14 research outputs found
Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity.
A High-throughput-compatible FRET-based Platform for Identification and Characterization of Botulinum Neurotoxin Light Chain Modulators
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Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity.
The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus
Copper transporters and chaperones CTR1, CTR2, ATOX1, and CCS as determinants of cisplatin sensitivity
The development of resistance to cisplatin (cDDP) is commonly accompanied by reduced drug uptake or increased efflux. Previous studies in yeast and murine embryonic fibroblasts have reported that the copper (Cu) transporters and chaperones participate in the uptake, efflux, and intracellular distribution of cDDP. However, there is conflicting data from studies in human cells. We used CRISPR-Cas9 genome editing to individually knock out the human copper transporters CTR1 and CTR2 and the copper chaperones ATOX1 and CCS. Isogenic knockout cell lines were generated in both human HEK-293T and ovarian carcinoma OVCAR8 cells. All knockout cell lines had slowed growth compared to parental cells, small changes in basal Cu levels, and varying sensitivities to Cu depending on the gene targeted. However, all of the knockouts demonstrated only modest 2 to 5-fold changes in cDDP sensitivity that did not differ from the range of sensitivities of 10 wild type clones grown from the same parental cell population. We conclude that, under basal conditions, loss of CTR1, CTR2, ATOX1, or CCS does not produce a change in cisplatin sensitivity that exceeds the variance found within the parental population, suggesting that they are not essential to the mechanism by which cDDP enters these cell lines and is transported to the nucleus
Synergistic effect of aptamers that inhibit exosites 1 and 2 on thrombin
Thrombin is a multifunctional protease that plays a key role in hemostasis, thrombosis, and inflammation. Most thrombin inhibitors currently used as antithrombotic agents target thrombin's active site and inhibit all of its myriad of activities. Exosites 1 and 2 are distinct regions on the surface of thrombin that provide specificity to its proteolytic activity by mediating binding to substrates, receptors, and cofactors. Exosite 1 mediates binding and cleavage of fibrinogen, proteolytically activated receptors, and some coagulation factors, while exosite 2 mediates binding to heparin and to platelet receptor GPIb-IX-V. The crystal structures of two nucleic acid ligands bound to thrombin have been solved. Previously Padmanabhan and colleagues solved the structure of a DNA aptamer bound to exosite 1 and we reported the structure of an RNA aptamer bound to exosite 2 on thrombin. Based upon these structural studies we speculated that the two aptamers would not compete for binding to thrombin. We observe that simultaneously blocking both exosites with the aptamers leads to synergistic inhibition of thrombin-dependent platelet activation and procoagulant activity. This combination of exosite 1 and exosite 2 inhibitors may provide a particularly effective antithrombotic approach
High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes
Botulism
is caused by potent and specific bacterial neurotoxins
that infect host neurons and block neurotransmitter release. Treatment
for botulism is limited to administration of an antitoxin within a
short time window, before the toxin enters neurons. Alternatively,
current botulism drug development targets the toxin light chain, which
is a zinc-dependent metalloprotease that is delivered into neurons
and mediates long-term pathology. Several groups have identified inhibitory
small molecules, peptides, or aptamers, although no molecule has advanced
to the clinic due to a lack of efficacy in advanced models. Here we
used a homogeneous high-throughput enzyme assay to screen three libraries
of drug-like small molecules for new chemotypes that modulate recombinant
botulinum neurotoxin light chain activity. High-throughput screening
of 97088 compounds identified numerous small molecules that activate
or inhibit metalloprotease activity. We describe four major classes
of inhibitory compounds identified, detail their structure–activity
relationships, and assess their relative inhibitory potency. A previously
unreported chemotype in any context of enzyme inhibition is described
with potent submicromolar inhibition (<i>K</i><sub>i</sub> = 200–300 nM). Additional detailed kinetic analyses and cellular
cytotoxicity assays indicate the best compound from this series is
a competitive inhibitor with cytotoxicity values around 4–5
μM. Given the potency and drug-like character of these lead
compounds, further studies, including cellular activity assays and
DMPK analysis, are justified
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Conformationally Selective RNA Aptamers Allosterically Modulate the β2-Adrenoceptor
G-protein-coupled receptor (GPCR) ligands function by stabilizing multiple, functionally distinct receptor conformations. This property underlies how “biased agonists” activate specific subsets of a given receptor’s signaling profile. However, stabilization of distinct active GPCR conformations to enable structural characterization of mechanisms underlying GPCR activation remains difficult. These challenges have accentuated the need for receptor tools that allosterically stabilize and regulate receptor function via unique, previously unappreciated mechanisms. Here, utilizing a highly diverse RNA library combined with advanced selection strategies involving state-of-the-art next-generation sequencing and bioinformatics analyses, we identify RNA aptamers that bind a prototypical GPCR, β2-adrenoceptor (β2AR). Using biochemical, pharmacological, and biophysical approaches, we demonstrate that these aptamers bind with nanomolar affinity at defined surfaces of the receptor, allosterically stabilizing active, inactive, and ligand-specific receptor conformations. The discovery of RNA aptamers as allosteric GPCR modulators significantly expands the diversity of ligands available to study the structural and functional regulation of GPCRs
Identification of Clinically Viable Quinolinol Inhibitors of Botulinum Neurotoxin A Light Chain
Botulinum
neurotoxins (BoNT) are the most potent toxins known and
a significant bioterrorist threat. Few small molecule compounds have
been identified that are active in cell-based or animal models, potentially
due to toxin enzyme plasticity. Here we screened commercially available
quinolinols, as well as synthesized hydroxyquinolines. Seventy-two
compounds had IC<sub>50</sub> values below 10 μM, with the best
compound exhibiting submicromolar inhibition (IC<sub>50</sub> = 0.8
μM). Structure–activity relationship trends showed that
the enzyme tolerates various substitutions at R<sub>1</sub> but has
a clear preference for bulky aryl amide groups at R<sub>2</sub>, while
methylation at R<sub>3</sub> increased inhibitor potency. Evaluation
of the most potent compounds in an ADME panel showed that these compounds
possess poor solubility at pH 6.8, but display excellent solubility
at low pH, suggesting that oral dosing may be possible. Our data show
the potential of quinolinol compounds as BoNT therapeutics due to
their good in vitro potencies and favorable ADME properties