Voltage-Gated K+ Channel Modulation by Marine Toxins: Pharmacological Innovations and Therapeutic Opportunities.

Bioactive compounds are abundant in animals originating from marine ecosystems. Ion channels, which include sodium, potassium, calcium, and chloride, together with their numerous variants and subtypes, are the primary molecular targets of the latter. Based on their cellular targets, these venom compounds show a range of potencies and selectivity and may have some therapeutic properties. Due to their potential as medications to treat a range of (human) diseases, including pain, autoimmune disorders, and neurological diseases, marine molecules have been the focus of several studies over the last ten years. The aim of this review is on the various facets of marine (or marine-derived) molecules, ranging from structural characterization and discovery to pharmacology, culminating in the development of some "novel" candidate chemotherapeutic drugs that target potassium channels.

In Silico Methods for the Discovery of Kv7.2/7.3 Channels Modulators: A Comprehensive Review.

The growing interest in Kv7.2/7.3 agonists originates from the involvement of these channels in several brain hyperexcitability disorders. In particular, Kv7.2/7.3 mutants have been clearly associated with epileptic encephalopathies (DEEs) as well as with a spectrum of focal epilepsy disorders, often associated with developmental plateauing or regression. Nevertheless, there is a lack of available therapeutic options, considering that retigabine, the only molecule used in clinic as a broad-spectrum Kv7 agonist, has been withdrawn from the market in late 2016. This is why several efforts have been made both by both academia and industry in the search for suitable chemotypes acting as Kv7.2/7.3 agonists. In this context, in silico methods have played a major role, since the precise structures of different Kv7 homotetramers have been only recently disclosed. In the present review, the computational methods used for the design of Kv.7.2/7.3 small molecule agonists and the underlying medicinal chemistry are discussed in the context of their biological and structure-function properties.

KCNT1 Channel Blockers: A Medicinal Chemistry Perspective.

Potassium channels have recently emerged as suitable target for the treatment of epileptic diseases. Among potassium channels, KCNT1 channels are the most widely characterized as responsible for several epileptic and developmental encephalopathies. Nevertheless, the medicinal chemistry of KCNT1 blockers is underdeveloped so far. In the present review, we describe and analyse the papers addressing the issue of KCNT1 blockers' development and identification, also evidencing the pros and the cons of the scientific approaches therein described. After a short introduction describing the epileptic diseases and the structure-function of potassium channels, we provide an extensive overview of the chemotypes described so far as KCNT1 blockers, and the scientific approaches used for their identification.

In Vitro and In Vivo Pharmacological Characterization of a Novel TRPM8 Inhibitor Chemotype Identified by Small-Scale Preclinical Screening.

Transient receptor potential melastatin type 8 (TRPM8) is a target for the treatment of different physio-pathological processes. While TRPM8 antagonists are reported as potential drugs for pain, cancer, and inflammation, to date only a limited number of chemotypes have been investigated and thus a limited number of compounds have reached clinical trials. Hence there is high value in searching for new TRPM8 antagonistic to broaden clues to structure-activity relationships, improve pharmacological properties and explore underlying molecular mechanisms. To address this, the EDASA Scientific in-house molecular library has been screened in silico, leading to identifying twenty-one potentially antagonist compounds of TRPM8. Calcium fluorometric assays were used to validate the in-silico hypothesis and assess compound selectivity. Four compounds were identified as selective TRPM8 antagonists, of which two were dual-acting TRPM8/TRPV1 modulators. The most potent TRPM8 antagonists (BB 0322703 and BB 0322720) underwent molecular modelling studies to highlight key structural features responsible for drug-protein interaction. The two compounds were also investigated by patch-clamp assays, confirming low micromolar potencies. The most potent compound (BB 0322703, IC50 1.25 ± 0.26 µM) was then profiled in vivo in a cold allodinya model, showing pharmacological efficacy at 30 µM dose. The new chemotypes identified showed remarkable pharmacological properties paving the way to further investigations for drug discovery and pharmacological purposes.

New Frontiers on ER Stress Modulation: Are TRP Channels the Leading Actors?

The endoplasmic reticulum (ER) is a dynamic structure, playing multiple roles including calcium storage, protein synthesis and lipid metabolism. During cellular stress, variations in ER homeostasis and its functioning occur. This condition is referred as ER stress and generates a cascade of signaling events termed unfolded protein response (UPR), activated as adaptative response to mitigate the ER stress condition. In this regard, calcium levels play a pivotal role in ER homeostasis and therefore in cell fate regulation since calcium signaling is implicated in a plethora of physiological processes, but also in disease conditions such as neurodegeneration, cancer and metabolic disorders. A large body of emerging evidence highlighted the functional role of TRP channels and their ability to promote cell survival or death depending on endoplasmic reticulum stress resolution, making them an attractive target. Thus, in this review we focused on the TRP channels' correlation to UPR-mediated ER stress in disease pathogenesis, providing an overview of their implication in the activation of this cellular response.

In Silico Identification and In Vitro Evaluation of New ABCG2 Transporter Inhibitors as Potential Anticancer Agents.

Different molecular mechanisms contribute to the development of multidrug resistance in cancer, including increased drug efflux, enhanced cellular repair mechanisms and alterations of drug metabolism or drug targets. ABCG2 is a member of the ATP-binding cassette superfamily transporters that promotes drug efflux, inducing chemotherapeutic resistance in malignant cells. In this context, the development of selective ABCG2 inhibitors might be a suitable strategy to improve chemotherapy efficacy. Thus, through a multidisciplinary approach, we identified a new ABCG2 selective inhibitor (8), highlighting its ability to increase mitoxantrone cytotoxicity in both hepatocellular carcinoma (EC50from 8.67 ± 2.65 to 1.25 ± 0.80 µM) and transfected breast cancer cell lines (EC50from 9.92 ± 2.32 to 2.45 ± 1.40 µM). Moreover, mitoxantrone co-administration in both transfected and non-transfected HEK293 revealed that compound 8 notably lowered the mitoxantrone EC50, demonstrating its efficacy along with the importance of the ABCG2 extrusion pump overexpression in MDR reversion. These results were corroborated by evaluating the effect of inhibitor 8 on mitoxantrone cell uptake in multicellular tumor spheroids and via proteomic experiments.

Lipid Nanoparticles Traverse Non-Corneal Path to Reach the Posterior Eye Segment: In Vivo Evidence.

Lipid-based nanocarriers (LNs) have made it possible to prolong corneal residence time and improve the ocular bioavailability of ophthalmic drugs. In order to investigate how the LNs interact with the ocular mucosa and reach the posterior eye segment, we have formulated lipid nanocarriers that were designed to bear a traceable fluorescent probe in the present work. The chosen fluorescent probe was obtained by a conjugation reaction between fluoresceinamine and the solid lipid excipient stearic acid, forming a chemically synthesized adduct (ODAF, N-(3',6'-dihydroxy-3-oxospiro [isobenzofuran-1(3H),9'-[9H] xanthen]-5-yl)-octadecanamide). The novel formulation (LN-ODAF) has been formulated and characterized in terms of its technological parameters (polydispersity index, mean particle size and zeta potential), while an in vivo study was carried out to assess the ability of LN-ODAF to diffuse through different ocular compartments. LN-ODAF were in nanometric range (112.7 nm ± 0.4), showing a good homogeneity and long-term stability. A TEM (transmission electron microscopy) study corroborated these results of characterization. In vivo results pointed out that after ocular instillation, LN ODAF were concentrated in the cornea (two hours), while at a longer time (from the second hour to the eighth hour), the fluorescent signals extended gradually towards the back of the eye. From the results obtained, LN-ODAF demonstrated a potential use of lipid-based nanoparticles as efficient carriers of an active pharmaceutical ingredient (API) involved in the management of retinal diseases.

l-Arginine Improves Solubility and ANTI SARS-CoV-2 Mpro Activity of Rutin but Not the Antiviral Activity in Cells.

The COVID-19 pandemic outbreak prompts an urgent need for efficient therapeutics, and repurposing of known drugs has been extensively used in an attempt to get to anti-SARS-CoV-2 agents in the shortest possible time. The glycoside rutin shows manifold pharmacological activities and, despite its use being limited by its poor solubility in water, it is the active principle of many pharmaceutical preparations. We herein report our in silico and experimental investigations of rutin as a SARS-CoV-2 Mpro inhibitor and of its water solubility improvement obtained by mixing it with l-arginine. Tests of the rutin/l-arginine mixture in a cellular model of SARS-CoV-2 infection highlighted that the mixture still suffers from unfavorable pharmacokinetic properties, but nonetheless, the results of this study suggest that rutin might be a good starting point for hit optimization.

Intraocular pressure reduction with once-a-day application of a new prostaglandin eye drop: a pilot placebo-controlled study in 12 patients.

PURPOSE: To assess the ocular hypotensive effect of 15-keto fluprostenol, the oxidized metabolite of travoprost, on glaucoma patients, through a randomized double-masked placebo-controlled study. METHODS: Twelve patients with ocular normal tension glaucoma (NTG) (intraocular pressure [IOP] < 22 mmHg) were enrolled. In order to ensure patient compliance to treatment, all study subjects were hospitalized. In each patient, the eye to be submitted to the treatments was randomly chosen. After hospital admission (day 1), those patients received for 5 days at 8 P.M. either one drop of 15-keto fluprostenol (35 µg/ml) or one drop of placebo. IOP evaluation was performed within 8 A.M. and 8 P.M. for 6 days. Furthermore, we performed a determination of cardiovascular parameters before and after the treatments. RESULTS: Starting with the first IOP measurement after the first treatment (8 A.M. on day 2), IOP was reduced of about 14% in the eyes treated 15-keto fluprostenol, in comparison with baseline IOP values of 15-keto fluprostenol-treated patients. The IOP reduction in the 15-keto fluprostenol-treated group was significantly compared to placebo group (p < 0.05) starting from day 3 till day 6 of the study. Except for mild hyperemia in one 15-keto fluprostenol-treated eye, no other side effects were observed or reported by the enrolled patients. CONCLUSIONS: The travoprost metabolite 15-keto fluprostenol was effective in decrease IOP and maintained IOP reduction along 5 days of treatment. The 15-keto fluprostenol can be developed as a good candidate for once-a-day NTG patients' treatment.

Anti-Inflammatory and Antioxidant Properties of Dehydrated Potato-Derived Bioactive Compounds in Intestinal Cells.

Inflammation and oxidative stress are always more recognized as responsible for chronic disease at the intestinal level. Currently, a growing interest is addressed to the discovery of diet-derived products which have anti-inflammatory and antioxidant properties. This work aims to characterize the pharmacological potential of dehydrated potatoes. For this purpose, a simulated gastrointestinal digestion was carried out. The bioaccessible peptides were fractionated on the basis of their molecular weight and tested on intestinal epithelial cells (IEC-6) under oxidative and inflammatory conditions. Our results demonstrate that the tested peptide fractions were able to significantly inhibit tumor necrosis factor-α release and cycloxygenase-2 and inducible nitric oxide synthase expression. The tested peptides also showed significant antioxidant activity, being able to both reduce reactive oxygen species (ROS) release, also from mitochondria, and nitrotyrosine formation, and increase the antioxidant response by heme oxygenase-1 and superoxide dismutase expression. Moreover, the peptide fractions were able to significantly increase the wound repair in IEC-6. The obtained results indicate the anti-inflammatory and antioxidant potential of dehydrated potatoes at the intestinal level.

Characterization of New TRPM8 Modulators in Pain Perception.

BACKGROUND: Transient Receptor Potential Melastatin-8 (TRPM8) is a non-selective cation channel activated by cold temperature and by cooling agents. Several studies have proved that this channel is involved in pain perception. Although some studies indicate that TRPM8 inhibition is necessary to reduce acute and chronic pain, it is also reported that TRPM8 activation produces analgesia. These conflicting results could be explained by extracellular Ca2+-dependent desensitization that is induced by an excessive activation. Likely, this effect is due to phosphatidylinositol 4,5-bisphosphate (PIP2) depletion that leads to modification of TRPM8 channel activity, shifting voltage dependence towards more positive potentials. This phenomenon needs further evaluation and confirmation that would allow us to understand better the role of this channel and to develop new therapeutic strategies for controlling pain. EXPERIMENTAL APPROACH: To understand the role of TRPM8 in pain perception, we tested two specific TRPM8-modulating compounds, an antagonist (IGM-18) and an agonist (IGM-5), in either acute or chronic animal pain models using male Sprague-Dawley rats or CD1 mice, after systemic or topical routes of administration. RESULTS: IGM-18 and IGM-5 were fully characterized in vivo. The wet-dog shake test and the body temperature measurements highlighted the antagonist activity of IGM-18 on TRPM8 channels. Moreover, IGM-18 exerted an analgesic effect on formalin-induced orofacial pain and chronic constriction injury-induced neuropathic pain, demonstrating the involvement of TRPM8 channels in these two pain models. Finally, the results were consistent with TRPM8 downregulation by agonist IGM-5, due to its excessive activation. CONCLUSIONS: TRPM8 channels are strongly involved in pain modulation, and their selective antagonist is able to reduce both acute and chronic pain.

Antioxidant Properties of Buffalo-Milk Dairy Products: A ß-Lg Peptide Released after Gastrointestinal Digestion of Buffalo Ricotta Cheese Reduces Oxidative Stress in Intestinal Epithelial Cells.

Redox signaling regulates different gastrointestinal (G.I.) epithelium functions. At the intestinal level, the loss of redox homeostasis in intestinal epithelial cells (IECs) is responsible for the pathogenesis and development of a wide diversity of G.I. disorders. Thus, the manipulation of oxidative stress in IECs could represent an important pharmacological target for different diseases. In this study, peptides released from in vitro gastro intestinal digestion of different buffalo-milk commercial dairy products were identified and evaluated for their bioactive properties. In particular, six G.I. digests of dairy products were tested in a model of oxidative stress for IECs. Among them, buffalo ricotta cheese was the most active and the presence of an abundant ß-lactoglobulin peptide (YVEELKPTPEGDL, f:60-72) was also revealed. The antioxidant potential of the identified peptide was also evaluated in a model of hydrogen peroxide (H2O2)-induced oxidative stress in the IEC-6 cell line. The peptide was able to reduce ROS release, while, on the other hand, it increased nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activation and the expression of antioxidant cytoprotective factors, such as heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), and superoxide dismutase (SOD). These results indicate that buffalo ricotta cheese-isolated peptide could have potential in the treatment of some gastrointestinal disorders.

Fast Profiling of Natural Pigments in Different Spirulina (Arthrospira platensis) Dietary Supplements by DI-FT-ICR and Evaluation of their Antioxidant Potential by Pre-Column DPPH-UHPLC Assay.

Arthrospira platensis, better known as Spirulina, is one of the most important microalgae species. This cyanobacterium possesses a rich metabolite pattern, including high amounts of natural pigments. In this study, we applied a combined strategy based on Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) and Ultra High-Performance Liquid Chromatography (UHPLC) for the qualitative/quantitative characterization of Spirulina pigments in three different commercial dietary supplements. FT-ICR was employed to elucidate the qualitative profile of Spirulina pigments, in both direct infusion mode (DIMS) and coupled to UHPLC. DIMS showed to be a very fast (4 min) and accurate (mass accuracy ≤ 0.01 ppm) tool. 51 pigments were tentatively identified. The profile revealed different classes, such as carotenes, xanthophylls and chlorophylls. Moreover, the antioxidant evaluation of the major compounds was assessed by pre-column reaction with the DPPH radical followed by fast UHPLC-PDA separation, highlighting the contribution of single analytes to the antioxidant potential of the entire pigment fraction. ß-carotene, diadinoxanthin and diatoxanthin showed the highest scavenging activity. The method took 40 min per sample, comprising reaction. This strategy could represent a valid tool for the fast and comprehensive characterization of Spirulina pigments in dietary supplements, as well as in other microalgae-based products.

Ring-Fused Cyclic Aminals from Tetrahydro-ß-carboline-Based Dipeptide Compounds.

An acid- and oxidant-promoted intramolecular cyclization of a tetrahydro-ß-carboline-based dipeptide has been developed to prepare new indole-fused aminoacetals. This approach involves N-acyliminium formation from readily available precursors and cyclization under mild reaction conditions. The diastereoselectivity in the formation of the products is influenced by the specific substituents of the starting reagents, which has been rationalized analyzing the energy profile of the related reactions and the relative stability of the proposed structures based on DFT computational methods.

Development of an improved online comprehensive hydrophilic interaction chromatography × reversed-phase ultra-high-pressure liquid chromatography platform for complex multiclass polyphenolic sample analysis.

In this study, an improved online comprehensive two-dimensional liquid chromatography platform coupled to tandem mass spectrometry was developed for the analysis of complex polyphenolic samples. A narrowbore hydrophilic interaction chromatography column (150 × 2.0 mm, 3.0 µm, cross-linked diol) was employed in the first dimension, while a reversed-phase column based on monodisperse sub-2 µm fully porous particles (50 × 3.0 mm, 1.9 µm d.p.) with high surface area (410 m2 /g) was employed in the second dimension. The combination of a trapping column modulation interface with the high retentive fully porous monodisperse reversed-phase column in the second dimension resulted in higher peak capacity values (1146 versus 867), increased sensitivity, sharper and more symmetrical peaks in comparison with a conventional loop-based method, with the same analysis time (70 min). The system was challenged against a complex polyphenolic extract of a typical Italian apple cultivar, enabling the simultaneous separation of multiple polyphenolic classes, including oligomeric procyanidins, up to degree of polymerization of 10. Hyphenation with an ion trap time-of-flight mass spectrometer led to the tentative identification of 121 analytes, showing how this platform could be a powerful analytical tool for the accurate profiling of complex polyphenolic samples.

Flavonoid Composition of Tarocco (Citrus sinensis L. Osbeck) Clone "Lempso" and Fast Antioxidant Activity Screening by DPPH-UHPLC-PDA-IT-TOF.

INTRODUCTION: Clonal selection and hybridisation are valid strategies to obtain fruits with enhanced sensorial and nutraceutical properties. Within Citrus sinensis varieties, Tarocco clone "Lempso" is a typical product of the Calabria region (Italy) characterised by its red pulp. This is the first report concerning its accurate profiling. OBJECTIVE: To characterise in detail the flavonoid composition of Lempso clone and to compare its antioxidant potential with other Citrus varieties by a fast screening method. METHODOLOGY: Extracts were subjected to solid phase extraction and the qualitative/quantitative profile was elucidated through ultra-high performance liquid chromatography (UHPLC) coupled to photodiode array (PDA) and ion trap time-of-flight (IT-TOF) mass spectrometry detection, and compared to both Cleopatra mandarin (Citrus reticulata) and blood orange (Citrus sinensis (L.) Osbeck) Sanguinello varieties. The antioxidant activity was assessed by pre-column 2,2'-diphenyl-1-picrylhydrazyl (DPPH) reaction coupled to UHPLC-PDA. RESULTS: Lempso is characterised by flavonoids (17) and anthocyanins (8). Flavanones content (Hesperidin: 57.19 ± 0.49, Vicenin-2: 4.59 ± 0.03, Narirutin: 5.78 ± 0.13 mg/100 mL) was considerably higher than Cleopatra and Sanguinello varieties. The developed DPPH-UHPLC-PDA method provides information regarding the single contributions to antioxidant activity, highlighting how Ferulic acid, Quercetin and Cyanidin derivatives possess considerable radical scavenging activity (> 50%). The total antioxidant activity was also evaluated and compared with positive controls, showing higher scavenging activity than Cleopatra and Sanguinello (IC50 : 333.76 ± 10.81 µg/mL vs. 452.62 ± 10.81 and 568.39 ± 26.98 µg/mL, respectively). CONCLUSION: These data evidence the nutraceutical potential of Lempso variety, which could be an ingredient for functional beverages. Copyright © 2017 John Wiley & Sons, Ltd.

Bioavailable Citrus sinensis Extract: Polyphenolic Composition and Biological Activity.

Citrus plants contain large amounts of flavonoids with beneficial effects on human health. In the present study, the antioxidant and anti-inflammatory potential of bioavailable polyphenols from Citrus sinensis was evaluated in vitro and ex vivo, using the murine macrophages cell line J774A.1 and primary peritoneal macrophages. Following simulated gastro-intestinal digestion, the in vitro bioavailability of Citrus sinensis polyphenolic extract was assessed using the human cell line Caco-2 grown as monolayers on a transwell membrane. Data demonstrated a relative permeation of its compounds (8.3%). Thus, the antioxidant and anti-inflammatory effect of polyphenolic Citrus sinensis fraction (Cs) was compared to the bioavailable one (CsB). Results revealed that Citrus extract were able to reduce macrophages pro-inflammatory mediators, including nitric oxide, iNOS, COX-2 and different cytokines. Moreover, the effect of Citrus sinensis polyphenols was associated with antioxidant effects, such as a reduction of reactive oxygen species (ROS) and heme-oxygenase-1 (HO-1) increased expression. Our results provide evidence that the bioavailable polyphenolic constituents of the Citrussinensis extract accumulate prevalently at intestinal level and could reach systemic circulation exerting their effect. The bioavailable fraction showed a higher anti-inflammatory and antioxidant potential compared to the initial extract, thus highlighting its potential nutraceutical value.

Design and Synthesis of New Cell Penetrating Peptides: Diffusion and Distribution Inside the Cornea.

The role of cell penetrating peptides (CPPs) has been challenged in recent years for drug delivery to ocular tissues for the targeting of both anterior and posterior segments. The enhancement of trans-corneal transport for anterior segment targeting is a very important issue possibly leading to important outcomes on efficacy and to the opportunity of topical administration of molecules with unfavorable penetration properties. The aim of the present work was the design and synthesis of new CPPs, deriving from the structure of PEP-1 peptide. Synthesized peptides were labeled with 5-carboxyfluorescein (5-FAM), and their diffusion behavior and distribution inside the cornea were evaluated by a validated ex vivo model and a confocal microscopy approach. Newly synthesized peptides showed similar corneal permeation profiles as PEP-1 (Papp = 0.75 ± 0.56 × 10-6 cm/s), about 2.6-fold higher than 5-FAM (Papp = 0.29 ± 0.08 × 10-6 cm/s) despite the higher molecular weight. Confocal microscopy experiments highlighted the tendency of PEP-1 and its derived peptides to localize in the intercellular space and/or in the plasma membrane. Noteworthy, using penetratin as positive control, a higher trans-corneal permeation (Papp = 6.18 ± 1.46 × 10-6 cm/s) was evidenced together with a diffusion by intracellular route and a different accumulation between wings and basal epithelial cells, probably depending on the stage of cell development. Finally, PEP-1 and pep-7 proved to be safe and well tolerated when tested on human conjuctival cell line.

Improvement of Topical Palmitoylethanolamide Anti-Inflammatory Activity by Pegylated Prodrugs.

A small library of polyethylene glycol esters of palmitoylethanolamide (PEA) was synthesized with the aim of improving the pharmacokinetic profile of the parent drug after topical administration. Synthesized prodrugs were studied for their skin accumulation, pharmacological activities, in vitro chemical stability, and in silico enzymatic hydrolysis. Prodrugs proved to be able to delay and prolong the pharmacological activity of PEA by modification of its skin accumulation profile. Pharmacokinetic improvements were particularly evident when specific structural requirements, such as flexibility and reduced molecular weight, were respected. Some of the synthesized prodrugs prolonged the pharmacological effects 5 days following topical administration, while a formulation composed by PEA and two pegylated prodrugs showed both rapid onset and long-lasting activity, suggesting the potential use of polyethylene glycol prodrugs of PEA as a suitable candidate for the treatment of skin inflammatory diseases.

In Silico Assisted Identification, Synthesis, and In Vitro Pharmacological Characterization of Potent and Selective Blockers of the Epilepsy-Associated KCNT1 Channel.

Gain-of-function (GoF) variants in KCNT1 channels cause severe, drug-resistant forms of epilepsy. Quinidine is a known KCNT1 blocker, but its clinical use is limited due to severe drawbacks. To identify novel KCNT1 blockers, a homology model of human KCNT1 was built and used to screen an in-house library of compounds. Among the 20 molecules selected, five (CPK4, 13, 16, 18, and 20) showed strong KCNT1-blocking ability in an in vitro fluorescence-based assay. Patch-clamp experiments confirmed a higher KCNT1-blocking potency of these compounds when compared to quinidine, and their selectivity for KCNT1 over hERG and Kv7.2 channels. Among identified molecules, CPK20 displayed the highest metabolic stability; this compound also blocked KCNT2 currents, although with a lower potency, and counteracted GoF effects prompted by 2 recurrent epilepsy-causing KCNT1 variants (G288S and A934T). The present results provide solid rational basis for future design of novel compounds to counteract KCNT1-related neurological disorders.