Formation Of Conjugates Research Articles

New Kids on the Block: Bile Salt Conjugates of Microbial Origin.

Biotransformation of host bile salts by gut microbes results in generation of secondary bile salt species that have biological and physicochemical properties that are distinct from the parent compounds. There is increased awareness that a bile salt–gut microbiome axis modulates various processes in the host, including innate and adaptive immunity, by interaction of microbial bile salt metabolites with host receptors. Omics and targeted approaches have vastly expanded the number and repertoire of secondary bile salt species. A new class of microbial bile salt metabolites was reported in 2020 and comprises bile salts that are conjugated by microbial enzymes. Amino acids other than those employed by host enzymes (glycine and taurine) are used as substrates in the formation of these microbial bile salt conjugates (MBSCs). Leucocholic acid, phenylalanocholic acid and tyrosocholic acid were the first MBSCs identified in mice and humans. The number of distinct MBSCs is now approaching 50, with variation both at the level of bile salt and amino acid employed for conjugation. Evidence is emerging that MBSC generation is a common feature of human gut bacteria, and initial links with disease states have been reported. In this review, we discuss this intriguing new class of secondary bile salts, with yet enigmatic function.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

In Vitro Metabolism of Helenalin Acetate and 11α,13-Dihydrohelenalin Acetate: Natural Sesquiterpene Lactones from Arnica.

Arnica tincture is a herbal medicinal preparation with anti-inflammatory activity which is used traditionally for the topical treatment of blunt injuries as well as rheumatic muscle and joint complaints. Its main bioactive constituents are sesquiterpene lactones (STLs) of the helenalin and 11α,13-dihydrohelenalin types. Besides the mentioned activity, the tincture and its isolated STLs have antileishmanial activity. In a recent in vivo study, a treatment with Arnica tincture cured cutaneous Leishmaniasis (CL) in a golden hamster model. CL is a neglected tropical disease affecting more than two million people every year, for which new treatments are urgently needed. In order to use Arnica tincture on open CL lesions of human patients, it is important to know how the constituents are metabolized. Therefore, in vitro metabolism experiments with liver microsomes of different species (rat, pig and human) were performed with the Arnica STLs helenalin acetate and 11α,13-dihydrohelenalin acetate. Phase I and phase II metabolism experiments were performed, as well as a combination of both. Glutathione conjugation plays a major role in the metabolism of these STLs, as could be expected based on previous reports on their reactivity. Besides glutathione conjugates, several other metabolites were formed, e.g., water conjugates and hydroxides. Our results show for the first time a detailed picture of the metabolism of Arnica STLs. The fast and extensive formation of glutathione conjugates makes it unlikely that low absorbed levels of these compounds, as expected after dermal absorption from Arnica tincture, could be of toxicological concern.

Enhanced Calcium Signal Induces NK Cell Degranulation but Inhibits Its Cytotoxic Activity.

Although the mechanism of NK cell activation is still unclear, the strict calcium dependence remains the hallmark for lytic granule secretion. A plethora of studies claiming that impaired Ca2+ signaling leads to severely defective cytotoxic granule exocytosis accompanied by weak target cell lysis has been published. However, there has been little discussion about the effect of induced calcium signal on NK cell cytotoxicity. In our study, we observed that small-molecule inhibitor UNC1999, which suppresses global H3K27 trimethylation (H3K27me3) of human NK cells, induced a PKD2-dependent calcium signal. Enhanced calcium entry led to unbalanced vesicle release, which resulted into fewer target cells acquiring lytic granules and subsequently being killed. Further analyses revealed that the ability of conjugate formation, lytic synapse formation, and granule polarization were normal in NK cells treated with UNC1999. Cumulatively, these data indicated that induced calcium signal exclusively enhances unbalanced degranulation that further inhibits their cytotoxic activity in human NK cells.

Microbial transglutaminase-mediated formation of erythropoietin-polyester conjugates

Erythropoietin (EPO) is a glycoprotein hormone that has been used to treat anemia in patients with chronic kidney disease and in cancer patients who are receiving chemotherapy. Here, we investigated the accessibility of the glutamine (Gln, Q) residues of recombinant human erythropoietin (rHuEPO) towards a thermoresistant variant microbial transglutaminase (mTGase), TG16 with the aim of developing novel rHuEPO conjugates that may potentially enhance its biological efficacy. As a model bioconjugation, we studied the reactivity of rHuEPO towards TG16 with a low molar mass amine group containing substrate, monodansyl cadaverine (MDC). The reactions were carried out at a Tm of 54.3 °C, the transition temperature of rHuEPO. Characterization by SDS-PAGE and mass spectrometry confirmed the conjugates formation. Then, we examined the conjugation of rHuEPO with a biodegradable and biocompatible polyester, poly(D-sorbitol adipate) (PDSA). To achieve this, PDSA was enzymatically synthesized using lipase B from Candida antartica (CAL-B), chemically modified with side chains having free primary amine (NH2) groups that can be acyl acceptor substrate of TG16, thoroughly characterized by 1H NMR spectroscopy, and then applied for the TG16-mediated conjugation reaction with rHuEPO. rHuEPO conjugates generated by this approach were identified by SDS-PAGE proving that the amine-grafted PDSA is accepted as a substrate for TG16. The successful conjugation was further verified by the detection of high molar mass fluorescent bands after labelling of amine-grafted PDSA with rhodamine B-isothiocyanate. Overall, this enzymatic procedure is considered as an effective approach to prepare biodegradable rHuEPO-polymer conjugates even in the presence of N- and O-glycans.

Crystal structure of the plant feruloyl–coenzyme A monolignol transferase provides insights into the formation of monolignol ferulate conjugates

Lignin is a highly complex phenolic polymer which is essential for plants, but also makes it difficult for industrial processing. Engineering lignin by introducing relatively labile linkages into the lignin backbone can render it more amenable to chemical depolymerization. It has been reported that introducing a feruloyl-coenzyme A monolignol transferase from Angelica sinensis (AsFMT) into poplar could incorporate monolignol ferulate conjugates (ML-FAs) into lignin polymers, suggesting a promising way to manipulate plants for readily deconstructing. FMT catalyzes a reaction between monolignols and feruloyl-CoA to produce ML-FAs and free CoA-SH. However, the mechanisms of substrate specificity and catalytic process of FMT remains poorly understood. Here we report the structure of AsFMT, which adopts a typical fold of BAHD acyltransferase family. Structural comparisons with other BAHD homologs reveal several unique structural features of AsFMT, different from those of the BAHD homologs. Further molecular docking studies showed that T375 in AsFMT may function as an oxyanion hole to stabilize the reaction intermediate and also proposed a role of H278 in the binding of the nucleophilic hydroxyl group of monolignols. Together, this study provides important structural insights into the reactions catalyzed by AsFMT and will shed light on its future application in lignin engineering.

Filamin A Is Required for NK Cell Cytotoxicity at the Expense of Cytokine Production via Synaptic Filamentous Actin Modulation.

Natural killer (NK) cells are innate cytotoxic lymphocytes that efficiently eliminate malignant and virus-infected cells without prior activation via the directed and focused release of lytic granule contents for target cell lysis. This cytolytic process is tightly regulated at discrete checkpoint stages to ensure the selective killing of diseased target cells and is highly dependent on the coordinated regulation of cytoskeletal components. The actin-binding protein filamin crosslinks cortical actin filaments into orthogonal networks and links actin filament webs to cellular membranes to modulate cell migration, adhesion, and signaling. However, its role in the regulation of NK cell functions remains poorly understood. Here, we show that filamin A (FLNa), a filamin isoform with preferential expression in leukocytes, is recruited to the NK cell lytic synapse and is required for NK cell cytotoxicity through the modulation of conjugate formation with target cells, synaptic filamentous actin (F-actin) accumulation, and cytotoxic degranulation, but not granule polarization. Interestingly, we also find that the loss of FLNa augments the target cell-induced expression of IFN-γ and TNF-α by NK cells, correlating with enhanced activation signals such as Ca2+ mobilization, ERK, and NF-κB, and a delayed down-modulation of the NKG2D receptor. Thus, our results identify FLNa as a new regulator of NK cell effector functions during their decision to kill target cells through a balanced regulation of NK cell cytotoxicity vs cytokine production. Moreover, this study implicates the cross-linking/bundling of F-actin mediated by FLNa as a necessary process coordinating optimal NK effector functions.

Evaluation of the nanosize and stability of aqueous dispersions of iodinecontaining conjugates based on carriers of plant origin, promising for iodine enrichment of foods

The high proportion of iodine deficiency diseases in the structure of general morbidity determines the relevance of creating new nontoxic iodine-containing compounds and improving the processes of iodization of foods of mass consumption. In recent years, to solve this problem, a number of iodine-containing compounds based on natural polymers have been proposed, including pectin (iodinepectin), casein (iodine-casein), inulin (iodine-inulin). It seems promising to create iodine-containing conjugates for food industry not only based on natural polymers, which are not individual compounds, but also based on low-molecular plant metabolites that have a high potential for correcting public health. The attention of researchers is attracted, in particular, by the glycosides of Stevia rebaudiana, which have positively proven themselves in the food industry for many years. Current trends in the development of industrial production of iodine-fortified products also include requirements for the compatibility of iodine-containing concentrates with food industry technologies, especially in the formation of time-stable micro-heterogeneous aqueous dispersions. In connection with the above, the purpose of the research was to confirm the formation of an iodine conjugate with a low molecular weight metabolite of Stevia rebaudiana (glycoside rebaudioside A) by infrared (IR) spectroscopy; and to assess the resistance to sedimentation of its aqueous micro-heterogeneous dispersions (iodine-glycoside) comparatively with iodine compounds with natural plant polymeric carriers - pectin (iodine-pectin) and inulin (iodine-inulin) by the method of nanostructural analysis. Material and methods. The IR spectra of the compounds were recorded on a Fourier spectrometer. Measurement of particle sizes in dispersions of iodine-containing substances was carried out on a laser analyzer, wave length 375 nm, in a quartz cuvette (7 ml) after 0, 2, 4, 6, 10, 24, 72 and 144 h. To prepare dispersions, a weighed portion of iodine-pectin (105 mg) was mixed in 100 cm3 of water (600 rpm) at a temperature of 35-40 °C for 60-70 min, weighed portions of iodine-glycoside (120 mg) and iodine-inulin (49.7 mg) were dissolved in 100 cm3 of water at room temperature (23 °C) for 3-4 min. Results. An IR spectroscopy study of the interaction of iodine with rebaudioside A indicated the stabilization of molecular iodine by the functional groups of the glycoside, leading to the formation of a stable iodine-glycoside conjugate. Assessment of the aqueous dispersions of particles of this conjugate by nanostructural analysis showed that the substance iodine-glycoside formed a stable microheterogeneous dispersion with a particle diameter of about 300 nm, which didn't undergo sedimentation during the entire observation period (6 days). Aqueous dispersions of the iodine-inulin with a particle size of 10 nm were comparable with true solutions and remained also stable throughout the entire observation period. Particles of the iodine-containing compound iodine-pectin formed moderately stable aquatic dispersed systems, the time of complete sedimentation of which was 6-10 h. Conclusion. The study of the IR spectra of rebaudioside A and the synthesized sample iodine-glycoside made it possible to confirm the conclusion about the inclusion of iodine molecules in the structure of glycoside molecules. Micro-heterogeneous stable dispersions of this conjugate, as well as iodine-inulin substances, can be convenient concentrates for enriching milk with iodine. Rapid aggregation of particles of iodine-pectin substances in concentrates can prevent their uniform distribution in the target environment, which reduces the effectiveness of industrial technologies for iodine-fortified foods for the mass prevention of endemic goiter.

In silico Study of the interaction of fucoidan with thrombolytic agents

Background: Tissue plasminogen activator (tPA) is one of the most widely used drugs in thrombolytic therapy. However, due to the inactivation of tPA in the bloodstream and increased risk of bleeding with increasing tPA dosages, the development of targeted delivery systems of tPA is required. For these purposes, it is possible to use fucoidan. The aim of the work was to study the possibility of forming of tPA-fucoidan conjugates and maintaining the activity of the enzyme using molecular docking. Methods: Docking simulations between tPA and fucoidan were performed by use of a docking software AutoDock tools version 1.5.7 and AutoDock 4.2.6. Using “blind docking” to identify the centers of molecular docking approaches of the enzyme (tPA) with the ligand (the active part of the fucoidan structure), as well as to establish the influence of the ligand on the active site of the enzyme. Results: Two “hot spots” of fucoidan binding to the enzyme were found: the region containing SER85-CYS97 residues and the region containing PHE217-TYR223 residues. This interaction can lead to the successful binding of the enzyme and polysaccharide to form a protein-polysaccharide complex. In this case, there may be a lack of suppression of the action of tPA. The interaction with the ligand was found to occur on the surface of the protein molecule. Conclusions: In this study, coupling simulations of interactions of tPA with fucoidan were conducted. The resulting conjugate can be used in the development of systems for the targeted delivery of a thrombolytic agent. This study predicts that the formation of tPA-fucoidan conjugate is a promising approach for optimizing treatment strategies for thrombosis.

Tissue-Specific Cell Penetrating Peptides for Targeted Delivery of Small Interfering RNAs

Cell penetrating peptides (CPPs) offer unique and promising solutions to overcome barriers to intracellular delivery of potential therapeutics for a variety of diseases, particularly when used as a delivery method for RNA interference agents such as siRNA. CPP mediated siRNA delivery provides promising therapeutic potential for various pathologies including many different types of cancer as well as other pathologies. Both cell-specific and non-cell-specific CPPs can play roles in transporting several types of cargo into cells, including but not limited to drugs, viral vectors, peptide nucleic acids, nanoparticles, liposomes, and siRNA. CPPs mediate siRNA delivery through a variety of delivery methods, including covalent conjugation and nanoparticle formation, and can be fashioned to facilitate endosomal escape. While no therapeutics currently utilize CPP-mediated siRNA delivery, two major approved therapeutics use RNA interference as a treatment modality, and many clinical trials are in progress testing the use of CPPs, again with an emphasis on the treatment of cancer. Further research is needed before the clinical use of CPP/siRNA complexes is commonplace, but advances in both CPP and siRNA technology appear promising for this method of treatment.

Evaluation of colloidal properties of amino-functionalized mesoporous silica nanoparticles conjugated with the plasmid vector pEGFP-N3 and its influence on the pDNA delivery to HeLa cells

Efforts are being made to develop gene therapy delivery systems capable of substituting the viral vectors with comparable efficiency but with fewer biosafety concerns, easier production on large-scale and improved shelf-stability. Among the potential delivery systems based on inorganic nanoparticles for plasmid DNA, mesoporous silica has been gaining significance due to its combination of biocompatibility with unique structural and chemical properties. In this study, mesoporous silica nanoparticles were grafted with different alkyl-amino groups to enable the formation of conjugates with the plasmid vector pEGFP-N3. The colloidal properties, cytotoxicity and transfection efficiency of conjugates to HeLa cells were evaluated. Spherical nanoparticles with around 90 nm of diameter were synthesized and then functionalized with propylamine, (2-aminoethyl)(ethyl)propylamine and 3-(diethylenetriamine)propyl groups. Dispersions prepared in different cell culture media and the zeta potential measurements revealed that their aggregation tendency was influenced by their functionalities. Besides, the structural integrity of the functionalized nanoparticles dispersed in Dulbecco's modified Eagle's medium was affected when sterilized in autoclave. The highest efficiency on the pEGFP-N3 conjugation was obtained with 3-(diethylenetriamine)propyl groups while the highest transfection efficiency was achieved with propyl amino groups onto the surface of mesoporous silica nanoparticles. The cytotoxicity of conjugates to HeLa cells was acceptable and the transfection efficiency was low possibly due to the aggregation observed in the cell culture medium.

A combination of a graphene quantum dots-cationic red dye donor-acceptor pair and cucurbit[7]uril as a supramolecular sensor for ultrasensitive detection of cancer biomarkers spermine and spermidine.

In a unique approach, the combination of a donor-acceptor pair of hydroxy graphene quantum dots (GQDs-OH) and a red-emissive donor-two-acceptor (D-2-A) type dye with pyridinium units (BPBP) and the well-known host cucurbit[7]uril (CB[7]) has been exploited as a supramolecular sensing assembly for the detection of cancer biomarkers spermine and spermidine in aqueous media at the sub-ppb level based on the affinity-driven exchange of guests from the CB[7] portal. In the binary conjugate, green fluorescent GQDs-OH transfers energy to trigger the emission of the dye BPBP and itself remains in the turn-off state. CB[7] withdraws the dye from the surface of GQDs-OH by strong host-guest interactions with its portal, making GQDs-OH fluoresce again to produce a ratiometric response. In the presence of spermine (SP) or spermidine (SPD), their strong affinity with CB[7] forces the ejection of the fluorophore to settle on the GQDs-OH surface, and the strong green emission of GQDs-OH turns off to device a supramolecular sensor for the detection of SP/SPD. The DFT studies revealed interesting excited-state charge-transfer conjugate formation between BPBP and GQDs leading to turn-on emission of the dye, and further supported the stronger binding modes of BPBP-CB[7], indicating the retrieval of the emission of GQDs. The assembly-disassembly based sensing mechanism was also established by Job's plot analysis, particle size analysis, zeta potential, time-resolved spectroscopy, ITC studies, microscopic studies, etc. The supramolecular sensing assembly is highly selective to SP and SPD, and showed nominal interference from other biogenic amines, amino acids, various metal ions, and anions. The limits of detection (LODs) were 0.1 ppb and 0.9 ppb for spermine and spermidine, respectively. The potential for the real-world application of this sensing assembly was demonstrated by spiking SP and SPD in human urine and blood serum with a high %recovery.

Self-assembly of supramolecular complexes of charged conjugated polymers and imidazolium-based ionic liquid crystals

The use of supramolecular interactions to construct mesogens represents a versatile means of accessing new macromolecular architectures, and material properties in the resulting mesophases. Here, we demonstrate the formation of thermotropic liquid crystalline supramolecular conjugates of sulfonated poly(alkyl thiophenes) backbones coupled to biphenyl-containing 1-alkyl-3-methylimidazolium salts with bromide counter anions. Two types of liquid crystalline imidazolium salts are studied with chemical structures differing in the terminal group of the biphenyl mesogenic unit. Both ionic liquid crystals exhibit multiple phase transitions with lamellar structures, which correspond to crystal and smectic liquid crystalline transitions. Supramolecular complexes of sulfonated polythiophene and ionic liquid crystals display thermotropic liquid crystalline phases with lamellar-like nanostructures. Detailed analysis show that the lamellar periodicity increases in the supramolecular complexes, compared to those of the ionic liquid crystals. Aligned textures were produced in thin films of supramolecular complexes by shear and magnetic field alignment of the liquid crystalline mesophases. The incorporation of ionic liquid crystal units into a conjugated polymer opens opportunities for fabrication of aligned thin films of mixed electron-ion conducting polymers. Such molecular materials could be of interest in electrochemical devices.

Translocation and metabolism of the chiral neonicotinoid cycloxaprid in oilseed rape (Brassica napus L.)

Neonicotinoids have been banned in some countries because of increased nontarget resistance and ecological toxicity. Cycloxaprid is a potentially promising substitute, but its metabolism in plants is still poorly understood. The study aims to clarify the translocation of cycloxaprid, identify its metabolites, propose possible metabolic pathways and compare differences between enantiomers in oilseed rape via 14C tracing technology and HPLC–QTOF–MS. The results showed that most cycloxaprid remained in the treated leaves, and only a small amount translocated to the anthers. Seven metabolites were identified, and the possible metabolic pathway was divided into two phases. Phase Ⅰ metabolism included two metabolites obtained via cleavage of the oxa-bridged seven-membered ring. Phase II metabolism was responsible for glucose conjugate formation. The possible metabolic pathways revealed that the proportion of phase I metabolites gradually decreased over time, and the phase II metabolites transformed from monosaccharide and disaccharide conjugates to trisaccharide and tetrasaccharide conjugates. The levels of metabolites were significantly different between the enantiomers. In particular, the main metabolite was M4, which has confirmed biological toxicity. M2 was the only metabolite detected in rapeseed. The results will promote the scientific application of cycloxaprid in agriculture and could have implications for assessing environmental risk.

Surface modification of Ag3PO4 using the alginate for highly active photocatalyst under visible light irradiation

Surface modification of Ag3PO4 using alginate has been successfully synthesized. Ag3PO4 was prepared by precipitation method using CaHPO4/hydroxyapatite composite as a source of phosphate ion and AgNO3 ethanol solution. Alginate was introduced on the surface of Ag3PO4 under the chemisorption method. Products were analyzed using XRD, DRS, PL spectra, FTIR, SEM, TEM, and XPS. A small band edge absorption at 729 nm was created after surface modification using alginate. XPS analysis showed that the binding energy shifts of 0.3 eV and 0.5 eV were observed after alginate treatment for Ag3d and P2p respectively indicating that the alginate was successfully chemically bound to the Ag3PO4 surface. The alginate-modified Ag3PO4 photocatalyst showed much higher photocatalytic activity than pure Ag3PO4. The high activity is caused by the formation of conjugates that can act as electron donors under visible light irradiation.

Cathepsin X Activity Does Not Affect NK-Target Cell Synapse but Is Rather Distributed to Cytotoxic Granules.

Cathepsin X is a lysosomal peptidase that is involved in tumour progression and represents a potential target for therapeutic interventions. In addition, it regulates important functions of immune cells and is implicated in the modulation of tumour cell–immune cell crosstalk. Selective cathepsin X inhibitors have been proposed as prospective antitumour agents to prevent cancer progression; however, their impact on the antitumour immune response has been overlooked. Previous studies indicate that the migration and adhesion of T cells and dendritic cells are affected by diminished cathepsin X activity. Meanwhile, the influence of cathepsin X inhibition on natural killer (NK) cell function has not yet been explored. Here, we examined the localization patterns of cathepsin X and the role of its inhibitors on the cytotoxicity of cell line NK-92, which is used for adoptive cellular immunotherapy in cancer patients. NK-92 cells depend on lymphocyte function-associated antigen 1 (LFA-1) to form stable immunoconjugates with target cells, providing, in this way, optimal cytotoxicity. Since LFA-1 is a substrate for cathepsin X activity in other types of cells, we hypothesized that cathepsin X could disturb the formation of NK-92 immunoconjugates. Thus, we employed cathepsin X reversible and irreversible inhibitors and evaluated their effects on the NK-92 cell interactions with target cells and on the NK-92 cell cytotoxicity. We show that cathepsin X inhibition does not impair stable conjugate formation or the lytic activity of NK-92 cells. Similarly, the conjugate formation between Jurkat T cells and target cells was not affected by cathepsin X activity. Unlike in previous migration and adhesion studies on T cells, in NK-92 cells cathepsin X was not co-localized with LFA-1 at the plasma membrane but was, rather, redistributed to the cytotoxic granules and secreted during degranulation.

Shifts in Backbone Conformation of Acetylcholinesterases upon Binding of Covalent Inhibitors, Reversible Ligands and Substrates

The influence of ligand binding to human, mouse and Torpedo californica acetylcholinesterase (EC 3.1.1.7; AChE) backbone structures is analyzed in a pairwise fashion by comparison with X-ray structures of unliganded AChEs. Both complexes with reversible ligands (substrates and inhibitors) as well as covalently interacting ligands leading to the formation of covalent AChE conjugates of tetrahedral and of trigonal-planar geometries are considered. The acyl pocket loop (AP loop) in the AChE backbone is recognized as the conformationally most adaptive, but not necessarily sterically exclusive, structural element. Conformational changes of the centrally located AP loop coincide with shifts in C-terminal α-helical positions, revealing interacting components for a potential allosteric interaction within the AChE backbone. The stabilizing power of the aromatic choline binding site, with the potential to attract and pull fitting entities covalently tethered to the active Ser, is recognized. Consequently, the pull can promote catalytic reactions or relieve steric pressure within the impacted space of the AChE active center gorge. These dynamic properties of the AChE backbone inferred from the analysis of static X-ray structures contribute towards a better understanding of the molecular template important in the structure-based design of therapeutically active molecules, including AChE inhibitors as well as reactivators of conjugated, inactive AChE.

Native CGRP Neuropeptide and Its Stable Analogue SAX, But Not CGRP Peptide Fragments, Inhibit Mucosal HIV-1 Transmission.

BackgroundThe vasodilator neuropeptide calcitonin gene-related peptide (CGRP) plays both detrimental and protective roles in different pathologies. CGRP is also an essential component of the neuro-immune dialogue between nociceptors and mucosal immune cells. We previously discovered that CGRP is endowed with anti-viral activity and strongly inhibits human immunodeficiency virus type 1 (HIV-1) infection, by suppressing Langerhans cells (LCs)-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission ex-vivo. This inhibition is mediated via activation of the CGRP receptor non-canonical NFκB/STAT4 signaling pathway that induces a variety of cooperative mechanisms. These include CGRP-mediated increase in the expression of the LC-specific pathogen recognition C-type lectin langerin and decrease in LC-T-cell conjugates formation. The clinical utility of CGRP and modalities of CGRP receptor activation, for inhibition of mucosal HIV-1 transmission, remain elusive.MethodsWe tested the capacity of CGRP to inhibit HIV-1 infection in-vivo in humanized mice. We further compared the anti-HIV-1 activities of full-length native CGRP, its metabolically stable analogue SAX, and several CGRP peptide fragments containing its binding C-terminal and activating N-terminal regions. These agonists were evaluated for their capacity to inhibit LCs-mediated HIV-1 trans-infection in-vitro and mucosal HIV-1 transmission in human mucosal tissues ex-vivo.ResultsA single CGRP intravaginal topical treatment of humanized mice, followed by HIV-1 vaginal challenge, transiently restricts the increase in HIV-1 plasma viral loads but maintains long-lasting higher CD4+ T-cell counts. Similarly to CGRP, SAX inhibits LCs-mediated HIV-1 trans-infection in-vitro, but with lower potency. This inhibition is mediated via CGRP receptor activation, leading to increased expression of both langerin and STAT4 in LCs. In contrast, several N-terminal and N+C-terminal bivalent CGRP peptide fragments fail to increase langerin and STAT4, and accordingly lack anti-HIV-1 activities. Finally, like CGRP, treatment of human inner foreskin tissue explants with SAX, followed by polarized inoculation with cell-associated HIV-1, completely blocks formation of LC-T-cell conjugates and HIV-1 infection of T-cells.ConclusionOur results show that CGRP receptor activation by full-length CGRP or SAX is required for efficient inhibition of LCs-mediated mucosal HIV-1 transmission. These findings suggest that formulations containing CGRP, SAX and/or their optimized agonists/analogues could be harnessed for HIV-1 prevention.

DESIGN, CHARACTERIZATION AND ENHANCED BIOAVAILABILITY OF HYDROXYPROPYLCELLULOSE-BASED NOVEL BIOCONJUGATES FOR INCLUSION OF A FLUOROQUINOLONE ANTIBIOTIC – GEMIFLOXACIN

Polysaccharides are beneficially used as drug carriers via prodrug formation and offer a mechanism for better effectiveness and delivery of the drug. The unique geometry of hydroxypropylcellulose (HPC), a polysaccharide, allows the attachment of drug molecules with a higher degree of substitution. Therefore, HPC-gemifloxacin conjugates, i.e., macromolecular prodrugs, were synthesized using acylation reagents, i.e., tosyl chloride and carbonyldiimidazole using N,N-dimethylacetamide as a solvent. The reactions were carried out at 80 °C under stirring for 24 h in inert environment. This strategy of reaction appeared efficient to obtain a high degree of drug substitution (DS = 0.42-1.34) on the polymer parent chain, as calculated by UV-visible spectrophotometry after hydrolysis of the samples. The method provides high efficacy as product yields were high (71-76%). Macromolecular prodrugs with different DS of gemifloxacin (GEM) designed were found soluble in organic solvents. The pharmacokinetic studies showed that the t1/2 and tmax values of GEM from HPC-GEM conjugate were considerably higher, which indicates improved bioavailability of the drug after conjugate formation.

Adhesion concept in cancer biology: local and central mechanisms (part 2)

The review presents the concept the key mechanism of the tumor process is a violation of adhesion interactions involving local and central mechanisms. Local features of adhesive dysregulation are demonstrated in the part 1. The second part describes the central processes. Features of local adhesive dysregulation which provides the main properties of the tumor (loss of tissue control of proliferation, anaplasia, invasion, metastasis, lack of immunological surveillance) can be controlled by central mechanisms involving the dopaminergic system which is able using immunoadhesional interactions to regulate the active phase of immune responses against the tumor interfering the process and thus interrupting the development of a malignant neoplasm initiated by a local mutation in the target tissue. The proposed concept of the adhesion key role dysregulation in the target tissue neoplasia and the processes of immunoreactivity involving the loss of central dopamine as an adhesive-damaging factor at the level of immune responses reveals among other things the stress mechanism of cancer etiology. At the same time, the central dopamine directly affects the level of dopamine in the peripheral body. The main reserves of peripheral dopamine in platelets and blood lymphocytes can serve as a guarantee of antitumor protection. Being the production of lymphocytes peripheral dopamine plays a role in the maturation of cytotoxic lymphocytes promoting their migration to tumor nodes, the formation of conjugates with tumor cells. So, dopamine participates in the active phase of immune responses against the tumor contributing to the support of adhesive interactions between immune effectors and target cells. The latter also helps to protect the body from tumor diseases which obviously shorten life.The adhesive concept of local and central control of tumor formation creates a certain perspective for improving the effectiveness of diagnosticis, prevention and treatment methods which can be a step towards solving the problem of malignant neoplasms.