Syrosingopine Enhances 20S Proteasome Activity and Degradation of α-Synuclein.

Cellular proteins are degraded by the 26S proteasome in the ubiquitin-proteasome system in an ATP-dependent manner, whereas intrinsically disordered proteins (IDPs) are degraded by the 20S proteasome independent of ATP and ubiquitin. The accumulation and aggregation of IDPs are considered to be the etiology of neurodegenerative diseases. Notably, the 20S proteasome has a cylindrical structure, and its gate on the α-ring is closed in the inactive form. The compounds that open the gate promote the degradation of IDPs and prevent their accumulation, and therefore, such compounds may be promising therapeutic agents for neurodegenerative diseases. After screening the Prestwick Phytochemical Library, several yohimbine-type and ergot alkaloids were identified that enhance the 20S proteasome activity. Among them, syrosingopine was the most potent activator of the 20S proteasome and enhanced the degradation of fluorogenic substrates and α-synuclein, an IDP. Furthermore, in HeLa cells, syrosingopine enabled the binding of a membrane-permeable fluorescent probe to the catalytic site of the 20S proteasome by opening the gate.

Decomposition Profile Data Analysis for Deep Understanding of Multiple Effects of Natural Products.

It is difficult to understand the entire effect of a natural product because such products generally have multiple effects. We propose a strategy to understand these effects effectively by decomposing them with a profile data analysis method we developed. A transcriptome profile data set was obtained from a public database and analyzed. Considering their high similarity in structure and transcriptome profile, we focused on rescinnamine and syrosingopine. Decomposed effects predicted clear differences between the compounds. Two of the decomposed effects, SREBF1 activation and HDAC inhibition, were investigated experimentally because the relationship between these effects and the compounds had not yet been reported. Analyses in vitro validated these effects, and their strength was consistent with predicted scores. Moreover, the number of outliers in decomposed effects per compound was higher in natural products than in drugs in the data set, which is consistent with the nature of the effects of natural products.

A Holistic Evolutionary and 3D Pharmacophore Modelling Study Provides Insights into the Metabolism, Function, and Substrate Selectivity of the Human Monocarboxylate Transporter 4 (hMCT4).

Monocarboxylate transporters (MCTs) are of great research interest for their role in cancer cell metabolism and their potential ability to transport pharmacologically relevant compounds across the membrane. Each member of the MCT family could potentially provide novel therapeutic approaches to various diseases. The major differences among MCTs are related to each of their specific metabolic roles, their relative substrate and inhibitor affinities, the regulation of their expression, their intracellular localization, and their tissue distribution. MCT4 is the main mediator for the efflux of L-lactate produced in the cell. Thus, MCT4 maintains the glycolytic phenotype of the cancer cell by supplying the molecular resources for tumor cell proliferation and promotes the acidification of the extracellular microenvironment from the co-transport of protons. A promising therapeutic strategy in anti-cancer drug design is the selective inhibition of MCT4 for the glycolytic suppression of solid tumors. A small number of studies indicate molecules for dual inhibition of MCT1 and MCT4; however, no selective inhibitor with high-affinity for MCT4 has been identified. In this study, we attempt to approach the structural characteristics of MCT4 through an in silico pipeline for molecular modelling and pharmacophore elucidation towards the identification of specific inhibitors as a novel anti-cancer strategy.

Nature and health.

Urbanization, resource exploitation, and lifestyle changes have diminished possibilities for human contact with nature in urbanized societies. Concern about the loss has helped motivate research on the health benefits of contact with nature. Reviewing that research here, we focus on nature as represented by aspects of the physical environment relevant to planning, design, and policy measures that serve broad segments of urbanized societies. We discuss difficulties in defining "nature" and reasons for the current expansion of the research field, and we assess available reviews. We then consider research on pathways between nature and health involving air quality, physical activity, social cohesion, and stress reduction. Finally, we discuss methodological issues and priorities for future research. The extant research does describe an array of benefits of contact with nature, and evidence regarding some benefits is strong; however, some findings indicate caution is needed in applying beliefs about those benefits, and substantial gaps in knowledge remain.

AC-73 and Syrosingopine Inhibit SARS-CoV-2 Entry into Megakaryocytes by Targeting CD147 and MCT4.

Coagulation disorders are described in COVID-19 and long COVID patients. In particular, SARS-CoV-2 infection in megakaryocytes, which are precursors of platelets involved in thrombotic events in COVID-19, long COVID and, in rare cases, in vaccinated individuals, requires further investigation, particularly with the emergence of new SARS-CoV-2 variants. CD147, involved in the regulation of inflammation and required to fight virus infection, can facilitate SARS-CoV-2 entry into megakaryocytes. MCT4, a co-binding protein of CD147 and a key player in the glycolytic metabolism, could also play a role in SARS-CoV-2 infection. Here, we investigated the susceptibility of megakaryocytes to SARS-CoV-2 infection via CD147 and MCT4. We performed infection of Dami cells and human CD34+ hematopoietic progenitor cells induced to megakaryocytic differentiation with SARS-CoV-2 pseudovirus in the presence of AC-73 and syrosingopine, respective inhibitors of CD147 and MCT4 and inducers of autophagy, a process essential in megakaryocyte differentiation. Both AC-73 and syrosingopine enhance autophagy during differentiation but only AC-73 enhances megakaryocytic maturation. Importantly, we found that AC-73 or syrosingopine significantly inhibits SARS-CoV-2 infection of megakaryocytes. Altogether, our data indicate AC-73 and syrosingopine as inhibitors of SARS-CoV-2 infection via CD147 and MCT4 that can be used to prevent SARS-CoV-2 binding and entry into megakaryocytes, which are precursors of platelets involved in COVID-19-associated coagulopathy.

Observation on the clinical effect of thunder-fire moxibustion combined with acupressure on ocular muscle spasm: A clinical randomized controlled trial.

INTRODUCTION: With the rapid development of social economy, peoples dependence on computers and mobile phones is increasing day by day. This causes people to often overuse. Therefore, the incidence of Ocular muscle spasm has been increasing year by year in recent years. The disease usually starts and hides, which seriously affects the patients social image, daily life, and work. METHODS/DESIGN: We will compare the clinical efficacy of thunder-fire moxibustion combined with acupressure with pure thunder-fire moxibustion on Ocular muscle spasm using random control method. DISCUSSION: We aim to find a simple, safe, simple and effective Chinese medicine nursing technology that relieves Ocular muscle spasm. TRIAL REGISTRATION: ClinicalTrials.gov,ChiCTR2000034187, Registered on 27 June 2020.

Effects of vitamin D supplementation on cardiovascular risk factors in shift workers: Study protocol for randomized, double-blind, placebo-controlled clinical trial.

OBJECTIVE: The present clinical study aims to describe protocol to evaluate the effects of vitamin D3 supplementation on the cardiovascular risk factors in a population of rotating shift workers. DESIGN: A randomized, double-blind, placebo-controlled, parallel group clinical trial testing 2 oral dosages of cholecalciferol (14,000 IU and 28,000 IU per week) for 12 months. SETTING: The primary outcome for evaluation is an 18% reduction in hypertriglyceridemia (≥150 mg/dL) between pre and postintervention measurements. Baseline characteristics of the study population will be summarized separately within each randomized group, and will use tests for continuous and categorical variables. For all tests, a P < .05 will be considered significant. The analysis of primary and secondary outcomes will use an intention-to-treat population and a per-protocol population. The primary and secondary outcomes will be compared separately between each treatment group and placebo, using binary logistic regression or regressão de Poisson for proportions (for binary outcomes) and using linear regression for differences in means (for continuous endpoints), with 95% confidence intervals. PARTICIPANTS: Rotating shift workers, adults aged between 18 and 60 years, with hypovitaminosis D and alterations in at least 1 of the following parameters: fasting glucose, high-density lipoprotein cholesterol, triglycerides, low-density lipoprotein cholesterol, blood pressure, and waist circumference. CONCLUSION: This clinical trial aims to contribute to the gap in knowledge about the potential, dose, and time of vitamin D supplementation to generate beneficial effects on triglycerides in a population at increased risk for hypertriglyceridemia and vitamin D deficiency.

Clinical and Regulatory Aspects of Companion Diagnostic Development in Oncology.

Nearly 20 years have passed since the US Food and Drug Administration (FDA) approved the first companion diagnostic and today this type of assay governs the use of 21 different anticancer drugs. The regulators deem these assays essential for the safe and effective use of a corresponding therapeutic product. The companion diagnostic assays are important both during the drug development process as well as essential treatment decision tools after the approval of the drugs.

Dual Inhibition of the Lactate Transporters MCT1 and MCT4 Is Synthetic Lethal with Metformin due to NAD+ Depletion in Cancer Cells.

Highly glycolytic cancer cells prevent intracellular acidification by excreting the glycolytic end-products lactate and H+ via the monocarboxylate transporters 1 (MCT1) and 4 (MCT4). We report that syrosingopine, an anti-hypertensive drug, is a dual MCT1 and MCT4 inhibitor (with 60-fold higher potency on MCT4) that prevents lactate and H+ efflux. Syrosingopine elicits synthetic lethality with metformin, an inhibitor of mitochondrial NADH dehydrogenase. NAD+, required for the ATP-generating steps of glycolysis, is regenerated from NADH by mitochondrial NADH dehydrogenase or lactate dehydrogenase. Syrosingopine treatment leads to high intracellular lactate levels and thereby end-product inhibition of lactate dehydrogenase. The loss of NAD+ regeneration capacity due to combined metformin and syrosingopine treatment results in glycolytic blockade, leading to ATP depletion and cell death. Accordingly, ATP levels can be partly restored by exogenously provided NAD+, the NAD precursor nicotinamide mononucleotide (NMN), or vitamin K2. Thus, pharmacological inhibition of MCT1 and MCT4 combined with metformin treatment is a potential cancer therapy.

Toxicity of diethylaminoreserpine to a transplantable tumor: the significance of the presence of hypoxic cells.

The survival of clonogenic cells from two transplantable mouse tumors has been measured following i.p. injection of diethylaminoreserpine (DL-152) to the tumor-bearing mouse. Reduction of surviving fraction was seen for both tumors following injection of the drug, minimal numbers of surviving cells being seen from 24 to 48 hr after injection. Greater cell kill was observed for the KHT fibrosarcoma (surviving fraction = 3 x 10(-2) at 48 hr) than for the EMT6 mammary carcinoma (surviving fraction = 2 x 10(-1) at 48 hr). Reduction in surviving fraction of the KHT tumor was already observed at 1 hr after injection of the drug, and survival at that time was reduced if tumor cells were acutely hypoxic prior to excision of the tumor. Results also indicated that chronically hypoxic radioresistant cells were more sensitive to the drug than were aerated cells. Significant reduction in surviving fraction was seen for doses of DL-152 as low as 5 mg/kg. Using KHT tumors growing as lung nodules, it was shown that toxicity of DL-152 was not apparent until 11 to 14 days after initiation of the tumor and that subsequent sensitivity of cells to DL-152 increased with increasing age of the lung tumor. Hypoxic cells were detectable in 10-day-old lung tumors, a time at which the tumors were still resistant to the toxic effects of DL-152. In vitro experiments using KHT cells in suspension showed that a high concentration of DL-152 was toxic to both hypoxic and aerated cells but that hypoxic cells were more sensitive to lower concentrations than were aerated cells.

Homology modeling, molecular dynamics, and virtual screening of NorA efflux pump inhibitors of Staphylococcus aureus.

Emerging drug resistance in clinical isolates of Staphylococcus aureus might be implicated to the overexpression of NorA efflux pump which is capable of extruding numerous structurally diverse compounds. However, NorA efflux pump is considered as a potential drug target for the development of efflux pump inhibitors. In the present study, NorA model was constructed based on the crystal structure of glycerol-3-phosphate transporter (PDBID: 1PW4). Molecular dynamics (MD) simulation was performed using NAMD2.7 for NorA which is embedded in the hydrated lipid bilayer. Structural design of NorA unveils amino (N)- and carboxyl (C)-terminal domains which are connected by long cytoplasmic loop. N and C domains are composed of six transmembrane α-helices (TM) which exhibits pseudo-twofold symmetry and possess voluminous substrate binding cavity between TM helices. Molecular docking of reserpine, totarol, ferruginol, salvin, thioxanthene, phenothiazine, omeprazole, verapamil, nalidixic acid, ciprofloxacin, levofloxacin, and acridine to NorA found that all the molecules were bound at the large hydrophobic cleft and indicated significant interactions with the key residues. In addition, structure-based virtual screening was employed which indicates that 14 potent novel lead molecules such as CID58685302, CID58685367, CID5799283, CID5578487, CID60028372, ZINC12196383, ZINC72140751, ZINC72137843, ZINC39227983, ZINC43742707, ZINC12196375, ZINC66166948, ZINC39228014, and ZINC14616160 have highest binding affinity for NorA. These lead molecules displayed considerable pharmacological properties as evidenced by Lipinski rule of five and prophecy of toxicity risk assessment. Thus, the present study will be helpful in designing and synthesis of a novel class of NorA efflux pump inhibitors that restore the susceptibilities of drug compounds.

Syrosingopine sensitizes cancer cells to killing by metformin.

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer.

Molecular and biochemical characterization of a benzenoid/phenylpropanoid meta/para-O-methyltransferase from Rauwolfia serpentina roots.

The monoterpenoid indole alkaloids, reserpine and rescinnamine contain 3, 4, 5-trimethoxybenzoate or 3, 4, 5-trimethoxycinnamate, respectively, within their structures and they accumulate in different plant organs and particularly within roots of Rauwolfia serpentina. This plant also accumulates acylated sugars substituted with 3, 4, 5-trimethoxybenzoate and 3, 4, 5-trimethoxycinnamate. In the present study, transcriptome and metabolome analyses of R. serpentina roots allowed the identification of 7 candidate O-methytransferase (OMT) genes that might be associated with the formation of 3, 4, 5-trimethoxybenzoate and 3, 4, 5-trimethoxycinnamate and led to the molecular cloning of 4 genes for functional expression and analysis. Two candidate genes were expressed in E. coli and were shown to use different phenolics as methyl acceptors. RsOMT1, a member of the caffeoyl CoA-OMT-like family of genes, converted 3, 5 dimethoxy-4-hydroxycinnamic, caffeic and 3, 4, 5 trihydroxybenzoic acids to trimethoxycinnamic-, ferulic/isoferulic- and 3-methoxy, 4, 5 dihydroxybenzoic or 4-methoxy, 3, 5 dihydroxybenzoic acids, respectively, when supplied with these substrates. RsOMT3, a member of the caffeic acid-OMT-like family of genes, only converted caffeic acid to ferulic acid. Both enzymes showed considerable promiscuity with respect to various flavonoid substrates that they accepted. The para-O-methylation activity of RsOMT1 is quite rare and unusual for plant OMTs. The involvement of RsOMT1 and RsOMT3 in the assembly of trimethoxybenzoic and trimethoxycinnamic acids is discussed.

Colorimetric determination of indolic drugs.

A colorimetric method has been developed for the quantitative determination of the rescinnamine, reserpine upto (-10(-4M)), Yohimbine on complexation with bromothymol blue. The coloured complexes exhibit absorption maxima in the region 415-416 nm. The RSD (Relative Standard Deviation) of the method is 2.02%. The method is simple, easy, rapid and convenient for routine analysis of the indolic drugs.

Reserpine as an uncoupler of oxidative phosphorylation and the relevance to its psychoactive properties.

Many drugs differing widely in chemical structure uncouple mitochondrial oxidative phosphorylation in vitro. This observation has led to the hypothesis that in vivo uncoupling is the basis of their pharmacological activity. Serpasil, a parenteral preparation of reserpine, recently has been shown to uncouple oxidative phosphorylation in vervet monkey kidney mitochondria. Although the drug exhibits some properties of a "classical" uncoupler, our studies show that it has a dual effect on energy conservation. Reserpine released respiratory control in rat liver mitochondria only when dissolved in organic solvents (as in Serpasil) or when deprotonated. Reserpine also released the oligomycin-induced respiratory control in beef heart submitochondrial particles, and inhibited energized uptake of Ca2- by rat liver mitochondria. Reserpine had a dual effect on mitochondrial ATPase: It (a) enhanced ATP hydrolysis by intact liver mitochondria, and (b) inhibited ATP hydrolysis by submitochondrial particles of beef heart. On a molar basis, reserpine was less effective than carbonyl cyanide 3-chlorophenylhydrazone in all bioenergetic reactions examined. Homogenates and mitochondria isolated from brain and liver of rats stuporous from intraperitoneally injections of Serpasil exhibited no detectable abnormalities in respiratory states and responded to known uncouplers in the expected manner. There was no evidence of in vivo uncoupling of oxidative phosphorylation as a basis of the pharmacological activity of reserpine, although interference with energy transfer may be involved in toxic manifestations of the drug. The results indicate the need for caution in interpreting the action of drugs formulated in complex pharmaceutical preparations and based solely on in vitro experiments.

Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma.

A method based on ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed for the determination of reserpine in equine plasma. A comparison was made of the isolation of reserpine from plasma by liquid-liquid extraction and by solid-phase extraction. A structural analog, rescinnamine, was used as the internal standard. The reconstituted extracts were analyzed by ionspray LC/MS/MS in the selected reaction monitoring (SRM) mode. The calibration graph for reserpine extracted from equine plasma obtained using liquid-liquid extraction was linear from 10 to 5000 pg ml-1 and that using solid-phase extraction from 100 to 5000 pg ml-1. The lower level of quantitation (LLQ) using liquid-liquid and solid-phase extraction was 50 and 200 pg ml-1, respectively. The lower level of detection for reserpine by LC/MS/MS was 10 pg ml-1. The intra-assay accuracy did not exceed 13% for liquid-liquid and 12% for solid-phase extraction. The recoveries for the LLQ were 68% for liquid-liquid and 58% for solid-phase extraction.

Structural characteristics of compounds that modulate P-glycoprotein-associated multidrug resistance.

Multidrug resistance is mediated by a membrane-bound protein, P-gp, that functions as an energy dependent efflux system to reduce the intracellular concentration of anticancer drugs by binding to these drugs and actively exporting them from the cell. Compounds that interact with P-gp and compete with anticancer drug binding modulate the degree of drug resistance and therefore enhance the cytotoxicity of anticancer drugs against the resistant cell. Effective modulators share certain physical and chemical properties including octanol/water partitioning and molecular size, but the physical properties of size and shape seem to correlate best with modulator effectiveness. Using a photoactivatable analog of vinblastine as a probe, together with a semi-synthetic series of structurally homologous reserpine and yohimbine analogs, the need for two planar aromatic domains and a basic nitrogen atom was established within the structural context of these compounds. The use of three-dimensional comparisons was extended to examine important structural features in other modulator types such as the condensed-ring aromatics. This approach indicates that structural similarities between different classes of compounds are present in compounds recognized by the MDR phenotype. These studies emphasize the importance of a ligand-receptor relationship for modulators of MDR, and begin to define the P-gp-binding pharmacophore. It is likely that this approach will be useful in directing the de novo synthesis of compounds that modulate MDR and help to further define the requirements for molecular recognition by this system.

Reserpine methonitrate, a novel quaternary analogue of reserpine augments urinary excretion of VMA and 5-HIAA without affecting HVA in rats.

BACKGROUND: Reserpine, an alkaloid from Rauwolfia serpentina was widely used for its antihypertensive action in the past. In later years, its use has been reduced because of precipitation of depression and extra pyramidal symptoms due to its central action. In the present investigation, reserpine methonitrate (RMN), a novel quaternary analogue of reserpine was synthesised and evaluated biochemically for its central and peripheral amine depleting actions in rats while its influence on the blood pressure was measured in anaesthetized rats in comparison with reserpine RESULTS: Reserpine treatment (5 mg/kg) produced a significant increase in the urinary excretion of VMA, 5-HIAA and HVA while RMN at doses of equal to and double the equimolar doses of reserpine (5 and 10 mg/kg) produced significant increase in VMA and 5-HIAA excretion without producing any effect on HVA excretion compared to control animals. Reserpine in the dose range of 0.5 to 15 microg/kg produced significant reduction in blood pressure compared to control. RMN was also found to produce significant decrease in blood pressure at doses of 10, 25 and 50 microg/kg body weight in comparison to control. The results indicated peripheral depletion of biogenic amines by RMN without affecting the central stores of the amines. CONCLUSIONS: The present study clearly indicated that the quaternization of reserpine restricts its transfer across the blood-brain barrier and could be the reason for its selective peripheral action. It is also clear that the hypotensive actions of RMN could be due to peripheral depletion of catecholamines.

Antibody specificity studies for reserpine, its metabolites, and synthetic reserpine congeners: radioimmunoassay.

Progress in the development of radioimmunoassay techniques for reserpine and related compounds is reported. A conjugate of reserpine with human serum albumin was prepared, involving linkage at the indole nitrogen atom of reserpine. Injection of the purified conjugate into sheep elicited antibodies of high titer, which bound reserpine selectively. Tritiated reserpine was employed in the procedure, and dextran-coated charcoal was utilized to separate free and bound forms of the drug. Antibodies exhibited a selectivity for reserpine and did not cross-react significantly with major human metabolites. Cross-reactivity of antibodies with other reserpine derivatives (i.e., syrosingopine, deserpidine, and rescinnamine) also was investigated. A stable tritiated or radioiodinated reserpine derivative of high specific activity is being sought to improve assay sensitivity for use in bioequivalence and bioavailability studies. In the absence of any extraction or concentration procedures, at least a 10-fold increase in immunoassay sensitivity would be required to follow reserpine levels in humans given normal doses of the drug. The methods show promise also for the assay of reserpine derivatives such as deserpidine, which exhibits cross-reactivity to reserpine antibodies.