EB1 Increases the Dynamics of Tau Droplets and Inhibits Tau Aggregation: Implications in Tauopathies.

EB1, a microtubule plus end-tracking protein (+TIP), regulates microtubule dynamics. Recent evidence indicates cross-talk between EB proteins and tau, a microtubule-associated neuronal protein that is important for the growth and stability of microtubules. We investigated the interaction between tau and EB1 and the effect of binding of EB1 on tau function and aggregation. EB1 colocalized with tau in SH-SY5Y cells and coimmunoprecipitated with tau. Further, purified EB1 impaired the ability of adult tau to induce tubulin polymerization in vitro. EB1 bound to tau with a dissociation constant of 2.5 ± 0.7 µM. EB1 reduced heparin-induced tau aggregation with a half-maximal inhibitory concentration of 4.3 ± 0.2 µM, and increased the dynamics of tau in phase-separated droplets. The fluorescence recovery rate in tau droplets increased from 0.02 ± 0.01 to 0.07 ± 0.03 s-1, while the half-time of recovery decreased from 44.5 ± 14 to 13.5 ± 6 s in the presence of 8 µM EB1, suggesting a delay in the transition of tau from the soluble to aggregated form in tau liquid-liquid phase separation. EB1 decreased the rate of aggregation and increased the critical concentration of tau aggregation. Dynamic light scattering, atomic force microscopy, dot blot assays, and SDS-PAGE analysis showed that EB1 inhibited the formation of oligomers and higher-order aggregates of tau. The data suggest a novel role for EB1 as a regulator of tau function and aggregation, and the findings indicated the role of the EB family proteins in neuronal function and neurodegeneration.

Identification of ethyl-6-bromo-2((phenylthio)methyl)imidazo[1,2-a]pyridine-3-carboxylate as a narrow spectrum inhibitor of Streptococcus pneumoniae and its FtsZ.

Filamentous temperature-sensitive mutant Z (FtsZ) is a key cell-division protein recognized as an important target for anti-bacterial drug discovery, especially in the context of rising multi-drug resistance. A respiratory pathogen, Streptococcus pneumoniae, is rapidly evolving antibiotic resistance, thus posing a clinical risk in the developing world. Inhibiting the conserved protein FtsZ, leading to the arrest of cell division, is an attractive alternative strategy for inhibiting S. pneumoniae. Previously, Vitamin K3 was identified as an FtsZ-targeting agent against S. pneumoniae. In the present work, docking studies were used to identify potential anti-FtsZ agents that bind to the Vitamin K3-binding region of a homology model generated for S. pneumoniae FtsZ. Compounds with imidazo[1,2-a]pyridine-3-carboxylate core were synthesized and screened for their anti-proliferative activity against S. pneumoniae. Remarkably, the hit compound IP-01 showed anti-bacterial action against S. pneumoniae without any activity on other bacteria. In S. pneumoniae, IP-01 showed similar inhibitory action on FtsZ and cell division as Vitamin K3. Sequence alignment identified three unique residues within S. pneumoniae FtsZ that IP-01 binds to, providing a structural basis for the observed specificity. IP-01 is one of the first narrow-spectrum agents identified against S. pneumoniae that targets FtsZ, and we present it as a promising lead for the design of narrow-spectrum anti-FtsZ anti-pneumococcal compounds.

An adamantyl-caffeoyl-anilide exhibits broad-spectrum antibacterial activity by inhibiting FtsZ assembly and Z-ring formation.

Several harmful bacteria have evolved resistance to conventional antibiotics due to their extensive usage. FtsZ, a principal bacterial cell division protein, is considered as an important drug target to combat resistance. We identified a caffeoyl anilide derivative, (E)-N-(4-(3-(3,4-dihydroxyphenyl)acryloyl)phenyl)-1-adamantylamide (compound 11) as a new antimicrobial agent targeting FtsZ. Compound 11 caused cell elongation in Mycobacterium smegmatis, Bacillus subtilis, and Escherichia coli cells, indicating that it inhibits cell partitioning. Compound 11 inhibited the assembly of Mycobacterium smegmatis FtsZ (MsFtsZ), forming short and thin filaments in vitro. Interestingly, the compound increased the rate of GTP hydrolysis of MsFtsZ. Compound 11 also impeded the assembly of Mycobacterium tuberculosis FtsZ. Fluorescence and absorption spectroscopic analysis suggested that compound 11 binds to MsFtsZ and produces conformational changes in FtsZ. The docking analysis indicated that the compound binds at the interdomain cleft of MsFtsZ. Further, it caused delocalization of the Z-ring in Mycobacterium smegmatis and Bacillus subtilis without affecting DNA segregation. Notably, compound 11 did not inhibit tubulin polymerization, the eukaryotic homolog of FtsZ, suggesting its specificity on bacteria. The evidence indicated that compound 11 exerts its antibacterial effect by impeding FtsZ assembly and has the potential to be developed as a broad-spectrum antimicrobial agent.

Natural-Product-Inspired Discovery of Trimethoxyphenyl-1,2,4-triazolosulfonamides as Potent Tubulin Polymerization Inhibitors.

An approach of natural product-inspired strategy and incorporation of an NP-privileged motif has been investigated for the discovery of new tubulin polymerization inhibitors. Two series, N-Arylsulfonyl-3-arylamino-5-amino-1,2,4-triazole derivatives, and their isomers were considered. The compounds were synthesized by construction of the N-aryl-1,2,4-triazole-3,5-diamine motif and sulfonylation. Although the chemo- and regioselectivity in sulfonylation were challenging due to multiple ring-tautomerizable-NH and exocyclic NH2 functionalities present in the molecular motifs, the developed synthetic method enabled the preparation of designed molecular skeletons with biologically important motifs. The approach also led to explore interesting molecular regio- and stereochemical aspects valuable for activity. The X-ray crystallography study indicated that the hydrogen bonding between the arylamine-NH and the arylsulfonyl-"O" unit and appropriate molecular-functionality topology allowed the cis-locking of two aryls, which is important for tubulin-binding and antiproliferative properties. All synthesized compounds majorly showed characteristic antiproliferative effects in breast cancer cells (MCF-7), and four compounds exhibited potent antiproliferative activity. One compound potently bound to tubulin at the colchicine site and inhibited tubulin polymerization in vitro. The compound significantly depolymerized microtubules in MCF-7 cells, arrested the cells at the G2/M phase, and induced cell death. This study represents the importance of the design strategy in medicinal chemistry and the molecular structural features relevant to anticancer anti-tubulin properties. The explored molecules have the potential for further development.

Development of QTMP: A promising anticancer agent through NP-Privileged Motif-Driven structural modulation.

In this study of creating new molecules from clinical trial agents, an approach of Combretastatin structural modulation with the installation of NP-privileged motifs was considered, and a series of trimethoxyphenyl-2-aminoimidazole with functionalized quinolines and isoquinolines was investigated. An exciting method of quinoline C3-H iodination coupled with imidazopyridine-C3-H arylation and hydrazine-mediated fused-ring cleavage enabled synthesizing a class of compounds with two specific unsymmetric aryl substitutions. Interestingly, three compounds (6, 11, and 13) strongly inhibited HeLa cell proliferation with a half-maximal inhibitory concentration (10-46 nM). Among the compounds, compound 6 (QTMP) showed stronger antiproliferative ability than CA-4 (a clinical trial agent) in various cancer cell lines, including cervical, lung, breast, highly metastatic breast, and melanoma cells. QTMP inhibited the assembly of purified tubulin, depolymerized microtubules of A549 lung carcinoma cells, produced defective spindles, and arrested the cells in the G2/M phase. Further, QTMP binds to the colchicine site in tubulin with a dissociation constant of 5.0 ± 0.6 µM. QTMP displayed higher aqueous stability than CA-4 at 37 °C. Further, in silico analysis of QTMP indicated excellent drug-like properties, including good aqueous solubility, balanced hydrophilicity-lipophilicity, and high GI-absorption ability. The results together suggest that QTMP has anticancer potential.

Hybridization of the Pharmacophoric Features of Discoipyrrole C and Combretastatin A-4 toward New Anticancer Leads.

Pharmacophore hybridization is an attractive strategy to identify new leads against multifactorial diseases such as cancer. Based on literature analysis of compounds possessing 'vicinal diaryl' fragment in their structure, we considered Discoipyrroles A-D and Combretastatin A-4 (CA-4) as possible components in hybrid design. Discoipyrrole C (Dis C) and CA-4 were used as reference compounds in these studies and their hybrids, in the form of 4,5-diaryl-1H-pyrrol-3(2H)-ones, were synthesized from suitable amino acid precursors though their ynone intermediates. Of these, the hybrid having exact substitution pattern as that of CA-4 showed better potency and selectivity than Dis C, but its activity was less compared to CA-4. This new analog disrupted interphase microtubules by inhibiting tubulin assembly by binding to the colchicine site, induced multipolar spindles, caused cell cycle block and apoptosis in HeLa cells. It also inhibited colony formation and migration of breast cancer cell lines.

3,5-bis(styryl)pyrazole inhibits mitosis and induces cell death independent of BubR1 and p53 levels by depolymerizing microtubules.

Here, we show that 3,5-bis[(1E)-2-(2,6-dichlorophenyl)ethenyl]-1H-pyrazole 2l depolymerizes microtubules and reduces the number of growing tips of microtubules. The fluorescence recovery after photobleaching experiment in live MCF-7 cells showed that pyrazole 2l suppresses spindle microtubule dynamics. Further, the compound inhibits chromosome movements, activates the spindle assembly checkpoint and blocks mitosis in MCF-7 cells. Pyrazole 2l treatment induced cell death in a variety of pathways. Pyrazole 2l induces cell death independent of BubR1 and p53 levels of MCF-7 cells upon microtubule depolymerization. Further, pyrazole 2l increases the interaction between NF-κB and microtubules and enhances the nuclear localization of NF-κB at its half-maximal proliferation inhibitory concentration while a high concentration of the compound reduced the nuclear localization of NF-κB. Interestingly, the compound exerted significantly stronger antiproliferative effects in cancerous cells than in non-cancerous cells. The results indicated that pyrazole 2l inhibits mitosis by targeting microtubules, induces several types of cell death stimuli and suggests its potential as a lead in developing anticancer agent.

Models versus pathogens: how conserved is the FtsZ in bacteria?

Combating anti-microbial resistance by developing alternative strategies is the need of the hour. Cell division, particularly FtsZ, is being extensively studied for its potential as an alternative target for anti-bacterial therapy. Bacillus subtilis and Escherichia coli are the two well-studied models for research on FtsZ, the leader protein of the cell division machinery. As representatives of gram-positive and gram-negative bacteria, respectively, these organisms have provided an extensive outlook into the process of cell division in rod-shaped bacteria. However, research on other shapes of bacteria, like cocci and ovococci, lags behind that of model rods. Even though most regions of FtsZ show sequence and structural conservation throughout bacteria, the differences in FtsZ functioning and interacting partners establish several different modes of division in different bacteria. In this review, we compare the features of FtsZ and cell division in the model rods B. subtilis and E. coli and the four pathogens: Staphylococcus aureus, Streptococcus pneumoniae, Mycobacterium tuberculosis, and Pseudomonas aeruginosa. Reviewing several recent articles on these pathogenic bacteria, we have highlighted the functioning of FtsZ, the unique roles of FtsZ-associated proteins, and the cell division processes in them. Further, we provide a detailed look at the anti-FtsZ compounds discovered and their target bacteria, emphasizing the need for elucidation of the anti-FtsZ mechanism of action in different bacteria. Current challenges and opportunities in the ongoing journey of identifying potent anti-FtsZ drugs have also been described.

Copper-Plumbagin Complex Produces Potent Anticancer Effects by Depolymerizing Microtubules and Inducing Reactive Oxygen Species and DNA Damage.

Here, we have synthesized a copper complex of plumbagin (Cu-PLN) and investigated its antiproliferative activities in different cancer cells. The crystal structure of Cu-PLN showed that the complex was square planar with a binding stoichiometry of 1:2 (Cu/Plumbagin). Cu-PLN inhibited the proliferation of human cervical carcinoma (HeLa), human breast cancer (MCF-7), and murine melanoma (B16F10) cells with half-maximal inhibitory concentrations (IC50) of 0.85 ± 0.05, 2.3 ± 0.1, and 1.1 ± 0.1 µM, respectively. Plumbagin inhibited the proliferation of HeLa, MCF-7, and B16F10 cells with IC50 of 7 ± 0.1, 8.2 ± 0.2, and 6.2 ± 0.4 µM, respectively, showing that Cu-PLN is a stronger antiproliferative agent than plumbagin. Interestingly, Cu-PLN showed much stronger toxicity against breast carcinoma and skin melanoma cells than noncancerous breast epithelial and skin fibroblast cells, indicating its specific cytotoxicity toward cancer cells. A short exposure of Cu-PLN triggered microtubule disassembly in cultured cancer cells, and the complex also inhibited the polymerization of purified tubulin much more strongly than plumbagin. Furthermore, Cu-PLN inhibited the binding of colchicine to tubulin. In addition to microtubule depolymerization, the antiproliferative mechanism of Cu-PLN involved induction of reactive oxygen species, reduction of the mitochondrial membrane potential, and DNA damage. Moreover, the cytotoxic effects of Cu-PLN reduced significantly in cells pre-treated with N-acetyl cysteine, suggesting that reactive oxygen species generation is crucial in Cu-PLN's mode of action. Thus, the complexation of plumbagin with copper yields a promising antitumor agent having a stronger antiproliferative activity than cisplatin, a widely used anticancer drug.

ß-III Tubulin Levels Determine the Neurotoxicity Induced by Colchicine-Site Binding Agent Indibulin.

Indibulin, a microtubule-depolymerizing agent, produces minimal neurotoxicity in animals. It is also less cytotoxic toward differentiated neuronal cells than undifferentiated cells. We found that the levels of ß-III tubulin, acetylated tubulin, and polyglutamylated tubulin were significantly increased in differentiated neuroblastoma cells (SH-SY5Y). Since neuronal cells express ß-tubulin isotypes differently from other cell types, we explored the binding of indibulin to different ß-tubulin isotypes. Our molecular docking analysis suggested that indibulin binds to ß-III tubulin with lower affinity than to other ß-tubulin isotypes. We therefore studied the implications of different ß-tubulin isotypes on the cytotoxic effects of indibulin, colchicine, and vinblastine in differentiated SH-SY5Y cells. Upon depletion of ß-III tubulin in the differentiated cells, the toxicity of indibulin and colchicine significantly increased, while sensitivity to vinblastine was unaffected. Using biochemical, bioinformatics, and fluorescence spectroscopic techniques, we have identified the binding site of indibulin on tubulin, which had not previously been established. Indibulin inhibited the binding of colchicine and C12 (a colchicine-site binder) to tubulin and also increased the dissociation constant of the interaction between tubulin and colchicine. Indibulin did not inhibit the binding of vinblastine or taxol to tubulin. Interestingly, indibulin antagonized colchicine treatment but synergized with vinblastine treatment in a combination study performed in MDA-MB-231 cells. The results indicate that indibulin is a colchicine-site binder and that the efficacy of colchicine-site binders is affected by the ß-III tubulin levels in the cells.

ß-II tubulin isotype directs stiffness and differentiation of neuroblastoma SH-SY5Y cells.

ß-tubulin isotypes regulate the structure and bundling of microtubule (MT) lattice, its dynamics, and resulting functions. They exhibit differential tissue expression, varying due to physical and biochemical cues. In this work, we investigated the effect of transient heat shock at 42 °C on the nuclear and cytoplasmic stiffness of SH-SY5Y neuroblastoma cells through atomic force microscopy. Moreover, the variations in the expression of ß-tubulin isotypes as a heat shock response were also monitored. The heat-exposed cells endured a recovery at 37 °C for 24 h and they manifested an increase of cytoplasmic stiffness by 130 ± 25% with respect to untreated controls. The expression of ß-II tubulin isotype in heat-recovered cells is augmented by 51 ± 5% whereas the levels of total tubulin and ß-III tubulin isotype remain unaltered. Upon depletion of ß-II tubulin isotype using shRNA, the increase in cytoplasmic stiffness was dampened. However, it remained unaffected upon depletion with ß-III tubulin isotype shRNA. This features the role of the ß-II tubulin isotype in regulating cellular stiffness. In addition, neuroblastoma SH-SY5Y cells undergo differentiation by initiating neuritogenesis and prior evidence suggests the indispensable role of ß-II tubulin isotype in this process. The heat-recovered cells which expressed higher levels of ß-II tubulin isotype expedited the differentiation process in 3-day which was around 5-day for control cells, however, upon depletion of ß-II tubulin isotype, the cells almost lost their differentiation potential. Altogether, this work highlights the role of ß-II tubulin isotype as a biomarker for cellular stiffness.

Benzo[d]thiazole-2-carboxamides as new antituberculosis chemotypes inhibiting mycobacterial ATP phosphoribosyl transferase.

Aims: The screening of antimycobacterial benzo[d]thiazole-2-carboxamides against ATP-phosphoribosyl transferase (ATP-PRTase) was conducted. Materials & methods: The antitubercular potential of compounds 1 and 2 against ATP-PRTase was assessed through the determination of half maximal effective concentration (EC50) and binding constant (Kd), as well as competitive inhibitory studies and studies of perturbation of secondary structure, molecular modeling and L-histidine complementation assay. Results & conclusion: Compounds 1n and 2a significantly inhibited ATP-PRTase as evidenced by their EC50 and Kd values and the perturbation of the secondary structure study. Compound 1n exhibited stronger competitive inhibition toward ATP compared with 2a. The inhibition of the growth of Mycobacterium tuberculosis by targeting the L-histidine biosynthesis pathway and molecular modeling studies further supported the inhibition of ATP-PRTase.

Discovery of 2',6-Bis(4-hydroxybenzyl)-2-acetylcyclohexanone, a Novel FtsZ Inhibitor.

Multi-drug resistance is increasing in the pathogenic bacterium S. pneumoniae, which is mainly responsible for meningitis and community-acquired pneumonia (CAP), highlighting the need for new anti-pneumococcal agents. We have identified a potential anti-pneumococcal agent, enol 3, which acts by hindering the cell division process by perturbing Z-ring dynamics inside the cell. Enol 3 was also shown to inhibit FtsZ polymerization and induce its aggregation in vitro but does not affect the activity of tubulin and alkaline phosphatase. Docking studies show that 3 binds near the T7 loop, which is the catalytic site of FtsZ. Similar effects on Z-ring and FtsZ assembly were observed in B. subtilis, indicating that 3 could be a broad-spectrum anti-bacterial agent useful in targeting Gram-positive bacteria. In conclusion, compound 3 shows strong anti-pneumococcal activity, prompting further pre-clinical studies to explore its potential.

Bacterial FtsZ inhibition by benzo[d]imidazole-2-carboxamide derivative with anti-TB activity.

Aims: The present study aimed to assess the mode of action of previously reported anti-Mycobacterium tuberculosis benzo[d]imidazole-2-carboxamides against FtsZ along with their antibacterial potential. Materials & methods: The anti-mycobacterial action of benzo[d]imidazole-2-carboxamides against FtsZ was evaluated using inhibition of Bacillus subtilis 168, light scattering assay, circular dichroism spectroscopy, in silico molecular docking and molecular dynamics simulations. Results & conclusion: Three compounds (1k, 1o and 1e) were active against isoniazid-resistant strains. Four compounds (1h, 1i, 1o and 4h) showed >70% inhibition against B. subtilis 168. Compound 1o was the most potent inhibitor (91 ± 5% inhibition) of B. subtilis 168 FtsZ and perturbed its secondary structure. Molecular docking and molecular dynamics simulation of complexed 1o suggested M. tuberculosis FtsZ as a possible target for antitubercular activity.

Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity.

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 µg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.

Computational study of interactions of anti-cancer drug eribulin with human tubulin isotypes.

Microtubules (MTs) are widely targeted for the treatment of various types of cancer due to their essential role in cell division. MTs are polymers made of αß-tubulin heterodimers. These α- and ß-tubulins have 8 and 10 different isotypes, respectively. It is known that a few tubulin isotypes have anti-cancer drug resistance properties, especially ßIII, which shows poor sensitivity to many potent anti-cancer drugs such as eribulin. However, the molecular-level understanding of drug-resistance due to tubulin isotype variation is poorly understood. This paper presents the study of differential binding affinities of different tubulin isotypes with the potent anti-cancer drug eribulin. Eribulin (MT destabilizer) binds at the inter-dimer interface of MTs near the vinca site and induces a lattice deformation, which results in catastrophic events in MT dynamics. In this study, sequence analysis has been done throughway and the binding sites and based on that 2α-tubulin isotypes (αI and αVIII) and 7ß tubulin isotypes (ßI, ßIIa, ßIII, ßIVa, ßVI, ßVII and ßVIII) were selected. In total, 14 combinations were prepared after building homology models of these selected isotypes. Molecular docking and molecular dynamics simulations were performed to deeply understand the binding mode of eribulin at different MT compositions. RMSD, RMSF, radius of gyration, SASA, ligand-protein interactions, and calculations of binding free energy were performed to investigate the eribulin binding variations to tubulin isotypes and it was found that αIßII showed the maximum binding affinity among all 14 systems to eribulin. The ßIII-tubulin isotype, which shows low sensitivity to eribulin in experimental results, had the least binding affinity in the system αVIIIßIII complex and the average binding affinity in the system αIßIII among all 14 systems. Additionally, we performed steered MD simulations and DynDom analysis of the systems with the lowest binding energy (αIßII) and the highest binding energy (αVIIIßIII) and extracted force, displacement, and H-bonding profiles during the pulling simulations to get a better insight.

Investigating Tubulin-Drug Interaction Using Fluorescence Spectroscopy.

Fluorescence spectroscopy is routinely used for the determination of the interaction of a ligand with a protein. The quick detection of the interaction between the ligand and the protein is one of the most significant advantages of fluorescence spectroscopic methods. In this chapter, we have described assays to monitor drug -tubulin interactions using several fluorescence spectroscopic techniques. We have provided detailed protocols for different assays for investigating tubulin-drug interactions with key practical considerations for performing the experiments. We have also discussed how to deduce the binding parameters by fitting the fluorescence change data in different binding isotherms. Further, we have described detailed protocols to monitor the binding site of a ligand on tubulin by competitive inhibition. Though the methods are described for tubulin, these methods can also be used to monitor any drug -protein interactions.

Monitoring the Disruptive Effects of Tubulin-Binding Agents on Cellular Microtubules.

Tubulin-binding agents are an important class of chemotherapeutic agents. This chapter describes detailed protocols to examine the effects of tubulin-binding agents on cellular microtubules. The methods can be utilized for the screening of novel chemotherapeutic agents targeting microtubules. These assays can also be extended to study the effects of various proteins on the stability of microtubules. We have described five assays, which together provides qualitative and quantitative information about the effects of tubulin-binding agents on microtubule stability and dynamics. The key steps and crucial information regarding different steps have been included along with the theory of each of the assays.

Shikonin impedes phase separation and aggregation of tau and protects SH-SY5Y cells from the toxic effects of tau oligomers.

Tauopathies such as Alzheimer's and Parkinson's diseases involve the abnormal deposition of tau aggregates in the brain and neuronal tissues. We report that a natural naphthoquinone, shikonin, impeded the oligomerization and fibrillization of tau. The compound strongly inhibited heparin, arachidonic acid, and RNA-induced tau aggregation. Atomic force microscopy, dynamic light scattering, SDS-PAGE, and dot blot assays revealed that shikonin diminished tau oligomerization and decreased the mean size of tau oligomers. Transmission electron microscopy and atomic force microscopy analysis further showed that shikonin could suppress tau fibrillization and shorten the tau filaments. Shikonin inhibited tau droplet formation. The compound significantly reduced the aggregation rate of a tryptophan mutant (Y310W-tau) of tau. In addition, shikonin disaggregated preformed tau filaments with a half-maximal disaggregation concentration (DC50) of 6.3 ± 0.4 µM. Pre-treatment of neuroblastoma cells (SH-SY5Y) with shikonin protected the cells from the toxicity induced by tau oligomers and increased their viability. The findings imply that shikonin inhibited several steps in the tau aggregation pathways, especially the early stages, such as liquid-liquid phase separation. Therefore, shikonin is an attractive candidate for developing a therapy against tauopathy.

Combinatorial cetuximab targeted polymeric nanocomplexes reduce PRC1 level and abrogate growth of metastatic hepatocellular carcinoma in vivo with efficient radionuclide uptake.

Hepatocellular carcinoma (HCC) is the most aggressive form of cancer with poor drug responses. Developing an effective drug treatment remains a major unmet clinical need for HCC. We report a comprehensive study of combinatorial Cetuximab (Cet) targeted polymeric poly(D, L-lactide-co-glycolide)-b-poly(ethylene glycol) nanocomplexes delivery of Combretastatin A4 (CA4) and 2-Methoxyestradiol (2ME) (Cet-PLGA-b-PEG-CA4 NP + Cet-PLGA-b-PEG-2ME NP) against metastatic HCC in SCID mice. 125I-Cet-PLGA-b-PEG NP showed potent accumulation and retention in HCC tumors with longer circulation time up to 48 h (18 ±â€¯1.0% ID/g, P < .0001). Combinatorial treatment with targeted polymeric nanocomplexes presented significant tumor growth inhibition (85%, P < .0001) than the free drug combinatorial counterpart, effectively inhibited orthotopic HCC and prevented lung metastasis. Combinatorial nanocomplexes treatment significantly blocked PRC1, a novel target of therapeutic response against HCC. Thus, the combinatorial cetuximab-targeted polymeric nanocomplexes possess superior antitumor activity against metastatic HCC and provide supports for the clinical translation ahead.