The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS) assay, we screened a 151,616-compound library for the ability to inhibit ε-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.
Current therapies to enhance CNS cholinergic function rely primarily on extracellular acetylcholinesterase (AChE) inhibition, a pharmacotherapeutic strategy that produces dose-limiting side effects. The Na + -dependent, high-affinity choline transporter (CHT) is an unexplored target for cholinergic medication development. Although functional at the plasma membrane, CHT at steady-state is localized to synaptic vesicles such that vesicular fusion can support a biosynthetic response to neuronal excitation. To identify allosteric potentiators of CHT activity, we mapped endocytic sequences in the C-terminus of human CHT, identifying transporter mutants that exhibit significantly increased transport function. A stable HEK-293 cell line was generated from one of these mutants (CHT LV-AA) and used to establish a high-throughput screen (HTS) compatible assay based on the electrogenic nature of the transporter. We established that the addition of choline to these cells, at concentrations appropriate for high-affinity choline transport at presynaptic terminals, generates a hemicholinium-3 (HC-3)-sensitive, membrane depolarization that can be used for the screening of CHT inhibitors and activators. Using this assay, we discovered that staurosporine increased CHT LV-AA choline uptake activity, an effect mediated by a decrease in choline K M with no change in V max . As staurosporine did not change surface levels of CHT, nor inhibit HC-3 binding, we propose that its action is directly or indirectly allosteric in nature. Surprisingly, staurosporine reduced choline-induced membrane depolarization, suggesting that increased substrate coupling to ion gradients, arising at the expense of nonstoichiometric ion flow, accompanies a shift of CHT to a higher-affinity state. Our findings provide a new approach for the identification of CHT modulators that is compatible with high-throughput screening approaches and presents a novel model by which small molecules can enhance substrate flux through enhanced gradient coupling.
Misregulation of the Wnt pathway has been shown to be responsible for a variety of human diseases, most notably cancers. Screens for inhibitors of this pathway have been performed almost exclusively using cultured mammalian cells or with purified proteins. We have previously developed a biochemical assay using Xenopus egg extracts to recapitulate key cytoplasmic events in the Wnt pathway. Using this biochemical system, we show that a recombinant form of the Wnt coreceptor, LRP6, regulates the stability of two key components of the Wnt pathway (β-catenin and Axin) in opposing fashion. We have now fused β-catenin and Axin to firefly and Renilla luciferase, respectively, and demonstrate that the fusion proteins behave similarly as their wild-type counterparts. Using this dual luciferase readout, we adapted the Xenopus extracts system for high-throughput screening. Results from these screens demonstrate signal distribution curves that reflect the complexity of the library screened. Of several compounds identified as cytoplasmic modulators of the Wnt pathway, one was further validated as a bona fide inhibitor of the Wnt pathway in cultured mammalian cells and Xenopus embryos. We show that other embryonic pathways may be amendable to screening for inhibitors/modulators in Xenopus egg extracts.
KCC2, a neuronal‐specific K‐Cl cotransporter plays a major role in maintaining intracellular Cl− concentration in neurons below its electrochemical equilibrium potential, thus favoring robust GABA hyperpolarizing or inhibitory responses. The pharmacology of the K‐Cl cotransporter is dominated by the loop diuretics furosemide and bumetanide. These molecules are used in clinical medicine as they inhibit the loop of Henle Na‐K‐2Cl cotransporter with much higher affinity. In order to identify novel molecules that affect KCC2 activity, we developed a fluorescent‐based assay suitable for high throughput screening (HTS) and used the assay to screen a library of 234,000 small molecules. We identified a large number of molecules that either decrease or increase the activity of the cotransporter. We characterized a small number of inhibitory compounds, some of which inhibit KCC2 activity in the micromolar or sub‐micromolar range, without substantially affecting NKCC1 activity. We synthesized a number of chemical variants, tested their effect on KCC2 function, and provide a first analysis of structure/activity relationship. We also used one of the compounds to demonstrate competitive inhibition in regard to external [K+], and non‐competitive inhibition in respect to external [Cl−].
Telemedicine for the hospitalist: Expanding telemedicine beyond outpatient and subspecialty useThe COVID pandemic overwhelmed many aspects of our healthcare infrastructure from an inability to deliver outpatient, inpatient, or surgical care to staffing shortages, or disruption in supply chains causing the inadequate supply of equipment. Times of crisis tend to propel innovation, welcome telemedicine. For years, telemedicine has increasingly been applied in the outpatient and subspecialty setting. Recent disruptions have spurred increasing inpatient utilization. It forces the question, is a new way better? In this issue of JHM, the review by Gutierez et al. 1 provides a systematic review of applications of telehealth services by hospitalists. It suggests that telehealth expands
Disclaimer In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. Purpose Hospital at home is an alternative means of providing inpatient care for a patient requiring prolonged liposomal amphotericin B therapy. Summary Hospital at home is a unique care model that allows patients to receive inpatient hospital care within the comfort of their home and can be seen as an alternative care site for patients with complex treatment regimens that may require prolonged hospitalization. Hospital systems have increasingly begun incorporating hospital at home programs into their inpatient service lines. We present the case of a patient with disseminated histoplasmosis requiring a prolonged course of intravenous liposomal amphotericin B therapy. Because of the complex administration and stability of this medication, care is often provided in an inpatient setting. The Vanderbilt University Medical Center Hospital at Home team was able to coordinate resources and services to allow for this patient to receive acute hospital care at home and continue to receive amphotericin B infusion. Conclusion This experience spotlights how hospital at home can be considered for patients requiring ongoing inpatient care for prolonged intravenous treatment courses.
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