Insight into the Interactions between Novel Coumarin Derivatives and Human A3 Adenosine Receptors

A study focused on the discovery of new chemical entities based on the 3‐arylcoumarin scaffold was performed with the aim of finding new adenosine receptor (AR) ligands. Thirteen synthesized compounds were evaluated by radioligand binding (A₁, A_2A, and A₃) and adenylyl cyclase activity (A_2B) assays in order to study their affinity for the four human AR (hAR) subtypes. Seven of the studied compounds proved to be selective A₃AR ligands, with 3‐(4′‐methylphenyl)‐8‐(2‐oxopropoxy)coumarin ( 12 ) being the most potent (K_i=634 nM ). None of the compounds showed affinity for the A_2B receptor, while four compounds were found to be nonselective AR ligands for the other three subtypes. Docking simulations were carried out to identify the hypothetical binding mode and to rationalize the interaction of these types of coumarin derivatives with the binding site of the three ARs to which binding was observed. The results allowed us to conclude that the 3‐arylcoumarin scaffold composes a novel and promising class of A₃AR ligands. ADME properties were also calculated, with the results suggesting that these compounds are promising leads for the identification of new drug candidates.     

The Versatile 2‐Substituted Imidazoline Nucleus as a Structural Motif of Ligands Directed to the Serotonin 5‐HT1A Receptor

The involvement of the serotonin 5‐HT_1A receptor (5‐HT_1A‐R) in the antidepressant effect of allyphenyline and its analogues indicates that ligands bearing the 2‐substituted imidazoline nucleus as a structural motif interact with 5‐HT_1A‐R. Therefore, we examined the 5‐HT_1A‐R profile of several imidazoline molecules endowed with a common scaffold consisting of an aromatic moiety linked to the 2‐position of an imidazoline nucleus by a biatomic bridge. Our aim was to discover other ligands targeting 5‐HT_1A‐R and to identify the structural features favoring 5‐HT_1A‐R interaction. Structure–activity relationships, supported by modeling studies, suggested that some structural cliché such as a polar function and a methyl group in the bridge, as well as proper steric hindrance in the aromatic area of the above scaffold, favored 5‐HT_1A‐R recognition and activation. We also highlighted the potent antidepressant‐like effect (mouse forced swimming test) of (S)‐(+)‐ 19 [(S)‐(+)‐naphtyline] at very low dose (0.01 mg kg^?1). This effect was clearly mediated by 5‐HT_1A, as it was significantly reduced by pretreatment with the 5‐HT_1A antagonist WAY100635.     

Design and synthesis of novel dual-action compounds targeting the adenosine A(2A) receptor and adenosine transporter for neuroprotection

A novel compound, N⁶‐(4‐hydroxybenzyl)adenosine, isolated from Gastrodia elata and which has been shown to be a potential therapeutic agent for preventing and treating neurodegenerative disease, was found to target both the adenosine A_2A receptor (A_2AR) and the equilibrative nucleoside transporter 1 (ENT1). As A_2AR and ENT1 are proximal in the synaptic crevice of striatum, where the mutant huntingtin aggregate is located, the dual‐action compounds that concomitantly target these two membrane proteins may be beneficial for the therapy of Huntington’s disease. To design the desired dual‐action compounds, pharmacophore models of the A_2AR agonists and the ENT1 inhibitors were constructed. Accordingly, potentially active compounds were designed and synthesized by chemical modification of adenosine, particularly at the N⁶ and C^5’ positions, if the predicted activity was within an acceptable range. Indeed, some of the designed compounds exhibit significant dual‐action properties toward both A_2AR and ENT1. Both pharmacophore models exhibit good statistical correlation between predicted and measured activities. In agreement with competitive ligand binding assay results, these compounds also prevent apoptosis in serum‐deprived PC12 cells, rendering a crucial function in neuroprotection and potential utility in the treatment of neurodegenerative diseases.     

METTL3 Inhibitors for Epitranscriptomic Modulation of Cellular Processes

The methylase METTL3 is the writer enzyme of the N⁶-methyladenosine (m⁶A) modification of RNA. Using a structure-based drug discovery approach, we identified a METTL3 inhibitor with potency in a biochemical assay of 280 nM, while its enantiomer is 100 times less active. We observed a dose-dependent reduction in the m⁶A methylation level of mRNA in several cell lines treated with the inhibitor already after 16 h of treatment, which lasted for at least 6 days. Importantly, the prolonged incubation (up to 6 days) with the METTL3 inhibitor did not alter levels of other RNA modifications (i. e., m¹A, m⁶A_m, m⁷G), suggesting selectivity of the developed compound towards other RNA methyltransferases.     

Heteromeric assembled polypeptidic artificial hydrolases with a six-helical bundle scaffold

Enzyme efficiency results from the cooperation of functional groups in the catalytic site. In order to mimic a natural enzyme, a definite 3D scaffold must be carefully designed so that the functional groups can work cooperatively. During the HIV‐1 fusion process, the gp41 N‐ and C‐terminal heptad repeat regions form a coiled‐coil six‐helical bundle (6HB) that brings the viral and target cell membranes into close proximity for fusion. We used 6HB as the molecular model for a novel scaffold for the design of an artificial enzyme, in which the modified C34 and N36 peptides formed a unique 6HB structure through specific molecular recognition, and the position and orientation of the side‐chain groups on this scaffold were predictable. The histidine modified 6HB C34_H13/20/N36_H15/22 showed enzyme‐like hydrolytic activity towards p‐nitrophenyl acetate (PNPA; k_cat/K_M=3.66 M ^?1 s^?1) through the cooperation of several inter‐ or intrahelical imidazole groups. Since the catalytic activity of 6HB depends on the C‐ and N‐peptide assembly, either HIV fusion inhibitors that can compete with the formation of catalytic 6HB or denaturants that can destroy the ordered structure were able to modulate its activity. Further engineering of the solvent‐exposing face with Glu^?‐Lys⁺ salt bridges enhanced the helicity and the stability of 6HB. As a result, the population and stability of cooperative catalytic units increased. In addition, the Glu^?‐Lys⁺‐stabilized 6HB SC35_H13/20/N36_H15/22 had increased catalytic efficiency (k_cat/K_M=6.30 M ^?1 s^?1). A unique 6HB system was specifically assembled and provided a scaffold sufficiently stable to mimic the function of enzymes or other biomolecules.     

7-Amidocoumarins as Multitarget Agents against Neurodegenerative Diseases: Substitution Pattern Modulation

This study explores the potential of 7-amidocoumarins as multitarget agents against Parkinson's and Alzheimer's diseases, by modulating the substitution patterns within the scaffold. Sixteen compounds were synthesized via 7-amino-4-methylcoumarin acylation, and in vitro evaluation of the molecules against hMAO-A, hMAO-B, hAChE, hBuChE and hBACE1 was performed. Five compounds turned out to be potent and selective hMAO-B inhibitors in the nanomolar range, six displayed inhibitory activity of hMAO-A in the low micromolar range, one showed hAChE inhibitory activity and another one hBACE1 inhibitory activity. MAO-B reversibility profile of 7-(4’-chlorobenzamido)-4-methylcoumarin ( 10 ) was investigated, with this compound being a reversible inhibitor. Neurotoxicity on motor cortex neurons and neuroprotection against H₂O₂ were also studied, corroborating the safety profile of these molecules. Finally, theoretical ADME properties were also calculated, showing these molecules as good candidates for the optimization of a lead compound. Results suggest that by modulating the substitution pattern at position 7 of the scaffold, selective or multitarget molecules can be achieved.     

Discovery of α‐Substituted Imidazole‐4‐acetic Acid Analogues as a Novel Class of ρ1 γ‐Aminobutyric Acid Type A Receptor Antagonists with Effect on Retinal Vascular Tone

The ρ‐containing γ‐aminobutyric acid type A receptors (GABA_ARs) play an important role in controlling visual signaling. Therefore, ligands that selectively target these GABA_ARs are of interest. In this study, we demonstrate that the partial GABA_AR agonist imidazole‐4‐acetic acid (IAA) is able to penetrate the blood–brain barrier in vivo; we prepared a series of α‐ and N‐alkylated, as well as bicyclic analogues of IAA to explore the structure–activity relationship of this scaffold focusing on the acetic acid side chain of IAA. The compounds were prepared via IAA from l ‐histidine by an efficient minimal‐step synthesis, and their pharmacological properties were characterized at native rat GABA_ARs in a [³H]muscimol binding assay and at recombinant human α₁β₂γ_2S and ρ₁ GABA_ARs using the FLIPR? membrane potential assay. The (+)‐α‐methyl‐ and α‐cyclopropyl‐substituted IAA analogues ((+)‐ 6 a and 6 c , respectively) were identified as fairly potent antagonists of the ρ₁ GABA_AR that also displayed significant selectivity for this receptor over the α₁β₂γ_2S GABA_AR. Both 6 a and 6 c were shown to inhibit GABA‐induced relaxation of retinal arterioles from porcine eyes.     

5‐(Piperidin‐4‐yl)‐3‐Hydroxypyrazole: A Novel Scaffold for Probing the Orthosteric γ‐Aminobutyric Acid Type A Receptor Binding Site

A series of bioisosteric N₁‐ and N₂‐substituted 5‐(piperidin‐4‐yl)‐3‐hydroxypyrazole analogues of the partial GABA_AR agonists 4‐PIOL and 4‐PHP have been designed, synthesized, and characterized pharmacologically. The unsubstituted 3‐hydroxypyrazole analogue of 4‐PIOL ( 2 a ; IC₅₀～300 μM ) is a weak antagonist at the α₁β₂γ₂ GABA_AR, whereas substituting the N₁‐ or N₂‐position with alkyl or aryl substituents resulted in antagonists with binding affinities in the high nanomolar to low micromolar range at native rat GABA_ARs. Docking studies using a α₁β₂γ₂ GABA_AR homology model along with the obtained SAR indicate that the N₁‐substituted analogues of 4‐PIOL and 4‐PHP, 2 a – k , and previously reported 3‐substituted 4‐PHP analogues share a common binding mode to the orthosteric binding site in the receptor. Interestingly, the core scaffold of the N₂‐substituted analogues of 4‐PIOL and 4‐PHP, 3 b – k , are suggested to flip 180° thereby adapting to the binding pocket and addressing a cavity situated above the core scaffold.     

Design,Synthesis, and Characterization of a Novel Fluoroprobe for Live Human Islet Cell Imaging of Serotonin 5-HT1A Receptor

Mounting evidence suggests that the serotonin system serves in signal transmission to regulate insulin secretion in pancreatic islets of Langerhans. Among the 5-HT receptor subtype found in pancreatic islets, serotonin receptor 1A (5-HT_1A) demonstrates a unique ability to inhibit β-cell insulin secretion. We report the design, synthesis, and characterization of two novel fluorescent probes for the 5-HT_1A receptor. The compounds were prepared by conjugating the scaffold of the 5-HT_1A receptor agonist 8-OH-DPAT with two fluorophores suitable for live-cell imaging. Compound 5a {5-(dimethylamino)-N-[5-[(8-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino]pentyl]naphtalen-1-sulfonammide} showed high affinity for the 5-HT_1A receptor (K_i=1.8 nM). Fluoroprobe 5a was able to label the 5-HT_1A receptor in pancreatic islet cell cultures in a selective manner, as the fluorescence emission was significantly attenuated by co-administration of the 5-HT_1A receptor antagonist WAY-100635. Thus, fluoroprobe 5a showed useful properties to further characterize this unique receptor‘s role.     

Biochemical,Structural, and Genetic Characterization of Tridecaptin A1, an Antagonist of Campylobacter jejuni

Bacillus circulans NRRL B‐30644 (now Paenibacillus terrae) was previously reported to produce SRCAM 1580, a bacteriocin active against the food pathogen Campylobacter jejuni. We have been unable to isolate SRCAM 1580, and did not find any genetic determinants in the genome of this strain. We now report the reassignment of this activity to the lipopeptide tridecaptin A₁. Structural characterization of tridecaptin A₁ was achieved through NMR, MS/MS and GC‐MS studies. The structure was confirmed through the first chemical synthesis of tridecaptin A₁, which also revealed the stereochemistry of the lipid chain. The impact of this stereochemistry on antimicrobial activity was examined. The biosynthetic machinery responsible for tridecaptin production was identified through bioinformatic analyses. P. terrae NRRL B‐30644 also produces paenicidin B, a novel lantibiotic active against Gram‐positive bacteria. MS/MS analyses indicate that this lantibiotic is structurally similar to paenicidin A.     

Collagen‐based scaffolds reinforced by chitosan fibres for bone tissue engineering

In this paper, porous bone scaffolds reinforced with chitosan fibres were prepared. The porosity and pore size of the reinforced scaffolds were both satisfactory. The reinforced scaffolds resembled natural bone in both components and crystal size. Only if the length of the fibres was no shorter than the critical length, could the fibres reinforce the material. We have proposed an empirical formula to calculate the critical length of the fibres for the porous materials and determined the modifying factor (F_l) for the porous bone scaffold investigated in this work. Along with the increase of the fibres' volume content, the compressive strength of the scaffold also increased. We have proposed a further empirical formula for calculating the compressive strength of the porous reinforced materials and determined the modifying factor (F_σ) for the porous reinforced bone scaffold examined in these studies. Along with the degradation in vitro, the decrease in strength of the reinforced scaffold was less than that of the unreinforced scaffold. The growth rate of osteoblast cells on the reinforced scaffold was higher than that on the unreinforced scaffold. These results suggest that the reinforced scaffold may be a promising candidate matrix for repairing large bone defects. Copyright © 2005 Society of Chemical Industry     

Novel β-TCP scaffold production using NaCl as a porogen for bone tissue applications

There are numerous methods for producing scaffolds to be applied in bone tissue engineering. However, the best method of scaffold production is essential to consider, with respect to their chemical composition and mechanical and structural properties, so that debris is not produced when the scaffolds are evaluated in vitro or in vivo.The primary aim of the present investigation was to produce six novel β-TCP scaffold compositions, using sodium chloride as a porogen, with two different particle sizes, measuring 1–2 mm and 750 mm-1mm, and at varied concentrations (30, 50, and 70 wt %). Physical, chemical, mechanical, and in vitro characterizations were then performed on each scaffold composition, using artificial saliva, for 7 and 14 days, with promising results. The XRD diffractograms showed the formation of two new crystalline phases (NaCaPO₄ and Ca₅[PO₄]₃Cl) in the scaffolds, after their production. In addition, scaffold porosity, Young's modulus, and the maximum resistance of compression values were in the trabecular bone range and the in vitro test, using artificial saliva, was favorable in relation to scaffold bioactivity.     

Protein Arginine N‐Methyltransferase Substrate Preferences for Different Nη‐Substituted Arginyl Peptides

Protein arginine N‐methyltransferases (PRMTs) catalyze methyl‐group transfer from S‐adenosyl‐L ‐methionine onto arginine residues in proteins. In this study, modifications were introduced at the guanidine moiety of a peptidyl arginine residue to investigate how changes to the PRMT substrate can modulate enzyme activity. We found that peptides bearing Nη‐hydroxy or Nη‐amino substituted arginine showed higher apparent k_cat values than for the monomethylated substrate when using PRMT1, whereas this catalytic preference was not observed for PRMT4 and PRMT6. Methylation by compromised PRMT1 variants E153Q and D51N further supports the finding that the N‐hydroxy substitution facilitates methyl transfer by tuning the reactivity of the guanidine moiety. In contrast, Nη‐nitro and Nη‐canavanine substituted substrates inhibit PRMT activity. These findings demonstrate that methylation of these PRMT substrates is dependent on the nature of the modification at the guanidine moiety.     

The Inactivation Mechanism of Human Group IIA Phospholipase A2 by Scalaradial

Secretory phospholipases A₂ (sPLA₂s) are implicated in the pathogenesis of several inflammation diseases, such as rheumatoid arthritis, septic shock, psoriasis, and asthma. Thus, an understanding of their inactivation mechanisms could be useful for the development of new classes of chemical selective inhibitors. In the marine environment, several bioactive terpenoids possess interesting anti‐inflammatory activity, often through covalent and/or noncovalent inactivation of sPLA₂. Herein, we report the molecular mechanism of human group IIA phospholipase A₂ (sPLA₂‐IIA) inactivation by Scalaradial (SLD), a marine 1,4‐dialdehyde terpenoid isolated from the sponge Cacospongia mollior and endowed with a significant anti‐inflammatory profile. Our results have been collected by a combination of biochemical approaches, advanced mass spectrometry, surface plasmon resonance, and molecular modeling. These suggest that SLD acts as a competitive inhibitor. Indeed, the sPLA₂‐IIA inactivation process seems to be driven by the noncovalent recognition process of SLD in the enzyme active site and by chelation of the catalytic calcium ion. In contrast, covalent modification of the enzyme by the SLD dialdehyde moiety emerges as only a minor side event in the ligand–enzyme interaction. These results could be helpful for the rational design of new PLA₂ inhibitors that would be able to selectively target the enzyme active site.     

Fabrication of LaCl3‐containing nanofiber scaffolds and their application in skin wound healing

Poly(?‐caprolactone) (PCL)/gelatin (GE) nanofiber scaffolds with varying concentrations of lanthanum chloride (LaCl₃, from 0 to 25 mM) were fabricated by electrospinning. The scaffolds were characterized by scanning electron microscopy, contact angle and porosity measurements, mechanical strength tests, and in vitro degradation studies. In vitro cytotoxicity and cell adhesion and proliferation studies were performed to assess the biocompatibility of the scaffolds, and in vivo wound healing studies were conducted to assess scaffold applications in the clinic. All prepared scaffolds were noncytotoxic, and the growth of adipose tissue–derived stem cells on LaCl₃‐containing scaffolds was better than on the pure PCL/GE scaffold. Cell proliferation studies showed the greatest cell growth in the PCL/GE/LaCl₃ scaffolds. Further, in vivo studies proved that the PCL/GE/LaCl₃ scaffolds can promote wound healing. The results suggest that nanofiber scaffolds containing LaCl₃ promote cell proliferation and have good biocompatibility, and thus potential for application in the treatment of skin wounds. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46672.     

Development of Polyvinyl Alcohol Fibrous Biodegradable Scaffolds for Nerve Tissue Engineering Applications: In Vitro Study

In this study, polyvinyl alcohol (PVA) fibers were modified through an effective cross linking method. Adequate porosity and surface area are widely recognized as important parameters in the design of scaffolds for tissue engineering and therefore measurement of porosity is very important. Herein, porosity measurement of various surface layers of scaffold was done through a new method, and image analysis was used for this purpose. Scanning electron microscopy micrographs of nanofibrous scaffolds were converted to binary images using different thresholds and porosity of scaffold was measured in various layers. In addition, for ascertaining of cross linking of the PVA nanofibrous scaffolds, Fourier transform infrared spectroscopy analysis was employed. Also, the in vitro biodegradability of the nanofibrous scaffold was evaluated. The PVA crosslinked nanofibrous scaffold was found to exhibit the most balanced properties to meet all the required specifications for nerve tissue and was used for in vitro culture of nerve stem cells (PC12 cells). Finally, the results of the swelling behavior of the samples revealed that the cross linked PVA scaffold has a strong swelling about 450%.     

New N-(2-carboxybenzyl)chitosan composite scaffolds containing nanoTiO2 or bioactive glass with enhanced cell proliferation for bone-tissue engineering applications

In the present study N-(2-carboxbenzyl)chitosan (CBCS) 3D macroporous hybrid scaffolds with interconnected pore system, containing 0.5, 2.5, and 5?wt% TiO₂ nanoparticles (nTiO₂) and 2.5?wt% Bioglass 45S5 (BG) have been synthesized using freeze-drying technique. Compressive strength values verified that the modification of chitosan combined with the presence of inorganic fillers can attribute significant mechanical stiffness to the scaffold. The in vitro biomineralization test confirmed that all samples were bioinert as mineral deposits were detected with X-ray diffractometry after incubation in SBF. Cytotoxicity and biocompatibility of all scaffolds were tested by using and Wharton’s jelly–derived mesenchymal stem cells (WJ-MSCs) and human embryonic kidney 293 (HEK 293) cell line. Metabolic activity, proliferation, migration, and attachment to the scaffolds were examined. Cells appeared to attach around the superficial pores and migrate in them. Cells also maintained their morphology, proliferated, and migrated across the scaffolds and showed consistent and proved compatibility.     

Plasma polymerization‐modified bacterial polyhydroxybutyrate nanofibrillar scaffolds

The design and the development of novel scaffold materials for tissue engineering have attracted much interest in recent years. Especially, the prepared nanofibrillar scaffold materials from biocompatible and biodegradable polymers by electrospinning are promising materials to be used in biomedical applications. In this study, we propose to produce low‐cost and cell‐friendly bacterial electrospun PHB polymeric scaffolds by using Alcaligenes eutrophus DSM 545 strain to PHB production. The produced PHB was characterized by Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared Spectroscopy (FTIR). Nanofibrous scaffolds were fabricated via electrospinning method that has a fiber diameter approximately 700–800 nm. To investigate cell attachment, cell growth, and antioxidant enzyme activity on positively and negatively charged PHB scaffold, PHB surface was modified by plasma polymerization technique using polyethylene glycol (PEG) and ethylenediamine (EDA). According to the results of superoxide dismutase (SOD) activity study, PEG‐modified nanofibrillar scaffolds indicated more cellular resistance against oxidative stress compared to the EDA modification. As can be seen in cell proliferation results, EDA modification enhanced the cell proliferation more than PEG modification, while PEG modification is better as compared with nonmodified scaffolds. In general, through plasma polymerization technique, surface modified nanofibrillar structures are effective substrates for cell attachment and outgrowth. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Bioactive Ceramic/Polyamide 6 Scaffold for Bone Regeneration: In vitro and in vivo Evaluation

A porous scaffold of bio-ceramic/polyamide 6 (PA 6) has been fabricated through a thermally induced phase separation technique. The scaffold was characterized by XPS, SEM and mechanical test. The results revealed that bio-ceramic/PA 6 scaffold had an interconnected porous structure with a porosity of about 78%. Moreover, bio-ceramic/PA 6 scaffolds were cultured with BMSCs to investigate their in vitro cytocompatibility, and they were implanted in subcutaneous sites of mice for 4 and 8 weeks to evaluate their in vivo histocompatibility. The result showed the composite scaffolds provided a favourable environment for initial cell adhesion, maintained cell viability and cell proliferation, and had good tissue compatibility.     

Novel,Dual Target-Directed Annelated Xanthine Derivatives Acting on Adenosine Receptors and Monoamine Oxidase B

Annelated purinedione derivatives have been shown to act as possible multiple-target ligands, addressing adenosine receptors and monoaminooxidases. In this study, based on our previous results, novel annelated pyrimido- and diazepino[2,1-f]purinedione derivatives were designed as dual-target-directed ligands combining A_2A adenosine receptor (AR) antagonistic activity with blocking monoamine oxidase B. A library of 19 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. This allowed 9-(2-chloro-6-fluorobenzyl)-3-ethyl-1-methyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione ( 13 e ; K_i human A_2AAR: 264 nM and IC₅₀ human MAO-B: 243 nM) to be identified as the most potent dual-acting ligand from this series. ADMET parameters were estimated in vitro, and analysis of the structure-activity relationships was complemented by molecular-docking studies based on previously published X-ray structures of the protein targets. Such dual-acting ligands, by selectively blocking A_2A AR, accompanied by the inhibition of dopamine metabolizing enzyme MAO-B, might provide symptomatic and neuroprotective effects in, among others, the treatment of Parkinson disease