Identification of a Novel Structural Class of HV1 Inhibitors by Structure-Based Virtual Screening.

The human voltage-gated proton channel, hHV1, is highly expressed in various cell types including macrophages, B lymphocytes, microglia, sperm cells and also in various cancer cells. Overexpression of HV1 has been shown to promote tumor formation by highly metastatic cancer cells, and has been associated with neuroinflammatory diseases, immune response disorders and infertility, suggesting a potential use of hHV1 inhibitors in numerous therapeutic areas. To identify compounds targeting this channel, we performed a structure-based virtual screening on an open structure of the human HV1 channel. Twenty selected virtual screening hits were tested on Chinese hamster ovary (CHO) cells transiently expressing hHV1, with compound 13 showing strong block of the proton current with an IC50 value of 8.5 µM. Biological evaluation of twenty-three additional analogs of 13 led to the discovery of six other compounds that blocked the proton current by more than 50% at 50 µM concentration. This allowed for an investigation of structure-activity relationships. The antiproliferative activity of the selected promising hHV1 inhibitors was investigated in the cell lines MDA-MB-231 and THP-1, where compound 13 inhibited growth with an IC50 value of 9.0 and 8.1 µM, respectively. The identification of a new structural class of HV1 inhibitors contributes to our understanding of the structural requirements for inhibition of this ion channel and opens up the possibility of investigating the role of HV1 inhibitors in various pathological conditions and in cancer therapy.

Assessing Changes in the Distribution Patterns of the European Wildcat in Hungary.

The European wildcat (Felis silvestris Schreber, 1777) is an endangered and elusive carnivore that is slowly recovering in Central Europe after persecution and a decline in its distribution over the past two centuries, and specific conservation plans are needed in most of its range. Knowledge of the continent-wide distribution and status of this species is still poor. Using an online questionnaire, we evaluated the nationwide distribution of wildcats across three time periods (2004, 2014, and 2022) in Hungary. The species' reported occurrence was analyzed according to binominal logistic regression using the percent cover of land cover categories as explanatory variables. We found that the spatial cover of broad-leaved forest was positively associated with the occurrence of wildcats, and the analysis revealed a positive trend in the larger 2004-2022 time frame. We also recorded that although wildcats have disappeared from areas of the central, southern, and western parts of Hungary, regions in the eastern, northern, and south-western areas appear to retain stable populations.

Elucidation of the binding mode of organic polysulfides on the human TRPA1 receptor.

Introduction: Previous studies have established that endogenous inorganic polysulfides have significant biological actions activating the Transient Receptor Potential Ankyrin 1 (TRPA1) receptor. Organic polysulfides exert similar effects, but they are much more stable molecules, therefore these compounds are more suitable as drugs. In this study, we aimed to better understand the mechanism of action of organic polysulfides by identification of their binding site on the TRPA1 receptor. Methods: Polysulfides can readily interact with the thiol side chain of the cysteine residues of the protein. To investigate their role in the TRPA1 activation, we replaced several cysteine residues by alanine via site-directed mutagenesis. We searched for TRPA1 mutant variants with decreased or lost activating effect of the polysulfides, but with other functions remaining intact (such as the effects of non-electrophilic agonists and antagonists). The binding properties of the mutant receptors were analyzed by in silico molecular docking. Functional changes were tested by in vitro methods: calcium sensitive fluorescent flow cytometry, whole-cell patch-clamp and radioactive calcium-45 liquid scintillation counting. Results: The cysteines forming the conventional binding site of electrophilic agonists, namely C621, C641 and C665 also bind the organic polysulfides, with the key role of C621. However, only their combined mutation abolished completely the organic polysulfide-induced activation of the receptor. Discussion: Since previous papers provided evidence that organic polysulfides exert analgesic and anti-inflammatory actions in different in vivo animal models, we anticipate that the development of TRPA1-targeted, organic polysulfide-based drugs will be promoted by this identification of the binding site.

5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human HV1 Channel.

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 µM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 µM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 µM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

ABT-333 (Dasabuvir) Increases Action Potential Duration and Provokes Early Afterdepolarizations in Canine Left Ventricular Cells via Inhibition of IKr.

ABT-333 (dasabuvir) is an antiviral agent used in hepatitis C treatment. The molecule, similarly to some inhibitors of hERG channels, responsible for the delayed rectifier potassium current (IKr), contains the methanesulfonamide group. Reduced IKr current leads to long QT syndrome and early afterdepolarizations (EADs), therefore potentially causing life-threatening arrhythmias and sudden cardiac death. Our goal was to investigate the acute effects of ABT-333 in enzymatically isolated canine left ventricular myocardial cells. Action potentials (APs) and ion currents were recorded with a sharp microelectrode technique and whole-cell patch clamp, respectively. Application of 1 µM ABT-333 prolonged the AP in a reversible manner. The maximal rates of phases 0 and 1 were irreversibly decreased. Higher ABT-333 concentrations caused larger AP prolongation, elevation of the early plateau potential, and reduction of maximal rates of phases 0, 1, and 3. EADs occurred in some cells in 3-30 µM ABT-333 concentrations. The 10 µM ABT-333-sensitive current, recorded with AP voltage clamp, contained a late outward component corresponding to IKr and an early outward one corresponding to transient outward potassium current (Ito). ABT-333 reduced hERG-channel-mediated ion current in a concentration-dependent, partially reversible manner with a half-inhibitory concentration of 3.2 µM. As the therapeutic plasma concentration of ABT-333 is 1 nM, the arrhythmic risk of ABT-333 is very low, even in the case of drug overdose.

Mapping the functional expression of auxiliary subunits of KCa1.1 in glioblastoma.

Glioblastoma (GBM) is the most aggressive glial tumor, where ion channels, including KCa1.1, are candidates for new therapeutic options. Since the auxiliary subunits linked to KCa1.1 in GBM are largely unknown we used electrophysiology combined with pharmacology and gene silencing to address the functional expression of KCa1.1/ß subunits complexes in both primary tumor cells and in the glioblastoma cell line U-87 MG. The pattern of the sensitivity (activation/inhibition) of the whole-cell currents to paxilline, lithocholic acid, arachidonic acid, and iberiotoxin; the presence of inactivation of the whole-cell current along with the loss of the outward rectification upon exposure to the reducing agent DTT collectively argue that KCa1.1/ß3 complex is expressed in U-87 MG. Similar results were found using human primary glioblastoma cells isolated from patient samples. Silencing the ß3 subunit expression inhibited carbachol-induced Ca2+ transients in U-87 MG thereby indicating the role of the KCa1.1/ß3 in the Ca2+ signaling of glioblastoma cells. Functional expression of the KCa1.1/ß3 complex, on the other hand, lacks cell cycle dependence. We suggest that the KCa1.1/ß3 complex may have diagnostic and therapeutic potential in glioblastoma in the future.

Multiple mechanisms contribute to fluorometry signals from the voltage-gated proton channel.

Voltage-clamp fluorometry (VCF) supplies information about the conformational changes of voltage-gated proteins. Changes in the fluorescence intensity of the dye attached to a part of the protein that undergoes a conformational rearrangement upon the alteration of the membrane potential by electrodes constitute the signal. The VCF signal is generated by quenching and dequenching of the fluorescence as the dye traverses various local environments. Here we studied the VCF signal generation, using the Hv1 voltage-gated proton channel as a tool, which shares a similar voltage-sensor structure with voltage-gated ion channels but lacks an ion-conducting pore. Using mutagenesis and lipids added to the extracellular solution we found that the signal is generated by the combined effects of lipids during movement of the dye relative to the plane of the membrane and by quenching amino acids. Our 3-state model recapitulates the VCF signals of the various mutants and is compatible with the accepted model of two major voltage-sensor movements.

Investigation of the Role of the TRPA1 Ion Channel in Conveying the Effect of Dimethyl Trisulfide on Vascular and Histological Changes in Serum-Transfer Arthritis.

Rheumatoid arthritis (RA) is one of the most prevalent autoimmune diseases. Its therapy is often challenging, even in the era of biologicals. Previously, we observed the anti-inflammatory effects of garlic-derived organic polysulfide dimethyl trisulfide (DMTS). Some of these effects were mediated by activation of the TRPA1 ion channel. TRPA1 was mostly expressed in a subset of nociceptor neurons. We decided to investigate the action of DMTS in K/BxN serum-transfer arthritis, which is a relevant model of RA. TRPA1 gene knockout (KO) and wild-type (WT) mice were used. The interaction of DMTS and TRPA1 was examined using a patch clamp in CHO cells. Arthritis was characterized by mechanical hyperalgesia, paw swelling, movement range of the ankle joint, hanging performance, plasma extravasation rate, myeloperoxidase activity, and histological changes in the tibiotarsal joint. DMTS activated TRPA1 channels dose-dependently. DMTS treatment reduced paw swelling and plasma extravasation in both TRPA1 WT and KO animals. DMTS-treated TRPA1 KO animals developed milder collagen deposition in the inflamed joints than WT ones. TRPA1 WT mice did not exhibit significant cartilage damage compared to ones administered a vehicle. We concluded that DMTS and related substances might evolve into novel complementary therapeutic aids for RA patients.

Functional Voltage-Gated Sodium Channels Are Present in the Human B Cell Membrane.

B cells express various ion channels, but the presence of voltage-gated sodium (NaV) channels has not been confirmed in the plasma membrane yet. In this study, we have identified several NaV channels, which are expressed in the human B cell membrane, by electrophysiological and molecular biology methods. The sensitivity of the detected sodium current to tetrodotoxin was between the values published for TTX-sensitive and TTX-insensitive channels, which suggests the co-existence of multiple NaV1 subtypes in the B cell membrane. This was confirmed by RT-qPCR results, which showed high expression of TTX-sensitive channels along with the lower expression of TTX-insensitive NaV1 channels. The biophysical characteristics of the currents also supported the expression of multiple NaV channels. In addition, we investigated the potential functional role of NaV channels by membrane potential measurements. Removal of Na+ from the extracellular solution caused a reversible hyperpolarization, supporting the role of NaV channels in shaping and maintaining the resting membrane potential. As this study was mainly limited to electrophysiological properties, we cannot exclude the possible non-canonical functions of these channels. This work concludes that the presence of voltage-gated sodium channels in the plasma membrane of human B cells should be recognized and accounted for in the future.

Multifocal Urinary Tract Metastasis of Colorectal Carcinoma.

INTRODUCTION: Secondary urinary tract tumors are uncommon findings and mainly evolve by direct invasion from adjacent organs. Actual metastatic involvement often develops in the urinary bladder, while the upper urinary tract is infrequently affected. In addition, the lungs, breast, and prostate gland are the usual primary sites. Colorectal carcinoma (CRC) may spread to the ureter directly or seeds via vascular or lymphatic channels. It may pose struggles in the differential diagnosis because CRC shares standard pathologic features with the primary adenocarcinoma of the urinary tract. CASE PRESENTATION: We describe the case of an 81-year-old man who was referred to our hospital with a distal ureteral tumor that was treated by a ureteronephrectomy. The histopathological and genetic analysis established the diagnosis of metastatic CRC along with 3 metastases in the renal pelvis. CONCLUSION: This rare case highlights the limitations of conventional histological processing, including immunohistochemistry, and it underlines the role of molecular investigations in certain circumstances.

Peptide Inhibitors of Kv1.5: An Option for the Treatment of Atrial Fibrillation.

The human voltage gated potassium channel Kv1.5 that conducts the IKur current is a key determinant of the atrial action potential. Its mutations have been linked to hereditary forms of atrial fibrillation (AF), and the channel is an attractive target for the management of AF. The development of IKur blockers to treat AF resulted in small molecule Kv1.5 inhibitors. The selectivity of the blocker for the target channel plays an important role in the potential therapeutic application of the drug candidate: the higher the selectivity, the lower the risk of side effects. In this respect, small molecule inhibitors of Kv1.5 are compromised due to their limited selectivity. A wide range of peptide toxins from venomous animals are targeting ion channels, including mammalian channels. These peptides usually have a much larger interacting surface with the ion channel compared to small molecule inhibitors and thus, generally confer higher selectivity to the peptide blockers. We found two peptides in the literature, which inhibited IKur: Ts6 and Osu1. Their affinity and selectivity for Kv1.5 can be improved by rational drug design in which their amino acid sequences could be modified in a targeted way guided by in silico docking experiments.

Microcirculatory effects of sildenafil in experimental testicular torsion in rats.

PURPOSE: Investigate the short-term effect of sildenafil on microcirculation, especially the velocity, the pattern of the flow and the recruitment of the leukocyte in postcapillaries. METHODS: In male Sprague-Dawley rats, the microcirculatory consequences of 60 min experimental testicular torsion, followed by 240 min of reperfusion, were examined. Using fluorescence intravital microscopy, changes in red blood cell velocity in post-capillary venules and rolling as well as adhesion of leukocytes in the postcapillary venules were examined before the torsion and every hour during the reperfusion period. Sildenafil was given 10 min prior to reperfusion (iv 0.7 mg/kg, n = 6), while control animals received saline vehicle (n = 5). RESULTS: The characteristic flow motion disappeared in the affected testicular during the torsion. Red blood cell velocity values were dramatically decreased (by > 50%) and both rolling and adhesion of leukocytes increased during the reperfusion phase. Sildenafil treatment resulted in significantly higher red blood cell velocity values during the entire reperfusion period, but exerted only a temporary positive effect on the plost-ischaemic leukocyte-endothelial interactions. CONCLUSIONS: Intraoperative administration of sildenafil during surgical detorsion may provide marked testicular microperfusion benefits, but failed to influence the overall leukocyte-driven microcirculatory inflammatory reactions.

[Nocturia]. / Nocturia.

Nocturia is a significantly underestimated disorder, resulting in general worsening of patients' quality of life, while morbidity and mortality are increasing. Several urologic and other pathologic causes can be described in the background including relatively severe conditions. Therefore, accurate evaluation and adequate treatment is recommended. In this review the authors summarize the international literature regarding nocturia. PubMed and ScienceDirect databases were accurately reviewed for the relevant information. Epidemiology, etiology, unfavourable effects, diagnosis and possible treatment options were analysed. They found that symptoms can be releaved by lifestyle changes and traditional therapy in several cases, but clinically significant improvement can be reached using desmopressin in patients suffering from nocturnal polyuria. The authors conclude that nocturia may have negative effects on patients' quality of life and also on the society. Early detection and proper treatment is essential. Orv. Hetil., 2016, 157(36), 1419-1426.