(S)-ML-SA1 ACTIVATES AUTOPHAGY VIA TRPML1-TFEB PATHWAY.

Autophagic flux plays a crucial role in various diseases. Recently, the lysosomal ion channel TRPML1 has emerged as a promising target in lysosomal storage diseases, such as mucolipidosis. The discovery of mucolipin synthetic agonist-1 (ML-SA1) has expanded our understanding of TRPML1's function and its potential therapeutic uses. However, ML-SA1 is a racemate with limited cellular potency and poor water solubility. In this study, we synthetized rac-ML-SA1, separated the enantiomers by chiral liquid chromatography and determined their absolute configuration by vibrational circular dichroism (VCD). In addition, we focused on investigating the impact of each enantiomer of ML-SA1 on the TRPML1-TFEB axis. Our findings revealed that (S)-ML-SA1 acts as an agonist for TRPML1 at the lysosomal membrane. This activation prompts transcription factor EB (TFEB) to translocate from the cytosol to the nucleus in a dose-dependent manner within live cells. Consequently, this signaling pathway enhances the expression of coordinated lysosomal expression and regulation (CLEAR) genes and activates autophagic flux. Our study presents evidence for the potential use of (S)-ML-SA1 in the development of new therapies for lysosomal storage diseases that target TRPML1.

The Brazilian octocoral Phyllogorgia dilatata as a source of cytotoxic compounds.

The extensive marine biodiversity has proved to be a promising source of substances with biomedical potential. In this study, the cytotoxicity of the Brazilian octocoral Phyllogorgia dilatata (Gorgoniidae) was evaluated against two tumor cell lines and three bacterial strains. The methanol/dichloromethane crude extract presented no antibacterial activity up to the highest concentration tested (512 µg/mL), however it revealed a noteworthy antiproliferative effect against HCT-116 (80%) and MCF-7 (54%) cell lines at 50 µg/mL. Therefore, guided by the cytotoxic activity, a multistep chemical fractionation of the extract provided the subfraction 5 (PDPH2-5) with IC50 values of 3.18 and 17.80 µg/mL against HCT-116 and MCF-7, respectively. The LC-HRMS/MS analysis of PDPH2-5 showed ions of m/z 219.1742 and 219.1743, characterized as (E,E) and (Z,E) germacrone, after a LC-DAD-SPE/NMR analysis of the hexanic fraction and comparisons of NMR data with the literature. Previously reported assessments to the cytotoxic activity of the (E,E)-diastereoisomer disclosed higher IC50 values than that obtained for the PDPH2-5 fraction, suggesting, herein, a potentiated effect of the diastereoisomeric mixture. Such remark encourage further bioactivity studies with stereoisomer mixtures and reduce the urge for compound isolation.

Enantioselectivity Effects in Clinical Metabolomics and Lipidomics.

Metabolomics and lipidomics have demonstrated increasing importance in underlying biochemical mechanisms involved in the pathogenesis of diseases to identify novel drug targets and/or biomarkers for establishing therapeutic approaches for human health. Particularly, bioactive metabolites and lipids have biological activity and have been implicated in various biological processes in physiological conditions. Thus, comprehensive metabolites, and lipids profiling are required to obtain further advances in understanding pathophysiological changes that occur in cells and tissues. Chirality is one of the most important phenomena in living organisms and has attracted long-term interest in medical and natural science. Enantioselective separation plays a pivotal role in understanding the distribution and physiological function of a diversity of chiral bioactive molecules. In this context, it has been the goal of method development for targeted and untargeted metabolomics and lipidomic assays. Herein we will highlight the benefits and challenges involved in these stereoselective analyses for clinical samples.

Determination of the Absolute Configuration of Bioactive Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-diones and Study of Their In Vitro Metabolic Profile.

In recent decades, fungi-derived naturally occurring quinazolines have emerged as potential drug candidates. Nevertheless, most studies are conducted for bioactivity assays, and little is known about their absorption, distribution, metabolism, and elimination (ADME) properties. To perform metabolic studies, the synthesis of the naturally occurring quinazolinone, fiscalin B (1), and its chloro derivative, 4-((1H-indol-3-yl)methyl)-8,10-dichloro-1-isobutyl-1,2-dihydro-6H-pyrazino[2,1-b]quinazoline-3,6(4H)-dione (2), disclosed as an antibacterial agent, was performed in a gram scale using a microwave-assisted polycondensation reaction with 22% and 17% yields, respectively. The structure of the non-natural (+)-fiscalin B was established, for the first time, by X-ray crystallography as (1R,4S)-1, and the absolute configuration of the naturally occurring fiscalin B (-)-1 was confirmed by comparison of its calculated and experimental electronic circular dichroism (ECD) spectra as (1S,4R)-1. in vitro metabolic studies were monitored for this class of natural products for the first time by ultra-high-performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). The metabolic characteristics of 1 and 2 in human liver microsomes indicated hydration and hydroxylation mass changes introduced to the parent drugs.

Scope of the 2(5H)-furanone helicity rule: a combined ECD, VCD, and DFT investigation.

One of the most widely used methods to assess the stereochemistry of chiral 2(5H)-furanones is an empirical electronic circular dichroism (ECD) helicity rule. In the present work, an extensive experimental and theoretical investigation of the scope of the above-mentioned empirical rule for acetogenins with a hydroxyl group substituted at C-4 revealed a possible exception to this rule. The underlying causes for this observation are discussed with respect to side chain substitutions, conformational requirements, chromophore handedness as well as a qualitative orbital analysis. Further investigation using vibrational circular dichroism (VCD) spectroscopy led to the identification of spectral markers that seem to be more localized and less affected by side chain substitutions. As the presence of a [capital Upsilon]-lactone ring and a hydroxyl group at C-4 is a very common structural feature of Annonaceous acetogenins, we recommend the combined use of ECD and VCD spectroscopy, along with quantum chemical computations, for the stereochemical analysis of structurally related molecules.

Enantiomeric Mixtures in Natural Product Chemistry: Separation and Absolute Configuration Assignment.

Chiral natural product molecules are generally assumed to be biosynthesized in an enantiomerically pure or enriched fashion. Nevertheless, a significant amount of racemates or enantiomerically enriched mixtures has been reported from natural sources. This number is estimated to be even larger since the enantiomeric purity of secondary metabolites is rarely checked in the natural product isolation pipeline. This latter fact may have drastic effects on the evaluation of the biological activity of chiral natural products. A second bottleneck is the determination of their absolute configurations. Despite the widespread use of optical rotation and electronic circular dichroism, most of the stereochemical assignments are based on empirical correlations with similar compounds reported in the literature. As an alternative, the combination of vibrational circular dichroism and quantum chemical calculations has emerged as a powerful and reliable tool for both conformational and configurational analysis of natural products, even for those lacking UV-Vis chromophores. In this review, we aim to provide the reader with a critical overview of the occurrence of enantiomeric mixtures of secondary metabolites in nature as well the best practices for their detection, enantioselective separation using liquid chromatography, and determination of absolute configuration by means of vibrational circular dichroism and density functional theory calculations.

Preparative chiral separation and absolute configuration of the synthetic pterocarpanquinone LQB-118.

The racemic pterocarpanquinone LQB-118 is active, in mice and hamsters, against tegumentary and visceral leishmaniasis. This compound also presents antiinflammatory and antineoplastic activity in mice. The low level of toxicity observed in these studies makes LQB-118 a promising drug candidate. In order to conduct further biological testing to investigate enantioselectivity in the above-mentioned activities, a multimilligram amount of each enantiomer of LQB-118 was produced. Furthermore, vibrational circular dichroism (VCD) and Density Functional Theory (DFT) calculations were used to determine unambiguously their absolute configurations. The comparison of experimental and calculated VCD data led to the assignment of (-)-LQB-118 as 7aR,12aR and, consequently, (+)-LQB-118 as 7aS12aS.

New chiral stationary phases based on xanthone derivatives for liquid chromatography.

Six chiral derivatives of xanthones (CDXs) were covalently bonded to silica, yielding the corresponding xanthonic chiral stationary phases (XCSPs). The new XCSPs were packed into stainless-steel columns with 150 x 4.6 mm i.d. Moreover, the greening of the chromatographic analysis by reducing the internal diameter (150 x 2.1 mm i.d.) of the liquid chromatography (LC) columns was also investigated. The enantioselective capability of these phases was evaluated by LC using different chemical classes of chiral compounds, including several types of drugs. A library of CDXs was evaluated in order to explore the principle of reciprocity as well as the chiral self-recognition phenomenon. The separation of enantiomeric mixtures of CDXs was investigated under multimodal elution conditions. The XCSPs provided high specificity for the enantiomeric mixtures of CDXs evaluated mainly under normal-phase elution conditions. Furthermore, two XCSPs were prepared with both enantiomers of the same xanthonic selector in order to confirm the inversion order elution.

9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors.

A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.

In vitro analysis of albendazole sulfoxide enantiomers shows that (+)-(R)-albendazole sulfoxide is the active enantiomer against Taenia solium.

Albendazole is an anthelmintic drug widely used in the treatment of neurocysticercosis (NCC), an infection of the brain with Taenia solium cysts. However, drug levels of its active metabolite, albendazole sulfoxide (ABZSO), are erratic, likely resulting in decreased efficacy and suboptimal cure rates in NCC. Racemic albendazole sulfoxide is composed of ABZSO (+)-(R)- and (-)-(S) enantiomers that have been shown to differ in pharmacokinetics and activity against other helminths. The antiparasitic activities of racemic ABZSO and its (+)-(R)- and (-)-(S) enantiomers against T. solium cysts were evaluated in vitro. Parasites were collected from naturally infected pigs, cultured, and exposed to the racemic mixture or to each enantiomer (range, 10 to 500 ng/ml) or to praziquantel as a reference drug. The activity of each compound against cysts was assayed by measuring the ability to evaginate and inhibition of alkaline phosphatase (AP) and parasite antigen release. (+)-(R)-ABZSO was significantly more active than (-)-(S)-ABZSO in suppressing the release of AP and antigen into the supernatant in a dose- and time-dependent manner, indicating that most of the activity of ABZSO resides in the (+)-(R) enantiomer. Use of this enantiomer alone may lead to increased efficacy and/or less toxicity compared to albendazole.

Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies.

The parasite Schistosoma mansoni (Sm) depends exclusively on the salvage pathway for its purine requirements. The enzyme purine nucleoside phosphorylase (PNP) is, therefore, a promising target for development of antischistosomal agents and an assay for screening of inhibitors. To enable this, immobilized SmPNP reactors were produced. By quantification of hypoxanthine by liquid chromatography, kinetic constants (K M) for the substrate inosine were determined for the free and immobilized enzyme as 110 ± 6.90 µmol L (-1) and 164 ± 13.4 µmol L (-1), respectively, indicating that immobilization did not affect enzyme activity. Furthermore, the enzyme retained 25 % of its activity after four months. Non-Michaelis kinetics for the phosphate substrate, and capacity for Pi-independent hydrolysis were also demonstrated, despite the low rate of enzymatic catalysis. Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated with a small library of 9-deazaguanine analogues. The method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.

Enantioselective disposition of omeprazole, pantoprazole, and lansoprazole in a same Brazilian subjects group.

This work reports the result of the enantioselective disposition of pantoprazole, omeprazole, and lansoprazole in a same group of Brazilian health subjects. Ten nongenotyped healthy subjects were used for this study. Each subject received a single oral dose of 80 mg of pantoprazole, 40 mg of omeprazole, and 30 mg of lansoprazole, and the plasma concentrations of the enantiomers were measured for 8 h postdose. For pantoprazole and omeprazole, among the 10 volunteers investigated, only one volunteer (Subject # 4) presented higher plasma concentrations of the (+)-enantiomer than those of (-)-enantiomer. Nevertheless, the area under the concentration-time curve of the (+)-lansoprazole was higher than those the (-)-lansoprazole for all subjects. The comparison of proton pump inhibitors' enantiomers disposition from a single group volunteer demonstrated that pantoprazole and omeprazole can be used to differentiate extensive from poor CYP2C19 metabolizer while lansoprazole cannot do it.

Chromatography Conditions Development by Design of Experiments for the Chemotype Differentiation of Four Bauhinia Species.

The extensive use of medicinal herbs to traditionally treat disease persists for generations, and scientific evidence on plant-derived extracts has indicated their numerous biological activities. The Bauhinia, popular known as cow's paw ("pata de vaca"), with more than 60 native species, are extensively used in Brazilian popular medicine for the control of diabetes. Therefore, in 2009, B. forficata, B. variegata and/or B. affinis were included in the Brazilian National List of Medicinal Plants of Interest to SUS (RENISUS - Brazil). In this context, this work reports the results of the chemical differentiation of B. forficata, B. variegata, B. longifolia, and B. affinis using liquid chromatography coupled to high-resolution mass spectrometry and unsupervised chemometric tools. Chromatographic conditions were optimized by using the design of experiments (DoE) and chromatographic knowledge. Furthermore, the chemical profile of the studied species was analyzed by principal component analysis (PCA) and hierarchical cluster analysis that differentiated the four species of Bauhinia, and 55 compounds were also inferred by MS2 experiments, some of them for the first time in B. affinis. In this manner, this work provides important information that could be used in quality control, development of new pharmaceuticals, and food products based on Bauhinia leaves, as well as to explain ethnomedicinal properties, pharmacological and toxicological actions.

Real-time investigation of mannosyltransferase function of a Xylella fastidiosa recombinant GumH protein using QCM-D.

Xylella fastidiosa is a gram-negative bacterium that causes serious diseases in economically important crops, including grapevine, coffee, and citrus fruits. X. fastidiosa colonizes the xylem vessels of the infected plants, thereby blocking water and nutrient transport. The genome sequence of X. fastidiosa has revealed an operon containing nine genes possibly involved in the synthesis of an exopolisaccharide (EPS) named fastidian gum that can be related with the pathogenicity of this bacterium. The α-1,3-mannosyltransferase (GumH) enzyme from X. fastidiosa is involved in fastidian gum production. GumH is responsible for the transfer of mannose from guanosine diphosphate mannose (GDP-man) to the cellobiose-pyrophosphate-polyprenol carrier lipid (CPP-Lip) during the assembly and biosynthesis of EPS. In this work, a method for real-time detection of recombinant GumH enzymatic activity was successfully developed using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). The QCM-D transducer was strategically modified with CPP-Lip by using a solid-supported lipid bilayer that makes use of a self-assembled monolayer of 1-undecanethiol. Monitoring the real-time CPP-Lip QCM-D transducer in the presence of GDP-man and GumH enzyme shows a mass increase, indicating the transfer of mannose. The real-time QCM-D determination of mannosyltransferase function was validated by a High Performance Liquid Chromatography (LC) method developed for determination of GDP produced by enzymatic reaction. LC results confirmed the activity of recombinant GumH protein, which is the first enzyme involved in the biosynthesis of the EPS from X. fastidiosa enzymatically characterized.

Structure elucidation and absolute stereochemistry of isomeric monoterpene chromane esters.

Six novel monoterpene chromane esters were isolated from the aerial parts of Peperomia obtusifolia (Piperaceae) using chiral chromatography. This is the first time that chiral chromane esters of this kind, ones with a tethered chiral terpene, have been isolated in nature. Due to their structural features, it is not currently possible to assess directly their absolute stereochemistry using any of the standard classical approaches, such as X-ray crystallography, NMR, optical rotation, or electronic circular dichroism (ECD). Herein we report the absolute configuration of these molecules, involving four chiral centers, using vibrational circular dichroism (VCD) and density functional theory (DFT) (B3LYP/6-31G*) calculations. This work further reinforces the capability of VCD to determine unambiguously the absolute configuration of structurally complex molecules in solution, without crystallization or derivatization, and demonstrates the sensitivity of VCD to specify the absolute configuration for just one among a number of chiral centers. We also demonstrate the sufficiency of using the so-called inexpensive basis set 6-31G* compared to the triple-ζ basis set TZVP for absolute configuration analysis of larger molecules using VCD. Overall, this work extends our knowledge of secondary metabolites in plants and provides a straightforward way to determine the absolute configuration of complex natural products involving a chiral parent moiety combined with a chiral terpene adduct.

LC-HRMS and acetylcholinesterase affinity assay as a workflow for profiling alkaloids in Annona salzmannii extract.

Structure-based molecular networking is useful as a dereplication strategy to identify known molecules, unknown close analogues, or compound families. On the other hand, the ligand fishing assay is a remarkable alternative to accelerate the screening process and to overcome the drawbacks of laborious experiments usually adopted in natural product research. The combination of these approaches contributes to high productivity in disclosing active metabolites and a decrease in lead time identification. To provide a valuable data base for the alkaloids of A. salzmannii bark herein we disclose thirty-one isoquinoline alkaloids including benzyltetrahydroisoquinolines, aporphines, proaporphines, and protoberberines. Among these, twenty-six have not been described for A. salzmannii including the unprecedented alkaloid N,O-dimethylcoclaurine N-oxide. In addition, norcoclaurine (1), norreticuline (13), N,O-dimethylcoclaurine N-oxide (15), and N-acetylasimilobine (24) are now reported for the first time as ligand for acetylcholinesterase.

Integrated analytical workflow for chromatographic profiling and metabolite annotation of a cytotoxic Phorbas amaranthus extract.

Phorbas is a widely studied genus of marine sponge and produce structurally rich cytotoxic metabolites. Still, only few studies have assessed metabolites present in Brazilian species. To circumvent redundancy, in this work, we applied and herein report the use of a scouting liquid chromatographic system associate to the design of experiment produced by the DryLab® software to obtain a fast and efficient chromatographic separation of the active hexane fraction, further enabling untargeted high-resolution mass spectrometry (HRMS) data. To this end, a crude hydroalcoholic extract of the sponge Phorbas amaranthus collected in Brazilian coast was prepared and partitioned. The cytotoxicity of the crude extract and the fractions was evaluated using tumor cell culture models. Fragmentation pathways assembled from HRMS data allowed the annotation of 18 known Phorbas metabolites, while 17 metabolites were inferred based on Global Natural Product Social Molecular Networking (GNPS), matching with a further 29 metabolites annotated through molecular subnetwork. The workflow employed demonstrates that chromatographic method development can be accelerated by the use of automated scouting systems and DryLab®, which is useful for profiling natural product libraries, as well as data curation by molecular clusters and should be incorporated to the tools of natural product chemists.

Phosphoenolpyruvate carboxykinase from T. cruzi magnetic beads affinity-based screening assays on crude plant extracts from Brazilian Cerrado.

In T. cruzi, a causative agent of Chagas disease, phosphoenolpyruvate carboxykinase (TcPEPCK) is associated with carbohydrate catabolism. Due to its importance in the metabolism of the parasite, it has become a promising target for the development of new drugs against Chagas disease. Aiming to investigate different approaches for ligands screening, TcPEPCK was immobilized on amine-terminated magnetic beads (TcPEPCK-MB) and kinetically characterized by liquid chromatography tandem mass spectrometry activity assay with a KMapp value of 10 ± 1 µM to oxaloacetate as substrate. Natural products library affords highly diverse molecular frameworks through their secondary metabolites, herein a ligand fishing TcPEPCK-MB assay is described for prospecting ligands in four ethanolic extracts of Brazilian Cerrado plants: Qualea grandiflora (Vochysiaceae), Diospyros burchellii (Ebenaceae), Anadenanthera falcata (Fabaceae) and Byrsonima coccolobifolia (Malpighiaceae). The chemical characterization of eleven identified ligands was carried out by liquid chromatography tandem high-resolution mass spectrometry experiments. Senecic acid, syneilesinolide A, phytosphingosine and vanillic acid 4-glucopyranoside are herein reported for the first time for Q. grandiflora, D. burchellii, A. falcata, respectively. In addition, the specificity of the assay was observed since only catechin was fished out from the ethanolic extract of B. coccolobifolia leaves, despite the presence of epicatechin epimer.

Two-dimensional chromatography method applied to the enantiomeric determination of lansoprazole in human plasma by direct sample injection.

A two-dimensional HPLC method based on the direct injection of biological samples has been developed and validated for the determination of lansoprazole enantiomers in human plasma. The lansoprazole enantiomers were extracted from the biological matrix using an octyl restricted access media bovine serum albumin column (C8 RAM BSA) and the enantioseparation was performed on an amylose tris(3,5-dimethoxyphenylcarbamate) chiral column using acetonitrile:water (35:65 v/v) and UV detection at 285 nm. Analysis time was 25 min with no time spent on sample preparation. The method was applied to the analysis of the plasma samples obtained from nine Brazilian volunteers who received a 30 mg oral dose of racemic lansoprazole and was able to quantify the enantiomers of lansoprazole in the clinical samples analyzed.

On-Flow LC-MS/MS method for screening of xanthine oxidase inhibitors.

The screening of compounds is the initial step in research for the development of new drugs. For this reason, the availability of fast and reliable tools for the screening of a large number of compounds becomes essential. Among the therapeutic targets, the enzyme xanthine oxidase (XO) is of great interest for its importance as a biological source of superoxide radicals, which contribute to the oxidative stress on organisms and are involved in many pathological processes. In the present study, we validated a new method using an immobilized capillary enzyme reactor in an LC system directly coupled to triple quadrupole mass spectrometry to screen for XO ligands. The use of mass spectrometry provided selectivity and speed to the system, eliminating the analytical separation step. The Michaelis-Menten constant (KM) value determined for the immobilized enzyme was 14.5 ±â€¯0.4 µmol L-1, which is consistent with the value previously reported for the XO-ICER with UV detection in a 2D LC method. The on-line approach was successfully applied to assay the XO inhibitory activities of thirty isolated compounds from different classes of natural products and provided greater productivity (288 analysis/day) than 2D LC method (84 analysis/day) of screened samples.