Drug-Like Small Molecules That Inhibit Expression of the Oncogenic MicroRNA-21.

We report the discovery of drug-like small molecules that bind specifically to the precursor of the oncogenic and pro-inflammatory microRNA-21 with mid-nanomolar affinity. The small molecules target a local structure at the Dicer cleavage site and induce distinctive structural changes in the RNA, which correlate with specific inhibition of miRNA processing. Structurally conservative single nucleotide substitutions eliminate the conformational change induced by the small molecules, which is also not observed in other miRNA precursors. The most potent of these compounds reduces cellular proliferation and miR-21 levels in cancer cell lines without inhibiting kinases or classical receptors, while closely related compounds without this specific binding activity are inactive in cells. These molecules are highly ligand-efficient (MW < 330) and display specific biochemical and cellular activity by suppressing the maturation of miR-21, thereby providing an avenue toward therapeutic development in multiple diseases where miR-21 is abnormally expressed.

Preferential solvation of carbohydrates in water-trifluoroethanol mixtures: a solvent detected heteronuclear NMR approach.

The present study aims to establish a simple approach involving multi-field multinuclear longitudinal relaxation (R1) analysis of the solvents to decipher solute-solvent interactions during the solvation of model carbohydrates in aqueous trifluoroethanol (TFE) co-solvent systems (TFE:D2O). The behavior of D2O and TFE is monitored around ß-CD (ß-cyclodextrin) and glucose through R1D (2H) and R1F (19F), respectively. Correlation times (τc) are estimated for D2O and TFE for various % (v/v) compositions of TFE:D2O mixtures. The differential trends of the R1 or τc ratio for D2O and TFE (in the presence and absence of carbohydrates) revealed that both ß-CD and glucose undergo selective solvation by TFE in comparison to D2O. Owing to its encapsulation properties, ß-CD exhibited a comparatively higher tendency to undergo solvation by TFE than glucose. The maximum transfer of solute bound water to bulk solvent appears in the 20-30% (v/v) TFE range. The current approach emerges as being straightforward in contrast to traditional methods that primarily focus on solute behavior to unravel the preferential solvation dynamics.

Solution-state NMR evaluation of molecular interaction between monoaromatic carboxylic acids and dissolved humic acid.

Understanding the nature of interactions between the aromatic organic pollutants with dissolved humic acid (HA) is fundamental for the prediction of their environmental fate and subsequent development of efficient remediation methods. The present study employs solution-state 1H/19F NMR methods to investigate the non-covalent interaction between aqueous peat humic acid (Aldrich HA) and monoaromatic carboxylic acids (CA), viz., 2, 6 diflourobenzoic acid (DFBA) and its non-fluorinated analog, benzoic acid (BA). NMR self-diffusion measurement of HA protons confirmed micellar nature indicating possibility of encapsulation of small molecules through host-guest interaction. 19F-1H and 1H-1H saturation transfer difference (STD) experiments reveal the mode of insertion of CA into HA superstructure. The strength of interaction has been evaluated by analyzing T1/T2 relaxation times and self-diffusion coefficients of CA as a function of HA concentration. Association constants extracted for CA-HA complexes from NMR diffusion experiments reflected that the association between DFBA-HA (2.34 mM-1) is significantly higher than that of BA-HA (0.97 mM-1). The experimental outcome reiterated that substitution of -H with halogen atoms (-F in specific) to aromatic ring plays a dominant role in modulating the strength of association and mode of insertion of organic pollutants into HA superstructure. The present study emphasizes that AHA can be a potential remediating agent for organic contaminants due to its superior binding affinity compared to less humified extracted HA (EHA) from Karwar, Rajasthan, India.

Assessment of the Role of 2,2,2-Trifluoroethanol Solvent Dynamics in Inducing Conformational Transitions in Melittin: An Approach with Solvent 19F Low-Field NMR Relaxation and Overhauser Dynamic Nuclear Polarization Studies.

2,2,2-Trifluoroethanol (TFE) is one of the fluoroalcohols that have been known to induce and stabilize an open helical structure in many proteins and peptides. The current study has benchmarked low-field 19F NMR relaxation and 19F Overhauser dynamic nuclear polarization (ODNP) by providing a brief account of TFE solvent dynamics in a model melittin (MLT, an antimicrobial peptide) solution with a TFE-D2O cosolvent mixture at pH 7.4. Further, this approach has been employed to reveal the solvation of MLT by TFE in a nonbuffered solution with pH 2.8 for the first time. The structural transition of MLT has been elucidated via solvent dynamics by measuring the 19F TFE relaxation rates at 0.34 T for various TFE-D2O compositions in the absence (bulk TFE) and in the presence of MLT at both the pH values. A complementary initial record of circular dichroism experiments on these aqueous MLT solutions with TFE as the cosolvent at two different pH conditions demonstrated the structural transition from a random coil to a helical or from a folded helical to an open helical structure. The molecular correlation time derived from the corresponding relaxation rates shows that TFE resides on the MLT surface in both pH conditions. However, the trends in the variation of molecular correlation time ratio as a function of TFE concentration represent that the mechanism and the extent to which TFE affects the MLT structural integrity are different at different pH values. The extraction of the DNP coupling parameter from steady-state 19F ODNP experiments performed in the presence of 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl at 0.34 T revealed changes in the solvation dynamics of TFE concomitant with the MLT structural transition. In summary, 19F relaxation and ODNP measurements made at a low field have allowed direct monitoring of TFE dynamics during the MLT structural transition in terms of preferential solvation. The choice of experiments performed at a moderately low field (0.34 T) enabled us to exploit on the one hand almost 1200-fold mitigation of the strong contribution of 19F chemical shift anisotropy at 11.76 T, whereas on the other hand, the ODNP experiment offered a window for probing molecular dynamics on timescales of the order of 10-1000 ps.

Binding Interaction of Organofluorine-Serum Albumin: A Comparative Ligand-Detected 19F NMR Analysis.

In the present study, we attempt to characterize fluorinated ligand-serum albumin interaction in solution by a set of one-dimensional 19F ligand-based experiments. In this regard, a model system diflunisal (DFL)-human serum albumin (HSA) has been chosen to benchmark the utility of 19F relaxation and diffusion-based experiments in deciphering ligand-protein interactions. Further, we extend the application of a similar set of 19F experiments to unravel the molecular interaction in an unexplored system of 2,6-difluorobenzoic acid (DFBA)-bovine serum albumin (BSA). Interaction analysis of DFBA-SA is of particular interest because DFBA is not only a stable metabolite of a number of pesticides but also used as the starting reagent of many fluorinated drugs. Observation of 19F-1H & 1H-1H saturation transfer difference effects confirmed binding of the ligands to SA. Further, these ligand-protein complexes were probed in terms of the dissociation constant ( KD), number of binding sites ( n), bound fraction of the ligand ( Pb), the complex lifetime (τres), and exchange rate ( Kex). Although Carr-Purcell-Meiboom-Gill (CPMG)-based transverse relaxation and diffusion analysis quantified the former three quantities, the latter two were determined by the constant time fast pulsing CPMG method. Additionally, 19F competition binding experiments performed with well-characterized BSA site markers and DFBA indicated nonspecific binding of DFBA to BSA, whereas similar measurements in the case of HSA with DFL and DFBA revealed superior binding interaction of DFL with SA.

Role of biofilm morphology, matrix content and surface hydrophobicity in the biofilm-forming capacity of various Candida species.

The present study aimed to evaluate the role of biofilm morphology, matrix content and surface hydrophobicity in the biofilm-forming capacity of Candida albicans and non-albicans Candida (NAC) spp. Biofilm formation was determined by microtitre plate assay and bright-field and scanning electron microscopy. The matrix carbohydrates, proteins and e-DNA were quantified by phenol-sulfuric acid, bicinchoninic acid and UV spectroscopy, respectively. Specific glycosyl residues were detected by dot blot. The cell-surface hydrophobicity was determined by hydrocarbon adhesion assay. Candida tropicalis was found to exhibit the highest adherence to polystyrene. It formed dense biofilms with extensive pseudohyphae and hyphal elements, high hydrophobicity and the greatest amount of matrix carbohydrates, proteins and e-DNA. C. albicans displayed higher adherence and a complex biofilm morphology with larger aggregates than Candida parapsilosis and Candida krusei, but had lower matrix content and hydrophobicity. Thus, the combinatorial effect of increased filamentation, maximum matrix content and high hydrophobicity contributes to the enhanced biofilm-forming capacity of C. tropicalis.

Solvent-dependent binding interactions of the organophosphate pesticide, chlorpyrifos (CPF), and its metabolite, 3,5,6-trichloro-2-pyridinol (TCPy), with Bovine Serum Albumin (BSA): A comparative fluorescence quenching analysis.

Analysis of the interaction of pesticides and their metabolites with the cellular proteins has drawn considerable attention in past several years to understand the effect of pesticides on environment and mankind. In this study, we have investigated the binding interaction of Bovine Serum Albumin (BSA) with a widely used organophosphorous insecticide chlorpyrifos (CPF), and its stable metabolite, 3,5,6-trichloro-2-pyridinol (TCPy) to provide a comparative analysis of the two molecules by employing various spectroscopic techniques viz., UV-vis absorption, Circular Dichroism (CD), and Fluorescence spectroscopy. The fluorescence quenching studies of BSA emission in two different solvents viz., water and methanol in presence of CPF and TCPy have led to the revelation of several interesting facts about the pesticide-protein interaction. It has been found that both the molecules cause static quenching of BSA emission as seen from the Stern-Volmer constant (Ksv) irrespective of the solvent used for the analysis. While TCPy is a stronger quencher in water, it exhibits comparable quenching capacity with CPF in methanol. The solvent dependent differential binding interaction of the two molecules finally indicates possibility of diverse bio-distribution of the pesticides within human body. The UV-vis and CD spectra of BSA in presence of the test molecules have unravelled that the molecules formed ground state complex that are highly reversible in nature and have minimal effect on the protein secondary structure. Furthermore it is also understood that structural changes of BSA in presence of CPF is significantly higher compared to that in presence of TCPY.