Flavonoids as Human Intestinal α-Glucosidase Inhibitors

Certain flavonoids can influence glucose metabolism by inhibiting enzymes involved in carbohydrate digestion and suppressing intestinal glucose absorption. In this study, four structurally-related flavonols (quercetin, kaempferol, quercetagetin and galangin) were evaluated individually for their ability to inhibit human α-glucosidases (sucrase, maltase and isomaltase), and were compared with the antidiabetic drug acarbose and the flavan-3-ol(−)-epigallocatechin-3-gallate (EGCG). Cell-free extracts from human intestinal Caco-2/TC7 cells were used as the enzyme source and products were quantified chromatographically with high accuracy, precision and sensitivity. Acarbose inhibited sucrase, maltase and isomaltase with IC50 values of 1.65, 13.9 and 39.1 µM, respectively. A similar inhibition pattern, but with comparatively higher values, was observed with EGCG. Of the flavonols, quercetagetin was the strongest inhibitor of α-glucosidases, with inhibition constants approaching those of acarbose, followed by galangin and kaempferol, while the weakest were quercetin and EGCG. The varied inhibitory effects of flavonols against human α-glucosidases depend on their structures, the enzyme source and substrates employed. The flavonols were more effective than EGCG, but less so than acarbose, and so may be useful in regulating sugar digestion and postprandial glycaemia without the side effects associated with acarbose treatment.

Alternative Approach for Specific Tyrosinase Inhibitor Screening: Uncompetitive Inhibition of Tyrosinase by Moringa oleifera

Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.

Functional and Pharmacological Comparison of Human and Mouse Na+/Taurocholate Cotransporting Polypeptide (NTCP)

The Na+/taurocholate cotransporting polypeptide (NTCP) is located in the basolateral membrane of hepatocytes, where it transports bile acids from the portal blood back into hepatocytes. Furthermore, NTCP has a role for the hepatic transport of some drugs. Extrapolation of drug transport data from rodents to humans is not always possible, because species differences in the expression level, localization, affinity, and substrate selectivity of relevant transport proteins must be considered. In the present study, a functional comparison of human NTCP (hNTCP) and mouse Ntcp (mNtcp) showed similar Km values of 67 ± 10 µM and 104 ± 9 µM for the probe substrate estrone-3-sulfate as well as of 258 ± 42 µM and 199 ± 13 µM for the drug rosuvastatin, respectively. IC50 values for the probe inhibitor cyclosporine A were 3.1 ± 0.3 µM for hNTCP and 1.6 ± 0.4 µM for mNtcp. In a drug and pesticide inhibitory screening on both transporters, 4 of the 15 tested drugs (cyclosporine A, benzbromarone, MK571, and fluvastatin) showed high inhibitory potency, but only slight inhibition was observed for the 13 tested pesticides. Among these compounds, only four drugs and three pesticides showed significant differences in their inhibition pattern on hNTCP and mNtcp. Most pronounced was the difference for benzbromarone with a fivefold higher IC50 for mNtcp (27 ± 10 µM) than for hNTCP (5.5 ± 0.6 µM). In conclusion, we found a strong correlation between the transport kinetics and inhibition pattern among hNTCP and mNtcp. However, specific compounds, such as benzbromarone, showed clear species differences. Such species differences have to be considered when pharmacokinetic data are transferred from rodent to humans.

Molecular docking studies of tea (Thea sinensis Linn.) polyphenols inhibition pattern with Rat P-glycoprotein

Since 3000 B.C., evergreen plant Thea sinensis (Theaceae) is used both as a social and medicinal beverage. Leaves of T. sinensis contain amino acids, vitamins, caffeine, polysaccharides and polyphenols. Most of the natural medicinal actions of tea are due to the availability and abundance of polyphenols mainly catechins. It has also been stated that some catechins were absorbed more rapidly than other compounds after the oral administration of tea and could increase the bio-enhancing activities of anticancer drugs by inhibiting P-glycoprotein (P-gp). The results of the molecular docking showed that polyphenols bind easily to the active P-gp site. All compounds exhibited fluctuating binding affinity ranged from −11.67 to −8.36 kcal/mol. Observed binding energy required for theaflavin to bind to P-gp was lowest (−11.67 kcal/mol). The obtained data that supports all the selected polyphenols inhibited P-gp and therefore may enhance the bioavailability of drugs. This study may play a vital role in finding hotspots in P-gp and eventually may be proved useful in designing compounds with high affinity and specificity to the protein.

All-Trans-Retinoic Acid Prevents Carfilzomib-Induced Cardiotoxicity By Decreasing Activation of the Renin-Angiotensin System

INTRODUCTION Bortezomib (BTZ), Carfilzomib (CFZ) and Ixazomib (IXA) are proteasome inhibitors (PI) approved for Multiple Myeloma (MM) treatment. By design, they all target the rate-limiting proteasome ß5 subunit. CFZ treatment increases the survival of patients with relapsed/refractory MM compared to BTZ. However, CFZ treatment is associated with heart failure not commonly observed for BTZ. The molecular basis for CFZ-induced cardiotoxicity is poorly understood. We have shown before that CFZ co-inhibits the ß5 and ß2 proteasome subunits compared to co-inhibition of the ß5/ß1 subunits by BTZ. We hypothesized that the unique ß5/ß2 subunit inhibition pattern of CFZ likewise explains the CFZ related acute cardiotoxicity. METHODS Isolated primary murine cardiomyocytes treated with BTZ, CFZ or ß5, ß2 and ß1 subunit-selective PI were used as in vitro model. Selectivity of proteasome subunit inhibition was confirmed with activity based chemical probes. Cardiomyocyte contractility was assessed in vitro using motion vector image analysis. The accumulation of individual proteins in cardiomyocytes undergoing 1h treatment with different PI was analyzed by liquid chromatography/mass spectrometry (LC/MS). In vivo analysis of the effects of differential proteasome subunit inhibition patterns was performed by electrocardiography (ECG) monitoring and untargeted metabolomics analysis from murine heart and plasma using R v.3.5.1 (2018-07-02) and Matlab 2020a. RESULTS Co-inhibition of ß5/ß2 proteasome subunits by CFZ or by respective subunit-specific PI for 1h resulted in increased proteasome inhibition and proteotoxic stress in cardiomyocytes compared to ß5/ß1 inhibition induced by BTZ. Likewise, CFZ or the ß5/ß2 type co-inhibition of the proteasome impaired contractility of cardiomyocytes in vitro, in contrast to BTZ or to ß5/ß1-selective proteasome inhibition. LC/MS-based proteome analysis of murine primary cardiomyocytes treated with either CFZ-type or BTZ-type proteasome inhibition identified accumulation of Retinal Dehydrogenase 1 (ALDH1A1) in PI-treated cardiomyocytes, with CFZ showing a slightly stronger effect. ALDH1A1 is involved in retinoic acid metabolism. Co-treatment of cardiomyocytes with its enzymatic end-product, all-trans-Retinoic-Acid (atRA), restored contractility of cardiomyocytes undergoing CFZ treatment in vitro. Leucyl-Cistinyl Aminopeptidase (LCAP) was identified as the protein that accumulated in CFZ-versus BTZ-treated cardiomyocytes (Log2 CFZ = 1.1, Log2 BTZ = -0.49, p=0.002). LCAP is involved in the Renin-Angiotensin System (RAS) and its increase suggests selective activation of RAS with increased degradation of RAS metabolites via LCAP in cardiomyocytes undergoing CFZ treatment. Indeed, mRNA expression for angiotensin converting enzyme 2 (Ace2) and angiotensin receptor 1 (Agtr1) was increased in beating cardiomyocytes treated with CFZ. Conversely, treatment with aminopeptidase inhibitor Bestatin prevented CFZ-induced cardiomyocyte toxicity in vitro. Consistent with our model, co-treatment of cardiomyocytes with CFZ and Valsartan, an Agtr1 inhibitor, likewise prevented CFZ- induced toxicity. In vivo, 1h CFZ treatment selectively led to acute bradycardia with local activation of the RAS and accumulation of angiotensin in the heart. CFZ/atRA co-treatment decreased local angiotensin levels and prevented CFZ-induced bradycardia. There were no differences in angiotensin plasma levels in between the treated mice, suggesting that the local RAS in the heart rather than systemic effects of the RAS mediates the cardiotoxicity of CFZ. CONCLUSION Our data suggest that CFZ specifically impairs cardiac contractility in a dose-dependent manner that is caused by its unique ß5/ß2 proteasome subunit co-inhibition pattern. CFZ leads to more efficient functional proteasome inhibition in cardiomyocytes, resulting in accumulation of LCAP that subsequently activates the local RAS and induce acute cardiotoxicity. Based on our findings, CFZ-induced cardiotoxicity may be prevented or mitigated by either blocking RAS activation at the angiotensin II receptor level using selective drugs like Valsartan, or by reducing angiotensin levels with atRA. This provides a rational strategy to prevent CFZ-induced acute cardiac toxicity by modulating the activity of the local RAS with approved drugs. Disclosures No relevant conflicts of interest to declare.

The head direction circuit of two insect species

Recent studies of the Central Complex in the brain of the fruit fly have identified neurons with activity that tracks the animal’s heading direction. These neurons are part of a neuronal circuit with dynamics resembling those of a ring attractor. The homologous circuit in other insects has similar topographic structure but with significant structural and connectivity differences. We model the connectivity patterns of two insect species to investigate the effect of these differences on the dynamics of the circuit. We illustrate that the circuit found in locusts can also operate as a ring attractor but differences in the inhibition pattern enable the fruit fly circuit to respond faster to heading changes while additional recurrent connections render the locust circuit more tolerant to noise. Our findings demonstrate that subtle differences in neuronal projection patterns can have a significant effect on circuit performance and illustrate the need for a comparative approach in neuroscience.

Amaryllidaceae Alkaloids of Belladine-Type from Narcissus pseudonarcissus cv. Carlton as New Selective Inhibitors of Butyrylcholinesterase

Thirteen known (1–12 and 16) and three previously undescribed Amaryllidaceae alkaloids of belladine structural type, named carltonine A-C (13–15), were isolated from bulbs of Narcissus pseudonarcissus cv. Carlton (Amaryllidaceae) by standard chromatographic methods. Compounds isolated in sufficient amounts, and not tested previously, were evaluated for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7), butyrylcholinesterase (BuChE; E.C. 3.1.1.8) and prolyl oligopeptidase (POP; E.C. 3.4.21.26) inhibition activities. Significant human BuChE (hBUChE) inhibitory activity was demonstrated by newly described alkaloids carltonine A (13) and carltonine B (14) with IC50 values of 913 ± 20 nM and 31 ± 1 nM, respectively. Both compounds displayed a selective inhibition pattern for hBuChE with an outstanding selectivity profile over AChE inhibition, higher than 100. The in vitro data were further supported by in silico studies of the active alkaloids 13 and 14 in the active site of hBuChE.

Carfilzomib Induces Cardiotoxicity Via ß5/ß2-Specific Proteasome Subunit Inhibition Pattern

BACKGROUND All proteasome inhibitors (PI) approved for Multiple Myeloma (MM) treatment, Bortezomib (BTZ), Ixazomib (IXA) and Carfilzomib (CFZ), by design target the rate-limiting ß5 proteolytic proteasome subunit, but differ in their co-inhibition of the ß1 or ß2 proteolytic subunits at higher doses. CFZ is unique in inducing ß5/ß2 co-inhibition in a dose-dependent manner, which results in increased proteotoxic stress and higher cytotoxicity compared to the ß5/ß1 co-inhibition pattern mediated by BTZ or IXA. CFZ treatment, in particular at higher doses, is not only associated with increased clinical activity, but also with significantly higher cardiac toxicity compared to BTZ. The molecular basis for this difference is poorly understood, and mechanism-based pharmacological alleviation strategies are lacking. We hypothesized that the acute cardiotoxicity of CFZ is related to its unique ß5/ß2 inhibition pattern. METHODS Isolated primary murine cardiomyocytes showing spontaneous rhythmical contractions were used to assess the functional cardiotoxicity of PI in vitro. Cardiomyocytes were treated with the PI BTZ, CFZ or with highly-selective, subunit (ß1, ß2, ß5)-specific PI for 1h. Proteasome inhibition was directly verified using subunit-selective activity-based chemical probes. Contractility was assessed with motion vector image analysis, and calcium transients were examined by confocal microscopy after transduction of cardiomyocytes with a GCaMP vector. Differentially accumulated proteins after 1h PI treatment were quantitatively compared by liquid chromatography/mass spectrometry (LC/MS). In vivo, electrical changes and alterations in heart rate were monitored by electrocardiography (ECG). Calcium transients, ECG signals and LC/MS data were analyzed with R software version 3.5.1 (2018-07-02). RESULTS Treatment of cardiomyocytes with CFZ or with the combination of ß5/ß2-targeting subunit-specific PI impaired contractility in vitro in contrast to BTZ, or to co-inhibition of ß5/ß1 proteasome subunits. In vivo, the CFZ-type proteasome inhibition triggered acute bradycardia. Treatment of cardiomyocytes with CFZ induced a shift of intracellular calcium pools from the endoplasmic reticulum (ER) to the cytosol, consistent with ER-to-cytosol translocation of calcium described upon ER stress induction. Co-treatment with CFZ and cycloheximide, an inhibitor of protein synthesis, rescued cardiomyocytes from CFZ-induced functional impairment in vitro, suggesting that the accumulation of specific proteins is involved in CFZ-induced cardiomyocyte dysfunction. Quantitative proteomic comparison of primary cardiomyocytes treated by either CFZ-type or BTZ-type proteasome inhibition for 1h identified a selective accumulation of proteins of the retinoic acid pathway in CFZ-treated cardiomyocytes. Interestingly, co-treatment of cardiomyocytes with all-trans-retinoic acid (ATRA) prevented CFZ-induced acute cardiotoxicity in vitro. CONCLUSION Our data suggests that CFZ specifically impairs cardiac contractility through its unique ß5/ß2 proteasome subunit inhibition pattern, which results in more effective functional proteasome inhibition, protein accumulation and proteotoxic stress. The shift of intracellular calcium pools in cardiomyocytes upon CFZ treatment mirrors contractility impairment, and the specific changes identified in the retinoic acid pathway suggest ATRA as a potential drug candidate to alleviate CFZ-induced cardiac toxicity. Disclosures No relevant conflicts of interest to declare.

STEADY STATE AND SENSITIVITY ANALYSIS OF A NOTCH–DELTA SIGNALING SYSTEM OF ONE SINGLE CELL INTERACTING WITH FIXED ENVIRONMENT

The Notch–Delta signaling pathway is a highly conserved signaling system that partakes in a diverse process of growth, patterns and differentiation. Experiments have shown that Delta from different cells activates this pathway (trans-activation) while Delta from the same cell inhibits this pathway (cis-inhibition). The Notch–Delta interactions could switch a cell to one of the two opposite fates: either Sender (high Delta/low Notch) or Receiver (low Delta/high Notch). We studied a Notch–Delta signaling model from Sprinzak et al., (2010), to investigate the cell fate through steady state analysis. The focus was placed on a fundamental case of one single cell with fixed external Delta and Notch supplies. First, we proved there exists a unique steady state which is asymptotically stable. Second, we derived the increasing/decreasing and asymptotic properties of the steady state with respect to all the parameters. Third, we studied the sensitivity and discovered the cell fate is only sensitive to the production rates of Notch and Delta under strong cis-inhibition. Finally, we applied this model to multi-cellular cases and found that the lateral inhibition pattern could be created with the spatially varied Delta production rate. The Hopf bifurcation is not observed in the current model.