ABCB1-dependent collateral sensitivity of multidrug-resistant colorectal cancer cells to the survivin inhibitor MX106-4C.

AIMS: To investigate the collateral sensitivity (CS) of ABCB1-positive multidrug resistant (MDR) colorectal cancer cells to the survivin inhibitor MX106-4C and the mechanism. METHODS: Biochemical assays (MTT, ATPase, drug accumulation/efflux, Western blot, RT-qPCR, immunofluorescence, flow cytometry) and bioinformatic analyses (mRNA-sequencing, reversed-phase protein array) were performed to investigate the hypersensitivity of ABCB1 overexpressing colorectal cancer cells to MX106-4C and the mechanisms. Synergism assay, long-term selection, and 3D tumor spheroid test were used to evaluate the anti-cancer efficacy of MX106-4C. RESULTS: MX106-4C selectively killed ABCB1-positive colorectal cancer cells, which could be reversed by an ABCB1 inhibitor, knockout of ABCB1, or loss-of-function ABCB1 mutation, indicating an ABCB1 expression and function-dependent mechanism. MX106-4C's selective toxicity was associated with cell cycle arrest and apoptosis through ABCB1-dependent survivin inhibition and activation on caspases-3/7 as well as modulation on p21-CDK4/6-pRb pathway. MX106-4C had good selectivity against ABCB1-positive colorectal cancer cells and retained this in multicellular tumor spheroids. In addition, MX106-4C could exert a synergistic anti-cancer effect with doxorubicin or re-sensitize ABCB1-positive cancer cells to doxorubicin by reducing ABCB1 expression in the cell population via long-term exposure. CONCLUSIONS: MX106-4C selectively kills ABCB1-positive MDR colorectal cancer cells via a novel ABCB1-dependent survivin inhibition mechanism, providing a clue for designing CS compound as an alternative strategy to overcome ABCB1-mediated colorectal cancer MDR.

Sphingolipid biosynthetic inhibitor L-Cycloserine prevents oxidative-stress-mediated death in an in vitro model of photoreceptor-derived 661W cells.

Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H2O2-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.

Discovery of agonist-antagonist pairs for the modulation of Ca [2]+ and voltage-gated K+ channels of large conductance that contain beta1 subunits.

Large conductance, calcium/voltage-gated potassium channels (BK) regulate critical body processes, including neuronal, secretory and smooth muscle (SM) function. While BK-forming alpha subunits are ubiquitous, accessory beta1 subunits are highly expressed in SM. This makes beta1 an attractive target for pharmaceutical development to treat SM disorders, such as hypertension or cerebrovascular spasm. Compounds activating BK via beta1 have been identified, yet they exhibit low potency and off-target effects while antagonists that limit agonist activity via beta 1 remain unexplored. Beta1-dependent BK ligand-based pharmacophore modeling and ZINC database searches identified 15 commercially available hits. Concentration-response curves on BK alpha + beta1 subunit-mediated currents were obtained in CHO cells. One potent (EC50 = 20 nM) and highly efficacious activator (maximal activation = ×10.3 of control) was identified along with a potent antagonist (KB = 3.02 nM), both of which were dependent on beta1. Our study provides the first proof-of-principle that an agonist/antagonist pair can be used to control beta1-containing BK activity.

Colchicine Binding Site Agent DJ95 Overcomes Drug Resistance and Exhibits Antitumor Efficacy.

Interfering with microtubule dynamics is a well-established strategy in cancer treatment; however, many microtubule-targeting agents are associated with drug resistance and adverse effects. Substantial evidence points to ATP-binding cassette (ABC) transporters as critical players in the development of resistance. Herein, we demonstrate the efficacy of DJ95 (2-(1H-indol-6-yl)-4-(3,4,5-trimethoxyphenyl)-1H-imidazo[4,5-c]pyridine), a novel tubulin inhibitor, in a variety of cancer cell lines, including malignant melanomas, drug-selected resistant cell lines, specific ABC transporter-overexpressing cell lines, and the National Cancer Institute 60 cell line panel. DJ95 treatment inhibited cancer cell migration, caused morphologic changes to the microtubule network foundation, and severely disrupted mitotic spindle formation of mitotic cells. The high-resolution crystal structure of DJ95 in complex with tubulin protein and the detailed molecular interactions confirmed its direct binding to the colchicine site. In vitro pharmacological screening of DJ95 using SafetyScreen44 (Eurofins Cerep-Panlabs) revealed no significant off-target interactions, and pharmacokinetic analysis showed that DJ95 was maintained at therapeutically relevant plasma concentrations for up to 24 hours in mice. In an A375 xenograft model in nude mice, DJ95 inhibited tumor growth and disrupted tumor vasculature in xenograft tumors. These results demonstrate that DJ95 is potent against a variety of cell lines, demonstrated greater potency to ABC transporter-overexpressing cell lines than existing tubulin inhibitors, directly targets the colchicine binding domain, exhibits significant antitumor efficacy, and demonstrates vascular-disrupting properties. Collectively, these data suggest that DJ95 has great potential as a cancer therapeutic, particularly for multidrug resistance phenotypes, and warrants further development. SIGNIFICANCE STATEMENT: Paclitaxel is a widely used tubulin inhibitor for cancer therapy, but its clinical efficacy is often limited by the development of multidrug resistance. In this study, we reported the preclinical characterization of a new tubulin inhibitor DJ95, and demonstrated its abilities to overcome paclitaxel resistance, disrupt tumor vasculature, and exhibit significant antitumor efficacy.

Bioavailability testing of a newly developed clindamycin oral suspension in a pediatric porcine model.

Background: Clindamycin's bitter taste and odor is known to affect treatment adherence in children. Recently, a formulation of clindamycin HCl complexed with ion exchange resin IRP 69 was shown to mask the bitter taste. Because of the potential benefit of this formulation for children, a pilot study using a porcine model was conducted to evaluate its relative bioavailability. Methods: A randomized two-way crossover study design using six (n = 6) healthy male piglets 10-12 kg was used to evaluate the absorption profiles and pharmacokinetic parameters of clindamycin from the resinate complex formulation (Test) compared to a commercialized reference suspension. A dose of 15 mg/kg was administered orally by gastric gavage to each piglet followed by repeated blood sampling over 12 h. A wash-out period of 48 h occurred between treatments. Plasma concentration vs. time data was analyzed by non-compartmental analysis. Results: The mean relative bioavailability of clindamycin from the resinate formulation was 78.8%. A two-tailed, paired Student t test yielded a p < 0.05 for AUC∞ and Tmax parameters. A two one-sided test (TOST) suggested a difference in AUC∞ and Cmax for the Test formulation compared to the reference formulation according to the FDA's criteria for bioequivalence. Conclusion: The bioavailability of clindamycin from this novel oral formulation supports continued evaluation of the drug in humans for potential pediatric applications.

Development and physicochemical characterization of clindamycin resinate for taste masking in pediatrics.

PURPOSE: To evaluate the physicochemical characteristics of clindamycin HCl in a complex form (resinate) with ion exchange resin (IER) (Amberlite IRP69). METHODS: Drug-resin complex was prepared by simple aqueous binding method. Drug binding study was carried out at different drug and resin concentrations. Several physicochemical characterization studies were conducted to evaluate the resinate complex. These studies included flow properties, in vitro drug release in SGF and SIF, DSC, TGA, mass spectroscopy and XPRD evaluations. In addition, stability study of resinate complex was conducted at 25 and 40 °C for up to 1 month. RESULTS: Clindamycin and Amberlite IRP69 have formed a complex (resinate) and have shown good flow properties, good thermal properties and chemical stability (short term over 4 weeks) at 25 and 40 °C. Clindamycin release profiles from resinate in SGF and SIF have shown immediate release characteristics and release in simulated saliva has shown dependence on water volume. CONCLUSION: The clindamycin stable complex with IER (Amberlite IRP69) has the potential for further development as a compatible pediatric liquid formulation (suspension) or a fast disintegrating tablet.

Cu(I)-mediated allosteric switching in a copper-sensing operon repressor (CsoR).

The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. A phylogenetic analysis of 227 DUF156 protein members, including biochemically or structurally characterized CsoR/RcnR repressors, reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)-bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2-helix between two conserved copper-ligating residues and folds an N-terminal tail (residues 12-19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo-state with these dynamics quenched upon Cu(I) binding. Small angle x-ray scattering experiments on an N-terminally truncated Gt CsoR (Δ2-10) reveal that the Cu(I)-bound tetramer is hydrodynamically more compact than is the apo-state. The implications of these findings for the allosteric mechanisms of other CsoR/RcnR repressors are discussed.

Design, Synthesis and Biological Evaluation of Novel 5H-Chromenopyridines as Potential Anti-Cancer Agents.

A novel series of 5H-chromenopyridines was identified as anticancer agents in our continuing effort to discover and develop new small molecule anti-proliferative agents. Based on our initial lead SP-6-27 compound, we designed and synthesized novel tricyclic 5H-thiochromenopyridine and 5H-chromenopyridine analogs to evaluate the impact of an additional ring, as well as conformational flexibility on cytotoxic activity against human melanoma and glioma cell lines. All of the 5H-thiochromenopyridines have been achieved in good yields (89%-93%) using a single-step, three-component cyclization without the need for purification. The 5H-chromenopyridine analog of the potent 5H-thiochromenopyride was obtained in a good yield upon purification. All newly-prepared 5H-thiochromenopyridines showed good to moderate cytotoxicity against three melanoma and two glioma cell lines (3-15 µM). However, the 5H-chromenopyridine analogue that we prepared in our laboratory lost cytotoxic activity. The moderate cytotoxic activity of 5H-thiochromenopyridines shows the promise of developing chromenopyridines as potential anticancer agents.

Experimental study on shearer traction vibration considering attitude disturbances.

Due to the influence of structural clearances, the shearer's oscillates and jumps concerning the scraper are frequent, which induces the collision and vibration impact of the traction components and exacerbates the traction failure of the shearer. Therefore, to explore the correlation between attitude disturbance and traction vibration, an experiment on the traction vibration is carried out, the spatial swaying of the shearer and vibration differences between two traction components are obtained, the influence of the lifting angle of the rocker arm is discussed, and the influence mechanism of the shearer attitude disturbance on traction vibration is elucidated. The results indicate that the rolling swing intensity of the shearer is the highest while the yawing swing intensity is the lowest, and the pitch swing intensity increases with the increase of the lifting angle of the rocker arm. Besides, the vibration impact indices of the two walking mechanisms have a competitive relationship of one decreasing but the other increasing, which can be used as a reference signal to judge the rolling swing and load-sharing performance of the traction part. Moreover, with the swing attitude, the competitive relationship of the average of vibration peaks is shown in the two support shoes, and it can be used as a reference signal to judge the pitching swing and the load-sharing performance of the traction part. This result reveals the impact mechanism of attitude disturbances on traction vibration and proposes a signal monitoring approach for judging the traction attitude disturbance and load-sharing performance, providing a reference for reducing traction faults.

Biological activity of a stable 6-aryl-2-benzoyl-pyridine colchicine-binding site inhibitor, 60c, in metastatic, triple-negative breast cancer.

BACKGROUND: Improving survival for patients diagnosed with metastatic disease and overcoming chemoresistance remain significant clinical challenges in treating breast cancer. Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by a lack of therapeutically targetable receptors (ER/PR/HER2). TNBC therapy includes a combination of cytotoxic chemotherapies, including microtubule-targeting agents (MTAs) like paclitaxel (taxane class) or eribulin (vinca class); however, there are currently no FDA-approved MTAs that bind to the colchicine-binding site. Approximately 70 % of patients who initially respond to paclitaxel will develop taxane resistance (TxR). We previously reported that an orally bioavailable colchicine-binding site inhibitor (CBSI), VERU-111, inhibits TNBC tumor growth and treats pre-established metastatic disease. To further improve the potency and metabolic stability of VERU-111, we created next-generation derivatives of its scaffold, including 60c. RESULTS: 60c shows improved in vitro potency compared to VERU-111 for taxane-sensitive and TxR TNBC models, and suppress TxR primary tumor growth without gross toxicity. 60c also suppressed the expansion of axillary lymph node metastases existing prior to treatment. Comparative analysis of excised organs for metastasis between 60c and VERU-111 suggested that 60c has unique anti-metastatic tropism. 60c completely suppressed metastases to the spleen and was more potent to reduce metastatic burden in the leg bones and kidney. In contrast, VERU-111 preferentially inhibited liver metastases and lung metastasis repression was similar. Together, these results position 60c as an additional promising CBSI for TNBC therapy, particularly for patients with TxR disease.

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.

Impact Response of the Tail Beam Jack Based on Bidirectional Fluid-Structure Coupling Simulation.

In top coal caving mining, the coal rock collapse will cause an irregular impact on the tail beam jack of the caving control mechanism. The severe impact will lead to jack failure. The bidirectional fluid-structure coupling model is built on Fluent and Mechanical software to study the impact response of the tail beam jack. The dynamic flow velocity streamlines, hydraulic pressure distribution, stress field, and strain field of the jack under impact load are extracted. The response characteristics of the jack in the stationary state and motion state are analyzed. The conclusions are as follows: the stress and strain of the rodless cavity are much larger than those of the rod cavity, which is more likely to be damaged. The hydraulic pressure in the jack cavity is in vertical layered distribution. The flow velocity streamlines present spiral shapes. The response degree of the hydraulic pressure signal in the rodless cavity is stronger than that in the rod cavity, and the response degree of the flow velocity signal in the rod cavity is stronger than that in the rodless cavity. The impact response of the jack in the motion state is more sensitive and stronger than that in the stationary state. The coal rock collapse situation can be most effectively identified only by comprehensively analyzing the rodless cavity's pressure signal and the rod cavity's velocity signal. This paper innovatively visualizes the flow velocity streamlines and pressure distribution together. The bidirectional fluid-structure coupling method is innovatively applied to the tail beam jack. The findings of this study can help for better understanding of the tail beam jack's structural design and failure prevention. This study provides a certain research basis for the intelligent coal rock identification technology in mining coal based on jack vibration signals.

Intranasal delivery of darunavir improves brain drug concentrations in mice for effective HIV treatment.

Despite the availability of combined antiretroviral therapy (cART), which reduces the HIV replication in chronically HIV-infected patients, HIV associated neurocognitive disorders (HAND) persists in the brain. The blood-brain barrier (BBB) is the major barrier for the penetration of drugs including antiretrovirals, limiting the drug penetration to the brain. In the present study, we have shown improved brain drug concentration in mice for darunavir (DRV), an FDA-approved drug, using an intranasal (IN) delivery method that bypasses the BBB. Here, we compared the time-dependent biodistribution of DRV at two different concentrations, high (25 mg/kg) and low (2.5 mg/kg), using two administration routes intravenous (IV) and intranasal (IN) in brain, liver, lungs, and plasma. Compared with IV administration, IN administration demonstrated a significantly improved DRV penetration in the brain at both low and high DRV concentrations (IV vs IN: at 2.5 mg/kg: 6.91 ± 1.69 ng/g vs 12.08 ± 2.91 ng/g, at 25 mg/kg: 12.84 ± 2.88 ng/g vs 19.74 ± 1.80 ng/g). As expected, IN administration showed significantly lower DRV concentrations in plasma (IV vs IN: at 2.5 mg/kg: 81.37 ± 22.04 ng/g vs 19.91 ± 12.65 ng/g, at 25 mg/kg: 899.12 ± 136.93 ng/g vs 320.56 ± 40.04 ng/g) and liver (IV vs IN: at 2.5 mg/kg: 118.39 ± 28.13 ng/g vs 29.27 ± 4.17 ng/g at 25 mg/kg: 1085.18 ± 255.0 ng/g vs 833.83 ± 242.4 ng/g). The IN administration did not show significant change in lungs compared to the IV administration. As a result, these findings suggest that the IN route can increase the DRV level in the brain, suppressing HIV in the brain reservoirs. Additionally, it could also reduce off-target effects, especially in peripheral organs.

The transiently ordered regions in intrinsically disordered ExsE are correlated with structural elements involved in chaperone binding.

Many Gram-negative bacteria utilize a type III secretion system (T3SS) to deliver protein effectors to target host cells. Transcriptional control of T3SS gene expression is generally coupled to secretion through the release of a regulatory protein. T3SS gene expression in Pseudomonas aeruginosa is regulated by extracellular secretion of ExsE. ExsE is a small 81 residue protein that appears to lack a stable structural core as indicated by previous studies. In this study, we employed various NMR methods to characterize the structure of ExsE alone and when bound to its secretion chaperone ExsC. We found that ExsE is largely unfolded throughout the polypeptide chain, belonging to a class of proteins that are intrinsically disordered. The unfolded, extended conformation of ExsE may expedite efficient secretion through the narrow path of the T3SS secretion channel to activate gene expression in a timely manner. We also found that the structurally flexible ExsE samples through conformations with localized structurally ordered regions. Importantly, these transiently ordered elements are related to the secondary structures involved in binding ExsC based on a prior crystal structure of the ExsC-ExsE complex. These findings support the notion that preexisting structured elements facilitate binding of intrinsically disordered proteins to their targets.

Probiotics in the treatment of infantile colic: a meta-analysis of randomized controlled trials.

Introduction: Introduction: infantile colic has always been a problem for caregivers, and research on probiotics in treating and preventing infant colic is still controversial. Material and methods: trials were performed before November 2021 and retrieved from the PubMed, Web of Science, The Cochrane Library, Medline, and Google Scholar databases. Data extraction and quality evaluation of the trials were performed independently by two investigators. A meta-analysis was performed using Review Manager 5.3. It includes nine randomized controlled trials in 587 infants with colic. Results: eight of these experiments described probiotics for the prevention and treatment of intestinal colic in infants, with 228 in the probiotics group and 227 in the placebo group, with a total effective rate (RR = 1.88, 95 % CI: 1.61 to 2.19, p < 0.00001). Conclusion: probiotics may improve therapeutic and preventive effects, especially within four weeks of probiotic treatment.

Introducción: Introducción: el cólico infantil siempre ha sido un problema para los cuidadores y la investigación sobre los probióticos para tratar y prevenir el cólico infantil sigue siendo controvertida. Material y métodos: los ensayos se realizaron antes de noviembre de 2021 y se recuperaron de las bases de datos PubMed, Web of Science, The Cochrane Library, Medline y Google Scholar. Dos investigadores realizaron de forma independiente la extracción de datos y la evaluación de la calidad de los ensayos. Se realizó un metaanálisis utilizando Review Manager 5.3. Incluye nueve ensayos controlados aleatorios en 587 lactantes con cólicos. Resultados: ocho de estos experimentos describieron probióticos para la prevención y el tratamiento del cólico intestinal en lactantes, con 228 en el grupo de probióticos y 227 en el grupo de placebo, con una tasa efectiva total (RR = 1,88, IC del 95 %: 1,61 a 2,19, p < 0,00001). Conclusión: los probióticos pueden mejorar los efectos terapéuticos y preventivos, especialmente dentro de las cuatro semanas posteriores al tratamiento con los mismos.

Analysis of the vibration characteristics of tail beam of the hydraulic support under random coal gangue particle slip conditions.

To study the influence of the gangue content of coal gangue particles on the vibration signal of the tail beam under sliding condition, this paper combines three-dimensional(3D) laser scanning technology with the finite element method, establishes a finite element model of the real shape of coal gangue particles and the hydraulic support in top coal caving in LS-DYNA, analyzes the influence of gangue content on some characteristics of the acceleration signal on the tail beam in the time and frequency domains, and then studies the influence of the size and total mass of the rock, and the angle of the tail beam on the characteristics. The following conclusions are obtained: when the coal gangue particles slip on the tail beam, an increase in gangue content significantly improves the effective value of the acceleration signal of the tail beam in the time domain and the average power and average amplitude in the frequency domain. With different sizes, total masses, and tail beam angles, the increase in gangue content always causes an increase in acceleration signal characteristics. In terms of the influence of various factors on the same gangue content, at the same total mass, the larger the rock mass size is, the faster the characteristic value increases with the increase in gangue content. The greater the total mass, the greater the value of the acceleration signal characteristics. A smaller angle between the tail beam and the ground increases the value of each characteristic. The results of this study provide a reference for further research on coal gangue identification based on vibrations.

Vibration Response Analysis of the Tail Beam of Hydraulic Support Impacted by Coal Gangue Particles with Different Shapes.

The existing research on coal gangue identification based on vibration usually assumes that coal gangue particles are ideal shapes. To understand the vibration response difference in hydraulic support caused by coal and gangue with real shapes, this paper uses a three-dimensional (3D) scanning technology to determine the real shape of coal particles. The process of coal and gangue impacting the tail beam at different angles was simulated in the LS-DYNA software package, and the effects of shape parameters, velocity, and coal strength on the difference in vibration signals caused by the two were analyzed statistically. The conclusions are as follows: the vibrational response of the tail beam is concentrated mainly in the area between the ribs. The regularity of the velocity signal caused by gangue is better than the regularity of the velocity signal caused by coal, and the attenuation speed of the acceleration signal of gangue is slower than the attenuation speed of the acceleration signal of coal. The probability distributions of the velocity and acceleration responses were analyzed statistically, and the results show that the results from coal can be well fitted by a logarithmic normal function, and the standard deviations of velocity and acceleration are 0.05591 and 489.8, respectively. The gangue results are fitted by the gamma function and the Weibull function, and the standard deviations are 0.13531 and 737.9, respectively, showing that the fitting function has the potential to be used as the basis for coal gangue identification. The change in coal strength has little effect on the vibration response of the tail beam. With increasingly falling velocity, the vibration signal intensity of the tail beam increases, but the discrimination between coal and gangue weakens; therefore, measures should be taken to reduce the falling velocity of the rock mass. The research results of this paper can provide a reference for further study of coal gangue identification methods based on vibration.

Colchicine-Binding Site Agent CH-2-77 as a Potent Tubulin Inhibitor Suppressing Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a highly aggressive type of breast cancer. Unlike other subtypes of breast cancer, TNBC lacks hormone and growth factor receptor targets. Colchicine-binding site inhibitors (CBSI) targeting tubulin have been recognized as attractive agents for cancer therapy, but there are no CBSI drugs currently FDA approved. CH-2-77 has been reported to have potent antiproliferative activity against a panel of cancer cells in vitro and efficacious antitumor effects on melanoma xenografts, yet, its anticancer activity specifically against TNBC is unknown. Herein, we demonstrate that CH-2-77 inhibits the proliferation of both paclitaxel-sensitive and paclitaxel-resistant TNBC cells with an average IC50 of 3 nmol/L. CH-2-77 also efficiently disrupts the microtubule assembly, inhibits the migration and invasion of TNBC cells, and induces G2-M cell-cycle arrest. The increased number of apoptotic cells and the pattern of expression of apoptosis-related proteins in treated MDA-MB-231 cells suggest that CH-2-77 induces cell apoptosis through the intrinsic apoptotic pathway. In vivo, CH-2-77 shows acceptable overall pharmacokinetics and strongly suppresses the growth of orthotopic MDA-MB-231 xenografts without gross cumulative toxicities when administered 5 times a week. The in vivo efficacy of CH-2-77 (20 mg/kg) is comparable with that of CA4P (28 mg/kg), a CBSI that went through clinical trials. Importantly, CH-2-77 prevents lung metastasis originating from the mammary fat pad in a dose-dependent manner. Our data demonstrate that CH-2-77 is a promising new generation of tubulin inhibitors that inhibit the growth and metastasis of TNBC, and it is worthy of further development as an anticancer agent.

NMR studies of a channel protein without membranes: structure and dynamics of water-solubilized KcsA.

Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics.

Design, Synthesis, and Biological Evaluation of Stable Colchicine-Binding Site Tubulin Inhibitors 6-Aryl-2-benzoyl-pyridines as Potential Anticancer Agents.

We previously reported a potent tubulin inhibitor CH-2-77. In this study, we optimized the structure of CH-2-77 by blocking metabolically labile sites and synthesized a series of CH-2-77 analogues. Two compounds, 40a and 60c, preserved the potency while improving the metabolic stability over CH-2-77 by 3- to 4-fold (46.8 and 29.4 vs 10.8 min in human microsomes). We determined the high-resolution X-ray crystal structures of 40a (resolution 2.3 Å) and 60c (resolution 2.6 Å) in complex with tubulin and confirmed their direct binding at the colchicine-binding site. In vitro, 60c maintained its mode of action by inhibiting tubulin polymerization and was effective against P-glycoprotein-mediated multiple drug resistance and taxol resistance. In vivo, 60c exhibited a strong inhibitory effect on tumor growth and metastasis in a taxol-resistant A375/TxR xenograft model without obvious toxicity. Collectively, this work showed that 60c is a promising lead compound for further development as a potential anticancer agent.