12 research outputs found
Anti-malarial activity of geldanamycin derivatives in mice infected with Plasmodium yoelii
Background
Geldanamycin (GA), a benzoquinone ansamycin antibiotic has been shown in vitro to possess anti-plasmodial activity. Pharmacological activity of this drug is attributed to its ability to inhibit PfHSP90. The parasite growth arrest has been shown to be due to drug-induced blockage of the transition from ring to trophozoite stage. To further evaluate the consequences of this pharmacodyamic feature, the anti-malarial activity of GA analogs with enhanced drug properties in a Plasmodium-infected animal model have been evaluated for their capacity to induce clearance of the parasite. In the process, a hypothesis was subsequently tested regarding the susceptibility of the cured animals to malaria reflected in an attenuated parasite load that may be evoked by a protective immune response in the host. Methods
Six weeks old Swiss mice were infected with a lethal Plasmodium yoelii (17XL) strain. On appearance of clinical symptoms of malaria, these animals were treated with two different GA derivatives and the parasite load was monitored over 15-16 days. Drug-treated animals cured of the parasite were then re-challenged with a lethal dose of P. yoelii 17XL. Serum samples from GA cured mice that were re-challenged with P. yoelii 17XL were examined for the presence of antibodies against the parasite proteins using western blot analysis. Results
Treatment of P. yoelii 17XL infected mice with GA derivatives showed slow recovery from clinical symptoms of the disease. Blood smears from drug treated mice indicated a dominance of ring stage parasites when compared to controls. Although, P. yoelii preferentially invades normocytes (mature rbcs), in drug-treated animals there was an increased invasion of reticulocytes. Cured animals exhibited robust protection against subsequent infection and serum samples from these animals showed antibodies against a vast majority of parasite proteins. Conclusions
Treatment with GA derivatives blocked the transition from ring to trophozoite stage presumably by the inhibition of HSP90 associated functions. Persistence of parasite in ring stage leads to robust humoral immune response as well as a shift in invasion specificity from normocytes to reticulocyte. It is likely that the treatment with the water-soluble GA derivative creates an attenuated state (less virulent with altered invasion specificity) that persists in the host system, allowing it to mount a robust immune response
Multimeric Rhodamine Dye-Induced Aggregation of Silver Nanoparticles for Surface-Enhanced Raman Scattering
Isotopic
variants of Rhodamine 6G (R6G) have previously been used
as a method of multiplexed detection for Surface Enhanced Raman Spectroscopy
(SERS), including protein detection and quantification. Challenges
exist, however, with producing long-term stable SERS signals with
exposure to silver or gold metal surfaces without the use of additional
protective coatings of nanomaterials. Here, novel rhodamine âdimersâ
and âtrimersâ have been created that demonstrate a higher
avidity for metal nanoparticles and induce aggregation to create plasmonic
âhotspotsâ as indicated by enhanced Raman scattering in situ. These aggregates can be formed in a colloid, on
surfaces, or membrane substrates such as polyÂ(vinylidene fluoride)
for applications in biosciences. The integrity of the materials and
Raman signals are maintained for months of time on different substrates.
These dye materials should provide avenues for simplified in situ generation of sensors for Raman-based assays especially
in settings requiring highly robust performance
Translation of a Protease Turnover Assay for Clinical Discrimination of Mucinous Pancreatic Cysts
The classification of pancreatic cyst fluids can provide a basis for the early detection of pancreatic cancer while eliminating unnecessary procedures. A candidate biomarker, gastricsin (pepsin C), was found to be present in potentially malignant mucinous pancreatic cyst fluids. A gastricsin activity assay using a magnetic bead-based platform has been developed using immobilized peptide substrates selective for gastricsin bearing a dimeric rhodamine dye. The unique dye structure allows quantitation of enzyme-cleaved product by both fluorescence and surface enhanced Raman spectroscopy (SERS). The performance of this assay was compared with ELISA assays of pepsinogen C and the standard of care, carcinoembryonic antigen (CEA), in the same clinical sample cohort. A retrospective cohort of mucinous (n = 40) and non-mucinous (n = 29) classes of pancreatic cyst fluid samples were analyzed using the new protease activity assay. For both assay detection modes, successful differentiation of mucinous and non-mucinous cyst fluid was achieved using 1 µL clinical samples. The activity-based assays in combination with CEA exhibit optimal sensitivity and specificity of 87% and 93%, respectively. The use of this gastricsin activity assay requires a minimal volume of clinical specimen, offers a rapid assay time, and shows improvements in the differentiation of mucinous and non-mucinous cysts using an accurate standardized readout of product formation, all without interfering with the clinical standard of care
Translation of a Protease Turnover Assay for Clinical Discrimination of Mucinous Pancreatic Cysts.
The classification of pancreatic cyst fluids can provide a basis for the early detection of pancreatic cancer while eliminating unnecessary procedures. A candidate biomarker, gastricsin (pepsin C), was found to be present in potentially malignant mucinous pancreatic cyst fluids. A gastricsin activity assay using a magnetic bead-based platform has been developed using immobilized peptide substrates selective for gastricsin bearing a dimeric rhodamine dye. The unique dye structure allows quantitation of enzyme-cleaved product by both fluorescence and surface enhanced Raman spectroscopy (SERS). The performance of this assay was compared with ELISA assays of pepsinogen C and the standard of care, carcinoembryonic antigen (CEA), in the same clinical sample cohort. A retrospective cohort of mucinous (n = 40) and non-mucinous (n = 29) classes of pancreatic cyst fluid samples were analyzed using the new protease activity assay. For both assay detection modes, successful differentiation of mucinous and non-mucinous cyst fluid was achieved using 1 ÂľL clinical samples. The activity-based assays in combination with CEA exhibit optimal sensitivity and specificity of 87% and 93%, respectively. The use of this gastricsin activity assay requires a minimal volume of clinical specimen, offers a rapid assay time, and shows improvements in the differentiation of mucinous and non-mucinous cysts using an accurate standardized readout of product formation, all without interfering with the clinical standard of care
Discovery of Inhibitors for Proliferating Cell Nuclear Antigen Using a Computational-Based Linked-Multiple-Fragment Screen
Proliferating cell nuclear antigen (PCNA) is a central factor in DNA replication and repair pathways that plays an essential role in genome stability. The functional roles of PCNA are mediated through an extensive list of protein-protein interactions, each of which transmits specific information in protein assemblies. The flexibility at the PCNA-protein interaction interfaces offers opportunities for the discovery of functionally selective inhibitors of DNA repair pathways. Current fragment-based drug design methodologies can be limited by the flexibility of protein interfaces. These factors motivated an approach to defining compounds that could leverage previously identified subpockets on PCNA that are suitable for fragment-binding sites. Methodologies for screening multiple connected fragment-binding events in distinct subpockets are deployed to improve the selection of fragment combinations. A flexible backbone based on; N; -alkyl-glycine amides offers a scaffold to combinatorically link multiple fragments for in silico screening libraries that explore the diversity of subpockets at protein interfaces. This approach was applied to discover new potential inhibitors of DNA replication and repair that target PCNA in a multiprotein recognition site. The screens of the libraries were designed to computationally filter ligands based upon the fragments and positions to <1%, which were synthesized and tested for direct binding to PCNA. Molecular dynamics simulations also revealed distinct features of these novel molecules that block key PCNA-protein interactions. Furthermore, a Bayesian classifier predicted 15 of the 16 new inhibitors to be modulators of protein-protein interactions, demonstrating the method's utility as an effective screening filter. The cellular activities of example ligands with similar affinity for PCNA demonstrate unique properties for novel selective synergy with therapeutic DNA-damaging agents in drug-resistant contexts
Selective Inhibition of STAT3 Phosphorylation Using a Nuclear-Targeted Kinase Inhibitor
The
discovery of compounds that selectively modulate signaling
and effector proteins downstream of EGFR could have important implications
for understanding specific roles for pathway activation. A complicating
factor for receptor tyrosine kinases is their capacity to be translocated
to the nucleus upon ligand engagement. Once localized in subcellular
compartments like the nucleus, the roles for EGFR take on additional
features, many of which are still being revealed. Additionally, nuclear
localization of EGFR has been implicated in downstream events that
have significance for therapy resistance and disease progression.
The challenges to addressing the differential roles for EGFR in the
nucleus motivated experimental approaches that can selectively modulate
its subcellular function. By adding modifications to the established
EGFR kinase inhibitor gefitinib, an approach to small molecule conjugates
with a unique nuclear-targeting peptoid sequence was tested in both
human and murine breast tumor cell models for their capacity to inhibit
EGF-stimulated activation of ERK1/2 and STAT3. While gefitinib alone
inhibits both of these downstream effectors, data acquired here indicate
that compartmentalization of the gefitinib conjugates allows for pathway
specific inhibition of STAT3 while not affecting ERK1/2 signaling.
The inhibitor conjugates offered a more direct route to evaluate the
role of EGF-stimulated epithelial-to-mesenchymal transition in these
breast cancer cell models. These conjugates revealed that STAT3 activation
is not involved in EGF-induced EMT, and instead utilization of the
cytoplasmic MAP kinase signaling pathway is critical to this process.
This is the first example of a conjugate kinase inhibitor capable
of partitioning to the nucleus and offers a new approach to enhancing
kinase inhibitor specificity
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Intramuscular administration of glyoxylate rescues swine from lethal cyanide poisoning and ameliorates the biochemical sequalae of cyanide intoxication
Cyanide-a fast-acting poison-is easy to obtain given its widespread use in manufacturing industries. It is a high-threat chemical agent that poses a risk of occupational exposure in addition to being a terrorist agent. FDA-approved cyanide antidotes must be given intravenously, which is not practical in a mass casualty setting due to the time and skill required to obtain intravenous access. Glyoxylate is an endogenous metabolite that binds cyanide and reverses cyanide-induced redox imbalances independent of chelation. Efficacy and biochemical mechanistic studies in an FDA-approved preclinical animal model have not been reported. Therefore, in a swine model of cyanide poisoning, we evaluated the efficacy of intramuscular glyoxylate on clinical, metabolic, and biochemical endpoints. Animals were instrumented for continuous hemodynamic monitoring and infused with potassium cyanide. Following cyanide-induced apnea, saline control or glyoxylate was administered intramuscularly. Throughout the study, serial blood samples were collected for pharmacokinetic, metabolite, and biochemical studies, in addition, vital signs, hemodynamic parameters, and laboratory values were measured. Survival in glyoxylate-treated animals was 83% compared with 12% in saline-treated control animals (pâ<â.01). Glyoxylate treatment improved physiological parameters including pulse oximetry, arterial oxygenation, respiration, and pH. In addition, levels of citric acid cycle metabolites returned to baseline levels by the end of the study. Moreover, glyoxylate exerted distinct effects on redox balance as compared with a cyanide-chelating countermeasure. In our preclinical swine model of lethal cyanide poisoning, intramuscular administration of the endogenous metabolite glyoxylate improved survival and clinical outcomes, and ameliorated the biochemical effects of cyanide
Identification of Platinum(II) Sulfide Complexes Suitable as Intramuscular Cyanide Countermeasures
The
development of rapidly acting cyanide countermeasures
using
intramuscular injection (IM) represents an unmet medical need to mitigate
toxicant exposures in mass casualty settings. Previous work established
that cisplatin and other platinumÂ(II) or platinumÂ(IV)-based agents
effectively mitigate cyanide toxicity in zebrafish. Cyanideâs in vivo reaction with platinum-containing materials was
proposed to reduce the risk of acute toxicities. However, cyanide
antidote activity depended on a formulation of platinum-chloride salts
with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered
saline (PBS). A working hypothesis to explain the DMSO requirement
is that the formation of platinumâsulfoxide complexes activates
the cyanide scavenging properties of platinum. Preparations of isolated
NaPtCl5âDMSO and Na (NH3)2PtClâDMSO complexes in the absence of excess DMSO provided
agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue
in zebrafish and mouse models. The enhancement of the cyanide scavenging
effects of the DMSO ligand could be attributed to the activation of
platinumÂ(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy
of DMSO complexes was not robust when administered IM. Alternative
PtÂ(II) materials containing sulfide and amine ligands in bidentate
complexes show enhanced reactivity toward cyanide addition. The cyanide
addition products yielded tetracyanoplatinateÂ(II), translating to
a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents
demonstrate a robust and enhanced potency over the DMSO-containing
complexes using IM administration in mouse and rabbit models of cyanide
toxicity. Using the zebrafish model with these PtÂ(II) complexes, no
acute cardiotoxicity was detected, and dose levels required to reach
lethality exceeded 100 times the effective dose. Data are presented
to support a general chemical design approach that can expand a new
lead candidate series for developing next-generation cyanide countermeasures
Glyoxylate protects against cyanide toxicity through metabolic modulation
Although cyanideâs biological effects are pleiotropic, its most obvious effects are as a metabolic poison. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations that are believed to cause lethality. From systematic screens of human metabolites using a zebrafish model of cyanide toxicity, we have identified the TCA-derived small molecule glyoxylate as a potential cyanide countermeasure. Following cyanide exposure, treatment with glyoxylate in both mammalian and non-mammalian animal models confers resistance to cyanide toxicity with greater efficacy and faster kinetics than known cyanide scavengers. Glyoxylate-mediated cyanide resistance is accompanied by rapid pyruvate consumption without an accompanying increase in lactate concentration. Lactate dehydrogenase is required for this effect which distinguishes the mechanism of glyoxylate rescue as distinct from countermeasures based solely on chemical cyanide scavenging. Our metabolic data together support the hypothesis that glyoxylate confers survival at least in part by reversing the cyanide-induced redox imbalances in the cytosol and mitochondria. The data presented herein represent the identification of a potential cyanide countermeasure operating through a novel mechanism of metabolic modulation