TIMP-1 Protects Tight Junctions of Brain Endothelial Cells From MMP-Mediated Degradation.

OBJECTIVE: The blood-brain barrier (BBB) plays a critical role in central nervous system homeostasis, and the integrity of BBB is disrupted in many neurodegenerative diseases. Matrix metalloproteinases (MMPs) degrade the tight junctions (TJs) of endothelial cells and basement membrane components essential to BBB integrity, which leads to increased BBB permeability and allows inflammatory cells and neurotoxic substances to enter the brain. Tissue inhibitors of metalloproteinases (TIMPs), endogenous inhibitors of MMPs, regulate MMP activity, thereby maintaining BBB integrity. METHODS: The disruptive impacts of MMP-3 and MMP-9 on BBB and protective effect of TIMP-1 were investigated in a simplified in vitro model of the BBB, which was generated using rat brain microvascular endothelial cells (RBMEC). The main features of BBB formation, including permeability and the trans-endothelial electrical resistance (TEER), were monitored over time after the addition of MMP-3 and MMP-9 and their complexes with TIMP-1 inhibitor. RESULTS: Our results indicated that MMP-3 and MMP-9 caused a dose-dependent disruption of the BBB, with 1.5 µM MMPs resulting in an over threefold increase in permeability, while TIMP-1 inhibition protected the integrity of the BBB model and recovered TEER and permeability of RBMECs. The disruption and recovery of tight junction proteins of RBMECs after MMP and TIMP treatment were also detected using fluorescent microscopy. CONCLUSION: MMP-9 and MMP-3 disrupt the BBB by degrading tight junctions in endothelial cells, and TIMP-1 could inhibit the disruptive effect of MMP-3 and MMP-9 by showing potential as therapeutic protein against MMP-related diseases where BBB disruption plays a role.

Cannabinoid and Opioid Receptor Affinity and Modulation of Cancer-Related Signaling Pathways of Machaeriols and Machaeridiols from Machaerium Pers.

Machaeriols and machaeridiols are unique hexahydrodibenzopyran-type aralkyl phytocannabinoids isolated from Machaerium Pers. Earlier studies of machaeriol A (1) and B (2) did not show any affinity for cannabinoid receptor 1 (CB1 or CNR1), although they are structural analogs of psychoactive hexahydrocannabinol. This study comprehensively reports on the affinities of isolated Machaerium Pers. compounds, namely machaeriol A-D (1-4) and machaeridiol A-C (5-7), against cannabinoid (CB1 and CB2) and opioid (κ, δ and µ) receptors. Among the isolated compounds, machaeriol D (4) and machaeridiol A-C (5-7) showed some selective binding affinity for the CB2 receptor, using a radioligand binding assay, with Ki values of >1.3, >1.77, >2.18 and >1.1 µM, respectively. On the other hand, none of the compounds showed any binding to the CB1 receptor. Due to recent reports on the anticancer potential of the endocannabinoid system, compounds 1-7 were tested against a battery of luciferase reporter gene vectors that assess the activity of many cancer-related signaling pathways, including Stat3, Smad2/3, AP-1, NF-κB, E2F, Myc, Ets, Notch, FoxO, Wnt, Hedgehog and pTK in HeLa and T98G glioblastoma cells. Complete dose-response curves have been determined for each compound in both of these cell lines, which revealed that machaeridiol 6 displayed activities (IC50 in µM in HeLa and T98G cells) towards Stat3 (4.7, 1.4), Smad2/3 (1.2, 3.0), AP-1 (5.9, 4.2), NF-κB (0.5, 4.0), E2F (5.7, 0.7), Myc (5.3, 2.0), ETS (inactive, 5.9), Notch (5.3, 4.6), Wnt (4.2, inactive) and Hedgehog (inactive, 5.0). Furthermore, a combination study between machaeriol C (3) and machaeridiol B (6) displayed additive effects for E2F, ETS, Wnt and Hedgehog pathways, where these compounds individually were either minimally active or inactive. None of the compounds inhibited luciferase expression driven by the minimal thymidine kinase promoter (pTK), indicating the lack of general cytotoxicity for luciferase enzyme inhibition at the 50 µM concentration in both of these cell lines. The significance of the inhibition of these signaling pathways via machaeridiol 5-7 and their cross-talk potential has been discussed.

In Vitro and In Silico Studies of Neolignans from Magnolia grandiflora L. Seeds against Human Cannabinoids and Opioid Receptors.

Magnolia grandiflora L. (Magnoliaceae) is a plant of considerable medicinal significance; its flowers and seeds have been used in various traditional remedies. Radioligand binding assays of n-hexane seeds extract showed displacement of radioligand for cannabinoid (CB1 and CB2) and opioid δ (delta), κ (kappa), and µ (mu) receptors. Bioactivity-guided fractionation afforded 4-O-methylhonokiol (1), magnolol (2), and honokiol (3), which showed higher binding to cannabinoid rather than opioid receptors in radioligand binding assays. Compounds 1-3, together with the dihydro analog of 2 (4), displayed selective affinity towards CB2R (Ki values of 0.29, 1.4, 1.94, and 0.99 µM, respectively), compared to CB1R (Ki 3.85, 17.82, 14.55, and 19.08 µM, respectively). An equal mixture of 2 and 3 (1:1 ratio) showed additive displacement activity towards the tested receptors compared to either 2 or 3 alone, which in turn provides an explanation for the strong displacement activity of the n-hexane extract. Due to the unavailability of an NMR or X-ray crystal structure of bound neolignans with the CB1 and CB2 receptors, a docking study was performed to predict ligand-protein interactions at a molecular level and to delineate structure-activity relationships (SAR) of the neolignan analogs with the CB1 and CB2 receptors. The putative binding modes of neolignans 1-3 and previously reported related analogs (4, 4a, 5, 5a, 6, 6a, and 6b) into the active site of the CB1 and CB2 receptors were assessed for the first time via molecular docking and binding free-energy (∆G) calculations. The docking and ∆G results revealed the importance of a hydroxyl moiety in the molecules that forms strong H-bonding with Ser383 and Ser285 within CB1R and CB2R, respectively. The impact of a shift from a hydroxyl to the methoxy group on experimental binding affinity to CB1R versus CB2R was explained through ∆G data and the orientation of the alkyl chain within the CB1R. This comprehensive SAR, influenced by the computational study and the observed in vitro displacement binding affinities, has indicated the potential of magnolia neolignans for developing new CB agonists for potential use as analgesics, anti-inflammatory agents, or anxiolytics.

Novel Machaeriol Analogues as Modulators of Cannabinoid Receptors: Structure-Activity Relationships of (+)-Hexahydrocannabinoids and Their Isoform Selectivities.

Machaeriols are an important class of compounds that structurally resemble tetrahydrocannabinol (Δ9-THC), with the major differences being inverted stereochemistry at the ring junction as [6aR, 10aR] and an additional stereocenter at the C9 position of the A-ring due to saturation. A previous study reported that machaeriols did not show any cannabinoid receptor activity, even though these hexahydrodibenzopyran analogues mimic a privileged (+)-tetrahydrocannabinoid scaffold. To unravel structural requisites for modulation of cannabinoid receptors, a simple late-stage divergent approach was undertaken to functionalize the machaeriol scaffold using the Suzuki coupling reaction. Fourteen hexahydro analogues were synthesized and screened against both cannabinoid receptor isoforms, CB1 and CB2. Interestingly, many of the analogues showed a significant binding affinity for both receptors; however, two analogues, 11H and 11J, were identified as possessing CB2 receptor-selective functional activity in the GTPγS assay; they were found to be micromolar-range agonists, with EC50 values of 5.7 and 16 µM, respectively. Furthermore, molecular dynamics simulations between the CB2 receptor and two novel analogues resulted in unique interaction profiles by tightly occupying the active ligand-binding domain of the CB2 receptor and maintaining stable interactions with the critical residues Phe94, Phe281, and Ser285. For the first time, with the aid of structure-activity relationships of (+)-hexahydrocannabinoids, CB2 selective agonists were identified with late-stage diversification using palladium-mediated C-C bond formation. By simply switching to (R)-citronellal as a chiral precursor, enantiomerically pure (-)-hexahydrocannabinoids with better CB1/CB2 receptor isoform selectivity can be obtained using the current synthetic approach.

In vitro opioid receptor affinity and in vivo behavioral studies of Nelumbo nucifera flower.

ETHNOPHARMACOLOGICAL RELEVANCE: Nelumbo nucifera Geartn., known as sacred lotus, has been used traditionally in South East Asia as a traditional medicine for various CNS disorders including stress, fever, depression, insomnia, and cognitive conditions. AIM OF THE STUDY: To investigate the in vitro cannabinoid and opioid receptor binding affinities, and in vivo behavioral actions of Nelumbo flower extracts and to isolate the potential compounds to treat CNS associated disorders. MATERIALS AND METHODS: The white and pink flowers of N. nucifera were extracted with 95% EtOH, followed by acid-base partitioning using CHCl3 to give acidic and basic partitions. These partitions were subjected to Centrifugal Preparative TLC (CPTLC) to yield benzyltetrahydroisoquinoline (BTIQ) alkaloids and long chain fatty acids, identified by physical and spectroscopic methods. In addition, EtOH extracts and partitions were analyzed for chemical markers by UHPLC/MS and GC/MS. In vitro neuropharmacological effects were evaluated by cannabinoid (CB1 and CB2) and opioid [delta (δ), kappa (ĸ), and mu (µ)] competitive radioligand binding and GTPγS functional assays. The in vivo behavioral effect was studied through the use of the mouse tetrad assay at 10, 30, 75 and 100mg/kg/ip doses that revealed the effect on locomotion, catalepsy, body temperature, and nociception of acidic and basic CHCl3 partitions, fractions, and compounds. RESULTS: Three aporphines, nuciferine (1), N-nor-nuciferine (2), asimilobine (3), and five BTIQs, armepavine (4), O-methylcoclaurine (5), N-methylcoclaurine (6), coclaurine (7), neferine (10), and a mixture of linoleic and palmitic acids (LA and PA), were identified and evaluated for cannabinoid and opioid receptor displacement activities. Compounds 5-7 showed binding affinities for the ĸ opioid receptor with equilibrium dissociation constant (Ki) values of 3.5 ± 0.3, 0.9 ± 0.1, 2.2 ± 0.2 µM, respectively. Compound 10 displayed affinities for δ-and µ- opioid receptors with Ki values of 0.7 ± 0.1 and 1.8 ± 0.2 µM, respectively, and was determined to be a weak δ agonist by GTPγS functional assay. The mixture of LA and PA (1:1) showed an affinity for δ opioid receptor with a Ki value of 9.2 ± 1.1 µM. The acidic and basic CHCl3 partitions, compounds 1 and 7, and 5-7 mixture were subjected to the tetrad assay, of which the acidic partition displayed decreased locomotion and increased catalepsy, antinociception, and hypothermia in animal at doses of 75-100 mg/kg/ip, and also showed clonic-tonic seizures upon touch at 100mg/kg. CONCLUSION: Bioassay-guided isolation revealed compounds 5-7, 10, and the mixture of LA and PA displayed various degrees of opioid receptor radioligand displacement affinities. The in vivo tetrad assay of acidic CHCl3 partition, enriched with aporphines 1 and 2, displayed actions on all four points of behavioral parameters. It can be concluded that the in vivo mild canabimimetic-type effect observed for the CHCl3 partition is likely mediated through other CNS mechanisms since the extracts, partitions, and isolated compounds had no affinity for the in vitro CB1 and CB2 receptors. This work, along with traditional use and the reported bioactivities of the BTIQ alkaloids, suggested further studies on N. nucifera are needed to understand the roles that the extracts and/or individual compounds might contribute to the behavioral effects.

Longitudinal analysis of vaginal microbiome dynamics in women with recurrent bacterial vaginosis: recognition of the conversion process.

Bacterial vaginosis (BV) affects ∼ 30% of women of reproductive age, has a high rate of recurrence, and is associated with miscarriage, preterm birth, and increased risk of acquiring other sexually transmitted infections, including HIV-1. Little is known of the daily changes in the vaginal bacterial composition as it progresses from treatment to recurrence, or whether any of these might be useful in its prediction or an understanding of its causes. We used phylogenetic branch-inclusive quantitative PCR (PB-qPCR) and Lactobacillus blocked/unblocked qPCR (Lb-qPCR) to characterize longitudinal changes in the vaginal microbiota in sequential vaginal self-swabs from five women with recurrent BV, from diagnosis through remission to recurrence. Both patients with acute BV samples dominated by G. vaginalis recurred during the study with similar profiles, whereas the three patients with acute BV samples dominated by other anaerobes did not recur or recurred to an intermediate Nugent score. L. iners dominated remission phases, with intermittent days of abnormal microbial profiles typically associated with menses. The exception was a newly discovered phenomenon, a sustained period of abnormal profiles, termed conversion, which preceded symptomatic acute BV. Species known to have antagonistic activity towards Lactobacillus were detected in pre-conversion samples, possibly contributing to the decline in Lactobacillus. Lb-qPCR scores define two categories of response in the initial post-treatment visit samples; scores <5 may correspond with poor response to treatment or rapid recurrence, whereas scores >8 may predict delayed or no recurrence. Amsel criteria or Nugent scores did not have this potential predictive capability. Larger studies are warranted to evaluate the prognostic potential of detecting conversion and poor Lb-qPCR scores at the post-treatment visit of recurrent BV patients.

Novel PCR-based methods enhance characterization of vaginal microbiota in a bacterial vaginosis patient before and after treatment.

Deep characterization, even by next-generation sequencing, of the vaginal microbiota in healthy women or posttreatment bacterial vaginosis patients is limited by the dominance of lactobacilli. To improve detection, we offer two approaches: quantitative PCR (qPCR) using phylogenetic branch-inclusive primers and sequencing of broad-spectrum amplicons generated with oligomers that block amplification of lactobacilli.