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Light-driven molecular motors convert light into mechanical energy via excited state reactions. In this work we follow sub-picosecond primary events in the cycle of a two-stroke unidirectional motor by fluorescence up-conversion and transient absorption.

ATP-activated P2X3 receptors play a pivotal role in chronic cough, affecting more than 10% of the population. Despite the challenges posed by the highly conserved structure of P2X receptors, efforts to develop selective drugs targeting P2X3 have led to the development of camlipixant, a potent, selective P2X3 antagonist. However, the mechanisms of receptor desensitization, ion permeation, and structural basis of camlipixant binding to P2X3 remain unclear. Here, we report a cryo-EM structure of camlipixant-bound P2X3, revealing a previously undiscovered selective drug-binding site in the receptor. Our findings also demonstrate that conformational changes in the upper body domain, including the turret and camlipixant-binding pocket, play a critical role: turret opening facilitates P2X3 channel closure to a radius of 0.7 Å, hindering cation transfer, whereas turret closure leads to channel opening. Structural and functional studies combined with molecular dynamics simulations provide a comprehensive understanding of camlipixant's selective inhibition of P2X3, offering a foundation for future drug development targeting this receptor., Competing Interests: Conflict of interest P. P. N., S. S., I. H., S. A., and J. S. work for ELANCO. All the other authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Purinergic P2X3 receptors form trimeric cation-gated channels, which are activated by extracellular ATP. P2X3 plays a crucial role in chronic cough and affects over 10% of the population. Despite considerable efforts to develop drugs targeting P2X3, the highly conserved structure within the P2X receptor family presents obstacles for achieving selectivity. Camlipixant, a potent and selective P2X3 antagonist, is currently in phase III clinical trials. However, the mechanisms underlying receptor desensitization, ion permeation, principles governing antagonism, and the structure of P2X3 when bound to camlipixant remain elusive. In this study, we established a stable cell line expressing homotrimeric P2X3 and utilized a peptide scaffold to purify the complex and determine its structure using cryo-electron microscopy (cryo-EM). P2X3 binds to camlipixant at a previously unidentified drug-binding site and functions as an allosteric inhibitor. Structure-activity studies combined with modeling and simulations have shed light on the mechanisms underlying the selective targeting and inhibition of P2X3 by camlipixant, distinguishing it from other members of the P2X receptor family., Competing Interests: Conflict of Interest declaration: PPN, SS, IH, SA, and JS work for ELANCO. This work was funded by a grant to RS by ELANCO.

Currently, only a few chemical drug classes are available to control the global burden of nematode infections in humans and animals. Most of these drugs exert their anthelmintic activity by interacting with proteins such as ion channels, and the nematode neuromuscular system remains a promising target for novel intervention strategies. Many commonly-used phenotypic readouts such as motility provide only indirect insight into neuromuscular function and the site(s) of action of chemical compounds. Electrophysiological recordings provide more specific information but are typically technically challenging and lack high throughput for drug discovery. Because drug discovery relies strongly on the evaluation and ranking of drug candidates, including closely related chemical derivatives, precise assays and assay combinations are needed for capturing and distinguishing subtle drug effects. Past studies show that nematode motility and pharyngeal pumping (feeding) are inhibited by most anthelmintic drugs. Here we compare two microfluidic devices ("chips") that record electrophysiological signals from the nematode pharynx (electropharyngeograms; EPGs) ─ the ScreenChip™ and the 8-channel EPG platform ─ to evaluate their respective utility for anthelmintic research. We additionally compared EPG data with whole-worm motility measurements obtained with the wMicroTracker instrument. As references, we used three macrocyclic lactones (ivermectin, moxidectin, and milbemycin oxime), and levamisole, which act on different ion channels. Drug potencies (IC 50 and IC 95 values) from concentration-response curves, and the time-course of drug effects, were compared across platforms and across drugs. Drug effects on pump timing and EPG waveforms were also investigated. These experiments confirmed drug-class specific effects of the tested anthelmintics and illustrated the relative strengths and limitations of the different assays for anthelmintic research., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

For more than four decades, the free-living nematode Caenorhabditis elegans has been extensively used in anthelmintic research. Classic genetic screens and heterologous expression in the C. elegans model enormously contributed to the identification and characterization of molecular targets of all major anthelmintic drug classes. Although these findings provided substantial insights into common anthelmintic mechanisms, a breakthrough in the treatment and control of parasitic nematodes is still not in sight. Instead, we are facing increasing evidence that the enormous diversity within the phylum Nematoda cannot be recapitulated by any single free-living or parasitic species and the development of novel broad-spectrum anthelmintics is not be a simple goal. In the present review, we summarize certain milestones and challenges of the C. elegans model with focus on drug target identification, anthelmintic drug discovery and identification of resistance mechanisms. Furthermore, we present new perspectives and strategies on how current progress in C. elegans research will support future anthelmintic research., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Lactate dehydrogenase A (LDHA) is frequently overexpressed in tumors, thereby sustaining high glycolysis rates, tumor growth, and chemoresistance. High-throughput screening resulted in the identification of phthalimide and dibenzofuran derivatives as novel lactate dehydrogenase inhibitors, selectively inhibiting the activity of the LDHA isoenzyme. Cocrystallization experiments confirmed target engagement in addition to demonstrating binding to a novel allosteric binding site present in all four LDHA subunits of the LDH5 homotetramer., Competing Interests: The authors declare the following competing financial interest(s): All authors are current or former employees of Bayer AG. All authors were members of the respective research labs involved in the generation of these results., (Copyright © 2020 American Chemical Society.)

Multiheme cytochromes attract much attention for their electron transport properties. These proteins conduct electrons across bacterial cell walls and along extracellular filaments and when purified can serve as bionanoelectronic junctions. Thus, it is important and necessary to identify and understand the factors governing electron transfer in this family of proteins. To this end we have used ultrafast transient absorbance spectroscopy, to define heme-heme electron transfer dynamics in the representative multiheme cytochrome STC from Shewanella oneidensis in aqueous solution. STC was photosensitized by site-selective labeling with a Ru(II)(bipyridine) 3 dye and the dynamics of light-driven electron transfer described by a kinetic model corroborated by molecular dynamics simulation and density functional theory calculations. With the dye attached adjacent to STC Heme IV, a rate constant of 87 × 10 6 s -1 was resolved for Heme IV → Heme III electron transfer. With the dye attached adjacent to STC Heme I, at the opposite terminus of the tetraheme chain, a rate constant of 125 × 10 6 s -1 was defined for Heme I → Heme II electron transfer. These rates are an order of magnitude faster than previously computed values for unlabeled STC. The Heme III/IV and I/II pairs exemplify the T-shaped heme packing arrangement, prevalent in multiheme cytochromes, whereby the adjacent porphyrin rings lie at 90° with edge-edge (Fe-Fe) distances of ∼6 (11) Å. The results are significant in demonstrating the opportunities for pump-probe spectroscopies to resolve interheme electron transfer in Ru-labeled multiheme cytochromes.

Background: The American Academy of Pediatrics Section on Transport recommends the use of portable ventilators during the transport of patients with advanced airways. We sought to identify knowledge gaps and evaluate the effectiveness of a transport ventilator competency boot camp., Methods: Electronic health records of children requiring ventilatory support during air and ground interfacility transport from January 1 through December 31, 2015, were reviewed to determine when manual ventilation was used in lieu of a portable ventilator, and simulations were constructed from commonly occurring scenarios. All registered respiratory therapists trained in air and ground critical-care transports participated. Demographic data were collected. We assessed performance on 3 facilitated simulated scenarios using a ventilator connected to a low-fidelity pediatric mannequin attached to breathing simulator. Scores were based on the participants' ability to correctly perform pre-use checks, select and optimize ventilator settings, set alarms, and complete safety checks. A 60-min interactive education intervention was conducted between the pre- and post-assessments. The pre-assessment, intervention, and post-assessment were conducted 6 weeks apart. De-identified assessments were scored, and results were shared after study completion. Descriptive statistics reported participant demographics. Paired t tests compared before and after assessments. Statistical significance was established at P < .05., Results: A total of 172 electronic health records were reviewed. Manual ventilation was used more frequently in toddlers requiring pressure control ventilation; noninvasive ventilation was rarely used. A total of 17 registered respiratory therapists participated; 18% were male, 41% had 6-9 years of tenure and 5 years of experience with our transport team. Completing ventilator pre-use check and engaging alarms provided the most opportunity for improvement. Improvements were greater with the use of noninvasive ventilation ( P = .006) than pressure control ventilation ( P = .10) and volume control ventilation use ( P = .07)., Conclusions: Quality data were useful in identifying areas requiring knowledge and competency assessment. Re-assessment results validated the need to conduct education and competency assessment at defined intervals., (Copyright © 2018 by Daedalus Enterprises.)

Despite the long-known fact that the facilitative glucose transporter GLUT1 is one of the key players safeguarding the increase in glucose consumption of many tumor entities even under conditions of normal oxygen supply (known as the Warburg effect), only few endeavors have been undertaken to find a GLUT1-selective small-molecule inhibitor. Because other transporters of the GLUT1 family are involved in crucial processes, these transporters should not be addressed by such an inhibitor. A high-throughput screen against a library of ∼3 million compounds was performed to find a small molecule with this challenging potency and selectivity profile. The N-(1H-pyrazol-4-yl)quinoline-4-carboxamides were identified as an excellent starting point for further compound optimization. After extensive structure-activity relationship explorations, single-digit nanomolar inhibitors with a selectivity factor of >100 against GLUT2, GLUT3, and GLUT4 were obtained. The most promising compound, BAY-876 [N 4 -[1-(4-cyanobenzyl)-5-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl]-7-fluoroquinoline-2,4-dicarboxamide], showed good metabolic stability in vitro and high oral bioavailability in vivo., (© 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Cancerous cells have an acutely increased demand for energy, leading to increased levels of human glucose transporter 1 (hGLUT1). This up-regulation suggests hGLUT1 as a target for therapeutic inhibitors addressing a multitude of cancer types. Here, we present three inhibitor-bound, inward-open structures of WT-hGLUT1 crystallized with three different inhibitors: cytochalasin B, a nine-membered bicyclic ring fused to a 14-membered macrocycle, which has been described extensively in the literature of hGLUTs, and two previously undescribed Phe amide-derived inhibitors. Despite very different chemical backbones, all three compounds bind in the central cavity of the inward-open state of hGLUT1, and all binding sites overlap the glucose-binding site. The inhibitory action of the compounds was determined for hGLUT family members, hGLUT1-4, using cell-based assays, and compared with homology models for these hGLUT members. This comparison uncovered a probable basis for the observed differences in inhibition between family members. We pinpoint regions of the hGLUT proteins that can be targeted to achieve isoform selectivity, and show that these same regions are used for inhibitors with very distinct structural backbones. The inhibitor cocomplex structures of hGLUT1 provide an important structural insight for the design of more selective inhibitors for hGLUTs and hGLUT1 in particular.

The compound class of 1H-pyrazolo[3,4-d]pyrimidines was identified using HTS as very potent inhibitors of facilitated glucose transporter 1 (GLUT1). Extensive structure-activity relationship studies (SAR) of each ring system of the molecular framework was established revealing essential structural motives (i.e., ortho-methoxy substituted benzene, piperazine and pyrimidine). The selectivity against GLUT2 was excellent and initial in vitro and in vivo pharmacokinetic (PK) studies are encouraging., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Mesothelin is a tumor differentiation antigen frequently overexpressed in tumors such as mesothelioma, ovarian, pancreatic, and lung adenocarcinomas while showing limited expression in nonmalignant tissues. Mesothelin is therefore an attractive target for cancer therapy using antibody-drug conjugates (ADC). This study describes the detailed characterization of anetumab ravtansine, here referred to as BAY 94-9343, a novel ADC consisting of a human anti-mesothelin antibody conjugated to the maytansinoid tubulin inhibitor DM4 via a disulfide-containing linker. Binding properties of the anti-mesothelin antibody were analyzed using surface plasmon resonance, immunohistochemistry, flow cytometry, and fluorescence microscopy. Effects of BAY 94-9343 on cell proliferation were first studied in vitro and subsequently in vivo using subcutaneous, orthotopic, and patient-derived xenograft tumor models. The antibody binds to human mesothelin with high affinity and selectivity, thereby inducing efficient antigen internalization. In vitro, BAY 94-9343 demonstrated potent and selective cytotoxicity of mesothelin-expressing cells with an IC(50) of 0.72 nmol/L, without affecting mesothelin-negative or nonproliferating cells. In vivo, BAY 94-9343 localized specifically to mesothelin-positive tumors and inhibited tumor growth in both subcutaneous and orthotopic xenograft models. In addition, BAY 94-9343 was able to induce a bystander effect on neighboring mesothelin-negative tumor cells. Antitumor efficacy of BAY 94-9343 correlated with the amount of mesothelin expressed and was generally superior to that of standard-of-care regimen resulting in complete tumor eradication in most of the models. BAY 94-9343 is a selective and highly potent ADC, and our data support its development for the treatment of patients with mesothelin-expressing tumors., (©2014 American Association for Cancer Research.)

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors., (© 2013 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Treatment of oral squamous cell carcinoma (OSCC) is currently based on surgery and radiotherapy. Prolongation of the survival time of patients with progressing tumors is infrequently achieved. To improve the therapeutic options, targeted therapies are a favorable alternative. Therefore, we analyzed the effect of a chimeric toxin (CT) named SE consisting of the epidermal growth factor and the plant protein toxin saporin from Saponaria officinalis. A second construct (SA2E) additionally contains a peptidic adapter designed to enhance efficacy of the CT in vivo and to reduce side effects. The IC(50) values for an OSCC cell line (BHY) were 0.27 nM and 0.73 nM for SE and SA2E, respectively, while fibroblasts remained unaffected. To investigate primary tumor cells, we developed a technique to analyze freshly prepared OSCC cells of 28 patients in a stem cell assay directly after surgery. Cells were treated for 1 h with the CTs, subsequently seeded into soft agar and colony growth determined after 1-2 weeks In spite of the short time of CT incubation, the amount of colonies was reduced to about 78% by 10 nM and to 69% by 100 nM of either toxin. A combined application of 10 nM SA2E with a saponin from Gypsophila paniculata reduced the amount of surviving cells to 68%. The results demonstrate the impact of the CTs on OSCC cells and depict that the stem cell assay is suitable to determine the potential of anti-tumor drugs before studies in vivo will be initiated.

Two of the main problems associated with administration of receptor-targeted toxins in tumor therapy are severe systemic side effects and low transfer of the toxins into the cytosol after binding to the tumor cell surface. To improve chimeric toxins in this respect we have developed a molecular adapter that links the toxic moiety and ligand. The adapter is designed to improve cytosolic uptake, retain the toxin inside the cytosol and detoxify the drug after cell death. The plant toxin saporin linked either directly or via the adapter to epidermal growth factor (EGF) served to evaluate efficacy to inhibit tumor growth and reduce side effects in vivo. The lethal dose for BALB/c mice was three times less for the adapter-containing toxin (SA2E) than for the adapter-free construct (SE). Furthermore, SE only reduced the average weight of induced tumors by 33% whereas SA2E-treated mice exhibited 71% reduction with an almost complete suppression in 60% of the cases. Additionally, severe side effects like hyperalgesia, alopecia and death were drastically reduced in SA2E-treated animals. Tumors without target receptor were only slightly affected by SA2E and the reduction in side effects less pronounced indicating specific depletion from the blood by target receptor expressing cells.

Saponins are a group of plant glycosides consisting of a steroid or triterpenoid aglycone to which one or more sugar chains are attached. They exhibit cell membrane-permeabilizing properties and, thus, have been investigated for their therapeutic potential. Recently, at a non-permeabilizing concentration saponinum album from Gypsophila paniculata L. has been described to enhance the cytotoxicity of a chimeric toxin in a cell culture model. To elucidate whether this enhancing effect is also mediated by other saponins, we analyzed the ability of seven different saponins to enhance the cytotoxicity of a targeted chimeric toxin. The chimeric toxin is composed of saporin, a plant ribosome-inactivating toxin, a cleavable adapter, and human epidermal growth factor (EGF). Cytotoxicity on EGF receptor (EGFR)-bearing cells was analyzed both alone and after combined application of saponin and chimeric toxin. Only two of the tested saponins, quillajasaponin and saponinum album, enhanced cytotoxicity by more than 1,000-fold, whereas the enhancement factors of the other saponins were only approximately 10-fold. In contrast to saponinum album, quillajasaponin enhanced the cytotoxicity both on control cells lacking EGFR and on target cells, indicating that, in this case, the enhancement is not target cell receptor specific. This is also the case for some of the saponins with low enhancement factors. Saponinum album resulted in a more than 13,600-fold receptor-specific enhancement, decreasing the 50% inhibitory concentration (IC(50)) from 2.4 nM to 0.18 pM, which renders it the best option to promote saporin-3-based drug uptake while retaining specificity for the EGFR.

Direct targeting to the cytoplasm and nucleus using protein transduction domains (PTD) has been described to be efficient but non-cell-type-specific, and only has clinical relevance when the molecule is active exclusively in the diseased cell. The use of PTDs is an attractive mechanism to improve drug delivery. In this work, we designed recombinant proteins that contain epidermal growth factor as ligand to render uptake target cell-specific. We evaluated the potential of several PTDs to induce the cytosolic uptake of the catalytic domain of diphtheria toxin by measuring cytotoxicity. Although PTD-dependent membrane transfer is very low, the proteins exhibited concentration-dependent cytotoxic activity. Higher binding at 4 degrees C compared to 37 degrees C suggests that uptake by the PTDs MTS and TLM occurs via an endocytic pathway. Non-specific binding is predominantly a function of the PTD and greatly increases by substitution of a non-polar glycine with a negatively charged glutamate in the PTD HA2.

Immunotoxins have to be administered in high doses due to low cytosolic uptake with the consequence of severe side effects. Recently we found that the cytotoxic activity from Agrostemma githago seeds can be attributed to a synergistic toxicity of a triterpenoid saponin and a ribosome-inactivating protein. Here we investigated whether saponins are able to enhance the efficacy of a receptor-specific chimeric toxin consisting of saporin-3, epidermal growth factor and a molecular adapter previously shown to reduce side effects on non-target cells. Pre-applied saponin enhances the target cell-specific cytotoxic effect, dependent on the cell line, between 3560- and 385,000-fold with an IC50 up to 0.67 pM. Non-target cells are not affected at the same concentration. At the optimal concentrations of the chimeric toxin and saponin application of either one of the components shows no cytotoxicity at all proving a synergistic effect. In the presence of saponin ligand-free saporin-3 does not exhibit any cytotoxic effect up to 0.1 nM providing further evidence for an increased specificity. This synergistic effect is in the same order of magnitude as in a mouse model. Our investigations clearly demonstrate that a combined administration of saponin and chimeric toxins opens up a promising perspective for tumor therapy.

One of the problems associated with the administration of immunotoxins is hypersensitivity reaction such as vascular leak syndrome. This may be prevented by decreasing the plasma half-life. To improve immunotoxins with respect to reduced side effects, we have previously described the development of a cleavable adapter. This adapter links the toxic moiety and ligand that are usually directly coupled. In our study, the cytotoxicity of saporin linked either directly or via the adapter to epidermal growth factor (EGF) was evaluated in vitro. The immunotoxins exhibited similar cytotoxic activity towards A-431 and HER14 cells (IC(50) < 10 nM). The supernatant from 6 hr cultures of HER14 cells incubated in the presence of the adapter-containing immunotoxin exhibited a significantly reduced cytotoxicity as compared to the directly coupled immunotoxin. Western blotting revealed that the adapter was cleaved, thus supporting our proposal that cleavable adapters may reduce nonspecific effects. A similar reduced half-life was detected in platelet-poor plasma. In contrast MCF-7 cells remain unaffected by the immunotoxins. This was shown to be due to the absence of detectable EGF-receptor in comparison to A-431 and HER14 cells as determined by Western blotting. Furthermore, we could show that the adapter does not exert an effect on the N-glycosidase activity of saporin. These results suggest that the use of cleavable adapters may be a useful tool in immunotoxins for reducing the killing of surrounding noncancerous cells due to nonspecific binding., (Copyright 2002 Wiley-Liss, Inc.)

Adenine quantitation is required for a variety of applications. To date, the prevalent method for quantifying free adenine, in a variety of applications, is the detection of fluorescent-derivatized adenine by HPLC. For the present study, we developed a high-throughput, nonradioactive, enzyme-based colorimetric adenine quantitation assay that is performed in one multireaction incubation step. The assay does not require adenine derivatization and is designed for microplates. The key step is the conversion of adenine to adenosine monophosphate by adenine phosphoribosyl transferase. Subsequent reactions finally produce three inorganic phosphate ions per adenine molecule. Phosphate is quantitated by the color-generating phosphorylysis of a particular purine derivate. Ribosome-inactivating proteins that release adenine from polynucleotides are often used to investigate intracellular protein trafficking and are important for the design of immunotoxins. We therefore used ricin, dianthin, saporin, and a variety of saporin fusion proteins to show that this method is suitable for quantifying adenine release using different substrates. The measured rate of adenine release and substrate specificity are comparable to those determined by HPLC and radioactive detection techniques.