Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: Design,synthesis, biological investigation and docking studies

In an attempt to develop potent anti-tubulin agents against most dreadful disease cancer, a library of 28 novel triazole tethered isatin-coumarin hybrids were synthesized by click chemistry approach. Synthesized hybrids were characterized and evaluated against a panel of human cancer cell lines viz. THP-1, COLO-205, HCT-116 and PC-3. Biological assay unveiled that, compounds A-1 to A-6, B-1 to B-4 and C-1 to C-3 displayed significant inhibitory potential against THP-1, COLO-205 and HCT-116 cell lines which were more sensitive towards the designed hybrids. PC-3 among these cell lines was found to be almost resistant. Established SAR revealed marked dependence of the cytotoxic activity on the type of substituent on isatin and the length of carbon-bridge connecting isatin moiety with triazole ring. Unsubstituted isatin and two carbon-bridge were found to be crucial for cytotoxicity. Three most potent hybrids (A-1, A-2 and B-1) were further tested for tubulin polymerization inhibition. Among these three compounds, A-1 found to be endowed with most prominent tubulin polymerization inhibition potential with IC₅₀ value of 1.06 µM which was further confirmed by using confocal microscopy. Possible binding interactions between the most potent hybrid molecule A-1 and tubulin were also divulged by molecular modeling studies.     

A systematic review and integrative approach to decode the common molecular link between levodopa response and Parkinson’s disease

Background

PD is a progressive neurodegenerative disorder commonly treated by levodopa. The findings from genetic studies on adverse effects (ADRs) and levodopa efficacy are mostly inconclusive. Here, we aim to identify predictive genetic biomarkers for levodopa response (LR) and determine common molecular link with disease susceptibility. A systematic review for LR was conducted for ADR, and drug efficacy, independently. All included articles were assessed for methodological quality on 14 parameters. GWAS of PD were also reviewed. Protein-protein interaction (PPI) analysis using STRING and functional enrichment using WebGestalt was performed to explore the common link between LR and PD.

Results

From 37 candidate studies on levodopa toxicity, 18 genes were found associated, of which, CA_n STR 13, 14 (DRD2) was most significantly associated with dyskinesia, followed by rs1801133 (MTHFR) with hyper-homocysteinemia, and rs474559 (HOMER1) with hallucination. Similarly, 8 studies on efficacy resulted in 4 genes in which rs28363170, rs3836790 (SLC6A3) and rs4680 (COMT), were significant. To establish the molecular connection between LR with PD, we identified 35 genes significantly associated with PD. With 19 proteins associated with LR and 35 with PD, two independent PPI networks were constructed. Among the 67 nodes (263 edges) in LR, and 62 nodes (190 edges) in PD pathophysiology, UBC, SNCA, FYN, SRC, CAMK2A, and SLC6A3 were identified as common potential candidates.

Conclusion

Our study revealed the genetically significant polymorphism concerning the ADRs and levodopa efficacy. The six common genes may be used as predictive markers for therapy optimization and as putative drug target candidates.

     

Activation of the P2X7 ion channel by soluble and covalently bound ligands

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7.     

Hexafluoroisopropanol-induced helix–sheet transition of stem bromelain: Correlation to function

Stem bromelain is a proteolytic phytoprotein with a variety of therapeutic effects. Understanding its structural properties could provide insight into the mechanisms underlying its clinical utility. Stem bromelain was evaluated for its conformational and folding properties at the pH conditions it encounters when administered orally. It exists as a partially folded intermediate at pH 2.0. The conformational changes to this intermediate state were evaluated using fluorinated alcohols known to induce changes similar to those seen in vivo. Studies using circular dichroism, fluorescence emission spectroscopy, binding of the hydrophobic dye 1-anilino-8-naphthalene sulfonic acid and mass spectrometry indicate that treatment with 10–30% hexafluoroisopropanol induces the partially folded intermediate to adopt much of the native protein's secondary structure, but only a rudimentary tertiary structure, characteristic of the molten globule state. Addition of slightly higher concentrations of hexafluoroisopropanol caused transformation from an α-helix to a β-sheet and induced formation of a compact nonnative structure. This nonnative form was more inhibitory of cell survival than either the native or the partially folded intermediate forms, as measured by enhanced suppression of proliferative cues (e.g., extracellular-signal-regulated kinase) and initiation of apoptotic events. The nonnative form also showed better antitumorigenic properties, as evaluated using an induced two-stage mouse skin papilloma model. In contrast, the nonnative state showed only a fraction of the proteolytic activity of the native form. This study demonstrates that hexafluoroisopropanol can induce a conformational change in stem bromelain to a form with potentially useful therapeutic properties different from those of the native protein.     

Trifluoroethanol stabilizes the molten globule state and induces non-amyloidic turbidity in stem bromelain near its isoelectric point

Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T_ms), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.     

Experimental Measurement of Settling Velocity of Spherical Particles in Unconfined and Confined Surfactant-based Shear Thinning Viscoelastic Fluids

An experimental study is performed to measure the terminal settling velocities of spherical particles in surfactant based shear thinning viscoelastic (VES) fluids. The measurements are made for particles settling in unbounded fluids and fluids between parallel walls. VES fluids over a wide range of rheological properties are prepared and rheologically characterized. The rheological characterization involves steady shear-viscosity and dynamic oscillatory-shear measurements to quantify the viscous and elastic properties respectively. The settling velocities under unbounded conditions are measured in beakers having diameters at least 25x the diameter of particles. For measuring settling velocities between parallel walls, two experimental cells with different wall spacing are constructed. Spherical particles of varying sizes are gently dropped in the fluids and allowed to settle. The process is recorded with a high resolution video camera and the trajectory of the particle is recorded using image analysis software. Terminal settling velocities are calculated from the data.The impact of elasticity on settling velocity in unbounded fluids is quantified by comparing the experimental settling velocity to the settling velocity calculated by the inelastic drag predictions of Renaud et al.¹ Results show that elasticity of fluids can increase or decrease the settling velocity. The magnitude of reduction/increase is a function of the rheological properties of the fluids and properties of particles. Confining walls are observed to cause a retardation effect on settling and the retardation is measured in terms of wall factors.     

Evidence for a GTP-binding protein coupling thrombin receptor to PIP2-phospholipase C in membranes of hamster fibroblasts

Two different methods were used to study directly alpha-thrombin modulation of polyphosphoinositide breakdown in membranes prepared from Chinese hamster lung (CHL) fibroblasts. In the first one we labelled the lipid pool by incubating the intact cells with myo-[3H]inositol prior to membrane isolation; in the other we used exogenous [3H]PIP2 with phosphatidylethanolamine (1:10) added as liposomes to freshly isolated membranes. A Ca2+-dependent PIP2 and PIP phospholipase C activity was characterized by measuring the rate of formation of inositol tris- and bisphosphate. Basal phospholipase C activity was stimulated up to 3-fold by GTP or GTP-gamma-S. Of the two mitogens, alpha-thrombin and EGF, known to stimulate DNA synthesis in Chinese hamster fibroblasts, only alpha-thrombin is a potent activator of PIP2 breakdown in intact cells. Consistent with this observation, alpha-thrombin but not EGF potentiated GTP-gamma-S-dependent phospholipase C activity in membrane preparations. These results strongly support the hypothesis that a GTP-binding protein couples alpha-thrombin receptor to PIP2 hydrolysis. Because both methods used to assay phospholipase C gave identical results, we conclude that the coupling is at the level of PIP2-phosphodiesterase activity.     

An immunoluminometric assay for cardiotrophin-1: a newly identified cytokine is present in normal human plasma and is increased in heart failure.

Cardiotrophin-1, a member of the interleukin-6 related cytokine family which acts via the glycoprotein 130 signalling pathway, may be involved in the process of ventricular remodelling. Its presence in human plasma has never been reported. We have devised a non-radioactive immunoluminometric sensitive and specific assay for CT-1 based on a competitive ligand binding principle. The chemiluminescent label 4-(2-succinimidyl-oxycarbonylethyl)phenyl-10-methylacridinium 9-carboxylate fluorosulfonate was used to label a peptide representing a domain in the middle section of CT-1. Assay of this domain of CT-1 (amino acids 105-120) in patients with heart failure revealed elevated CT-1 values median 87 [range 74.3-182.8] fmol/ml) compared to normal controls (CT-1 median 29.55 [range 6.9-48.3] fmol/ml, P<0.0005). The molecular weight of human CT-1 was estimated to be 26.7 kD from sodium dodecyl sulphate polyacrylamide gel electrophoresis. This is the first quantitative assessment of CT-1 in humans. Furthermore, this is the first demonstration of significant elevation of plasma CT-1 in patients with heart failure.