15 research outputs found
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A New Spin on Antibody–Drug Conjugates: Trastuzumab-Fulvestrant Colloidal Drug Aggregates Target HER2-Positive Cells
While
the formation of colloidal aggregates leads to artifacts in early
drug discovery, their composition makes them attractive as nanoparticle
formulations for targeted drug delivery as the entire nanoparticle
is composed of drug. The typical transient stability of colloidal
aggregates has inhibited exploiting this property. To overcome this
limitation, we investigated a series of proteins to stabilize colloidal
aggregates of the chemotherapeutic, fulvestrant, including the following:
bovine serum albumin, a generic human immunoglobulin G, and trastuzumab,
a therapeutic human epidermal growth factor receptor 2 antibody. Protein
coronas reduced colloid size to <300 nm and improved their stability
to over 48 h in both buffered saline and media containing serum protein.
Unlike colloids stabilized with other proteins, trastuzumab-fulvestrant
colloids were taken up by HER2 overexpressing cells and were cytotoxic.
This new targeted formulation reimagines antibody–drug conjugates,
delivering mM concentrations of drug to a cell
Stable Colloidal Drug Aggregates Catch and Release Active Enzymes
Small
molecule aggregates are considered nuisance compounds in
drug discovery, but their unusual properties as colloids could be
exploited to form stable vehicles to preserve protein activity. We
investigated the coaggregation of seven molecules chosen because they
had been previously intensely studied as colloidal aggregators, coformulating
them with bis-azo dyes. The coformulation reduced colloid sizes to
<100 nm and improved uniformity of the particle size distribution.
The new colloid formulations are more stable than previous aggregator
particles. Specifically, coaggregation of Congo Red with sorafenib,
tetraiodophenolphthalein (TIPT), or vemurafenib produced particles
that are stable in solutions of high ionic strength and high protein
concentrations. Like traditional, single compound colloidal aggregates,
the stabilized colloids adsorbed and inhibited enzymes like β-lactamase,
malate dehydrogenase, and trypsin. Unlike traditional aggregates,
the coformulated colloid-protein particles could be centrifuged and
resuspended multiple times, and from resuspended particles, active
trypsin could be released up to 72 h after adsorption. Unexpectedly,
the stable colloidal formulations can sequester, stabilize, and isolate
enzymes by spin-down, resuspension, and release
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Internal Structure and Preferential Protein Binding of Colloidal Aggregates
Colloidal
aggregates of small molecules are the most common artifact
in early drug discovery, sequestering and inhibiting target proteins
without specificity. Understanding their structure and mechanism has
been crucial to developing tools to control for, and occasionally
even exploit, these particles. Unfortunately, their polydispersity
and transient stability have prevented exploration of certain elementary
properties, such as how they pack. Dye-stabilized colloidal aggregates
exhibit enhanced homogeneity and stability when compared to conventional
colloidal aggregates, enabling investigation of some of these properties.
By small-angle X-ray scattering and multiangle light scattering, pair
distance distribution functions suggest that the dye-stabilized colloids
are filled, not hollow, spheres. Stability of the coformulated colloids
enabled investigation of their preference for binding DNA, peptides,
or folded proteins, and their ability to purify one from the other.
The coformulated colloids showed little ability to bind DNA. Correspondingly,
the colloids preferentially sequestered protein from even a 1600-fold
excess of peptides that are themselves the result of a digest of the
same protein. This may reflect the avidity advantage that a protein
has in a surface-to-surface interaction with the colloids. For the
first time, colloids could be shown to have preferences of up to 90-fold
for particular proteins over others. Loaded onto the colloids, bound
enzyme could be spun down, resuspended, and released back into buffer,
regaining most of its activity. Implications of these observations
for colloid mechanisms and utility will be considered
Hyperthermia influences fate determination of neural stem cells with lncRNAs alterations in the early differentiation - Fig 3
<p>A. Flow cytometric analysis of differentiated cells B. Western blot analysis of Tuj-1, GFAP and O4 of NSCs cultured on 1%FBS-DF<sub>12</sub> medium of 37NSCs and 40NSCs for 3 days. C. Quantitation of protein bands.*P<0.05.</p
The morphological changes of NSCs at different time point after differentiation.
<p>A-C: Morphological changes in 37NSC group during 0h-72h differentiation, most differentiated cells were small, round and triangular or polygonal with 2 to 3 processes. D-F: In 40NSCs group, the differentiated cell exhibited large, flat and elongated shape with longer, wider processes. Scale bar: 100um.</p
Characterization and differentiation of NSCs.
<p>A: phase-contrast image of NSCs globes cultured 5d in NSCs culture medium. B: Immunostaining of NSCs with Nestin antibody. C-F: immunostaining of differentiated cells with astrocyte marker GFAP, neuron marker Tuj-1 and oligodendrocyte marker O4 in 10% FBS-DF<sub>12</sub> for 5 days. Scale bar: A-B 400 um; C-F 20 um.</p
Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from <i>Clostridium perfringens</i>
<div><p><i>Clostridium perfringens</i> is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the <i>Cp</i>GH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s.</p></div
Oligonucleotide primers used for cloning of the <i>Cp</i>GH31 CBM32s.
<p>Oligonucleotide primers used for cloning of the <i>Cp</i>GH31 CBM32s.</p
GalNAc binding determinants of <i>Cp</i>GH31 CBM32-2.
<p>(A) Backbone cartoon representation of CBM32-2 (grey) in complex with GalNAc (green), solved to a resolution of 2.00 Å. The associated calcium ion is depicted as a blue sphere. <i>F</i><sub><i>obs</i></sub><i>-F</i><sub><i>calc</i></sub> electron density maps of GalNAc (green) bound to peptide chains A and B of the CBM32-2:GalNAc structure are shown in green mesh and contoured to 3.0 σ. (B) GalNAc (green) is bound to CBM32-2 by a several aromatic and polar residues (orange) via direct and water-mediated hydrogen bonds. Associated water molecules are shown as cyan spheres and hydrogen bonds are depicted by dashed lines. The stacking interaction is mediated by Trp1359. (C) The shallow GalNAc-specific binding site of CBM32-2 (shown in grey) accommodates the O4 hydroxyl group of the ligand in an axial position only. The sugar associates with the side chain of Trp1359 (light purple) and forms numerous hydrogen-bonding interactions (magenta) that target the O6 hydroxyl and 2-acetamido groups on either end of the sugar.</p
Amino acid sequence comparison of GalNAc-binding <i>C</i>. <i>perfringens</i> CBM32s.
<p>Sequence alignment of the three <i>Cp</i>GH31 CBM32 modules with other functionally characterized CBM32 modules from the following family 33, family 84, and family 89 glycoside hydrolases with specificity for galacto- or gluco-configured sugars: <i>Cp</i>GH33 CBM32, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref021" target="_blank">21</a>]; <i>Cp</i>GH84A CBM32-1, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref024" target="_blank">24</a>]; <i>Cp</i>GH84A CBM32-2, GlcNAc specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref023" target="_blank">23</a>]; <i>Cp</i>GH84C CBM32, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref022" target="_blank">22</a>]; <i>Cp</i>GH89 CBM32-3 and CBM32-4, GlcNAc-α-1,4-Gal specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref020" target="_blank">20</a>]; <i>Cp</i>GH89 CBM32-5, galacto-configured sugar specificity [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref020" target="_blank">20</a>]. Positions comprising conserved amino acid residues are identified by white single-letter code and highlighted in red while positions displaying amino acid residues of similar physicochemical properties are identified by red-single letter code. Sugar-coordinating amino acid residues in each CBM32 seequence are identified by black boxes. The sequence alignment was created using CLUSTAL OMEGA [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref046" target="_blank">46</a>] and displayed using ESPript [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171606#pone.0171606.ref047" target="_blank">47</a>].</p