Design of novel ion channel modulators

Function and modulation of neuronal sodium channels are critical for the neuromodulation of electrical excitability and synaptic transmission in neurons - the basis for many aspects of signal transduction, learning, memory and physiological regulation. Mutations in neuronal voltage-gated sodium channel genes are responsible for various human neurological disorders. Furthermore, human neuronal voltage-gated sodium channels are primary targets of therapeutic drugs used as local anesthetics and for treatment of neurological and cardiac disorders. Yarov-Yarovoy\u27s lab is working on rational design of novel therapeutically useful blockers of voltage-gated sodium channels for treatment of pain and epilepsy. Serious, chronic pain affects at least 116 million Americans each year and epilepsy affects nearly 3 million Americans and 50 million people Worldwide. However, the treatment of chronic pain and epilepsy remains a major unmet medical need because the use of currently available drugs is limited due to incomplete efficacy and/or significant side effects. Considerable efforts by pharmaceutical industry toward identifying selective inhibitors of one or more of voltage-gated sodium channels subtypes did not generate any genuinely subtype selective blockers. Yarov-Yarovoy\u27s laboratory uses an innovative approach to design novel subtype selective voltage-gated sodium channel blocking peptides, small molecules, and antibodies. This project will provide key structural information on the molecular basis of neuronal voltage-gated sodium channels function and its interaction with therapeutically useful subtype-specific modulators

The MX-Helix of Muscle nAChR Subunits Regulates Receptor Assembly and Surface Trafficking.

Nicotinic acetylcholine receptors (AChRs) are pentameric channels that mediate fast transmission at the neuromuscular junction (NMJ) and defects in receptor expression underlie neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndrome (CMS). Nicotinic receptor expression at the NMJ is tightly regulated and we previously identified novel Golgi-retention signals in the β and δ subunit cytoplasmic loops that regulate trafficking of the receptor to the cell surface. Here, we show that the Golgi retention motifs are localized in the MX-helix, a juxta-membrane alpha-helix present in the proximal cytoplasmic loop of receptor subunits, which was defined in recent crystal structures of cys-loop receptor family members. First, mutational analysis of CD4-MX-helix chimeric proteins showed that the Golgi retention signal was dependent on both the amphipathic nature of the MX-helix and on specific lysine residues (βK353 and δK351). Moreover, retention was associated with ubiquitination of the lysines, and βK353R and δK351R mutations reduced ubiquitination and increased surface expression of CD4-β and δ MX-helix chimeric proteins. Second, mutation of these lysines in intact β and δ subunits perturbed Golgi-based glycosylation and surface trafficking of the AChR. Notably, combined βK353R and δK351R mutations increased the amount of surface AChR with immature forms of glycosylation, consistent with decreased Golgi retention and processing. Third, we found that previously identified CMS mutations in the ε subunit MX-helix decreased receptor assembly and surface levels, as did an analogous mutation introduced into the β subunit MX-helix. Together, these findings indicate that the subunit MX-helix contributes to receptor assembly and is required for normal expression of the AChR and function of the NMJ. In addition, specific determinants in the β and δ subunit MX-helix contribute to quality control of AChR expression by intracellular retention and ubiquitination of unassembled subunits, and by facilitating the appropriate glycosylation of assembled surface AChR

Selective disruption of high sensitivity heat activation but not capsaicin activation of TRPV1 channels by pore turret mutations.

The capsaicin receptor transient receptor potential vanilloid (TRPV)1 is a highly heat-sensitive ion channel. Although chemical activation and heat activation of TRPV1 elicit similar pungent, painful sensation, the molecular mechanism underlying synergistic activation remains mysterious. In particular, where the temperature sensor is located and whether heat and capsaicin share a common activation pathway are debated. To address these fundamental issues, we searched for channel mutations that selectively affected one form of activation. We found that deletion of the first 10 amino acids of the pore turret significantly reduced the heat response amplitude and shifted the heat activation threshold, whereas capsaicin activation remained unchanged. Removing larger portions of the turret disrupted channel function. Introducing an artificial sequence to replace the deleted region restored sensitive capsaicin activation in these nonfunctional channels. The heat activation, however, remained significantly impaired, with the current exhibiting diminishing heat sensitivity to a level indistinguishable from that of a voltage-gated potassium channel, Kv7.4. Our results demonstrate that heat and capsaicin activation of TRPV1 are structurally and mechanistically distinct processes, and the pore turret is an indispensible channel structure involved in the heat activation process but is not part of the capsaicin activation pathway. Synergistic effect of heat and capsaicin on TRPV1 activation may originate from convergence of the two pathways on a common activation gate

The Trials and Tribulations of Structure Assisted Design of KCa Channel Activators.

Calcium-activated K+ channels constitute attractive targets for the treatment of neurological and cardiovascular diseases. To explain why certain 2-aminobenzothiazole/oxazole-type KCa activators (SKAs) are KCa3.1 selective we previously generated homology models of the C-terminal calmodulin-binding domain (CaM-BD) of KCa3.1 and KCa2.3 in complex with CaM using Rosetta modeling software. We here attempted to employ this atomistic level understanding of KCa activator binding to switch selectivity around and design KCa2.2 selective activators as potential anticonvulsants. In this structure-based drug design approach we used RosettaLigand docking and carefully compared the binding poses of various SKA compounds in the KCa2.2 and KCa3.1 CaM-BD/CaM interface pocket. Based on differences between residues in the KCa2.2 and KCa.3.1 models we virtually designed 168 new SKA compounds. The compounds that were predicted to be both potent and KCa2.2 selective were synthesized, and their activity and selectivity tested by manual or automated electrophysiology. However, we failed to identify any KCa2.2 selective compounds. Based on the full-length KCa3.1 structure it was recently demonstrated that the C-terminal crystal dimer was an artefact and suggested that the "real" binding pocket for the KCa activators is located at the S4-S5 linker. We here confirmed this structural hypothesis through mutagenesis and now offer a new, corrected binding site model for the SKA-type KCa channel activators. SKA-111 (5-methylnaphtho[1,2-d]thiazol-2-amine) is binding in the interface between the CaM N-lobe and the S4-S5 linker where it makes van der Waals contacts with S181 and L185 in the S45A helix of KCa3.1

Сучасні принципи і засоби медикаментозного лікування при генералізованому пародонтиті: (огляд літератури)

Summary. Treatment of generalized periodontitis is based on the principles of the integrated approach, taking into account the individual characteristics of dental and somatic status, the prevalence of certain local and general periodontal factors in each clinical situation. Etiotropic treatment involves rational general and local antimicrobial therapy to eliminate periodontal pathogenic bacteria and block the inflammatory cascade with the production of proinflammatory cytokines, eicosanoids, matrix metalloproteinases, etc. Pathogenetic treatment is aimed at alleviating the leading links in the development of the disease. The prescription of nonsteroidal anti-inflammatory drugs, use of exogenous inhibitors of matrix metalloproteinases; immunocorrective therapy; cytokine therapy; use of antioxidants; appointment of drugs to stimulate the process of bone formation are justified. The correct direction of etiopathogenetic drug treatment is confirmed by numerous data from the literature on its high clinical efficacy. However, the development of differentiated approaches to the choice of drugs for general and local use depending on exo- and endoparodontopathogenic factors, that influence the pathogenesis of generalized periodontitis most, still remains relevant.Резюме. Лечение генерализованного пародонтита базируется на принципах комплексного подхода с учетом индивидуальных особенностей стоматологического и соматического статуса, превалирования действия тех или общих пародонтопатогенных факторов в каждой конкретной клинической ситуации. Этиотропное лечение предполагает проведения рациональной общей и местной антимикробной терапии с целью элиминации пародонтопатогенных бактерий и блокады воспалительного каскада с продукцией противовоспалительных цитокинов, эйкозаноидов, матриксных металлопротеиназ и т. д. Патогенетическое лечение направленно на купирование ведущих звеньев развития заболевания. Обоснованным является назначение нестероидных противовоспалительных средств; применение экзогенных ингибиторов матриксных металлопротеиназ; иммунокоригирующая терапия; цитокинотерапия; применение антиоксидантов; назначение препаратов для стимулирования процесса формирования кости. Правильная направленность этиопатогенетического медикаментозного лечения подтверждена многочисленными данными литературы об их высокой клинической эффективности. Однако актуальной остается разработка дифференцированных подходов к выбору комплекса медикаментозных средств общего и местного применения в зависимости от экзо- и эндопародонтопатогенных факторов, которые наиболее влияют на патогенез генерализованного пародонтита.Резюме. Лікування генералізованого пародонтиту базується на принципах комплексного підходу з урахуванням індивідуальних особливостей стоматологічного та соматичного статусу, превалювання дії тих чи інших місцевих і загальних пародонтопатогенних факторів у кожній конкретній клінічній ситуації. Етіотропне лікування передбачає проведення раціональної загальної і місцевої антимікробної терапії з метою елімінації пародонтопатогенних бактерій і блокади запального каскаду з продукцією прозапальних цитокінів, ейкозаноїдів, матриксних металопротеїназ і т. д. Патогенетичне лікування спрямоване на купірування провідних ланок розвитку захворювання. Обґрунтованим є призначення нестероїдних протизапальних засобів; застосування екзогенних інгібіторів матриксних металопротеїназ; імунокоригуюча терапія; цитокінотерапія; застосування антиоксидантів; призначення препаратів для стимулювання процесу формування кістки. Правильна спрямованість етіопатогенетичного медикаментозного лікування підтверджена численними даними літератури про їх високу клінічну ефективність. Однак актуальними залишається розробка диференційованих підходів до вибору комплексу медикаментозних засобів загального та місцевого застосування  залежно від екзо- й ендопародонтопатогенних факторів, які найбільше впливають на патогенез генералізованого пародонтиту

Assessment of the effectiveness of differential management of patients in the early postoperative period following the dental implant surgery

Development of the modern dental implantology as a science is rapidly advancing, making impressive gains. Despite the undoubted achievements, an important issue of the dental implantation is the risk of inflammatory post-surgical complications. Aim of the study. To assess the effectiveness of the proposed differential medicamentous therapy after dental implant surgery based on the results of clinical picture dynamic monitoring in the early postoperative period (up to 3 months). Materials and methods. 124 somatically healthy people (54 men and 70 women) aged from 18 to 34 years were examined, among them: 25 patients with intact periodontium, 35 patients with chronic catarrhal gingivitis (CCG), 30 patients with generalized periodontitis (GP) of the initial, initial-І degrees of severity, 34 patients with GP of the І and ІІІ degree of severity. Patients were divided into 2 equal study groups: the proposed differential therapy was used for the experimental group (62 patients) in the early post-surgical period depending on the determined initial oral hygienic status; the traditional medicamentous treatment was applied for the control group (62 patients). Since the special medicamentous therapy was not advisable for the patients with healthy periodontium (25 patients) after the implant surgery, they made up the comparison group. Immunomodulating monotherapy (6 “Imudon” lozenges per day for 2 weeks) was prescribed to patients with CCG in the postoperative period. Immunomodulating therapy (“Imudon” by the same scheme) combined with a universal broad-spectrum antimicrobial drug “Miramistin” was prescribed to patients with GP of the initial, initial-I, І and І–ІІ degrees of severity for 2 weeks. Control examinations were performed one week after removing the stitches, then weekly for 3 months before the second stage of surgery. Results. One week after removing the stitches a significantly better oral hygienic status was observed in the main group of patients with CCG compared to the control (P < 0.05). When assessing the mucosa state above the intraosseous element in CCG patients, cases of peri-implantitis were detected neither in the main nor in the control group in the postoperative period. In the main group of patients with GP of the initial, initial-I degrees of severity the oral cavity hygienic status was significantly better in all periods of observation, starting from the first week after removing the stitches and after 3 months compared with the control (P < 0.05). When assessing the mucosa state above the intraosseous element in patients with GP of the initial, initial-I degree of severity in the postoperative period, one case of peri-implantitis was revealed in the control group. It manifested as hyperemia and edema of the mucosa above the intraosseous element during the 1st week after removing the stitches and granulation tissue growth during the 2nd week. The main group of patients with GP of the I, I–II degrees of severity were characterized by significantly better oral hygienic status – 1.90 times on average (P < 0.05), compared with the control, from the 1st week after removing the stitches and over the follow-up period up to 3 months. When assessing the mucosa state above the intraosseous element in patients with GP of the I, I–II degrees of severity in the postoperative period, one case of peri-implantitis was revealed in the control, similarly to patients with GP of the initial, initial-I degrees of severity. Conclusions. Analysis of the clinical parameters dynamics in the patients after dental implant surgery suggests the effectiveness of the proposed methods in addition to the traditional maintenance therapy in the early period of 1 week after removing the stitches to 3 months. The obtained results allow to recommend the proposed methods of differential medicamentous therapy in the early postoperative period following the dental implant surgery for a wide application in dental practice

A Gating Hinge in Na+ Channels A Molecular Switch for Electrical Signaling

AbstractVoltage-gated sodium channels are members of a large family with similar pore structures. The mechanism of opening and closing is unknown, but structural studies suggest gating via bending of the inner pore helix at a glycine hinge. Here we provide functional evidence for this gating model for the bacterial sodium channel NaChBac. Mutation of glycine 219 to proline, which would strongly favor bending of the α helix, greatly enhances activation by shifting its voltage dependence −51 mV and slowing deactivation by 2000-fold. The mutation also slows voltage-dependent inactivation by 1200-fold. The effects are specific because substitutions of proline at neighboring positions and substitutions of other amino acids at position 219 have much smaller functional effects. Our results fit a model in which concerted bending at glycine 219 in the S6 segments of NaChBac serves as a gating hinge. This gating motion may be conserved in most members of this large ion channel protein family