Directed evolution of cyclic peptides for inhibition of autophagy

In recent decades it has become increasingly clear that induction of autophagy plays an important role in the development of treatment resistance and dormancy in many cancer types. Unfortunately, chloroquine (CQ) and hydroxychloroquine (HCQ), two autophagy inhibitors in clinical trials, suffer from poor pharmacokinetics and high toxicity at therapeutic dosages. This has prompted intense interest in the development of targeted autophagy inhibitors to re-sensitize disease to treatment with minimal impact on normal tissue. We utilized Scanning Unnatural Protease Resistant (SUPR) mRNA display to develop macrocyclic peptides targeting the autophagy protein LC3. The resulting peptides bound LC3A and LC3B—two essential components of the autophagosome maturation machinery—with mid-nanomolar affinities and disrupted protein–protein interactions (PPIs) between LC3 and its binding partners in vitro. The most promising LC3-binding SUPR peptide accessed the cytosol at low micromolar concentrations as measured by chloroalkane penetration assay (CAPA) and inhibited starvation-mediated GFP-LC3 puncta formation in a concentration-dependent manner. LC3-binding SUPR peptides re-sensitized platinum-resistant ovarian cancer cells to cisplatin treatment and triggered accumulation of the adapter protein p62 suggesting decreased autophagic flux through successful disruption of LC3 PPIs in cell culture. In mouse models of metastatic ovarian cancer, treatment with LC3-binding SUPR peptides and carboplatin resulted in almost complete inhibition of tumor growth after four weeks of treatment. These results indicate that SUPR peptide mRNA display can be used to develop cell-penetrating macrocyclic peptides that target and disrupt the autophagic machinery in vitro and in vivo.

SUPR peptide mRNA display was used to evolve a cell-permeable, macrocyclic peptide for autophagy inhibition.     

Crystallographic report: Chlorodiphenyltin(IV) piperidine‐1‐carbodithioate

A trigonal bipyramidal C₂ClS₂ coordination geometry for tin is found in Ph₂Sn(S₂CN(CH₂)₅)Cl. Copyright © 2005 John Wiley & Sons, Ltd.     

Landau damping of ion-acoustic waves with simultaneous effects of non-extensivity and non-thermality in the presence of hybrid Cairns-Tsallis distributed electrons

The dispersion properties and Landau damping rate of ion-acoustic waves (IAWs) with the hybrid Cairns-Tsallis distributed (CTD) electrons and Maxwellian ions are investigated using the plasma kinetic model based on Vlasov-Poisson's equations. For both super-extensive (q < 1) and sub-extensive (q > 1) plasmas, the dielectric response function, real frequency, and Landau damping rate of IAWs are derived. By taking the effect of θ_{i, e} (ion-to-electron temperature ratio) into account, it is found that with the increase of ion temperature, the real frequency and wave dispersion effects increase as well (for both super-extensive and sub-extensive cases). Exploring the properties of the Landau damping rate of IAWs with the simultaneous presence of non-thermal parameter α and non-extensive parameter q, a comparison of numerical and analytical results is presented. It is found that in different ranges of θ_{e, i} (electron-to-ion temperature ratio), on decreasing the values of the non-extensive parameter and increasing values of the non-thermal parameter, the weak damping rate is observed (vice versa) in super-extensive or super-thermal plasma, although the trend of the damping rate in sub-thermal plasma is similar (as in the case of super-thermal plasma) but is less weak. It is further revealed that the damping rate of IAWs in thermal plasmas (Maxwellian) is stronger than the damping rate of IAWs in the case of non-thermal plasmas (CTD). The current study is applicable to provide deep insight and further allow the exploration of electrostatic plasma modes in different space and laboratory plasma environments where the hybrid CTD plasma exists.     

Synthesis,Spectroscopy, Semi‐empirical and Biological Activities of Organotin(IV) Complexes with o‐Isopropyl Carbonodithioic Acid

New organotin(IV) complexes have been synthesized by treating potassium o‐isopropyl carbonodithioate with R₂SnCl₂/R₃SnCl in 1 : 2/1 : 1 M/L ratio. All complexes have been characterized by IR and NMR (¹H, ¹³C) spectroscopy. IR results shows that ligand acts as bidentate which is also confirmed by semi‐empirical study. NMR data reveals four coordinated geometry in solution. Computed positive heat of formation shows that complex 5 is thermodynamically unstable. UV/visible spectroscopy was used to assess the mode of interaction and binding of the complexes with DNA which shows that complex 5 exhibits higher binding constant as compared to complex 3 . In protein kinase inhibition assay, compound 3 was found most active, while other biological activities shows that triorganotin(IV) complexes are biologically more active as compared to diorganotin(IV) complexes.     

Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl-O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na⁺ ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides.

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Theoretical and experimental studies of ion imprinted polymer for nitrate detection

Molecular imprinting is an approach to synthesize receptors with specific molecular recognition properties. A computational method was carried out to study interaction between template and monomer in prepolymerization mixture. The functional monomer and template complexes were optimized, at the minimum energy confirmation using Austin Model 1 semi empirical method within Restricted Hartree Fock formalism. The theoretical results showed that allylthiourea (functional monomer) has the largest interaction energy towards template (sodium nitrate) with the mole ratio of 4 : 1; functional monomer : template. The resulting polymers were characterized using Fourier Transform infrared spectroscopy, thermogravimetry analysis and field emission scanning electron microscopy. Rebinding experiments were carried out to evaluate binding capacity of the polymer. The adsorption data of ion imprinted polymer (IIP) were fitted with Langmuir-Freundlich isotherm model. Pseudo-second order kinetic model was used to describe the kinetic adsorption behavior of IIP. The experimental binding result showed good agreement with theoretical computation and the IIP was further used for nitrate ion detection. The results of membrane optimization indicated that the sensor, which composed of 30% polyvinylchloride, 60% nitrophenyl octyl ether as a plasticizer, 2% sodium tetraphenyl borate, and 10% IIP as ionophore exhibited an almost Nernstian slope with the limit of detection 3.9 × 10^-6 M. The fabricated sensor had shown good potential in nitrate detection with wide linear range, low limit of detection and found to have good selectivity towards nitrate ion over other anion.