Mapping the molecular surface of the analgesic NaV1.7-selective peptide Pn3a reveals residues essential for membrane and channel interactions

Compelling human genetic studies have identified the voltage-gated sodium channel NaV1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider venom-derived peptide µtheraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of NaV1.7, however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed seventeen Pn3a analogues and determined their activity at hNaV1.7 using patchclamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNaV1.7. Removal of hydrophobic residues Y4, Y27 and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at NaV1.7 (20-fold), whilst maintaining >100-fold selectivity over the major off-targets NaV1.4, NaV1.5 and NaV1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of post-surgical pain at doses 3-fold lower than wild-type Pn3a, without causing motor adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic NaV1.7 inhibitors for the development of more efficacious and safer analgesics but also to further investigate the involvement of NaV1.7 in pain

The Tarantula Venom Peptide Eo1a Binds to the Domain II S3-S4 Extracellular Loop of Voltage-Gated Sodium Channel NaV1.8 to Enhance Activation

Venoms from cone snails and arachnids are a rich source of peptide modulators of voltage-gated sodium (NaV) channels, however relatively few venom-derived peptides with activity at the mammalian NaV1.8 subtype have been isolated. Here, we describe the discovery and functional characterisation of β-theraphotoxin-Eo1a, a peptide from the venom of the Tanzanian black and olive baboon tarantula Encyocratella olivacea that modulates NaV1.8. Eo1a is a 37-residue peptide that increases NaV1.8 peak current (EC50 894 ± 146 nM) and causes a large hyperpolarising shift in both the voltage-dependence of activation (ΔV50-20.5 ± 1.2 mV) and steady-state fast inactivation (ΔV50-15.5 ± 1.8 mV). At a concentration of 10 μM, Eo1a has varying effects on the peak current and channel gating of NaV1.1-NaV1.7, although its activity is most pronounced at NaV1.8. Investigations into the binding site of Eo1a using NaV1.7/NaV1.8 chimeras revealed a critical contribution of the DII S3-S4 extracellular loop of NaV1.8 to toxin activity. Results from this work may form the basis for future studies that lead to the rational design of spider venom-derived peptides with improved potency and selectivity at NaV1.8

Modulatory features of the novel spider toxin μ-TRTX-Df1a isolated from the venom of the spider Davus fasciatus

BACKGROUND AND PURPOS

Novel venom-derived inhibitors of the human EAG channel, a putative antiepileptic drug target

Recently, we and other groups revealed that gain-of-function mutations in the human ether à go-go voltage-gated potassium channel hEAG1 (K10.1) lead to developmental disorders with associated infantile-onset epilepsy. However, the physiological role of hEAG1 in the central nervous system remains elusive. Potent and selective antagonists of hEAG1 are therefore much sought after, both as pharmacological tools for studying the (patho)physiological functions of this enigmatic channel and as potential leads for development of anti-epileptic drugs. Since animal venoms are a rich source of potent ion channel modifiers that have been finely tuned by millions of year of evolution, we screened 108 arachnid venoms for hEAG1 inhibitors using electrophysiology. Two hit peptides (Aa1a and Ap1a) were isolated, sequenced, and chemically synthesised for structure-function studies. Both of these hEAG1 inhibitors are C-terminally amidated peptides containing an inhibitor cystine knot motif, which provides them with exceptional stability in both plasma and cerebrospinal fluid. Aa1a and Ap1a are the most potent peptidic inhibitors of hEAG1 reported to date, and they present a novel mode of action by targeting both the activation and inactivation gating of the channel. These peptides should be useful pharmacological tools for probing hEAG1 function as well as informative leads for the development of novel anti-epileptic drugs

A tetrapeptide class of biased analgesics from an Australian fungus targets the μ-opioid receptor

An Australian estuarine isolate ofPenicilliumsp. MST-MF667 yielded3 tetrapeptides named the bilaids with an unusual alternating LDLDchirality. Given their resemblance to known short peptide opioidagonists, we elucidated that they were weak (Kilow micromolar)μ-opioid agonists, which led to the design of bilorphin, a potent andselectiveμ-opioid receptor (MOPr) agonist (Ki1.1 nM). In sharp con-trast to all-natural product opioid peptides that efficaciously recruitβ-arrestin, bilorphin is G protein biased, weakly phosphorylatingthe MOPr and marginally recruitingβ-arrestin, with no receptorinternalization. Importantly, bilorphin exhibits a similar G proteinbias to oliceridine, a small nonpeptide with improved overdosesafety. Molecular dynamics simulations of bilorphin and thestrongly arrestin-biased endomorphin-2 with the MOPr indicatedistinct receptor interactions and receptor conformations thatcould underlie their large differences in bias. Whereas bilorphinis systemically inactive, a glycosylated analog, bilactorphin, isorally active with similar in vivo potency to morphine. Bilorphinis both a unique molecular tool that enhances understanding ofMOPr biased signaling and a promising lead in the development ofnext generation analgesics

Information content of a sports undertaking’s statements serving different purposes – particularly with reference to the player rights

In the 21st century, sport is not just a fun, social cohesive force but also a business; it has become an independent industry by now and several countries possess developed sport markets. According to estimates, sport accounts for 4% of the EU’s GDP. The actuality of our research is given by the fact that the economic aspect of sports develops continuously which is also due to that more and more amounts already stream into sports in our days. In Hungary, sport is mainly state aided and has mostly financing problems while the sport businesses existing in the more developed Western Europe are principally sponsored by the private sector. The government considers sport as a strategic branch (HERCZEG et al, 2015) and manages as such because they see the international breakthrough potencies in sport as well. Sport companies must also adapt the business-based thinking, which requires the strategic planning and operation (BECSKY, 2011). The research covers the subject of economic approach of the players’ rights. The task of accounting is to give a true and fair image about the property, income and financial situation of an undertaking. Information provided by accounting is essential for both the management decisionmaking and the market operators. In Hungary, the sports undertakings, as each managing entity, have to prepare their statements according to the Act C of 2000 on Accounting (AoA.) (NAGY – BÁCSNÉ BÁBA, 2014). The purpose of this research is to examine how a domestic sports undertaking demonstrates the value of available players in the books and how the incomes and expenditures incurred with the players are accounted for, based on the regulations of the Hungarian, international associations and the Union of European Football Associations (hereinafter: UEFA). In order that the leaders of the businesses can make quick and appropriate economic decisions, it is essential in this intensively changing world that an enterprise should have a well-functioning accounting system based on up-to-date information. International Financial Reporting Standards (hereinafter: IFRS) are intended to provide the comparability across borders. Firstly, we deal with the accounting reporting system, both the Hungarian, international financial reporting standards and, relating to UEFA, the investigation of the intangible assets to a great extent during analysing the balance sheets. Then, we examine the income statements from the viewpoint player transfers. To what extent the rules of a statement laid down by UEFA differ from the ones of a statement prepared according to AoA? What is the difference in domestic and international relations? In this study, we search after the answers for questions mentioned before

The relevance of sports financing

According to estimations sports exploit approximately two percent of world GDP. In addition sports represent an important function in culture, socialization and also in education, act an outstanding role in economy, as well, though academic researchers cannot find positive relationship between sports financing and economic growth. Sports were originally civil actions, with non-profit nature, where they did not aspire to reach high returns, but fulfilment of sport itself. However returns can be realized and reinvested in sports. This paper tries to collect the different aspects of sports financing in order to highlight its relevance

Financing and operating questions of sports facilities

This paper tends to present financing and operating questions of sports facilities. Infrastructure is very important for the sport businesses. Sports facilities and sports institutions, infrastructure development, and their legal, financial, accounting conditionality can be defined by the investors and the government (subsidies, taxes, etc.). Financial questions and IT background of facility management can be crucial for the enterprises interested in operating sports businesses. The paper focuses on these kinds of aspects of facility management based on practical examples

Total synthesis of human hepcidin through regioselective disulfide-bond formation by using the safety-catch cysteine protecting group 4,4'-dimethylsulfinylbenzhydryl

A safety-catch cysteine protecting group, S-4,4'-dimethylsulfinylbenzhydryl (Msbh), was designed and developed to expand the capabilities of synthetic strategies for the regioselective formation of disulfide bonds in cysteine-rich peptides. The directed regioselective synthesis of human hepcidin, which contains four disulfide bonds, was undertaken and led to a high-resolution NMR structure under more physiologically relevant conditions than previously. Conversely, hepcidin synthesized with the formerly assigned vicinal disulfide-bond connectivity displayed significant conformational heterogeneity under similar conditions. The two synthetic forms of human hepcidin induced ferroportin internalization with apparent EC50 values of 2.0 (native fold, 1) and 4.4 nm (non-native fold, 2), with 2 undergoing isomerization to 1 in the presence of ferroportin expressing cells

Isolation, characterization and total regioselective synthesis of the novel μo-conotoxin MfVIA from Conus magnificus that targets voltage-gated sodium channels

The μO-conotoxins are notable for their unique selectivity for Nav1.8 over other sodium channel isoforms, making them attractive drug leads for the treatment of neuropathic pain. We describe the discovery of a novel μO-conotoxin, MfVIA, from the venom of Conus magnificus using high-throughput screening approaches. MfVIA was found to be a hydrophobic 32-residue peptide (amino acid sequence RDCQEKWEYCIVPILGFVYCCPGLICGPFVCV) with highest sequence homology to μO-conotoxin MrVIB. To overcome the synthetic challenges posed by μO-conotoxins due to their hydrophobic nature and difficult folding, we developed a novel regioselective approach for the synthesis of μO-conotoxins. Performing selective oxidative deprotections of the cysteine side-chain protecting groups of the fully protected peptide allowed manipulations in organic solvents with no chromatography required between steps. Using this approach, we obtained correctly folded MfVIA with increased synthetic yields. Biological activity of MfVIA was assessed using membrane potential-sensitive dyes and electrophysiological recording techniques. MfVIA preferentially inhibits Nav1.8 (IC50 95.9 ± 74.3 nM) and Nav1.4 (IC50 81 ± 16 nM), with significantly lower affinity for other Nav subtypes (IC50 431-6203 nM; Nav1.5 \u3e 1.6 ∼ 1.7 ∼ 1.3 ∼ 1.1 ∼ 1.2). This improved approach to μO-conotoxin synthesis will facilitate the optimization of μO-conotoxins as novel analgesic molecules to improve pain management