkably decreases the growth of tumor spheroid thereby affirming its cytotoxicity. The 2D cell viability assay shows significant morphological alteration that possibly caused by the cytoskeleton disturbances. Hence the binding interaction of HTDQ with cytoskeleton protein tubulin, its effect on tubulin polymerisation as well as depolymerisation of preformed microtubules along with the conformational alternation in the protein itself have been investigated in detail. Moreover, the apoptotic effects of HTDQ have been examined using a range of apoptotic markers. HTDQ-treated cancer cells showed increased expression of cleaved PARP-1 and pro-caspase-3, suggesting activation of the apoptosis process. HTDQ also upregulated pro-apoptotic Bax expression while inhibiting anti-apoptotic Bcl2 expression, supporting its ability to induce apoptosis in cancer cells. Hence the consolidated biochemical and spectroscopic research described herein may provide enormous information to use azapodophyllotoxin as promising anticancer therapeutics for TNBC cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The present study deals with an anti-cancer investigation of an array of phthalimide-1,2,3-triazole molecular conjugates with various sulfonamide fragments against human breast MCF-7 and prostate PC3 cancer cell lines. The targeted 1,2,3-triazole derivatives 4a-l and 6a-c were synthesized from focused phthalimide-based alkyne precursors using a facile click synthesis approach and were thoroughly characterized using several spectroscopic techniques (IR, 1 H, 13 C NMR, and elemental analysis). The hybrid click adducts 4b, 4 h, and 6c displayed cytotoxic potency (IC 50 values of 1.49, 1.07, and 0.56 μM, respectively) against MCF-7 cells. On the contrary, none of the synthesized compounds showed apparent cytotoxic efficacy for PC3 cells (IC 50 ranging from 9.87- >100 μM). As a part of the mechanism analysis, compound 6c demonstrated a potent inhibitory effect (78.3 % inhibition) of tubulin polymerization in vitro with an IC 50 value of 6.53 µM. In addition, biological assays showed that compound 6c could prompt apoptotic cell death and induce G2/M cell cycle arrest in MCF-7 cells. Accordingly, compound 6c can be further developed as an anti-breast cancer agent through apoptosis-induction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The dual-specificity tyrosine-phosphorylation-regulated kinase 1 A (DYRK1A) presents a promising therapeutic target for neurological diseases. However, current inhibitors lack selectivity, which can lead to unexpected side effects and increase the difficulty of studying DYRK1A. Therefore, identifying selective inhibitors targeting DYRK1A is essential for reducing side effects and facilitating neurological disease research. This study aimed to discover DYRK1A inhibitors through a screening pipeline incorporating a deep neural network (DNN) model. Herein, we report an optimized model with an accuracy of 0.93 on a testing set. The pipeline was then performed to identify potential DYRK1A inhibitors from the National Cancer Institute (NCI) library. Four novel DYRK1A inhibitors were identified, and compounds NSC657702 and NSC31059 were noteworthy for their potent inhibition, with IC 50 values of 50.9 and 39.5 nM, respectively. NSC31059 exhibited exceptional selectivity across 70 kinases. The compounds also significantly reduced DYRK1A-induced tau phosphorylation at key sites associated with the pathology of neurodegenerative diseases. Moreover, they promoted tubulin polymerization, suggesting a role in microtubule stabilization. Cytotoxicity assessments further confirmed the neuronal safety of the compounds. Together, the results demonstrated a promising screening pipeline and novel DYRK1A inhibitors as candidates for further optimization and development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Methyl benzimidazole-2-carbamate anthelmintics are a class of oral drugs to treat parasitic worm infections via microtubule disruption for non-systemic indications and currently in use. In order to use for anticancer treatment, the new benzimidazoles needs to improve solubility and pharmacokinetic parameters while maintaining its cellular potency as for systemic drug. Structure-activity-relationship on the benzimidazole is thoroughly examined and a novel benzimidazole-2 propionamide BNZ-111 is identified having good oral exposure and bioavailability in rat. Molecular docking study suggests BNZ-111 have a specific binding mode to the β subunit of curved tubulin. BNZ-111 is potent to cancer cells and possesses good drug-like properties as oral drug. Especially, BNZ-111 is not a P-gp substrate and it demonstrates its efficacy over Paclitaxel-resistance tumor in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

A series of novel 2-substituted 2, 3-dihydroquinazolin-4(1H)-one derivatives were designed, synthesized and estimated for their in vitro antiproliferative activities against HepG2, U251, PANC-1, A549 and A375 cell lines. Among them, compound 32 was the most promising candidate, and displayed strong broad-spectrum anticancer activity. The mechanism studies revealed that compound 32 inhibited tubulin polymerization in vitro, disrupted cell microtubule networks, arrested the cell cycle at G2/M phase, and induced apoptosis by up-regulating the expression of cleaved PARP-1 and caspase-3. Furthermore, molecular docking analysis suggested that compound 32 well occupied the binding site of tubulin. In addition, compound 32 exhibited no significant activity against 30 different kinases respectively, indicating considerable selectivity. Moreover, compound 32 significantly inhibited the tumour growth of the HepG2 xenograft in a nude mouse model by oral gavage without apparent toxicity. These results demonstrated that some 2-substituted 2, 3- dihydroquinazolin-4(1H)-one derivatives bearing phenyl, biphenyl, naphthyl or indolyl side chain at C 2 -position might be potentially novel antitumor agents as tubulin polymerization inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Guided by the X-ray cocrystal structure of the lead compound 4a, we developed a series of thieno[3,2-d]pyrimidine and heterocyclic fused pyrimidines demonstrating potent antiproliferative activity against four tumor cell lines. Two analogs, 13 and 25d, exhibited IC 50 values around 1 nM and overcame P-glycoprotein (P-gp)-mediated multidrug resistance (MDR). At low concentrations, 13 and 25d inhibited both the colony formation of SKOV3 cells in vitro and tubulin polymerization. Furthermore, mechanistic studies showed that 13 and 25d induced G 2 /M phase arrest and apoptosis in SKOV3 cells, as well as dose-dependent inhibition of tumor cell migration and invasion at low concentrations. Most notably, the X-ray cocrystal structures of compounds 4a, 25a, and the optimal molecule 13 in complex with tubulin were elucidated. This study identifies thieno[3,2-d]pyrimidine and heterocyclic fused pyrimidines as representatives of colchicine-binding site inhibitors (CBSIs) with potent antiproliferative activity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Microtubules are recognized as an appealing target for cancer treatment. We designed and synthesized of novel tubulin colchicine binding site inhibitors based on millepachine. Biological evaluation revealed compound 5h exhibited significant antiproliferative activity against osteosarcoma cell U2OS and MG-63. And compound 5h also remarkably inhibited tubulin polymerization. Further investigations indicated compound 5h not only arrest U2OS cells cycle at the G2/M phases, but also induced U2OS cells apoptosis in dose-dependent manners. Moreover, compound 5h was verified to inhibit cell migration and angiogenesis of HUVECs, induce mitochondrial membrane potential decreased and promoted the elevation of ROS levels. Furthermore, compound 5h exhibited remarkable effects on tumor growth in vivo, and the TGI rate was up to 84.94 % at a dose of 20 mg/kg without obvious toxicity. These results indicated that 5h may be an appealing tubulin inhibitor for treatment of osteosarcoma., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liming Wang reports financial support was provided by Nanjing Medical University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Dual-target agents have more advantages than drug combinations for cancer treatment. Here, we designed and synthesized a series of novel VEGFR-2/tubulin dual-target inhibitors through a molecular hybridization strategy, and the activities of all the synthesized compounds were tested against tubulin and VEGFR-2. Among which, compound 19 exhibited strong potency against tubulin and VEGFR-2, with IC 50 values of 0.76 ± 0.11 μM and 15.33 ± 2.12 nM, respectively. Additionally, compound 19 not only had significant antiproliferative effects on a series of human cancer cell lines, especially MGC-803 cells (IC 50  = 0.005 ± 0.001 μM) but also overcame drug resistance in Taxol-resistant MGC-803 cells, with an RI of 1.8. Further studies showed that compound 19 could induce tumor cell apoptosis by reducing the mitochondrial membrane potential, increasing the level of ROS, facilitating the induction of G2/M phase arrest, and inhibiting the migration and invasion of tumor cells in a dose-dependent manner. In addition, compound 19 also exhibits potent antiangiogenic effects by blocking the VEGFR-2/PI3K/AKT pathway and inhibiting the tubule formation, invasion, and migration of HUVECs. More importantly, compound 19 demonstrated favorable pharmacokinetic profiles, robust in vivo antitumor efficacy, and satisfactory safety profiles. Overall, compound 19 can be used as a lead compound for the development of tubulin/VEGFR-2 dual-target inhibitors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microtubules are core components of the neuronal cytoskeleton, providing structural support for the complex cytoarchitecture of neurons and serving as tracks for long-distance transport. The properties and functions of neuronal microtubules are controlled by tubulin isoforms and a variety of post-translational modifications, collectively known as the "tubulin code." The tubulin code exerts direct control over the intrinsic properties of neuronal microtubules and regulates the repertoire of proteins that read the code, which in turn, has a significant impact on microtubule stability and dynamics. Here, we review progress in the understanding of the tubulin code in the nervous system, with a particular focus on tubulin post-translational modifications that have been proposed as potential contributors to the development and maintenance of the mammalian nervous system. Furthermore, we also discuss the potential links between disruptions in the tubulin code and neurological disorders, including neurodevelopmental abnormalities and neurodegenerative diseases., Competing Interests: Declaration of Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The author Eun-Mi Hur is an Editorial Board Member for Molecules and Cells and was not involved in the editorial review or the decision to publish this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)