One-Step Synthesis of Functionalized Pyrazolo[3,4-b]pyridines via Ring Opening of the Pyrrolinium Ion.

Herein, we report a highly regioselective one-pot synthesis of pyrazolo[3,4-b]pyridines via the reaction of 3-arylidene-1-pyrrolines with aminopyrazoles. The reaction proceeds through the sequential nucleophilic addition/electrophilic substitution/C-N bond cleavage and provides easy access to pyrazolo[3,4-b]pyridine derivatives featuring a primary amino group. Moreover, the reaction can be terminated at the electrophilic substitution stage, thus providing convenient entry to the hardly accessible pyrazolopyrrolopyridine scaffold.

Silicas with Polyoxyethylene Branches for Modification of Membranes Based on Microporous Block Copolymers.

We have synthesized cubic and linear polysiloxanes containing polyoxyethylene branches (ASiP-Cu) using tetraethoxysilane, polyoxyethylene glycol, and copper chloride as precursors; the products are stable to self-condensation. The effect of copper chloride content on the chemical structure of ASiP-Cu has been established. A special study was aimed at defining the modifying effect of ASiP-Cu on the sorption characteristics of membranes based on microporous, optically transparent block copolymers (OBCs). These OBCs were produced using 2,4-toluene diisocyanate and block copolymers of ethylene and propylene oxides. The study demonstrated significantly increased sorption capacity of the modified polymers. On the basis of the modified microporous block copolymers and 1-(2-pyridylazo)-2-naphthol (PAN) analytical reagent, an analytical test system has been developed. Additionally, the modified OBCs have the benefit of high diffusion permeability for molecules of organic dyes and metal ions. It has been shown that the volume of voids and structural features of their internal cavities contribute to the complex formation reaction involving PAN and copper chloride.

Binding of Different Cyclosporin Variants to Micelles Evidenced by NMR and MD Simulations.

Peptides play a critical role in the life of organisms, performing completely different functions. The biological activity of some peptides, such as cyclosporins, can be determined by the degree of membrane permeability. Thus, it becomes important to study how the molecule interacts with lipid bilayers. Cyclosporins C, E, H and L were characterised molecular dynamics simulation; NMR spectroscopy studies were also carried out for cyclosporins C and E. The comparison of one- and two-dimensional spectra revealed certain similarities between spatial structures of the studied cyclosporin variants. Upon dissolving in water containing DPC micelles, which serve as model membranes, subtle changes in the NMR spectra appear, but in a different way for different cyclosporins. In order to understand whether observed changes are related to any structural modifications, simulation of the interaction of the peptide with the phospholipid micelle was performed. The onset of the interaction was observed, when the peptide is trapped to the surface of the micelle. Simulations of this kind are also of interest in the light of the well-known membrane permeability of cyclosporin, which is important for its biological action.

Conformational Screening of Arbidol Solvates: Investigation via 2D NOESY.

Understanding of the nucleation process's fundamental principles in saturated solutions is an urgent task. To do this task, it is necessary to control the formation of polymorphic forms of biologically active compounds. In certain cases, a compound can exist in a single polymorphic form, but have several solvates which can appear in different crystal forms, depending on the medium and conditions of formation, and show different pharmaceutical activity. In the present paper, we report on the analysis of Arbidol conformational preferences in two solvents of different polarities-deuterated chloroform and dimethyl sulfoxide-at 25 °C, using the 2D NOESY method. The Arbidol molecule has various solvate forms depending on the molecular conformation. The method based on the nuclear Overhauser effect spectroscopy was shown to be efficient in the analysis of complex heterocyclic compounds possessing conformation-dependent pseudo-polymorphism. It is one of the types of polymorphism observed in compounds forming crystal solvates. Combined use of NMR methods and X-ray data allowed determining of conformer populations of Arbidol in CDCl3 and DMSO-d6 which were found to be 8/92% and 37/63%, respectively. The preferred conformation in solution is the same that appears in stable crystal solvates of Arbidol.

Pervaporation Polyurethane Membranes Based on Hyperbranched Organoboron Polyols.

On the basis of aminoethers of boric acid (AEBA), polyurethane vapor-permeable and pervaporative membranes were obtained. AEBAs, the structure of which is modified by bulk adducts (EM) of diphenylol propane diglycidyl ether and ethanolamine, were studied. It turned out that AEBA exists in the form of clusters, and the use of EM as a result of partial destruction of associative interactions leads to a significant decrease in the size of AEBA-EM particles and their viscosity compared to unmodified AEBA. The introduction of EM into the composition of AEBA leads to a threefold increase in the vapor permeability of polyurethanes obtained on their basis. The observed effect is explained by the fact that a decrease in the size of clusters leads to loosening of their dense packing. Areas of clustering due to associative interactions of hydroxyl groups, together with the hydrophilic nature of polyoxyethylene glycol, create channels through which water molecules can penetrate. The increase in vapor permeability is accompanied by a multiple increase in the permeability coefficients in the pervaporative dehydration of isopropanol.

The Role of Hidden Conformers in Determination of Conformational Preferences of Mefenamic Acid by NOESY Spectroscopy.

Mefenamic acid has been used as a non-steroidal anti-inflammatory drug for a long time. However, its practical use is quite limited due to a number of side effects on the intestinal organs. Conformational polymorphism provides mefenamic acid with unique properties regarding possible modifications obtained during the micronization process, which can improve pharmacokinetics and minimize side effects. Micronization can be performed by decompression of supercritical fluids; methods such as rapid expansion of the supercritical solution have proven their efficiency. However, this group of methods is poorly applicable for compounds with low solubility, and the modification of the method using a pharmaceutically suitable co-solvent may be useful. In our case, addition of only 2 mol% dimethyl sulfoxide increased the solubility remarkably. Information on the conformational state may be critically important for carrying out micronization. In this work, structural analysis and estimate of conformational preferences of mefenamic acid in dimethyl sulfoxide-d6 (at 25 °C and 0.1 MPa) and in a mixed solvent supercritical carbon dioxide + dimethyl sulfoxide-d6 (45 °C, 9 MPa) were performed based on nuclear Overhauser effect spectroscopy. Results show changes in the conformation fractions depending on the medium used. The importance of allowing for hidden conformers in estimating the conformational state was demonstrated in the analysis. Obtained results may be useful for improving micronization parameters.

Catalytic Etherification of ortho-Phosphoric Acid for the Synthesis of Polyurethane Ionomer Films.

The etherification reaction of ortho-phosphoric acid (OPA) with polyoxypropylene glycol in the presence of tertiary amines was studied. The reaction conditions promoting the catalytic activity of triethanolamine (TEOA) and triethylamine (TEA) in the low-temperature etherification of OPA were established. The catalytic activity of TEOA and TEA in the etherification reaction of phosphoric acid is explained by the hydrophobic-hydrophilic interactions of TEA with PPG, leading, as a result of collective interactions, to a specific orientation of polyoxypropylene chains around the tertiary amine. When using triethylamine, complete etherification of OPA occurs, accompanied by the formation of branched OPA ethers terminated by hydroxyl groups and even the formation of polyphosphate structures. When triethanolamine is used as a catalyst, incomplete etherification of OPA with polyoxypropylene glycol occurs and as a result, part of the phosphate anions remain unreacted in the composition of the resulting aminoethers of ortho-phosphoric acid (AEPA). In this case, the hydroxyl groups of triethanolamine are completely involved in the OPA etherification reaction, but the catalytic activity of the tertiary amine weakens due to a decrease in its availability in the branched structure of AEPA. The kinetics of the etherification reaction of OPA by polyoxypropylene glycol catalyzed by TEOA and TEA were studied. It was shown that triethanolamine occupies a central position in the AEPA structure. The physico-mechanical and thermomechanical properties of polyurethane ionomer films obtained on the basis of AEPA synthesized in a wide temperature range were studied.

Williams-Beuren Syndrome Related Methyltransferase WBSCR27: From Structure to Possible Function.

Williams-Beuren syndrome (WBS) is a genetic disorder associated with the hemizygous deletion of several genes in chromosome 7, encoding 26 proteins. Malfunction of these proteins induce multisystemic failure in an organism. While biological functions of most proteins are more or less established, the one of methyltransferase WBSCR27 remains elusive. To find the substrate of methylation catalyzed by WBSCR27 we constructed mouse cell lines with a Wbscr27 gene knockout and studied the obtained cells using several molecular biology and mass spectrometry techniques. We attempted to pinpoint the methylation target among the RNAs and proteins, but in all cases neither a direct substrate has been identified nor the protein partners have been detected. To reveal the nature of the putative methylation substrate we determined the solution structure and studied the conformational dynamic properties of WBSCR27 in apo state and in complex with S-adenosyl-L-homocysteine (SAH). The protein core was found to form a canonical Rossman fold common for Class I methyltransferases. N-terminus of the protein and the ß6-ß7 loop were disordered in apo-form, but binding of SAH induced the transition of these fragments to a well-formed substrate binding site. Analyzing the structure of this binding site allows us to suggest potential substrates of WBSCR27 methylation to be probed in further research.

Comparison of structural properties of cyclosporin A and its analogue alisporivir and their effects on mitochondrial bioenergetics and membrane behavior.

The paper considers the effect of the MPT pore inhibitor cyclosporin A (CsA) and its non-immunosuppressive analogue alisporivir (Ali) on the functioning of rat skeletal muscle mitochondria. We have shown that both agents at a standard in vitro concentration of 1 µM increase the calcium capacity of organelles and have no effect on the parameters of oxidative phosphorylation. However, an increase in their concentration to 5 µM leads to the suppression of oxygen consumption by mitochondria, which is more pronounced in the case of Ali. This effect is accompanied by a decrease in the membrane potential of organelles and, apparently, is based on the inhibition of electron transport along the mitochondrial respiratory chain due to limited mobility of coenzyme Q. We have noted that both agents do not affect the production of hydrogen peroxide by isolated mitochondria. NMR spectroscopy and molecular dynamics simulation did not reveal significant differences in the structure and backbone flexibility of CsA and Ali. Both agents decrease the overall fluidity of the membrane of DPPC liposomes, inducing an increase in laurdan generalized polarization parameter. A similar effect was also found in the case of mitochondrial membranes. We suggested that these effects of CsA and Ali, associated with their lipophilic nature and the ability to accumulate in the lipid phase of membranes, may cause a decrease in the efficiency of electron transport in the respiratory chain of mitochondria and suppression of the bioenergetics of these organelles.

Design, Spectral Characteristics, Photostability, and Possibilities for Practical Application of BODIPY FL-Labeled Thioterpenoid.

This paper presents the design and a comparative analysis of the structural and solvation factors on the spectral and biological properties of the BODIPY biomarker with a thioterpene fragment. Covalent binding of the thioterpene moiety to the butanoic acid residue of meso-substituted BODIPY was carried out to find out the membranotropic effect of conjugate to erythrocytes, and to assess the possibilities of its practical application in bioimaging. The molecular structure of the conjugate was confirmed via X-ray, UV/vis-, NMR-, and MS-spectra. It was found that dye demonstrates high photostability and high fluorescence quantum yield (to ~100%) at 514-519 nm. In addition, the marker was shown to effectively penetrate the erythrocytes membrane in the absence of erythrotoxicity. The conjugation of BODIPY with thioterpenoid is an excellent way to increase affinity dyes to biostructures, including blood components.

Open-label use of an aliphatic polyamine immunomodulator in patients hospitalized with COVID-19.

Background: Evidence-based therapies used to treat coronavirus disease (COVID-19) remain limited. Azoximer bromide (AZB; Polyoxidonium®) is an immunomodulating molecule frequently used in the Russian Federation. It offers demonstrable therapeutic benefit in upper respiratory tract infections. This study evaluated the safety and efficacy of AZB when used in combination with standard of care treatment in patients hospitalized with COVID-19. Methods: Hospitalized patients with COVID-19 (n=81; nine sites) received AZB 12 mg intravenously once daily for 3 days then intramuscularly every other day until day 17. The primary endpoint included clinical status at day 15 versus baseline. Historical control data of 100 patients from a randomized, controlled, open-label trial conducted in China were included to serve as a direct control group. Results: Notable clinical improvement, assessed by seven-point ordinal scale (OS) score and National Early Warning Score, was observed. Mean duration of hospitalization was 19.3 days. Indicators of pneumonia and lung function showed gradual recovery to normalization. No patients died but, by day 28, one patient still required respiratory support; this patient died on day 34. A higher proportion of patients receiving AZB required invasive or non-invasive ventilation (OS 5 or 6) at baseline compared with the historical control group. Improvement in mean OS score by day 14/15 was not notable in the control group (OS 3.99-3.87) but was clear in the AZB group (OS 4.36-2.90). Mean duration of hospitalization was similar in the control group (16.0 days); however, day 28 mortality was higher, at 25.0% (n=25). Conclusion: AZB combined with standard of care was safe and well tolerated. An apparent clinical improvement could not be fully evaluated due to the lack of a direct control group; further assessment of AZB for the treatment of COVID-19 in a randomized, placebo-controlled study is warranted.

Conjugate of meso-carboxysubstituted-BODIPY with thioterpenoid as an effective fluorescent probe: Synthesis, structure, spectral characteristics, and molecular docking.

This paper is devoted to the design of a fluorescent probe based on meso-carboxysubstituted-BODIPY with a thioterpene fragment. The functional replacement of the methoxy group in the BODIPY molecule on a thioterpene fragment was carried out in order to find out the antiplatelet and anticoagulant action mechanisms of thioterpenoids and to assess the membrane and receptor factors contributions. The molecular structure of the conjugate was confirmed via UV/vis-, NMR- and MS-spectra. It is found that the probe is a high fluorescence quantum yield (to âˆ¼ 100%) in the blue-green region at 509-516 nm. Molecular docking of all studied molecules showed that the BODIPY with terpenoid conjugation is an excellent way to increase their affinity to platelet receptor P2Y12.

The effect of azoximer bromide (Polyoxidonium®) in patients hospitalized with coronavirus disease (COVID-19): an open-label, multicentre, interventional clinical study.

A clinical need for aetiotropic coronavirus disease (COVID-19) treatments is required. The immune modulator azoximer bromide (AZB; Polyoxidonium®) is indicated in Russia for use against acute viral infections and during remission. In this study, adults hospitalized with COVID-19 (n=32) received AZB and standard of care in an open-label, multicentre, interventional study. All patients were symptomatic; 22 had severe disease (National Early Warning Score ≥5) and required mechanical ventilation or oxygen saturation (SpO2) and 19 patients had co-morbidities. Patients received AZB 12 mg intravenously once daily for 3 days, then intramuscularly every other day (approximately ten injections) until discharge. The primary endpoint was the patient's clinical status (7-point Ordinal Scale; OS) on day 15 versus that at baseline. The mean duration of hospitalization was 20 days. All patients were alive and discharged with normal SpO2 with no secondary infections or delayed mortality reported by the end-of-study visit (on day 28-72). A decrease in the mean OS and National Early Warning Score values was observed following treatment with AZB. A decrease in OS score was marked in patients identified as severe. Both sets of patients achieved similar scores, which can be classified as an improvement by day 9-10; SpO2 levels trended to normalization over time. By day 11-12, all patients had a normal body temperature. Serum C-reactive protein levels decreased in patients with severe and mild disease. Most patients had signs of pneumonia at baseline (n=27), with the majority recovering by days 10-12. No major toxicities were observed. AZB was safe and well tolerated when administered in addition to standard of care treatment for COVID-19. Further randomized, placebo-controlled studies are needed to elucidate any potential therapeutic effect in COVID-19.

Design, Spectral Characteristics, and Possibilities for Practical Application of BODIPY FL-Labeled Monoterpenoid.

This article describes the design and biological properties of a BODIPY FL-labeled monoterpenoid BF2-meso-(4-((1″R)-6″,6″-dimethylbicyclo[3.1.1]hept-2″-ene-2″)yl-methoxycarbonylpropyl)-3,3',5,5'-tetramethyl-2,2'-dipyrromethene conjugate (BODIPYmyrt). The fluorophore was characterized using X-ray, NMR, MS, and UV/vis spectroscopy. The conjugate exhibits a high quantum yield (to ∼100%) in the region 515-518 nm. BODIPYmyrt effectively penetrates the membranes of the bacterial and fungal cells and therefore can be used to examine the features of a broad spectrum of Gram-positive and Gram-negative bacteria and pathogenic fungi as well. Moreover, BODIPYmyrt exhibits a moderate tropism to the subcellular structures in mammalian cells (e.g., mitochondria), thereby providing an attractive scaffold for fluorophores to examine these particular organelles.

Insights into the structure and function of Est3 from the Hansenula polymorpha telomerase.

Telomerase is a ribonucleoprotein enzyme, which maintains genome integrity in eukaryotes and ensures continuous cellular proliferation. Telomerase holoenzyme from the thermotolerant yeast Hansenula polymorpha, in addition to the catalytic subunit (TERT) and telomerase RNA (TER), contains accessory proteins Est1 and Est3, which are essential for in vivo telomerase function. Here we report the high-resolution structure of Est3 from Hansenula polymorpha (HpEst3) in solution, as well as the characterization of its functional relationships with other components of telomerase. The overall structure of HpEst3 is similar to that of Est3 from Saccharomyces cerevisiae and human TPP1. We have shown that telomerase activity in H. polymorpha relies on both Est3 and Est1 proteins in a functionally symmetrical manner. The absence of either Est3 or Est1 prevents formation of a stable ribonucleoprotein complex, weakens binding of a second protein to TER, and decreases the amount of cellular TERT, presumably due to the destabilization of telomerase RNP. NMR probing has shown no direct in vitro interactions of free Est3 either with the N-terminal domain of TERT or with DNA or RNA fragments mimicking the probable telomerase environment. Our findings corroborate the idea that telomerase possesses the evolutionarily variable functionality within the conservative structural context.

Backbone resonance assignment and dynamics of 110 kDa hexameric inorganic pyrophosphatase from Mycobacterium tuberculosis.

Family I soluble inorganic pyrophosphatases (PPases; EC 3.6.1.1) are enzymes essential for all organisms. They hydrolyze inorganic pyrophosphate, thus providing the driving force for numerous biosynthetic reactions. Soluble PPases retain enzymatic activity only in multimeric forms. PPases from various organisms are extensively studied by X-ray crystallography but until now there was no information on their structure and dynamics in solution. Hexameric 110 kDa (6 × 18.3 kDa) PPase from Mycobacterium tuberculosis (Mt-PPase) is a promising target for the rational design of potential anti-tuberculosis agents. In order to use NMR techniques in functional studies of Mt-PPase and rational design of the inhibitors for this enzyme, it is necessary to have information on the backbone 1H, 13C and 15N resonance assignments. Samples of Mt-PPase enriched with 99% of 13C and 15N isotopes, and 95% of 2H were obtained using recombinant protein expression in an isotopically-labeled medium and effective heat-shock protocol for the deuterium-to-hydrogen exchange of the amide groups. Backbone resonance assignment was achieved for more than 95% of the residues. It was found that the secondary structure of Mt-PPase in solution corresponds well to the crystal structure of this protein. Protein backbone dynamics were studied using 15N NMR relaxation experiments. Determined resonance assignments and dynamic properties provide the basis for the subsequent structure-based design of novel inhibitors of Mt-PPase-potential anti-tuberculosis drugs.

Williams-Beuren syndrome-related methyltransferase WBSCR27: cofactor binding and cleavage.

Williams-Beuren syndrome, characterized by numerous physiological and mental problems, is caused by the heterozygous deletion of chromosome region 7q11.23, which results in the disappearance of 26 protein-coding genes. Protein WBSCR27 is a product of one of these genes whose biological function has not yet been established and for which structural information has been absent until now. Using NMR, we investigated the structural and functional properties of murine WBSCR27. For protein in the apo form and in a complex with S-(5'-adenosyl)-l-homocysteine (SAH), a complete NMR resonance assignment has been obtained and the secondary structure has been determined. This information allows us to attribute WBSCR27 to Class I methyltransferases. The interaction of WBSCR27 with the cofactor S-(5'-adenosyl)-l-methionine (SAM) and its metabolic products - SAH, 5'-deoxy-5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'dAdo) - was studied by NMR and isothermal titration calorimetry. SAH binds WBSCR27 much tighter than SAM, leaving open the question of cofactor turnover in the methylation reaction. One possible answer to this question is the presence of weak but detectable nucleosidase activity for WBSCR27. We found that the enzyme catalyses the cleavage of the adenine moiety from SAH, MTA and 5'dAdo, similar to the action of bacterial SAH/MTA nucleosidases. We also found that the binding of SAM or SAH causes a significant change in the structure of WBSCR27 and in the conformational mobility of the protein fragments, which can be attributed to the substrate recognition site. This indicates that the binding of the cofactor modulates the folding of the substrate-recognizing region of the enzyme.

NMR assignments of the WBSCR27 protein related to Williams-Beuren syndrome.

Williams-Beuren syndrome is a genetic disorder characterized by physiological and mental abnormalities, and is caused by hemizygous deletion of several genes in chromosome 7. One of the removed genes encodes the WBSCR27 protein. Bioinformatic analysis of the sequence of WBSCR27 indicates that it belongs to the family of SAM-dependent methyltransferases. However, exact cellular functions of this protein or phenotypic consequences of its deficiency are still unknown. Here we report nearly complete 1H, 15N, and 13C chemical shifts assignments of the 26 kDa WBSCR27 protein from Mus musculus in complex with the cofactor S-adenosyl-L-methionine (SAM). Analysis of the assigned chemical shifts allowed us to characterize the protein's secondary structure and backbone dynamics. The topology of the protein's fold confirms the assumption that the WBSCR27 protein belongs to the family of class I methyltransferases.

Structure and function of the N-terminal domain of the yeast telomerase reverse transcriptase.

The elongation of single-stranded DNA repeats at the 3'-ends of chromosomes by telomerase is a key process in maintaining genome integrity in eukaryotes. Abnormal activation of telomerase leads to uncontrolled cell division, whereas its down-regulation is attributed to ageing and several pathologies related to early cell death. Telomerase function is based on the dynamic interactions of its catalytic subunit (TERT) with nucleic acids-telomerase RNA, telomeric DNA and the DNA/RNA heteroduplex. Here, we present the crystallographic and NMR structures of the N-terminal (TEN) domain of TERT from the thermotolerant yeast Hansenula polymorpha and demonstrate the structural conservation of the core motif in evolutionarily divergent organisms. We identify the TEN residues that are involved in interactions with the telomerase RNA and in the recognition of the 'fork' at the distal end of the DNA product/RNA template heteroduplex. We propose that the TEN domain assists telomerase biological function and is involved in restricting the size of the heteroduplex during telomere repeat synthesis.

Chemical shift assignments and the secondary structure of the Est3 telomerase subunit in the yeast Hansenula polymorpha.

Telomerase is a multisubunit ribonucleoprotein enzyme that is essential for continuous cellular proliferation. A key role of telomerase in cancer and ageing makes it a promising target for the development of cancer therapies and treatments of other age-associated diseases, since telomerase allows unlimited proliferation potential of cells in the majority of cancer types. However, the structure and molecular mechanism of telomerase action are still poorly understood. In budding yeast, telomerase consists of the catalytic subunit, the telomerase reverse transcriptase or Est2 protein, telomerase RNA (TLC1) and two regulatory subunits, Est1 and Est3. Each of the four subunits is essential for in vivo telomerase function. Est3 interacts directly with Est1 and Est2, and stimulates Est2 catalytic activity. However, the exact role of the Est3 protein in telomerase function is still unknown. Determination of the structure, dynamic and functional properties of Est3 can bring new insights into the molecular mechanism of telomerase activity. Here we report nearly complete 1H, 13C and 15N resonance assignments of Est3 from the yeast Hansenula polymorpha. Analysis of the assigned chemical shifts allowed us to identify the protein's secondary structure and backbone dynamic properties. Structure-based sequence alignment revealed similarities in the structural organization of yeast Est3 and mammalian TPP1 proteins.