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Glutamate dehydrogenases (GDHs) are key enzymes at the crossroads of N and C metabolism in plants. Legumes, whose N metabolism is particularly intricate, possess a unique type of GDH. This study presents an analysis of a legume-type GDH (isoform 2) from Medicago truncatula (MtGDH2). We measured MtGDH2 activity in both the Glu â 2-oxoglutarate (2OG) and 2OG â Glu reaction directions and obtained kinetic parameters for Glu, 2OG, NAD+, and NADH. Inhibition assays revealed that compounds possessing di- or tricarboxylates act as inhibitors of plant GDHs. Interestingly, 2,6-pyridinedicarboxylate (PYR) weakly inhibits MtGDH2 compared to Arabidopsis thaliana homologs. Furthermore, we explored tetrazole derivatives to discover 3-(1H-tetrazol-5-yl)benzoic acid (TBA) as an MtGDH2 inhibitor. The kinetic experiments are supported by six crystal structures, solved as: (i) unliganded enzyme, (ii) trapping the reaction intermediate 2-amino-2-hydroxyglutarate and NAD+, and also complexed with NAD+ and inhibitors such as (iii) citrate, (iv) PYR, (v) isophthalate, and (vi) TBA. The complex with TBA revealed a new mode of action that, in contrast to other inhibitors, prevents domain closure. This discovery points to TBA as a starting point for the development of novel GDH inhibitors to study the functions of GDH in plants and potentially boost biomass production.
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KDM4 histone demethylases became an exciting target for inhibitor development as the evidence linking them directly to tumorigenesis mounts. In this study, we set out to better understand the binding cavity using an X-ray crystallographic approach to provide a detailed landscape of possible interactions within the under-investigated region of KDM4. Our design strategy was based on utilizing known KDM binding motifs, such as nicotinic acid and tetrazolylhydrazides, as core motifs that we decided to enrich with flexible tails to map the distal histone binding site. The resulting X-ray structures of the novel compounds bound to KDM4D, a representative of the KDM4 family, revealed the interaction pattern with distal residues in the histone-binding site. The most prominent protein rearrangement detected upon ligand binding is the loop movement that blocks the accessibility to the histone binding site. Apart from providing new sites that potential inhibitors can target, the novel compounds may prove helpful in exploring the capacity of ligands to bind in sites distal to the cofactor-binding site of other KDMs or 2-oxoglutarate (2OG)-dependent oxygenases. The case study proves that combining a strong small binding motif with flexible tails to probe the binding pocket will facilitate lead discovery in classical drug-discovery campaigns, given the ease of accessing X-ray quality crystals.
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Histonas , Histona Demetilasas con Dominio de Jumonji , Piridinas , Tetrazoles , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/química , Tetrazoles/química , Tetrazoles/farmacología , Tetrazoles/metabolismo , Tetrazoles/síntesis química , Piridinas/química , Piridinas/farmacología , Piridinas/metabolismo , Piridinas/síntesis química , Humanos , Sitios de Unión , Cristalografía por Rayos X , Relación Estructura-Actividad , Histonas/metabolismo , Histonas/química , Estructura Molecular , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Modelos Moleculares , Relación Dosis-Respuesta a DrogaRESUMEN
3,4-bridged indoles are underrepresented among the vast number of indoles described in the literature. Attempts to access 3,4-macrocyclized indoles led to the unexpected formation of a novel tetracyclic indole through intramolecular acid-catalyzed ring contraction. The herein-established one-step synthetic route provides an excellent medicinal chemistry platform for the construction of screening libraries covering a unique chemical space of indoles.
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The TGFß type II receptor (TßRII) is a central player in all TGFß signaling downstream events, has been linked to cancer progression, and thus, has emerged as an auspicious anti-TGFß strategy. Especially its targeted degradation presents an excellent goal for effective TGFß pathway inhibition. Here, cellular structure-activity relationship (SAR) data from the TßRII degrader chemotype 1 was successfully transformed into predictive ligand-based pharmacophore models that allowed scaffold hopping. Two distinct 3,4-disubstituted indoles were identified from virtual screening: tetrahydro-4-oxo-indole 2 and indole-3-acetate 3. Design, synthesis, and screening of focused amide libraries confirmed 2r and 3n as potent TGFß inhibitors. They were validated to fully recapitulate the ability of 1 to selectively degrade TßRII, without affecting TßRI. Consequently, 2r and 3n efficiently blocked endothelial-to-mesenchymal transition and cell migration in different cancer cell lines while not perturbing the microtubule network. Hence, 2 and 3 present novel TßRII degrader chemotypes that will (1) aid target deconvolution efforts and (2) accelerate proof-of-concept studies for small-molecule-driven TßRII degradation in vivo.
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The flow dynamics of red blood cells in vivo in blood capillaries and in vitro in microfluidic channels is complex. Cells can obtain different shapes such as discoid, parachute, slipper-like shapes and various intermediate states depending on flow conditions and their viscoelastic properties. We use artificial intelligence based analysis of red blood cells (RBCs) in an oscillating microchannel to distinguish healthy red blood cells from red blood cells treated with formaldehyde to chemically modify their viscoelastic behavior. We used TensorFlow to train and validate a deep learning model and achieved a testing accuracy of over 97%. This method is a first step to a non-invasive, label-free characterization of diseased red blood cells and will be useful for diagnostic purposes in haematology labs. This method provides quantitative data on the number of affected cells based on single cell classification.
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BACKGROUND: Mechanical thrombectomy has been shown to reduce thrombus burden and pulmonary artery pressure (PAP) and to improve right ventricular (RV) function in patients with high-risk or intermediate-high-risk pulmonary embolism (PE). As hemodynamic data after mechanical thrombectomy for PE are scarce, we aimed to assess the hemodynamic effects of mechanical thrombectomy in acute PE with right heart overload. METHODS: In this prospective, open-label study, patients with acute symptomatic, computed tomography-documented PE with signs of right heart overload underwent mechanical thrombectomy using the FlowTriever System. Right heart catheterization was performed immediately before and after thrombectomy and after three months. Transthoracic echocardiography was performed before thrombectomy, discharge, and at three months. This analysis was done after 20 patients completed three months of follow-up. RESULTS: Twenty-nine patients (34% female) underwent mechanical thrombectomy, of which 20 completed three months follow-up with right heart catheterization. Most patients were at high (17%) or intermediate-high (76%) risk and had bilateral PE (79%). Before thrombectomy, systolic PAP (sPAP) was severely elevated (mean 51.3 ± 11.6 mmHg). Mean sPAP dropped by -15.0 mmHg (95% confidence interval [CI]: -18.9 to -11.0; p < 0.001) immediately after the procedure and continued to decrease from post-thrombectomy to three months (-6.4 mmHg, 95% CI: -10-0 to -2.9; p = 0.002). RV/left ventricular (LV) ratio immediately reduced within two days by -0.37 (95% CI: -0.47 to -0.27; p < 0.001). The proportion of patients with a tricuspid annular plane systolic excursion (TAPSE)/sPAP ratio < 0.31 mm/mmHg decreased from 28% at baseline to 0% before discharge and at three months (p = 0.007). There were no procedure-related major adverse events. CONCLUSIONS: Mechanical thrombectomy for acute PE was safe and immediately reduced PAP and improved right heart function. The reduction in PAP was maintained at three months follow-up.
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Embolia Pulmonar , Trombosis , Disfunción Ventricular Derecha , Humanos , Femenino , Masculino , Estudios Prospectivos , Embolia Pulmonar/diagnóstico por imagen , Embolia Pulmonar/cirugía , Trombectomía/efectos adversos , Trombectomía/métodos , Hemodinámica , Resultado del TratamientoRESUMEN
Groundwater-dependent vegetation (GDV) is essential for maintaining ecosystem functions and services, providing critical habitat for species, and sustaining human livelihoods. However, climate and land-use change are threatening GDV, highlighting the need for harmonised, global mapping of the distribution and extent of GDV. This need is particularly crucial in vulnerable biodiversity hotspots such as the Mediterranean biome. This study presents a novel multicriteria index to identify areas in the Mediterranean biome that provide suitable environmental conditions to support potentially groundwater-dependent vegetation (pGDV) where vegetation behaviour is also indicative of groundwater use. Global datasets targeting 1) groundwater vegetation interaction; 2) soil water holding capacity; 3) topographical landscape wetness potential; 4) land use land cover and 5) hydraulic conductivity of rocks have been combined for the first time in an easy-to-use index. Layer weightings from Analytical Hierarchy Process and Random Forest showed limited applicability on biome scale, but an unweighted overlay of eleven thematic layers produced plausible results. The final pGDV map indicates that 31 % of the natural vegetation in the Mediterranean biome likely depend on groundwater. Moreover, moderate to good agreement was found compared to actual GDV locations in Campania, Italy (91 % with at least moderate potential) and California, USA (87 % with at least moderate potential). The results provide valuable information for identifying regions with a substantial presence of pGDV in the Mediterranean biome and can be used for decision making, e.g. to prioritise field surveys and high-resolution remote sensing for GDV mapping. It can therefore support effective groundwater resource management and the conservation of biodiversity hotspots.
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Ecosistema , Agua Subterránea , Humanos , Biodiversidad , Clima , SueloRESUMEN
Cold physical plasma is a partially ionized gas operated at body temperature and utilized for heat-sensitive technical and medical purposes. Physical plasma is a multi-component system consisting of, e.g., reactive species, ions and electrons, electric fields, and UV light. Therefore, cold plasma technology is an interesting tool for introducing biomolecule oxidative modifications. This concept can be extended to anticancer drugs, including prodrugs, which could be activated in situ to enhance local anticancer effects. To this end, we performed a proof-of-concept study on the oxidative prodrug activation of a tailor-made boronic pinacol ester fenretinide treated with the atmospheric pressure argon plasma jet kINPen operated with either argon, argon-hydrogen, or argon-oxygen feed gas. Fenretinide release from the prodrug was triggered via Baeyer-Villiger-type oxidation of the boron-carbon bond based on hydrogen peroxide and peroxynitrite, which were generated by plasma processes and chemical addition using mass spectrometry. Fenretinide activation led to additive cytotoxic effects in three epithelial cell lines in vitro compared to the effects of cold plasma treatment alone regarding metabolic activity reduction and an increase in terminal cell death, suggesting that cold physical plasma-mediated prodrug activation is a new direction for combination cancer treatment studies.
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Flupirtine and retigabine were essential drugs to combat pain and epilepsy. However, the Kv 7 potassium channel openers are fraught with hepatotoxicity and tissue discoloration, respectively, limiting their therapeutic value. Both adverse events are likely due to reactive metabolites arising from oxidative metabolism. Designing safer analogues lacking the structural elements leading to described side effects is an active area of current research. One of the main metabolites of flupirtine is the biologically inactive 4-fluorohippuric acid. Hitherto unexplained, the proposed metabolic pathway leading to the formation of 4-fluorohippuric acid from flupirtine is verified here. Through the use of eighteen flupirtine analogues, mechanistic details of this pathway could be elucidated. A possible connection with the inâ vitro hepatotoxicity of the flupirtine analogues and the levels of 4-fluorobenzoic acid formed in enzyme incubations was examined by correlation analysis. These findings provide important information for the design of new flupirtine analogues as potential drug candidates.
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Enfermedad Hepática Inducida por Sustancias y Drogas , Esterasas , Humanos , Analgésicos/farmacología , Aminopiridinas/toxicidad , Aminopiridinas/química , Relación Estructura-ActividadRESUMEN
For the characterization of Kv 7.2/3 channel activators, several analytical methods are available that vary in effort and cost. In addition to the technically elaborate patch-clamp method, which serves as a reference method, there exist several medium to high-throughput screening methods including a rubidium efflux flame-atomic absorption spectrometry (F-AAS) assay and a commercial thallium uptake fluorescence-based assay. In this study, the general suitability of a graphite furnace atomic absorption spectrometry (GF-AAS)-based rubidium efflux assay as a screening method for Kv 7.2/3 channel activators was demonstrated. With flupirtine serving as a reference compound, 16 newly synthesizedcompounds and the known Kv 7.2/3 activator retigabine were first classified as either active or inactive by using the GF-AAS-based rubidium (Rb) efflux assay. Then, the results were compared with a thallium (Tl) uptake fluorescence-based fluorometric imaging plate reader (FLIPR) potassium assay. Overall, 16 of 17 compounds were classified by the GF-AAS-based assay in agreement with their channel-activating properties determined by the more expensive Tl uptake, fluorescence-based assay. Thus, the performance of the GF-AAS-based Rb assay for primary drug screening of Kv 7.2/3-activating compounds was clearly demonstrated, as documented by the calculated Z'-factor of the GF-AAS-based method. Moreover, method development included optimization of the coating of the microtiter plates and the washing procedure, which extended the range of this assay to poorly adherent cells such as the HEK293 cells used in this study.
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Grafito , Rubidio , Humanos , Espectrofotometría Atómica/métodos , Talio , Células HEK293 , Relación Estructura-ActividadRESUMEN
INTRODUCTION: The size and complexity of virtual screening libraries in drug discovery have skyrocketed in recent years, reaching up to multiple billions of accessible compounds. However, virtual screening of such ultra-large libraries poses several challenges associated with preparing the libraries, sampling, and pre-selection of suitable compounds. The utilization of artificial intelligence (AI)-assisted screening approaches, such as deep learning, poses a promising countermeasure to deal with this rapidly expanding chemical space. For example, various AI-driven methods were recently successfully used to identify novel small molecule inhibitors of the SARS-CoV-2 main protease (Mpro). AREAS COVERED: This review focuses on presenting various kinds of virtual screening methods suitable for dealing with ultra-large libraries. Challenges associated with these computational methodologies are discussed, and recent advances are highlighted in the example of the discovery of novel Mpro inhibitors targeting the SARS-CoV-2 virus. EXPERT OPINION: With the rapid expansion of the virtual chemical space, the methodologies for docking and screening such quantities of molecules need to keep pace. Employment of AI-driven screening compounds has already been shown to be effective in a range from a few thousand to multiple billion compounds, furthered by de novo generation of drug-like molecules without human interference.
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COVID-19 , SARS-CoV-2 , Humanos , Inteligencia Artificial , Simulación del Acoplamiento Molecular , Descubrimiento de Drogas/métodos , Inhibidores de Proteasas/farmacología , Antivirales/farmacología , Antivirales/químicaRESUMEN
KV 7 channel openers have proven their therapeutic value in the treatment of pain as well as epilepsy and, moreover, they hold the potential to expand into additional indications with unmet medical needs. However, the clinically validated but meanwhile discontinued KV 7 channel openers flupirtine and retigabine bear an oxidation-sensitive triaminoraryl scaffold, which is suspected of causing adverse drug reactions via the formation of quinoid oxidation products. Here, we report the design and synthesis of nicotinamide analogs and related compounds that remediate the liability in the chemical structure of flupirtine and retigabine. Optimization of a nicotinamide lead structure yielded analogs with excellent KV 7.2/3 opening activity, as evidenced by EC50 values approaching the single-digit nanomolar range. On the other hand, weighted KV 7.2/3 opening activity data including inactive compounds allowed for the establishment of structure-activity relationships and a plausible binding mode hypothesis verified by docking and molecular dynamics simulations.
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Aminopiridinas , Canales de Potasio KCNQ , Canales de Potasio KCNQ/metabolismo , Relación Estructura-Actividad , Aminopiridinas/químicaRESUMEN
Reduced river discharge and flow regulation are significant threats to freshwater biodiversity. An accurate representation of potential damage of water consumption on freshwater biodiversity is required to quantify and compare the environmental impacts of global value chains. The effect of discharge reduction on fish species richness was previously modeled in life cycle impact assessment, but models were limited by the restricted geographical scope of underlying species-discharge relationships and the small number of species data. Here, we propose a model based on a novel regionalized species-discharge relationship (SDR). Our SDR-based model covers 88 % of the global landmass (2320 river basins worldwide excluding deserts and permanently frozen areas) and is based on a global dataset of 11,450 riverine fish species, simulated river discharge, elevation, and climate zones. We performed 10-fold cross-validation to select the best set of predictors and validated the obtained SDRs based on observed discharge data. Our model performed better than previous SDRs employed in life cycle impact assessment (Kling-Gupta efficiency coefficient about 4 times larger). We provide both marginal and average models with their uncertainty ranges for assessing scenarios of small and large-scale water consumption, respectively, and include regional and global species loss. We conducted an illustrative case study to showcase the method's applicability and highlight the differences with the currently used approach. Our models are useful for supporting sustainable water consumption and riverine fish biodiversity conservation decisions. They enable a more specific, reliable, and complete impact assessment by differentiating impacts on regional riverine fish species richness and irreversible global losses, including up-to-date species data, and providing spatially explicit values with high geographical coverage.
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Ingestión de Líquidos , Agua Dulce , Animales , Ríos , Biodiversidad , Peces/fisiología , Estadios del Ciclo de Vida , EcosistemaRESUMEN
Droplet microfluidics allows one to address the ever-increasing demand to screen large libraries of biological samples. Absorbance spectroscopy complements the golden standard of fluorescence detection by label free target identification and providing more quantifiable data. However, this is limited by speed and sensitivity. In this paper we increase the speed of sorting by including acoustofluidics, achieving sorting rates of target droplets of 1 kHz. We improved the device design for detection of absorbance using fibre-based interrogation of samples with integrated lenses in the microfluidic PDMS device for focusing and collimation of light. This optical improvement reduces the scattering and refraction artefacts, improving the signal quality and sensitivity. The novel design allows us to overcome limitations based on dielectrophoresis sorting, such as droplet size dependency, material and dielectric properties of samples. Our acoustic activated absorbance sorter removes the need for offset dyes or matching oils and sorts about a magnitude faster than current absorbance sorters.
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Técnicas Analíticas Microfluídicas , Microfluídica , Acústica , Análisis Espectral , Aceites/química , ColorantesRESUMEN
Replica exchange molecular dynamics simulations are one of the most popular approaches to enhance conformational sampling of molecular systems. Applications range from protein folding to protein-protein or other host-guest interactions, as well as binding free energy calculations. While these methods are computationally expensive, highly accurate results can be obtained. We recently developed TIGER2hs, an improved version of the temperature intervals with global exchange of replicas (TIGER2) algorithm. This method combines the replica-based enhanced sampling in an explicit solvent with a hybrid solvent energy evaluation. During the exchange attempts, bulk water is replaced by an implicit solvent model, allowing sampling with significantly less replicas than parallel tempering (REMD). This enables accurate enhanced sampling calculations with only a fraction of computational resources compared to REMD. Our latest results highlight several issues with sampling imbalance and parameter sensitivity within the original TIGER2 exchange algorithms that affect the overall state populations. A high sensitivity on replica number and maximum temperature is eliminated by changing to a pairwise exchange kernel (PE) without additional sorting. Simulations are controlled by adjusting the average temperature change per exchange ⟨ΔT/χ⟩ to below 30 K to mimic a controlled temperature mixing of replicas similar to REMD. Thus, this parameter provides an applicable property for selecting combinations of replica number and maximum temperature to adjust simulations for best accuracy, with flexible resource investment. This increases the robustness of the method and ensures results in excellent agreement with REMD, as demonstrated for three different peptides.
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Simulación de Dinámica Molecular , Proteínas , Péptidos/química , Pliegue de Proteína , Proteínas/química , Solventes/química , TemperaturaRESUMEN
The KV 7 potassium channel openers flupirtine and retigabine have been valuable options in the therapy of pain and epilepsy. However, as a result of adverse reactions, both drugs are currently no longer in therapeutic use. The flupirtine-induced liver injury and the retigabine linked tissue discolouration do not appear related at first glance; nevertheless, both events can be attributed to the triaminoaryl scaffold, which is affected by oxidation leading to elusive reactive quinone diimine or azaquinone diimine metabolites. Since the mechanism of action, i. e. KV 7 channel opening, seems not to be involved in toxicity, this study aimed to further develop safer replacements for flupirtine and retigabine. In a ligand-based design strategy, replacing amino substituents of the triaminoaryl core with alkyl substituents led to carba analogues with improved oxidation resistance and negligible risk of quinoid metabolite formation. In addition to these improved safety features, some of the novel analogues exhibited significantly improved KV 7.2/3 channel opening activity, indicated by an up to 13-fold increase in potency and an efficacy of up to 176 % compared to flupirtine, thus being attractive candidates for further development.
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Carbamatos , Fenilendiaminas , Aminopiridinas/farmacología , Aminopiridinas/uso terapéutico , Carbamatos/farmacología , Canales de Potasio KCNQ/metabolismo , Fenilendiaminas/farmacologíaRESUMEN
Reactive oxygen species (ROS) are known to trigger drug release from arylboronate-containing ROS-responsive prodrugs. In cancer cells, elevated levels of ROS can be exploited for the selective activation of prodrugs via Baeyer-Villiger type oxidation rearrangement sequences. Here, we report a proof of concept to demonstrate that these cascades can as well be initiated by cold physical plasma (CPP). An analog of a recently reported fluorouracil prodrug based on the less toxic drug 5-fluorocytosine (5-FC) was synthesized with a view to laboratory safety reasons and used as a model compound to prove our hypothesis that CPP is suitable as a trigger for the prodrug activation. Although the envisioned oxidation and rearrangement with successive loss of boronic acid species could be achieved by plasma treatment, the anticipated spontaneous liberation of 5-FC was inefficient in the model case. However, the obtained results suggest that custom-tailored CPP-responsive prodrugs might become an evolving research field.
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Gases em Plasma , Profármacos , Línea Celular Tumoral , Flucitosina/farmacología , Profármacos/farmacología , Profármacos/uso terapéutico , Especies Reactivas de Oxígeno , Relación Estructura-ActividadRESUMEN
The potassium channel opening drugs flupirtine and retigabine have been withdrawn from the market due to occasional drug-induced liver injury (DILI) and tissue discoloration, respectively. While the mechanism underlying DILI after prolonged flupirtine use is not entirely understood, evidence indicates that both drugs are metabolized in an initial step to reactive ortho- and/or para-azaquinone diimines or ortho- and/or para-quinone diimines, respectively. Aiming to develop safer alternatives for the treatment of pain and epilepsy, we have attempted to separate activity from toxicity by employing a drug design strategy of avoiding the detrimental oxidation of the central aromatic ring by shifting oxidation toward the formation of benign metabolites. In the present investigation, an alternative retrometabolic design strategy was followed. The nitrogen atom, which could be involved in the formation of both ortho- or para-quinone diimines of the lead structures, was shifted away from the central ring, yielding a substitution pattern with nitrogen substituents in the meta position only. Evaluation of KV7.2/3 opening activity of the 11 new specially designed derivatives revealed surprisingly steep structure-activity relationship data with inactive compounds and an activity cliff that led to the identification of an apparent "magic methyl" effect in the case of N-(4-fluorobenzyl)-6-[(4-fluorobenzyl)amino]-2-methoxy-4-methylnicotinamide. This flupirtine analogue showed potent KV7.2/3 opening activity, being six times as active as flupirtine itself, and by design is devoid of the potential for azaquinone diimine formation.