Molecular determinants of the binding of various endogenous modulators to transient receptor potential (TRP) channels are crucial for the understanding of necessary cellular pathways, as well as new paths for rational drug designs. The aim of this study was to characterise interactions between the TRP cation channel subfamily melastatin member 4 (TRPM4) and endogenous intracellular modulators-calcium-binding proteins (calmodulin (CaM) and S100A1) and phosphatidylinositol 4, 5-bisphosphate (PIP2). We have found binding epitopes at the N- and C-termini of TRPM4 shared by CaM, S100A1 and PIP2. The binding affinities of short peptides representing the binding epitopes of N- and C-termini were measured by means of fluorescence anisotropy (FA). The importance of representative basic amino acids and their combinations from both peptides for the binding of endogenous TRPM4 modulators was proved using point alanine-scanning mutagenesis. In silico protein-protein docking of both peptides to CaM and S100A1 and extensive molecular dynamics (MD) simulations enabled the description of key stabilising interactions at the atomic level. Recently solved cryo-Electron Microscopy (EM) structures made it possible to put our findings into the context of the entire TRPM4 channel and to deduce how the binding of these endogenous modulators could allosterically affect the gating of TRPM4. Moreover, both identified binding epitopes seem to be ideally positioned to mediate the involvement of TRPM4 in higher-order hetero-multimeric complexes with important physiological functions.

• MeSH
• akvaporiny chemie   metabolismus   MeSH
• interakční proteinové domény a motivy * MeSH
• kalmodulin chemie   metabolismus   MeSH
• kationtové kanály TRPM chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární modely MeSH
• multiproteinové komplexy chemie   metabolismus   MeSH
• peptidové fragmenty MeSH
• proteiny S100 chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The transient receptor potential channel of melastatin 4 (TRPM4) belongs to a group of large ion receptors that are involved in countless cell signalling cascades. This unique member is ubiquitously expressed in many human tissues, especially in cardiomyocytes, where it plays an important role in cardiovascular processes. Transient receptor potential channels (TRPs) are usually constituted by intracellular N- and C- termini, which serve as mediators affecting allosteric modulation of channels, resulting in the regulation of the channel function. The TRPs tails contain a number of conserved epitopes that specifically bind the intracellular modulators. Here, we identify new binding sites for the calmodulin (CaM) and S100 calcium-binding protein A1 (S100A1), located in the very distal part of the TRPM4 N terminus. We have used chemically synthesized peptides of the TRPM4, mimicking the binding epitopes, along with fluorescence methods to determine and specify CaM- and S100A1-binding sites. We have found that the ligands binding epitopes at the TRPM4 N terminus overlap, but the interacting mechanism of both complexes is probably different. The molecular models supported by data from the fluorescence method confirmed that the complexes formations are mediated by the positively charged (R139, R140, R144) and hydrophobic (L134, L138, V143) residues present at the TRPM4 N terminus-binding epitopes. The data suggest that the molecular complexes of TRPM4/CaM and TRPM4/S100A1 would lead to the modulation of the channel functions.

• MeSH
• databáze proteinů MeSH
• epitopy MeSH
• expertní systémy MeSH
• fluorescenční polarizace MeSH
• interakční proteinové domény a motivy MeSH
• kalmodulin chemie   genetika   metabolismus   MeSH
• kationtové kanály TRPM chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• ligandy MeSH
• molekulární modely * MeSH
• mutace MeSH
• peptidové fragmenty chemická syntéza   chemie   genetika   metabolismus   MeSH
• proteiny S100 chemie   genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• simulace molekulového dockingu MeSH
• substituce aminokyselin MeSH
• vazebná místa MeSH
• výpočetní biologie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Herein, we report the synthesis, structure-activity relationship study, and biological evaluation of neurosteroid inhibitors of N-methyl-D-aspartate receptors (NMDARs) receptors that employ an amide structural motif, relative to pregnanolone glutamate (PAG) - a compound with neuroprotective properties. All compounds were found to be more potent NMDAR inhibitors (IC50 values varying from 1.4 to 21.7 μM) than PAG (IC50 = 51.7 μM). Selected compound 6 was evaluated for its NMDAR subtype selectivity and its ability to inhibit AMPAR/GABAR responses. Compound 6 inhibits the NMDARs (8.3 receptors (8.3 ± 2.1 μM) more strongly than it does at the GABAR and AMPARs (17.0 receptors (17.0 ± 0.2 μM and 276.4 ± 178.7 μM, respectively). In addition, compound 6 (10 μM) decreases the frequency of action potentials recorded in cultured hippocampal neurons. Next, compounds 3, 5-7, 9, and 10 were not associated with mitotoxicity, hepatotoxicity nor ROS induction. Lastly, we were able to show that all compounds have improved rat and human plasma stability over PAG.

• Publikační typ
• časopisecké články MeSH

Mebendazole (MBZ) was developed as a broad-spectrum anthelmintic but has recently shown efficacy as an anticancer agent. The use of MBZ for cancer, however, is challenging due to its poor solubility leading to poor bioavailability. Herein, we developed a prodrug approach with various N-linked promoieties including acyloxymethyl, aminoacyloxymethyl, and substituted phosphonooxymethyl in attempt to improve these characteristics. Compound 12, containing an (((((isopropoxycarbonyl)oxy)methoxy)phosphoryl)oxy)methyl promoiety, showed a >10 000-fold improvement in aqueous solubility. When evaluated in mice, 12 displayed a 2.2-fold higher plasma AUC0- t and a 1.7-fold improvement in brain AUC0- t with a calculated oral bioavailability of 52%, as compared to 24% for MBZ-polymorph C (MBZ-C), the most bioavailable polymorph. In dogs, 12 showed a 3.8-fold higher plasma AUC0- t with oral bioavailability of 41% compared to 11% for MBZ-C. In summary, we have identified a prodrug of MBZ with better physicochemical properties and enhanced bioavailability in both mice and dog.

• MeSH
• anthelmintika metabolismus   MeSH
• aplikace orální MeSH
• biologická dostupnost MeSH
• dusík chemie   MeSH
• mebendazol metabolismus   MeSH
• myši MeSH
• prekurzory léčiv aplikace a dávkování   chemie   metabolismus   farmakokinetika   MeSH
• psi MeSH
• rozpustnost MeSH
• stabilita léku MeSH
• tkáňová distribuce MeSH
• voda chemie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

4-Carboxy-α-[3-(hydroxyamino)-3-oxopropyl]-benzenepropanoic acid 1 is a potent hydroxamate-based inhibitor of glutamate carboxypeptidase II. In an attempt to improve its poor oral pharmacokinetics, we synthesized a series of prodrugs by masking its hydrophilic hydroxamate group. Prodrugs were evaluated for oral availability in mice and showed varying degree of plasma exposure to 1. Of these, para-acetoxybenzyl-based, 4-(5-(((4-acetoxybenzyl)oxy)amino)-2-carboxy-5-oxopentyl)benzoic acid, 12, provided 5-fold higher plasma levels of 1 compared to oral administration of 1 itself. Subsequently, para-acetoxybenzyl-based prodrugs with additional ester promoiety(ies) on carboxylate(s) were examined for their ability to deliver 1 to plasma. Isopropyloxycarbonyloxymethyl (POC) ester 30 was the only prodrug that achieved substantial plasma levels of 1. In vitro metabolite identification studies confirmed stability of the ethyl ester of benzoate while the POC group was rapidly hydrolyzed. At oral daily dose-equivalent of 3 mg/kg, 12 exhibited analgesic efficacy comparable to dose of 10 mg/kg of 1 in the rat chronic constrictive injury model of neuropathic pain.

• MeSH
• analgetika chemie   farmakokinetika   farmakologie   terapeutické užití   MeSH
• aplikace orální MeSH
• esterifikace MeSH
• glutamátkarboxypeptidasa II antagonisté a inhibitory   metabolismus   MeSH
• inhibitory enzymů chemie   farmakokinetika   farmakologie   terapeutické užití   MeSH
• krysa rodu rattus MeSH
• kyseliny hydroxamové chemie   farmakokinetika   farmakologie   terapeutické užití   MeSH
• lidé MeSH
• myši MeSH
• neuralgie farmakoterapie   enzymologie   MeSH
• objevování léků MeSH
• potkani Sprague-Dawley MeSH
• prekurzory léčiv chemie   farmakokinetika   farmakologie   terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

HYL-20 (GILSSLWKKLKKIIAK-NH2) is an analogue of a natural antimicrobial peptide (AMP) previously isolated from the venom of wild bee. We examined its antimicrobial activity against three strains of Enterococcus faecalis while focusing on its susceptibility to proteolytic degradation by two known proteases-gelatinase (GelE) and serine protease (SprE)-which are secreted by these bacterial strains. We found that HYL-20 was primarily deamidated at its C-terminal which made the peptide susceptible to consecutive intramolecular cleavage by GelE. Further study utilising 1,10-phenanthroline, a specific GelE inhibitor and analogous peptide with D-Lys at its C-terminus (HYL-20k) revealed that the C-terminal deamidation of HYL-20 is attributed to not yet unidentified protease which also cleaves internal peptide bonds of AMPs. In contrast to published data, participation of SprE in the protective mechanism of E. faecalis against AMPs was not proved. The resistance of HYL-20k to C-terminal deamidation and subsequent intramolecular cleavage has resulted in increased antimicrobial activity against E. faecalis grown in planktonic and biofilm form when compared to HYL-20.

• MeSH
• antibakteriální látky chemická syntéza   metabolismus   farmakologie   MeSH
• bakteriální proteiny antagonisté a inhibitory   chemie   metabolismus   MeSH
• biofilmy účinky léků   růst a vývoj   MeSH
• Enterococcus faecalis účinky léků   enzymologie   růst a vývoj   ultrastruktura   MeSH
• fenantroliny farmakologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• kationické antimikrobiální peptidy chemická syntéza   metabolismus   farmakologie   MeSH
• mikrobiální testy citlivosti MeSH
• plankton účinky léků   enzymologie   růst a vývoj   ultrastruktura   MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• serinové endopeptidasy chemie   metabolismus   MeSH
• substituce aminokyselin MeSH
• včely chemie   fyziologie   MeSH
• vztahy mezi strukturou a aktivitou MeSH
• želatinasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Capillary ITP (CITP) and CZE were applied to the determination of effective charges and ionic mobilities of polycationic antimicrobial peptides (AMPs). Twelve AMPs (deca- to hexadecapeptides) containing three to seven basic amino acid residues (His, Lys, Arg) at variable positions of peptide chain were investigated. Effective charges of the AMPs were determined from the lengths of their ITP zones, ionic mobilities, and molar concentrations, and from the same parameters of the reference compounds. Lengths of the ITP zones of AMPs and reference compounds were obtained from their CITP analyses in cationic mode using leading electrolyte (LE) composed of 10 mM NH4OH, 40 mM AcOH (acetic acid), pH 4.1, and terminating electrolyte (TE) containing 40 mM AcOH, pH 3.2. Ionic mobilities of AMPs and singly charged reference compounds (ammediol or arginine) were determined by their CZE analyses in the BGE of the same composition as the LE. The effective charges numbers of AMPs were found to be in the range 1.65-5.04, i.e. significantly reduced as compared to the theoretical charge numbers (2.86-6.99) calculated from the acidity constants of the analyzed AMPs. This reduction of effective charge due to tightly bound acetate counterions (counterion condensation) was in the range 17-47% depending on the number and type of the basic amino acid residues in the AMPs molecules. Ionic mobilities of AMPs achieved values (26.5-38.6) × 10-9 m2V-1s-1and in most cases were in a good agreement with the ratio of their effective charges and relative molecular masses.

• MeSH
• elektroforéza kapilární MeSH
• elektrolyty chemie   MeSH
• fyzikální chemie MeSH
• izotachoforéza MeSH
• kationické antimikrobiální peptidy analýza   MeSH
• koncentrace vodíkových iontů MeSH
• osmolární koncentrace MeSH
• Publikační typ
• časopisecké články MeSH

Capillary electrophoresis (CE) was employed for the determination of thermodynamic acidity constants (pKa ) and actual ionic mobilities of polycationic antimicrobial peptides (AMPs). The effective electrophoretic mobilities of AMPs were measured by CE in a series of the background electrolytes within a wide pH range (2.00-12.25), at constant ionic strength (25 mM) and ambient temperature, using polybrene coated fused silica capillaries to suppress sorption of cationic AMPs to the capillary wall. Eventually, Haarhoff-Van der Linde peak fitting function was used for the determination of correct migration times of some AMPs peaks that were distorted by electromigration dispersion. The measured effective mobilities were corrected to 25°C. Mixed acidity constants, pKa,i mix , and actual ionic mobilities, mi , of AMPs were determined by the nonlinear regression analysis of pH dependence of their effective mobilities. The pKa,i mix values were recalculated to thermodynamic pKa s using the Debye-Hückel theory. Thermodynamic pKa of imidazolium group of histidine residues was found to be in the range 3.72-4.98, pKa of α-NH3(+) group was in the range 6.14-6.93, and pKa of ε-NH3(+) group of lysine spanned the interval 7.26-9.84, depending on the particular amino acid sequence of the AMPs. Actual ionic mobilities of AMPs with positive charges from one to six elementary units achieved values (9.8 - 36.5) × 10(-9) m(2) V(-1) s(-1) .

• MeSH
• aminokyseliny chemie   MeSH
• elektroforéza kapilární metody   MeSH
• kationické antimikrobiální peptidy analýza   chemie   MeSH
• koncentrace vodíkových iontů MeSH
• nelineární dynamika MeSH
• osmolární koncentrace MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH

The glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON, 1) has shown robust anticancer efficacy in preclinical and clinical studies, but its development was halted due to marked systemic toxicities. Herein we demonstrate that DON inhibits glutamine metabolism and provides antitumor efficacy in a murine model of glioblastoma, although toxicity was observed. To enhance DON's therapeutic index, we utilized a prodrug strategy to increase its brain delivery and limit systemic exposure. Unexpectedly, simple alkyl ester-based prodrugs were ineffective due to chemical instability cyclizing to form a unique diazo-imine. However, masking both DON's amine and carboxylate functionalities imparted sufficient chemical stability for biological testing. While these dual moiety prodrugs exhibited rapid metabolism in mouse plasma, several provided excellent stability in monkey and human plasma. The most stable compound (5c, methyl-POM-DON-isopropyl-ester) was evaluated in monkeys, where it achieved 10-fold enhanced cerebrospinal fluid to plasma ratio versus DON. This strategy may provide a path to DON utilization in glioblastoma multiforme patients.

• MeSH
• diazooxonorleucin terapeutické užití   MeSH
• glioblastom farmakoterapie   metabolismus   MeSH
• glutamin metabolismus   MeSH
• Haplorrhini MeSH
• lidé MeSH
• myši nahé MeSH
• myši MeSH
• nádory mozku farmakoterapie   metabolismus   MeSH
• prekurzory léčiv farmakokinetika   terapeutické užití   MeSH
• protinádorové antimetabolity terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Transient receptor potential melastatin-1 channel (TRPM1) is an important mediator of calcium influx into the cell that is expressed in melanoma and ON-bipolar cells. Similar to other members of the TRP channel family, the intracellular N- and C- terminal domains of TRPM1 are expected to play important roles in the modulation of TRPM1 receptor function. Among the most commonly occurring modulators of TRP channels are the cytoplasmically expressed calcium binding proteins calmodulin and S100 calcium-binding protein A1 (S100A1), but the interaction of TRPM1 with S100A1 has not been described yet. Here, using a combination of biophysical and bioinformatics methods, we have determined that the N-terminal L242-E344 region of TRPM1 is a S100A1 binding domain. We show that formation of the TRPM1/S100A1 complex is calcium-dependent. Moreover, our structural model of the complex explained data obtained from fluorescence spectroscopy measurements revealing that the complex formation is facilitated through interactions of clusters positively charged (K271A, R273A, R274A) and hydrophobic (L263A, V270A, L276A) residues at the N-terminus of TRPM1. Taken together, our data suggest a molecular mechanism for the potential regulation of TRPM1 by S100A1.

• MeSH
• hydrofobní a hydrofilní interakce MeSH
• kationtové kanály TRPM chemie   metabolismus   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• proteinové domény MeSH
• proteiny S100 chemie   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• simulace molekulového dockingu MeSH
• vápník metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH