18720184
Synthesis and evaluation of aza-peptidyl inhibitors of the lysosomal asparaginyl endopeptidase, legumain.
Legumain or asparaginly endopeptidase (AEP) is a lysosomal cysteine protease with a high level of specificity for cleavage of protein substrates after an asparagine residue. It is also capable of cleaving after aspartic acids sites when in the acidic environment of the lysosome. Legumain expression and activity is linked to a number of pathological conditions including cancer, atherosclerosis and inflammation, yet its biological role in these pathologies is not well-understood. Highly potent and selective inhibitors of legumain would not only be valuable for studying the functional roles of legumain in these conditions, but may have therapeutic potential as well. We describe here the design, synthesis and in vitro evaluation of selective legumain inhibitors based on the aza-asparaginyl scaffold. We synthesized a library of aza-peptidyl inhibitors with various non-natural amino acids and different electrophilic warheads, and characterized the kinetic properties of inactivation of legumain. We also synthesized fluorescently labeled inhibitors to investigate cell permeability and selectivity of the compounds. The inhibitors have second order rate constants of up to 5 × 10(4)M(-1)s(-1) and IC(50) values as low as 4 nM against recombinant mouse legumain. In addition, the inhibitors are highly selective toward legumain and have little or no cross-reactivity with cathepsins. Overall, we have identified several valuable new inhibitors of legumain that can be used to study legumain function in multiple disease models.
981469
Development of near-infrared fluorophore (NIRF)-labeled activity-based probes for in vivo imaging of legumain.
Asparaginyl endopeptidase, or legumain, is a lysosomal cysteine protease that was originally identified in plants and later found to be involved in antigen presentation in higher eukaryotes. Legumain is also up-regulated in a number of human cancers, and recent studies suggest that it may play important functional roles in the process of tumorigenesis. However, detailed functional studies in relevant animal models of human disease have been hindered by the lack of suitably selective small molecule inhibitors and imaging reagents. Here we present the design, optimization, and in vivo application of fluorescently labeled activity-based probes (ABPs) for legumain. We demonstrate that optimized aza-peptidyl Asn epoxides are highly selective and potent inhibitors that can be readily converted into near-infrared fluorophore-labeled ABPs for whole body, noninvasive imaging applications. We show that these probes specifically label legumain in various normal tissues as well as in solid tumors when applied in vivo. Interestingly, addition of cell-penetrating peptides to the probes enhanced cellular uptake but resulted in increased cross-reactivity toward other lysosomal proteases as the result of their accumulation in lysosomes. Overall, we find that aza-peptidyl Asn ABPs are valuable new tools for the future study of legumain function in more complex models of human disease.
7778854
Target deconvolution techniques in modern phenotypic profiling.
The past decade has seen rapid growth in the use of diverse compound libraries in classical phenotypic screens to identify modulators of a given process. The subsequent process of identifying the molecular targets of active hits, also called 'target deconvolution', is an essential step for understanding compound mechanism of action and for using the identified hits as tools for further dissection of a given biological process. Recent advances in 'omics' technologies, coupled with in silico approaches and the reduced cost of whole genome sequencing, have greatly improved the workflow of target deconvolution and have contributed to a renaissance of 'modern' phenotypic profiling. In this review, we will outline how both new and old techniques are being used in the difficult process of target identification and validation as well as discuss some of the ongoing challenges remaining for phenotypic screening.
26956563
Water quantitatively induces the mucoadhesion of liquid crystalline phases of glyceryl monooleate.
The possible role of water in the mucoadhesion phenomenon exhibited by the liquid crystalline phases of glyceryl monooleate was investigated using an in-vitro tensile strength technique. The mucoadhesion of the liquid crystalline phases of glyceryl monooleate was found to occur following uptake of water. The mucoadhesive force of the cubic phase was consistent since it is not capable of taking up additional water. An increase in pre-load period greatly facilitated the mucoadhesion of glyceryl monooleate (0% w/w initial water content), suggesting that the mucoadhesion is dependent upon the extent of the dehydration of the substrate. A good linear relationship between initial water content of the liquid crystalline phases and mucoadhesive force led to the conclusion that the mucoadhesive force increased with decreasing initial water concentration. Rheological properties of the liquid crystalline phases were also studied to allow a correlation between physical changes and mucoadhesion of the liquid crystalline phases, revealing that higher water concentrations in the liquid crystalline phases led to a more ordered structure that showed less mucoadhesion. The results of this study indicated that the mucoadhesive force of the liquid crystalline phases of glyceryl monooleate is determined by the capability to take up water from a water-rich environment. It may, therefore, be advantageous to use the lamellar phase as a buccal drug carrier as opposed to the relatively less mucoadhesive cubic phase.
4366904
Principles of locomotion for simple-shaped cells
MOVING cells display a variety of shapes and modes of locomotion1, but it is not clear how motility at the molecular level relates to the locomotion of a whole cell, a problem compounded in studies of cells with complex shapes2–5. A striking feature of fish epidermal keratocyte locomotion is its apparent simplicity6. Here we present a kinematic description of locomotion which is consistent with the semicircular shape and persistent 'gliding'; motion of fish epidermal keratocytes. We propose that extension of the front and retraction of the rear of these cells occurs perpendicularly to the cell edge, and that a graded distribution of extension and retraction rates along the cell margin maintains cell shape and size during locomotion. Evidence for this description is provided by the predicted circumferential motion of lamellar features and the curvature of 'photo-marked' lines within specific molecular components of moving keratocytes. Our description relates the dynamics of molecular assemblies to the movement of a whole cell.
23509495
FgVelB globally regulates sexual reproduction, mycotoxin production and pathogenicity in the cereal pathogen Fusarium graminearum.
The velvet genes are conserved in ascomycetous fungi and function as global regulators of differentiation and secondary metabolism. Here, we characterized one of the velvet genes, designated FgVelB, in the plant-pathogenic fungus Fusarium graminearum, which causes fusarium head blight in cereals and produces mycotoxins within plants. FgVelB-deleted (ΔFgVelB) strains produced fewer aerial mycelia with less pigmentation than those of the wild-type (WT) during vegetative growth. Under sexual development conditions, the ΔFgVelB strains produced no fruiting bodies but retained male fertility, and conidiation was threefold higher compared with the WT strain. Production of trichothecene and zearalenone was dramatically reduced compared with the WT strain. In addition, the ΔFgVelB strains were incapable of colonizing host plant tissues. Transcript analyses revealed that FgVelB was highly expressed during the sexual development stage, and may be regulated by a mitogen-activated protein kinase cascade. Microarray analysis showed that FgVelB affects regulatory pathways mediated by the mating-type loci and a G-protein alpha subunit, as well as primary and secondary metabolism. These results suggest that FgVelB has diverse biological functions, probably by acting as a member of a possible velvet protein complex, although identification of the FgVelB-FgVeA complex and the determination of its roles require further investigation.
15555862
Hydrotropic Solubilization of Paclitaxel: Analysis of Chemical Structures for Hydrotropic Property
AbstractPurpose. To identify hydrotropic agents that can increase aqueous paclitaxel (PTX) solubility and to study the chemical structures necessary for hydrotropic properties so that polymeric hydrotropic agents can be synthesized.
Methods. More than 60 candidate hydrotropic agents (or hydro- tropes) were tested for their ability to increase the aqueous PTX solubility. A number of nicotinamide analogues were synthesized based on the observation that nicotinamide showed a favorable hydrotropic property. The identified hydrotropes for PTX were used to examine the structure-activity relationship.Results. N,N-Diethylnicotinamide (NNDENA) was found to be the most effective hydrotropic agent for PTX. The aqueous PTX solubility was 39 mg/ml and 512 mg/ml at NNDENA concentrations of 3.5 M and 5.95 M, respectively. These values are 5-6 orders of magnitude greater than the intrinsic solubility of 0.30 ± 0.02 μg/ml. N-Picolylnicotinamide, N-allylnicotinamide, and sodium salicylate were also excellent hydrotropes for PTX. Solubility data showed that an effective hydrotropic agent should be highly water soluble while maintaining a hydrophobic segment.
Conclusions. The present study identified several hydrotropic agents effective for increasing aqueous solubility of PTX and analyzed the structural requirements for this hydrotropic property. This information can be used to find other hydrotropic compounds and to synthesize polymeric hydrotropes that are effective for PTX and other poorly water-soluble drugs.
30413815
Bioconjugated Ag nanoparticles and CdTe nanowires: metamaterials with field-enhanced light absorption.
The hybrid assembly of inorganic and metallic nanomaterials by means of chemical and biological bonding to yield manifold optical and electromagnetic properties has received widespread attention. Currently available highly monodispersed nanomaterials such as semiconductors and noble metals that can be conjugated by ligand–receptor, antigen– antibody reactions, polymer tethering, and DNA hybridization are used as building blocks for 2D or 3D superstructures in which new collective properties of these artificial assemblies have been obtained. They represent a large class of newmaterials (i.e., metamaterials) in which the properties are determined not only by classical atomic composition, but also by nanoscale organization of structural components. Recently, metamaterials based on metallic composites have received special attention as these are expected to display negative values for permittivity and refractive index. This property should lead to a multitude of unique optical effects. Many of these effects are related to surface plasmons on Au or Ag nanoparticles (NPs), which generate exceptionally high localized electromagnetic fields, and have been exploited in surface-enhanced Raman spectroscopy and some optoelectronic devices. 22] Metamaterials from semiconductors and their combinations with metals can also produce optical effects that are useful for development of advanced sensing and imaging technologies. Recently, we