The benzophenanthridine alkaloid sanguinarine perturbs microtubule assembly dynamics through tubulin binding: a possible mechanism for its antiproliferative activity

Sanguinarine has been shown to inhibit proliferation of several types of human cancer cell including multidrug-resistant cells, whereas it has minimal cytotoxicity against normal cells such as neutrophils and keratinocytes. By analyzing the antiproliferative activity of sanguinarine in relation to its effects on mitosis and microtubule assembly, we found that it inhibits cancer cell proliferation by a novel mechanism. It inhibited HeLa cell proliferation with a half-maximal inhibitory concentration of 1.6±0.1μm. In its lower effective inhibitory concentration range, sanguinarine depolymerized microtubules of both interphase and mitotic cells and perturbed chromosome organization in mitotic HeLa cells. At concentrations of 2μm, it induced bundling of interphase microtubules and formation of granular tubulin aggregates. A brief exposure of HeLa cells to sanguinarine caused irreversible depolymerization of the microtubules, inhibited cell proliferation, and induced cell death. However, in contrast with several other microtubule-depolymerizing agents, sanguinarine did not arrest cell cycle progression at mitosis. In vitro, low concentrations of sanguinarine inhibited microtubule assembly. At higher concentrations (>40μm), it altered polymer morphology. Further, it induced aggregation of tubulin in the presence of microtubule-associated proteins. The binding of sanguinarine to tubulin induces conformational changes in tubulin. Together, the results suggest that sanguinarine inhibits cell proliferation at least in part by perturbing microtubule assembly dynamics

Modeling the Effects of Drug Binding on the Dynamic Instability of Microtubules

We propose a stochastic model that accounts for the growth, catastrophe and rescue processes of steady state microtubules assembled from MAP-free tubulin. Both experimentally and theoretically we study the perturbation of microtubule dynamic instability by S-methyl-D-DM1, a synthetic derivative of the microtubule-targeted agent maytansine and a potential anticancer agent. We find that to be an effective suppressor of microtubule dynamics a drug must primarily suppress the loss of GDP tubulin from the microtubule tip.Comment: 17 pages, 11 figures, to appear in Phys. Bio

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Cooperative stabilization of microtubule dynamics by EB1 and CLIP-170 involves displacement of stably bound P i at microtubule ends

End binding protein 1 (EB1) and cytoplasmic linker protein of 170 kDa (CLIP-170) are two well-studied microtubule plus-end-tracking proteins (+TIPs) that target growing microtubule plus ends in the form of comet tails and regulate microtubule dynamics. However, the mechanism by which they regulate microtubule dynamics is not well understood. Using full-length EB1 and a minimal functional fragment of CLIP-170 (ClipCG12), we found that EB1 and CLIP-170 cooperatively regulate microtubule dynamic instability at concentrations below which neither protein is effective. By use of small-angle X-ray scattering and analytical ultracentrifugation, we found that ClipCG12 adopts a largely extended conformation with two noninteracting CAP-Gly domains and that it formed a complex in solution with EB1. Using a reconstituted steady-state mammalian microtubule system, we found that at a low concentration of 250 nM, neither EB1 nor ClipCG12 individually modulated plus-end dynamic instability. Higher concentrations (up to 2 μM) of the two proteins individually did modulate dynamic instability, perhaps by a combination of effects at the tips and along the microtubule lengths. However, when low concentrations (250 nM) of EB1 and ClipCG12 were present together, the mixture modulated dynamic instability considerably. Using a pulsing strategy with [γ 32P]GTP, we further found that unlike EB1 or ClipCG12 alone, the EB1-ClipCG12 mixture partially depleted the microtubule ends of stably bound 32P i. Together, our results suggest that EB1 and ClipCG12 act cooperatively to regulate microtubule dynamics. They further indicate that stabilization of microtubule plus ends by the EB1-ClipCG12 mixture may involve modification of an aspect of the stabilizing cap.Supported by USPHS NS13560 (L.W.), by a FEBS Fellowship, Juan de la Cierva postdoctoral contracts, and a Marie Curie Career Integration Grant EB-SxIP: 293831 (R.M.B), and by grants from the Swiss National Science Foundation (M.O.S.).Peer Reviewe

Synthesis of nano-sized C<SUB>3</SUB>-symmetric 2,4,6-triphenyl-1,3,5-s-triazine and 1,3,5-triphenylbenzene derivatives via the trimerization followed by Suzuki-Miyaura cross-coupling or O-alkylation reactions and their biological evaluation

Various C3-symmetric 2,4,6-triphenyl-1,3-5-s-triazine and 1,3,5-triphenylbenzene derivatives have been prepared using cyclotrimerization, Suzuki-Miyaura cross-coupling and O-alkylation reactions as key steps. The biological activity of O-alkylated triazine derivatives has been studied towards the HeLa cell proliferation. The resulting C3-symmetric derivatives can also be useful in materials chemistry

Lemert Charles C., French sociology. Rupture and renewal since 1968.

Krämer Hans Leo. Lemert Charles C., French sociology. Rupture and renewal since 1968.. In: Revue française de sociologie, 1982, 23-4. pp. 707-710

Biochemical and <i>in silico</i> analysis of the binding mode of erastin with tubulin

Erastin (ERN) is a small molecule that induces different forms of cell death. For example, it has been reported to induce ferroptosis by disrupting tubulin subunits that maintain the voltage-dependent anion channels (VDACs) of mitochondria. Although its possible binding to tubulin has been suggested, the fine details of the interaction between ERN and tubulin are poorly understood. Using a combination of biochemical, cell-model and in silico approaches, we elucidate the interactions of ERN with tubulin and their biological manifestations. After confirming ERN's antiproliferative efficacy (IC50, 20 ± 3.2 M) and induction of cell death in the breast cancer cell line MDA-MB-231, the binding interactions of ERN with tubulin were examined. ERN bound to tubulin in a concentration-dependent manner, disorganizing the structural integrity of the protein, as substantiated via the tryptophan-quenching assay and the aniline-naphthalene sulfonate binding assay, respectively. In silico studies based on molecular docking revealed a docking score of −5.863 kcal/mol, suggesting strong binding interactions of ERN with tubulin. Additionally, molecular dynamics simulation and Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) analyses evinced the binding free energy (ΔGbinding) of −31.235 kcal/mol, substantiating strong binding affinity of ERN with tubulin. Ligplot analysis showed hydrogen bonding with specific amino acids (Asn A226, Thr A223, Gln B247 and Val B355). QikProp-based ADME (absorption, distribution, metabolism and excretion) assessment showed considerable therapeutic potential for ERN. Communicated by Ramaswamy H. Sarma</p

Elucidation of the anticancer potential and tubulin isotype-specific interactions of β-sitosterol

<p>Beta-sitosterol (β-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the ‘SITO-site’) compared to the colchicine binding site at the interface of α- and β-tubulin. In this study, we investigated the anticancer efficacy of β-SITO against invasive breast carcinoma using MCF-7 cells. Since ‘isotypes’ of β-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of β-tubulin in αβ-tubulin dimer. β-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of β-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to β-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that β-SITO preferentially binds with specific β-tubulin isotypes (β_II and β_III) in the αβ-tubulin dimer. Both these β-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of β-SITO, and its potential clinical utility against β_II and β_III isotype-overexpressing neoplasms.</p

Rational design, synthesis, and biological evaluation of third generation α-noscapine analogues as potent tubulin binding anti-cancer agents.

Systematic screening based on structural similarity of drugs such as colchicine and podophyllotoxin led to identification of noscapine, a microtubule-targeted agent that attenuates the dynamic instability of microtubules without affecting the total polymer mass of microtubules. We report a new generation of noscapine derivatives as potential tubulin binding anti-cancer agents. Molecular modeling experiments of these derivatives 5a, 6a-j yielded better docking score (-7.252 to -5.402 kCal/mol) than the parent compound, noscapine (-5.505 kCal/mol) and its existing derivatives (-5.563 to -6.412 kCal/mol). Free energy (ΔG bind ) calculations based on the linear interaction energy (LIE) empirical equation utilizing Surface Generalized Born (SGB) continuum solvent model predicted the tubulin-binding affinities for the derivatives 5a, 6a-j (ranging from -4.923 to -6.189 kCal/mol). Compound 6f showed highest binding affinity to tubulin (-6.189 kCal/mol). The experimental evaluation of these compounds corroborated with theoretical studies. N-(3-brormobenzyl) noscapine (6f) binds tubulin with highest binding affinity (KD, 38 ± 4.0 µM), which is ~ 4.0 times higher than that of the parent compound, noscapine (KD, 144 ± 1.0 µM) and is also more potent than that of the first generation clinical candidate EM011, 9-bromonoscapine (KD, 54 ± 9.1 µM). All these compounds exhibited substantial cytotoxicity toward cancer cells, with IC50 values ranging from 6.7 µM to 72.9 µM; compound 6f showed prominent anti-cancer efficacy with IC50 values ranging from 6.7 µM to 26.9 µM in cancer cells of different tissues of origin. These compounds perturbed DNA synthesis, delayed the cell cycle progression at G2/M phase, and induced apoptotic cell death in cancer cells. Collectively, the study reported here identified potent, third generation noscapinoids as new anti-cancer agents