Competitive binding of maltodextrin and pectin at the interface of whey protein hydrolyzate-based fish oil emulsion under high temperature sterilization: Effects on storage stability and in vitro digestion.

Whey protein hydrolysate (WPH), maltodextrin (MD), low methoxy pectin (LMP) and high methoxy pectin (HMP) were used to study the interface binding under high temperature sterilization conditions (121 °C, 15 min). The effect of competitive binding of MD and pectin with interface protein on the storage stability and gastrointestinal fate of fish oil emulsion was studied. The low-molecular-weight MD and the interface protein undergo a wide range of covalent binding through the Maillard reaction, while a small amount of high-molecular-weight pectin can form a protective shell with the interface protein through electrostatic interaction to inhibit the covalent reaction of MD, which was called competitive binding. However, due to the bridging and depletion flocculation of pectin, the emulsification stability of fish oil emulsion reduced. After 13 days of storage, compared with the particle size of the WPH fish oil emulsion (459.18 nm), the fish oil emulsion added with LMP and HMP reached 693.58 nm and 838.54 nm, respectively. In vitro digestion proved that WPH fish oil emulsion flocculated rapidly in the stomach (1.76 µm), while WPH-MD and WPH-MD-pectin fish oil emulsions flocculated slightly (less than800 nm). WPH-MD-pectin delayed digestion in the gastrointestinal tract, and HMP exhibited a better slow-release effect. This study provides reference for the design of multi-component functional drinks and other bioactive ingredient delivery system.

Baicalin ameliorates alcohol-induced hepatic steatosis by suppressing SREBP1c elicited PNPLA3 competitive binding to ATGL.

Baicalin is a flavonoid compound abundant in multiple edible and medicinal plants such as Scutellaria baicalensis Georgi. In this study, we provide evidence to support the fact that baicalin ameliorates alcohol-induced hepatic steatosis via regulating SREBP1c elicited PNPLA3 competitive binding to ATGL. Results showed that baicalin significantly attenuated the development of metabolic disorders and hepatic steatosis in alcohol-induced rats after four weeks of treatment. It was evident that baicalin treatment significantly normalized the serous contents of hepatic triglyceride (TG), alanine aminotransferase (ALT), and aspartate aminotransferase (AST), and attenuated the increase of hepatic vacuolization and Oil Red O staining area caused by alcohol. Meanwhile, baicalin relieves alcohol-induced hepatic fibrosis by masson staining and RT-qPCR analysis. Mechanistically, alcohol aggravated the nuclear expression of SREBP1c, which contributed to the high expression of PNPLA3 and FASN, thereby enhancing the binding of PNPLA3 to ABHD5, and indirectly impairing the binding ability between ATGL and ABHD5, ultimately causing a decline in the hydrolysis capacity in liver lipid droplets. As expected, these alcohol-induced pathobolism were reversed by baicalin treatment both in vivo and in vitro. In conclusion, this study has demonstrated that baicalin can protect against alcohol-induced hepatic lipid accumulation by activating hepatic lipolysis via suppressing SREBP1c elicited PNPLA3 competitive binding to ATGL. Baicalin is a promising natural product for preventing alcohol-induced hepatic steatosis.

PHA-680626 Is an Effective Inhibitor of the Interaction between Aurora-A and N-Myc.

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcription factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation by directly binding to a highly conserved N-Myc region. As a result, elevated levels of N-Myc are observed. During recent years, it has been demonstrated that some ATP competitive inhibitors of AURKA also cause essential conformational changes in the structure of the activation loop of the kinase that prevents N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complexes with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results identify PHA-680626 as an amphosteric inhibitor both in vitro and in MYCN overexpressing cell lines, thus expanding the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex and confirming that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

Antidepressant activity of Spathodea campanulata in mice and predictive affinity of spatheosides towards type A monoamine oxidase.

The antidepressant activity of Spathodea campanulata flowers was evaluated in mice and in silico. When tested at doses of 200 and 400 mg/kg, the methanol extract of S. campanulata (MESC) showed dose-dependent antidepressant activity in the force swim test (FST), tail suspension test (TST), lithium chloride-induced twitches test and the open field test. In FST and TST, animals treated with MESC demonstrated a significant decrease in the immobility period compared to the control group. The lithium chloride-induced head twitches were significantly reduced following administration of MESC. The latter, at the dose of 400 mg/kg, also significantly reduced locomotor activity. Following administration of MESC, changes in the levels of serum corticosterone, and of norepinephrine, dopamine, serotonin, 4-hydroxy-3-methoxyphenylglycol (MHPG), 4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) were measured in different brain regions using HPLC. The presence of spatheoside A (m/z 541) and spatheoside B (m/z 559) in MESC was detected using HPLC/ESI-MS. These two iridoids demonstrated a high predictive binding affinity for the active site of the type A monoamine oxidase (MAO-A) enzyme with scores of 99.40 and 93.54, respectively.  These data suggest that S. campanulata flowers warrants further investigation as a source of novel templates for antidepressive drugs.

Globally Approved EGFR Inhibitors: Insights into Their Syntheses, Target Kinases, Biological Activities, Receptor Interactions, and Metabolism.

Targeting the EGFR with small-molecule inhibitors is a confirmed valid strategy in cancer therapy. Since the FDA approval of the first EGFR-TKI, erlotinib, great efforts have been devoted to the discovery of new potent inhibitors. Until now, fourteen EGFR small-molecule inhibitors have been globally approved for the treatment of different types of cancers. Although these drugs showed high efficacy in cancer therapy, EGFR mutations have emerged as a big challenge for these drugs. In this review, we focus on the EGFR small-molecule inhibitors that have been approved for clinical uses in cancer therapy. These drugs are classified based on their chemical structures, target kinases, and pharmacological uses. The synthetic routes of these drugs are also discussed. The crystal structures of these drugs with their target kinases are also summarized and their bonding modes and interactions are visualized. Based on their binding interactions with the EGFR, these drugs are also classified into reversible and irreversible inhibitors. The cytotoxicity of these drugs against different types of cancer cell lines is also summarized. In addition, the proposed metabolic pathways and metabolites of the fourteen drugs are discussed, with a primary focus on the active and reactive metabolites. Taken together, this review highlights the syntheses, target kinases, crystal structures, binding interactions, cytotoxicity, and metabolism of the fourteen globally approved EGFR inhibitors. These data should greatly help in the design of new EGFR inhibitors.

Isobavachalcone as an Active Membrane Perturbing Agent and Inhibitor of ABCB1 Multidrug Transporter.

Isobavachalcone (IBC) is an active substance from the medicinal plant Psoralea corylifolia. This prenylated chalcone was reported to possess antioxidative, anti-inflammatory, antibacterial, and anticancer activities. Multidrug resistance (MDR) associated with the over-expression of the transporters of vast substrate specificity such as ABCB1 (P-glycoprotein) belongs to the main causes of cancer chemotherapy failure. The cytotoxic, MDR reversing, and ABCB1-inhibiting potency of isobavachalcone was studied in two cellular models: human colorectal adenocarcinoma HT29 cell line and its resistant counterpart HT29/Dx in which doxorubicin resistance was induced by prolonged drug treatment, and the variant of MDCK cells transfected with the human gene encoding ABCB1. Because MDR modulators are frequently membrane-active substances, the interaction of isobavachalcone with model phosphatidylcholine bilayers was studied by means of differential scanning calorimetry. Molecular modeling was employed to characterize the process of membrane permeation by isobavachalcone. IBC interacted with ABCB1 transporter, being a substrate and/or competitive inhibitor of ABCB1. Moreover, IBC intercalated into model membranes, significantly affecting the parameters of their main phospholipid phase transition. It was concluded that isobavachalcone interfered both with the lipid phase of cellular membrane and with ABCB1 transporter, and for this reason, its activity in MDR cancer cells was presumptively beneficial.

Identification of natural compounds as SARS-CoV-2 entry inhibitors by molecular docking-based virtual screening with bio-layer interferometry.

Coronavirus Disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enter the host cells through the interaction between its receptor binding domain (RBD) of spike glycoprotein with angiotensin-converting enzyme 2 (ACE2) receptor on the plasma membrane of host cell. Neutralizing antibodies and peptide binders of RBD can block viral infection, however, the concern of accessibility and affordability of viral infection inhibitors has been raised. Here, we report the identification of natural compounds as potential SARS-CoV-2 entry inhibitors using the molecular docking-based virtual screening coupled with bilayer interferometry (BLI). From a library of 1871 natural compounds, epigallocatechin gallate (EGCG), 20(R)-ginsenoside Rg3 (RRg3), 20(S)-ginsenoside Rg3 (SRg3), isobavachalcone (Ibvc), isochlorogenic A (IscA) and bakuchiol (Bkc) effectively inhibited pseudovirus entry at concentrations up to 100 µM. Among these compounds, four compounds, EGCG, Ibvc, salvianolic acid A (SalA), and isoliensinine (Isl), were effective in inhibiting SARS-CoV-2-induced cytopathic effect and plaque formation in Vero E6 cells. The EGCG was further validated with no observable animal toxicity and certain antiviral effect against SARS-CoV-2 pseudovirus mutants (D614G, N501Y, N439K & Y453F). Interestingly, EGCG, Bkc and Ibvc bind to ACE2 receptor in BLI assay, suggesting a dual binding to RBD and ACE2. Current findings shed some insight into identifications and validations of SARS-CoV-2 entry inhibitors from natural compounds.

Novel curcumin analog (cis-trans curcumin) as ligand to adenosine receptors A2A and A2B: potential for therapeutics.

All four of the adenosine receptor (AR) subtypes mediate pain and have been targeted by pharmacologists to generate new therapeutics for chronic pain. The vanilloid phytochemicals, which include curcumin, capsaicin, and gingerol, have been shown to alleviate pain. However, there is little to no literature on the interaction of vanilloid phytochemicals with ARs. In this study, photochemical methods were used to generate a novel isomer of curcumin (cis-trans curcumin or CTCUR), and the interactions of both curcumin and CTCUR with the two Gs-linked AR subtypes were studied. Competitive binding assays, docking analysis, and confocal fluorescence microscopy were performed to measure binding affinity; cell survival assays were used to measure toxicity; and cAMP assays were performed to measure receptor activation. Competitive binding results indicated that CTCUR binds to both AR A2A and AR A2B with Ki values of 5 µM and 7 µM, respectively, which is consistent with our docking results. Fluorescence microscopy data also shows binding for A2B and A2A. Cell survival results show that CTCUR and CUR are nontoxic at the tested concentrations in these cell lines. Overall, our results suggest that vanilloid phytochemicals may be slightly modified to increase interaction with Gs-ARs, and thereby can be further explored to provide a novel class of non-opioid antinociceptives.

Pharmacological characterisation of novel adenosine A3 receptor antagonists.

The adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics-Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery.

Computational Studies towards the Identification of Novel Rhodopsin-Binding Compounds as Chemical Chaperones for Misfolded Opsins.

Accumulation of misfolded and mistrafficked rhodopsin on the endoplasmic reticulum of photoreceptor cells has a pivotal role in the pathogenesis of retinitis pigmentosa and a subset of Leber's congenital amaurosis. One potential strategy to reduce rhodopsin misfolding and aggregation in these conditions is to use opsin-binding compounds as chemical chaperones for opsin. Such molecules have previously shown the ability to aid rhodopsin folding and proper trafficking to the outer cell membranes of photoreceptors. As means to identify novel chemical chaperones for rhodopsin, a structure-based virtual screening of commercially available drug-like compounds (300,000) was performed on the main binding site of the visual pigment chromophore, the 11-cis-retinal. The best 24 virtual hits were examined for their ability to compete for the chromophore-binding site of opsin. Among these, four small molecules demonstrated the ability to reduce the rate constant for the formation of the 9-cis-retinal-rhodopsin complex, while five molecules surprisingly enhanced the formation of this complex. Compound 7, 13, 20 and 23 showed a weak but detectable increase in the trafficking of the P23H mutant, widely used as a model for both retinitis pigmentosa and Leber's congenital amaurosis, from the ER to the cell membrane. The compounds did not show any relevant cytotoxicity in two different human cell lines, with the only exception of 13. Based on the structures of these active compounds, a series of in silico studies gave important insights on the potential structural features required for a molecule to act either as chemical chaperone or as stabiliser of the 11-cis-retinal-rhodopsin complex. Thus, this study revealed a series of small molecules that represent a solid foundation for the future development of novel therapeutics against these severe inherited blinding diseases.

Sensitive and reversible perylene derivative-based fluorescent probe for acetylcholinesterase activity monitoring and its inhibitor.

A reversible fluorescence probe for acetylcholinesterase activity detection was developed based on water soluble perylene derivative, N,N'-di(2-aspartic acid)-perylene-3,4,9,10-tetracarboxylic diimide (PASP). Based on the photo-induced electron transfer (PET), PASP fluorescence in aqueous is quenched after combining with copper ions (Cu2+). Acetylcholinesterase (AChE) is well known to catalyze the hydrolysis of acetylcholine (ATCh) to produce thiocholine, whose affinity is strong enough to capture Cu2+ by thiol (-SH) group from the complex PASP-Cu, resulting in the fluorescence signal of PASP recovers up to 90%. This optical switch is highly sensitive depended on the coordination and dissociation between PASP and Cu2+. We proposed its application for AChE activity detection, as well as its inhibitor screening. According to the change of fluorescence intensity, quantifying the detection limit of AChE was 1.78 mU·mL-1. Classical inhibitors, tacrine and organophosphate pesticide diazinon, were further evaluated for drug screening. The IC50 value of tacrine was calculated to be 0.43 µM, and the detection limit of diazinon was 0.22 µM. Both of these performances were much better than previous results, revealing our probe is sensitive and reversible for screening applications.

Mechanism of synergistic DNA damage induced by caffeic acid phenethyl ester (CAPE) and Cu(II): Competitive binding between CAPE and DNA with Cu(II)/Cu(I).

Caffeic acid phenethyl ester (CAPE) is an active polyphenol of propolis from honeybee hives, and exhibits antioxidant and interesting pharmacological activities. However, in this study, we found that in the presence of Cu(II), CAPE exhibited pro-oxidative rather than antioxidant effect: synergistic DNA damage was induced by the combination of CAPE and Cu(II) together as measured by strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, which is dependent on the molar ratio of CAPE:Cu(II). Production of Cu(I) and H2O2 from the redox reaction between CAPE and Cu(II), and subsequent OH formation was found to be responsible for the synergistic DNA damage. DNA sequencing investigations provided more direct evidence that CAPE/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Interestingly, we found there are competitive binding between CAPE and DNA with Cu(II)/Cu(I), which changed the redox activity of Cu(II)/Cu(I), via complementary applications of different analytical methods. The observed DNA damage was mainly attributed to the formation of DNA-Cu(II)/Cu(I) complexes, which is still redox active and initiated the redox reaction near the binding site between copper and DNA. Based on these data, we proposed that the synergistic DNA damage induced by CAPE/Cu(II) might be due to the competitive binding between CAPE and DNA with Cu, and site-specific production of OH near the binding site of copper with DNA. Our findings may have broad biological implications for future research on the pro-oxidative effects of phenolic compounds in the presence of transition metals.

Synthesis and Biological Activity of 2,22-Dimethylene Analogues of 19-Norcalcitriol and Related Compounds.

Continuing our search for vitamin D analogues, we explored the modification of the steroidal side chain and inserted a methylene moiety in position C-22 together with either lengthening the side chain or introducing a ring at the terminal end. Our conformational studies confirmed that the presence of a methylene group attached to C-22 restricts the conformational flexibility of the side chain, which can result in changes in biological characteristics of a molecule. All synthesized 1α,25-dihydroxy-2,22-dimethylene-19-norvitamin D3 analogues proved equal to calcitriol in their ability to bind to the vitamin D receptor, and most of them exert significantly higher differentiation and transcriptional activity than calcitriol. The most active compounds were characterized by the presence of an elongated side chain or 26,27-dimethylene bridge. The synthetic strategy was based on the Wittig-Horner coupling of the known A-ring phosphine oxide with the corresponding Grundmann ketones prepared from a 20-epi-Inhoffen-Lythgoe diol derived from vitamin D2.

Effect of ethanolic extract of Solanum virginianum Linn. on neuropathic pain using chronic constriction injury rat model and molecular docking studies.

The present research work was designed to examine the neuroprotective effect of ethanolic extract of Solanum virginianum Linn. (SV) in chronic construction injury (CCI) of sciatic nerve-induced neuropathic pain in rats. The extract was initially standardized by high-performance thin-layer chromatography using solasodine as a biomarker and was then subjected to assess the degree of mechanical allodynia, thermal allodynia, mechanical hyperalgesia, thermal hyperalgesia and biochemical evaluations. Administration of SV (100 and 200 mg/kg; p.o.) and pregabalin (10 mg/kg; p.o.) as a reference standard significantly debilitated hyperalgesia and allodynia and notably restored the altered antioxidant level and pro-inflammatory cytokine (IL-1ß and TNF-α) expression in a dose-dependent manner. Further, to appraise the mechanistic approach of solasodine, docking simulation studies were done on the 3D structure of the voltage-gated N-type calcium channel (Cav 2.2), R-type calcium channel (Cav 2.3) and sodium channel (Nav 1.7), and the results revealed that solasodine properly positioned into Phe 19, Leu 32, Met 51 and Met 71 (FLMM pocket) of Cav 2.2 and Cav 2.3 and being a competitor of Ca2+/N-lobe it may inactivate these calcium channels but did not bind into the desired binding pocket of Nav 1.7. Thus, the study confirmed the role of solasodine as a major biomarker for the observed neuroprotective nature of Solanum virginianum.

In Vitro Investigations of Acetohexamide Binding to Glycated Serum Albumin in the Presence of Fatty Acid.

The interaction of drugs with human serum albumin (HSA) is an important element of therapy. Albumin affects the distribution of the drug substance in the body, as well as its pharmacokinetic and pharmacodynamic properties. On the one hand, inflammation and protein glycation, directly associated with many pathological conditions and old age, can cause structural and functional modification of HSA, causing binding disorders. On the other hand, the widespread availability of various dietary supplements that affect the content of fatty acids in the body means that knowledge of the binding activity of transporting proteins, especially in people with chronic diseases, e.g., diabetes, will achieve satisfactory results of the selected therapy. Therefore, the aim of the present study was to evaluate the effect of a mixture of fatty acids (FA) with different saturated and unsaturated acids on the affinity of acetohexamide (AH), a drug with hypoglycaemic activity for glycated albumin, simulating the state of diabetes in the body. Based on fluorescence studies, we can conclude that the presence of both saturated and unsaturated FA disturbs the binding of AH to glycated albumin. Acetohexamide binds more strongly to defatted albumin than to albumin in the presence of fatty acids. The competitive binding of AH and FA to albumin may influence the concentration of free drug fraction and thus its therapeutic effect.

Hops compounds modulatory effects and 6-prenylnaringenin dual mode of action on GABAA receptors.

Hops (Humulus lupulus L.), a major component of beer, contain potentially neuroactive compounds that made it useful in traditional medicine as a sleeping aid. The present study aims to investigate the individual components in hops acting as allosteric modulators in GABAA receptors and bring further insight into the mode of action behind the sedative properties of hops. GABA-potentiating effects were measured using [3H]ethynylbicycloorthobenzoate (EBOB) radioligand binding assay in native GABAA receptors. Flumazenil sensitivity of GABA-potentiating effects, [3H]Ro 15-4513, and [3H]flunitrazepam binding assays were used to examine the binding to the classical benzodiazepines site. Humulone (alpha acid) and 6-prenylnaringenin (prenylflavonoid) were the most potent compounds displaying a modulatory activity at low micromolar concentrations. Humulone and 6-prenylnaringenin potentiated GABA-induced displacement of [3H]EBOB binding in a concentration-dependent manner where the IC50 values for this potentiation in native GABAA receptors were 3.2 µM and 3.7 µM, respectively. Flumazenil had no significant effects on humulone- or 6-prenylnaringenin-induced displacement of [3H]EBOB binding. [3H]Ro 15-4513 and [3H]flunitrazepam displacements were only minor with humulone but surprisingly prominent with 6-prenylnaringenin despite its flumazenil-insensitive modulatory activity. Thus, we applied molecular docking methods to investigate putative binding sites and poses of 6-prenylnaringenin at the GABAA receptor α1ß2γ2 isoform. Radioligand binding and docking results suggest a dual mode of action by 6-prenylnaringenin on GABAA receptors where it may act as a positive allosteric modulator at α+ß- binding interface as well as a null modulator at the flumazenil-sensitive α+γ2- binding interface.

Pharmacological and biotransformation studies of 1-acyl-substituted derivatives of d-lysergic acid diethylamide (LSD).

The ergoline d-lysergic acid diethylamide (LSD) is one of the most potent psychedelic drugs. 1-Acetyl-LSD (ALD-52), a derivative of LSD containing an acetyl group on the indole nitrogen, also produces psychedelic effects in humans and has about the same potency as LSD. Recently, several other 1-acyl-substitued LSD derivatives, including 1-propanoyl-LSD (1P-LSD) and 1-butanoyl-LSD (1B-LSD), have appeared as designer drugs. Although these compounds are assumed to act as prodrugs for LSD, studies have not specifically tested this prediction. The present investigation was conducted to address the gap of information about the pharmacological effects and mechanism-of-action of 1-acyl-substituted LSD derivatives. Competitive binding studies and calcium mobilization assays were performed to assess the interaction of ALD-52, 1P-LSD, and 1B-LSD with serotonin 5-HT2 receptor subtypes. A receptorome screening was performed with 1B-LSD to assess its binding to other potential targets. Head twitch response (HTR) studies were performed in C57BL/6J mice to assess in vivo activation of 5-HT2A (the receptor thought to be primarily responsible for hallucinogenesis). Finally, liquid chromatography/ion-trap mass spectrometry (LC/MS) was used to quantify plasma levels of LSD in Sprague-Dawley rats treated with ALD-52 and 1P-LSD. 1-Acyl-substitution reduced the affinity of LSD for most monoamine receptors, including 5-HT2A sites, by one to two orders of magnitude. Although LSD acts as an agonist at 5-HT2 subtypes, ALD-52, 1P-LSD and 1B-LSD have weak efficacy or act as antagonists in Ca2+-mobilization assays. Despite the detrimental effect of 1-acyl substitution on 5-HT2A affinity and efficacy, 1-acyl-substitued LSD derivatives induce head twitches in mice with relatively high potency. High levels of LSD were detected in the plasma of rats after subcutaneous administration of ALD-52 and 1P-LSD, demonstrating these compounds are rapidly and efficiently deacylated in vivo. These findings are consistent with the prediction that ALD-52, 1P-LSD and 1B-LSD serve as prodrugs for LSD. This article is part of the special issue entitled 'Serotonin Research: Crossing Scales and Boundaries'.

A Kinetic Process to Determine the Interaction Type Between Two Compounds, One of Which Is a Reaction Product, Using Alkaline Phosphatase Inhibition as a Case Study.

This study describes the development of a new methodology based on a new integrated equation which allows the determination of the kinetic parameters for two mutually non-exclusive inhibitors when one of which is produced during the time-course reaction. Alkaline phosphatase simultaneously inhibited by phosphate and urea is used to illustrate this methodology, including the evaluation of interaction effects between them. Data analyses were carried out using two integrated velocity equations: exclusive linear mixed inhibition (EMI) and non-exclusive linear mixed inhibition (NEMI). Kinetic parameters are estimated using non-linear regression and results show that (i) the interaction between enzyme and the inhibitors urea and phosphate exhibit a mutually non-exclusive behavior; (ii) more specifically, these inhibitors are non-exclusive only in free enzyme (E) species; (iii) the inhibitors also show an interaction with enzyme classified as facilitation; (iv) phosphate is a competitive inhibitor and urea a mixed inhibitor; (v) the inhibition constant for phosphate is much lower than that determined for urea. In addition, a functional Excel Spreadsheet which can be adapted to any kinetic study is also included as a supplement.

Pharmacologic Characterization of JNJ-42226314, [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone, a Reversible, Selective, and Potent Monoacylglycerol Lipase Inhibitor.

The serine hydrolase monoacylglycerol lipase (MAGL) is the rate-limiting enzyme responsible for the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. Inhibition of 2-AG degradation leads to elevation of 2-AG, the most abundant endogenous agonist of the cannabinoid receptors (CBs) CB1 and CB2. Activation of these receptors has demonstrated beneficial effects on mood, appetite, pain, and inflammation. Therefore, MAGL inhibitors have the potential to produce therapeutic effects in a vast array of complex human diseases. The present report describes the pharmacologic characterization of [1-(4-fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone (JNJ-42226314), a reversible and highly selective MAGL inhibitor. JNJ-42226314 inhibits MAGL in a competitive mode with respect to the 2-AG substrate. In rodent brain, the compound time- and dose-dependently bound to MAGL, indirectly led to CB1 occupancy by raising 2-AG levels, and raised norepinephrine levels in cortex. In vivo, the compound exhibited antinociceptive efficacy in both the rat complete Freund's adjuvant-induced radiant heat hypersensitivity and chronic constriction injury-induced cold hypersensitivity models of inflammatory and neuropathic pain, respectively. Though 30 mg/kg induced hippocampal synaptic depression, altered sleep onset, and decreased electroencephalogram gamma power, 3 mg/kg still provided approximately 80% enzyme occupancy, significantly increased 2-AG and norepinephrine levels, and produced neuropathic antinociception without synaptic depression or decreased gamma power. Thus, it is anticipated that the profile exhibited by this compound will allow for precise modulation of 2-AG levels in vivo, supporting potential therapeutic application in several central nervous system disorders. SIGNIFICANCE STATEMENT: Potentiation of endocannabinoid signaling activity via inhibition of the serine hydrolase monoacylglycerol lipase (MAGL) is an appealing strategy in the development of treatments for several disorders, including ones related to mood, pain, and inflammation. [1-(4-Fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone is presented in this report to be a novel, potent, selective, and reversible noncovalent MAGL inhibitor that demonstrates dose-dependent enhancement of the major endocannabinoid 2-arachidonoylglycerol as well as efficacy in models of neuropathic and inflammatory pain.

Design, synthesis and biological evaluation of new embelin derivatives as CK2 inhibitors.

A new series of furan embelin derivatives was synthesized and characterized as ATP-competitive CK2 inhibitors. The new compounds were efficiently synthesized using a multicomponent approach from embelin (1), aldehydes and isonitriles through a Knoevenagel condensation/Michael addition/heterocyclization. Several compounds with inhibitory activities in the low micromolar or even submicromolar were identified. The most active derivative was compound 4l (2-(tert-butylamino)-3-(furan-3-yl)-5-hydroxy-6-undecylbenzofuran-4,7-dione) with an IC50 value of 0.63 µM. It turned out to be an ATP competitive CK2 inhibitor with a Ki value determined to be 0.48 µM. Docking studies allowed the identification of key ligand-CK2 interactions, which could help to further optimize this family of compounds as CK2 inhibitors.