RESUMEN
Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bisBIAs in yeast.
Asunto(s)
Bencilisoquinolinas/metabolismo , Isoquinolinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Alcaloides/biosíntesis , Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Fermentación , Ingeniería Metabólica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas , Racemasas y Epimerasas/genética , Racemasas y Epimerasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/metabolismoRESUMEN
Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.
Asunto(s)
Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Simulación de Dinámica Molecular , Tetrahidroisoquinolinas/metabolismo , Antibacterianos/biosíntesis , Antibacterianos/química , Bacterias/genética , Proteínas Bacterianas/genética , Biocatálisis , Cromatografía Líquida de Alta Presión , Farmacorresistencia Microbiana/genética , Humanos , Isoquinolinas/química , Isoquinolinas/metabolismo , Espectrometría de Masas/métodos , Estructura Molecular , NADP/química , NADP/metabolismo , Naftiridinas/química , Naftiridinas/metabolismo , Oxidación-Reducción , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Tetrahidroisoquinolinas/químicaRESUMEN
Sigma receptors are transmembrane proteins with two different subtypes: σ1 and σ2. Because of its overexpression in tumors, the σ2 receptor (σ2R) is a well-known biomarker for cancer cells. A large number of small-molecule ligands for the σ2Rs have been identified and tested for imaging the proliferative status of tumors using single photon emission computed tomography (SPECT) and positron emission tomography (PET). These small molecules include derivatives of bicyclic amines, indoles, cyclohexylpiperazines and tetrahydroisoquinolines. This review discusses various aspects of small molecule ligands, such as chemical composition, labeling strategy, affinity for σ2Rs, and in vitro/in vivo investigations. The recent studies described here could be useful for the development of σ2R radioligands as potential tumor imaging agents.
Asunto(s)
Ligandos , Neoplasias/diagnóstico por imagen , Radiofármacos/química , Receptores sigma/química , Humanos , Indoles/química , Indoles/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Tomografía de Emisión de Positrones , Radiofármacos/metabolismo , Receptores sigma/metabolismo , Relación Estructura-Actividad , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/metabolismoRESUMEN
Herein we report an assessment of 24 1,2,3,4-tetrahydroisoquinoline derivatives for potential DNaseâ I (deoxyribonuclease I) inhibitory properties inâ vitro. Four of them inhibited DNaseâ I with IC50 values below 200â µM. The most potent was 1-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl)propan-2-one (2) (IC50 =134.35±11.38â µM) exhibiting slightly better IC50 value compared to three other active compounds, 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]-1-phenylethan-1-one (15) (IC50 =147.51±14.87â µM), 2-[2-(4-fluorophenyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (18) (IC50 =149.07±2.98â µM) and 2-[6,7-dimethoxy-2-(p-tolyl)-1,2,3,4-tetrahydroisoquinolin-1-yl]cyclohexan-1-one (22) (IC50 =148.31±2.96â µM). Cytotoxicity assessment of the active DNaseâ I inhibitors revealed a lack of toxic effects on the healthy cell lines MRC-5. Molecular docking and molecular dynamics simulations suggest that interactions with Glu 39, His 134, Asn 170, Tyr 211, Asp 251 and His 252 are an important factor for inhibitors affinity toward the DNaseâ I. Observed interactions would be beneficial for the discovery of new active 1,2,3,4-tetrahydroisoquinoline-based inhibitors of DNaseâ I, but might also encourage researchers to further explore and utilize potential therapeutic application of DNaseâ I inhibitors, based on a versatile role of DNaseâ I during apoptotic cell death.
Asunto(s)
Desoxirribonucleasa I/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Tetrahidroisoquinolinas/química , Apoptosis/efectos de los fármacos , Sitios de Unión , Dominio Catalítico , Línea Celular , Desoxirribonucleasa I/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Tetrahidroisoquinolinas/metabolismo , Tetrahidroisoquinolinas/farmacologíaRESUMEN
The σ2 receptor (transmembrane protein 97), which is involved in cholesterol homeostasis, is of high relevance for neoplastic processes. The upregulated expression of σ2 receptors in cancer cells and tissue in combination with the antiproliferative potency of σ2 receptor ligands motivates the research in the field of σ2 receptors for the diagnosis and therapy of different types of cancer. Starting from the well described 2-(4-(1H-indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline class of compounds, we synthesized a novel series of fluorinated derivatives bearing the F-atom at the aromatic indole/azaindole subunit. RM273 (2-[4-(6-fluoro-1H-pyrrolo[2,3-b]pyridin-1-yl)butyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline) was selected for labelling with 18F and evaluation regarding detection of σ2 receptors in the brain by positron emission tomography. Initial metabolism and biodistribution studies of [18F]RM273 in healthy mice revealed promising penetration of the radioligand into the brain. Preliminary in vitro autoradiography on brain cryosections of an orthotopic rat glioblastoma model proved the potential of the radioligand to detect the upregulation of σ2 receptors in glioblastoma cells compared to healthy brain tissue. The results indicate that the herein developed σ2 receptor ligand [18F]RM273 has potential to assess by non-invasive molecular imaging the correlation between the availability of σ2 receptors and properties of brain tumors such as tumor proliferation or resistance towards particular therapies.
Asunto(s)
Encéfalo/metabolismo , Radioisótopos de Flúor/química , Radioisótopos de Flúor/metabolismo , Radiofármacos/química , Radiofármacos/metabolismo , Receptores sigma/metabolismo , Animales , Femenino , Humanos , Ligandos , Masculino , Ratones , Neoplasias/metabolismo , Ratas , Ratas Endogámicas F344 , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/metabolismoRESUMEN
P-glycoprotein (P-gp)-mediated multidrug resistance (MDR) is a phenomenon in which cells become resistant to structurally and mechanistically unrelated drugs resulting in low intracellular drug concentrations. It is one of the noteworthy problems in malignant tumor clinical therapeutics. So P-gp protein is one of the ideal targets to solve MDR. Based on the lead compound 5m obtained from our previous work, a series of furan derivatives featuring alkyl-substituted phenols and 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline were designed and synthesized as reversal agents against P-gp in this paper. Compound 16 containing isopropoxy possessed good potency against P-gp mediated MDR in MCF-7/ADR (IC50 (doxorubicin) = 0.73 µM, RF = 69.6 with 5 µM 16 treated). Western blot results and Rh123 accumulation assays showed that 16 effectively inhibited P-gp efflux function but not its expression. The preliminary structure-activity relationship and docking studies demonstrated that compound 16 would be a potential P-gp inhibitor. Most worthy of mention is that compound 16 has achieved satisfactory results in combination with a variety of anti-tumor drugs, such as doxorubicin, paclitaxel, and vincristine. This study forwards a hopeful P-gp inhibitor for withstanding malignant tumor cell with multidrug resistance setting the basis for further studies.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Diseño de Fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Furanos/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Doxorrubicina/farmacología , Furanos/metabolismo , Furanos/farmacología , Humanos , Simulación del Acoplamiento Molecular , Paclitaxel/farmacología , Relación Estructura-Actividad , Tetrahidroisoquinolinas/química , Tetrahidroisoquinolinas/metabolismo , Tetrahidroisoquinolinas/farmacologíaRESUMEN
For decades, plants have represented an inexhaustible source of natural products used in various sectors such as health and industry. However, one recurring problem is the low accumulation of these compounds in planta and, therefore, their production costs and supply. In recent years, unprecedented hope has been brought by the metabolic engineering of microorganisms, which opens up prospects for supply of these molecules at lower cost with high added value. However, many of these productions remained at a laboratory scale. In a recent article published in Nature Communication, Vincent J. J. Martin's team has developed an optimized yeast strain capable of synthesizing not only a huge amount of (S)-reticuline, a major precursor of the plant tetrahydroisoquinoline alkaloid series, but also a whole range of new-to-nature compounds from this prominent family of natural products. This synthesis, reaching industrial scales, thus paves the way to efficient production in microbial cell factories.
Asunto(s)
Ingeniería Metabólica , Saccharomyces cerevisiae/metabolismo , Tetrahidroisoquinolinas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
17ß-Hydroxysteroid dehydrogenases catalyse interconversion at the C17 position between oxidized and reduced forms of steroidal nuclear receptor ligands. The type 1 enzyme, expressed in malignant cells, catalyses reduction of the less-active estrone to estradiol, and inhibitors have therapeutic potential in estrogen-dependent diseases such as breast and ovarian cancers and in endometriosis. Synthetic decoration of the nonsteroidal N-phenyl-1,2,3,4-tetrahydroisoquinoline (THIQ) template was pursued by using Pomeranz-Fritsch-Bobbitt, Pictet-Spengler and Bischler-Napieralski approaches to explore the viability of this scaffold as a steroid mimic. Derivatives were evaluated biologically inâ vitro as type 1 enzyme inhibitors in a bacterial cell homogenate as source of recombinant protein. Structure-activity relationships are discussed. THIQs possessing a 6-hydroxy group, lipophilic substitutions at the 1- or 4-positions in combination with N-4'-chlorophenyl substitution were most favourable for activity. Of these, one compound had an IC50 of ca. 350â nM as a racemate, testifying to the applicability of this novel approach.
Asunto(s)
17-Hidroxiesteroide Deshidrogenasas/antagonistas & inhibidores , Diseño de Fármacos , Tetrahidroisoquinolinas/química , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Receptor alfa de Estrógeno/antagonistas & inhibidores , Receptor alfa de Estrógeno/metabolismo , Humanos , Concentración 50 Inhibidora , Conformación Molecular , Relación Estructura-Actividad , Tetrahidroisoquinolinas/metabolismoRESUMEN
A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine-citrulline dipeptide linkers via heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02 µmol/kg).
Asunto(s)
Anticuerpos Monoclonales/química , Antineoplásicos/farmacología , Benzodiazepinas/química , Diseño de Fármacos , Inmunoconjugados/farmacología , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Neoplasias Gástricas/tratamiento farmacológico , Tetrahidroisoquinolinas/química , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Antineoplásicos/química , Apoptosis , Benzodiazepinas/metabolismo , Proliferación Celular , Femenino , Gangliósido G(M1)/análogos & derivados , Gangliósido G(M1)/inmunología , Proteínas Ligadas a GPI/inmunología , Humanos , Inmunoconjugados/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Mesotelina , Ratones , Ratones SCID , Carcinoma Pulmonar de Células Pequeñas/patología , Neoplasias Gástricas/patología , Relación Estructura-Actividad , Tetrahidroisoquinolinas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ABCB1 detoxifies cells by exporting diverse xenobiotic compounds, thereby limiting drug disposition and contributing to multidrug resistance in cancer cells. Multiple small-molecule inhibitors and inhibitory antibodies have been developed for therapeutic applications, but the structural basis of their activity is insufficiently understood. We determined cryo-EM structures of nanodisc-reconstituted, human ABCB1 in complex with the Fab fragment of the inhibitory, monoclonal antibody MRK16 and bound to a substrate (the antitumor drug vincristine) or to the potent inhibitors elacridar, tariquidar, or zosuquidar. We found that inhibitors bound in pairs, with one molecule lodged in the central drug-binding pocket and a second extending into a phenylalanine-rich cavity that we termed the "access tunnel." This finding explains how inhibitors can act as substrates at low concentration, but interfere with the early steps of the peristaltic extrusion mechanism at higher concentration. Our structural data will also help the development of more potent and selective ABCB1 inhibitors.
Asunto(s)
Acridinas/metabolismo , Anticuerpos Monoclonales/metabolismo , Microscopía por Crioelectrón/métodos , Tetrahidroisoquinolinas/metabolismo , Vincristina/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos Fitogénicos/metabolismo , Proliferación Celular , Resistencia a Múltiples Medicamentos , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The opioid receptors are members of the G-protein-coupled receptor (GPCR) family and are known to modulate a variety of biological functions, including pain perception. Despite considerable advances, the mechanisms by which opioid agonists and antagonists interact with their receptors and exert their effect are still not completely understood. In this report, six new hybrids of the Dmt-Tic pharmacophore and cyclic peptides, which were shown before to have a high affinity for the µ-opioid receptor (MOR) were synthesized and characterized pharmacologically in calcium mobilization functional assays. All obtained ligands turned out to be selective antagonists of the δ-opioid receptor (DOR) and did not activate or block the MOR. The three-dimensional structural determinants responsible for the DOR antagonist properties of these analogs were further investigated by docking studies. The results indicate that these compounds attach to the DOR in a slightly different orientation with respect to the Dmt-Tic pharmacophore than Dmt-TicΨ[CH2-NH]Phe-Phe-NH2 (DIPP-NH2[Ψ]), a prototypical DOR antagonist peptide. Key pharmacophoric contacts between the DOR and the ligands were maintained through an analogous spatial arrangement of pharmacophores, which could provide an explanation for the predicted high-affinity binding and the experimentally observed functional properties of the novel synthetic ligands.
Asunto(s)
Dipéptidos/metabolismo , Péptidos Opioides/metabolismo , Péptidos Cíclicos/metabolismo , Receptores Opioides delta/metabolismo , Tetrahidroisoquinolinas/metabolismo , Analgésicos Opioides/antagonistas & inhibidores , Animales , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Antagonistas de Narcóticos/química , Antagonistas de Narcóticos/metabolismo , Péptidos Opioides/síntesis química , Péptidos Cíclicos/síntesis química , Receptores Opioides/química , Relación Estructura-ActividadRESUMEN
The tetrahydroisoquinoline (THIQ) moiety is a privileged substructure of many bioactive natural products and semi-synthetic analogs. Plants manufacture more than 3,000 THIQ alkaloids, including the opioids morphine and codeine. While microbial species have been engineered to synthesize a few compounds from the benzylisoquinoline alkaloid (BIA) family of THIQs, low product titers impede industrial viability and limit access to the full chemical space. Here we report a yeast THIQ platform by increasing production of the central BIA intermediate (S)-reticuline to 4.6 g L-1, a 57,000-fold improvement over our first-generation strain. We show that gains in BIA output coincide with the formation of several substituted THIQs derived from amino acid catabolism. We use these insights to repurpose the Ehrlich pathway and synthesize an array of THIQ structures. This work provides a blueprint for building diverse alkaloid scaffolds and enables the targeted overproduction of thousands of THIQ products, including natural and semi-synthetic opioids.
Asunto(s)
Alcaloides/biosíntesis , Bencilisoquinolinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Tetrahidroisoquinolinas/metabolismo , Alcaloides/química , Analgésicos Opioides/química , Analgésicos Opioides/metabolismo , Bencilisoquinolinas/química , Productos Biológicos/química , Productos Biológicos/metabolismo , Vías Biosintéticas/genética , Ingeniería Genética , Modelos Químicos , Estructura Molecular , Saccharomyces cerevisiae/genética , Tetrahidroisoquinolinas/químicaRESUMEN
We broadened the usage of DNA transposon technology by demonstrating its capacity for the rapid creation of expression libraries for long biochemical pathways, which is beyond the classical application of building genome-scale knockout libraries in yeasts. This strategy efficiently leverages the readily available fine-tuning impact provided by the diverse transcriptional environment surrounding each random integration locus. We benchmark the transposon-mediated integration against the nonhomologous end joining-mediated strategy. The latter strategy was demonstrated for achieving pathway random integration in other yeasts but is associated with a high false-positive rate in the absence of a high-throughput screening method. Our key innovation of a nonreplicable circular DNA platform increased the possibility of identifying top-producing variants to 97%. Compared to the classical DNA transposition protocol, the design of a nonreplicable circular DNA skipped the step of counter-selection for plasmid removal and thus not only reduced the time required for the step of library creation from 10 to 5 d but also efficiently removed the "transposition escapers", which undesirably represented almost 80% of the entire population as false positives. Using one endogenous product (i.e., shikimate) and one heterologous product (i.e., (S)-norcoclaurine) as examples, we presented a streamlined procedure to rapidly identify high-producing variants with titers significantly higher than the reported data in the literature. We selected Scheffersomyces stipitis, a representative nonconventional yeast, as a demo, but the strategy can be generalized to other nonconventional yeasts. This new exploration of transposon technology, therefore, adds a highly versatile tool to accelerate the development of novel species as microbial cell factories for producing value-added chemicals.
Asunto(s)
Reactores Biológicos , Elementos Transponibles de ADN/genética , Ingeniería Metabólica/métodos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Alcaloides/metabolismo , Reparación del ADN por Unión de Extremidades , ADN Circular/genética , ADN Circular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Fúngico , Biblioteca Genómica , Ensayos Analíticos de Alto Rendimiento , Mutagénesis Insercional , Plásmidos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Ácido Shikímico/metabolismo , Tetrahidroisoquinolinas/metabolismoRESUMEN
Metabolic profiling of Streptomyces sp. IB2014/016-6 led to the identification of three new tetrahydroisoquinoline natural products, perquinolinesâ A-C (1-3). Labelled precursor feeding studies and the cloning of the pqr biosynthetic gene cluster revealed that 1-3 are assembled by the action of several unusual enzymes. The biosynthesis starts with the condensation of succinyl-CoA and l-phenylalanine catalyzed by the amino-7-oxononanoate synthase-like enzyme PqrA, representing rare chemistry in natural product assembly. The second condensation and cyclization events are conducted by PqrG, an enzyme resembling an acyl-CoA ligase. Last, ATP-grasp RimK-type ligase PqrI completes the biosynthesis by transferring a γ-aminobutyric acid or ß-alanine moiety. The discovered pathway represents a new route for assembling the tetrahydroisoquinoline cores of natural products.
Asunto(s)
Productos Biológicos/metabolismo , Streptomyces/metabolismo , Tetrahidroisoquinolinas/metabolismo , Acilcoenzima A/metabolismo , Aciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Vías BiosintéticasRESUMEN
RATIONALE: Retroactive analysis of previously tested urine samples has become an important sports anti-doping tool. Retroactive reprocessing of old data files acquired from a generic screening procedure can reveal detection of initially unknown substances, like illegal drugs and newly identified metabolites. METHODS: To be able to efficiently search through hundreds to thousands of liquid chromatography high-resolution full-scan Orbitrap mass spectrometry data files of anti-doping samples, a combination of MetAlign and HR_MS_Search software has been developed. MetAlign reduced the data size ca 100-fold making possible local storage of a massive volume of data. RESULTS: The newly developed HR_MS_Search module can search through the reduced data files for new compounds (mass or isotope pattern) defined by mass windows and retention time windows. A search for 33 analytes in 940 reduced data files lasted 10 s. The output of the automatic search was compared to the standard manual routine evaluation. The results of searching were evaluated in terms of false negatives and false positives. The newly banned b2-agonist higenamine and its metabolite coclaurine were successfully searched in reduced data files originating from a testing period for which these substances were not banned, as an example of retroactive analysis. CONCLUSIONS: The freeware MetAlign software and its automatic searching module HR_MS_Search facilitated the retroactive reprocessing of reduced full-scan high-resolution liquid chromatography/mass spectrometry screening data files and created a new tool in anti-doping laboratories' network.
Asunto(s)
Agonistas Adrenérgicos beta/orina , Alcaloides/orina , Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Tetrahidroisoquinolinas/orina , Agonistas Adrenérgicos beta/metabolismo , Alcaloides/metabolismo , Doping en los Deportes/prevención & control , Humanos , Isoquinolinas/orina , Detección de Abuso de Sustancias , Tetrahidroisoquinolinas/metabolismo , UrinálisisRESUMEN
The 1,2,3,4-tetrahydroisoquinolines (TIQs) are compounds frequently described as alkaloids that can be found in the human body fluids and/or tissues including the brain. In most circumstances, TIQs may be originated as a consequence of reactions, known as Pictet-Spengler condensations, between biogenic amines and electrophilic carbonyl compounds, including ethanol's main metabolite, acetaldehyde. Several TIQs may also be synthesized enzymatically whilst others may be formed in the body as by-products of other compounds including TIQs themselves. The biological actions of TIQs appear critically dependent on their metabolism, and nowadays, among TIQs, 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol), N-methyl-1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (N-methyl-(R)-salsolinol), 1-[(3,4-dihydroxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol (norlaudanosoline or tetrahydropapaveroline or THP) and 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1BnTIQ) are considered as those endowed with the most potent neurotoxic actions. However, it remains to be established whether a continuous exposure to TIQs or to their metabolites might carry toxicological consequences in the short- or long-term period. Remarkably, recent findings suggest that some TIQs such as (1-[(4-hydroxyphenyl)methyl]-1,2,3,4-tetrahydroisoquinoline-6,7-diol) (higenamine) and 1-methyl-1,2,3,4-tetrahydroisoquinoline (1-MeTIQ) as well as N-methyl-tetrahydroisoquinoline (N-methyl-TIQ) exert unique neuroprotective and neurorestorative actions. The present review article provides an overview on these aspects of TIQs and summarizes those that presently appear the most significant highlights on this puzzling topic.
Asunto(s)
Encéfalo/efectos de los fármacos , Etanol/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Fármacos Neuroprotectores/toxicidad , Tetrahidroisoquinolinas/administración & dosificación , Tetrahidroisoquinolinas/toxicidad , Animales , Apoptosis/efectos de los fármacos , Encéfalo/metabolismo , Dopamina/metabolismo , Humanos , Isoquinolinas/metabolismo , Fármacos Neuroprotectores/metabolismo , Tetrahidroisoquinolinas/metabolismoRESUMEN
Marine sponges often house small-molecule-producing symbionts extracellularly in their mesohyl, providing the host with a means of chemical defence against predation and microbial infection. Here, we report an intriguing case of chemically mediated symbiosis between the renieramycin-containing sponge Haliclona sp. and its herein discovered renieramycin-producing symbiont Candidatus Endohaliclona renieramycinifaciens. Remarkably, Ca. E. renieramycinifaciens has undergone extreme genome reduction where it has lost almost all necessary elements for free living while maintaining a complex, multi-copy plasmid-encoded biosynthetic gene cluster for renieramycin biosynthesis. In return, the sponge houses Ca. E. renieramycinifaciens in previously uncharacterized cellular reservoirs (chemobacteriocytes), where it can acquire nutrients from the host and avoid bacterial competition. This relationship is highly specific to a single clade of Haliclona sponges. Our study reveals intracellular symbionts as an understudied source for defence chemicals in the oldest-living metazoans and paves the way towards discovering similar systems in other marine sponges.
Asunto(s)
Gammaproteobacteria/fisiología , Haliclona/química , Haliclona/microbiología , Simbiosis , Tetrahidroisoquinolinas/metabolismo , Animales , Gammaproteobacteria/clasificación , Gammaproteobacteria/genética , Gammaproteobacteria/metabolismo , Tamaño del Genoma , Haliclona/citología , Haliclona/genética , Especificidad del Huésped , Metagenoma , Estructura Molecular , Familia de Multigenes , Filogenia , Plásmidos/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Simbiosis/genética , Tetrahidroisoquinolinas/químicaRESUMEN
Lorlatinib, a novel generation oral anaplastic lymphoma kinase (ALK) and ROS1 inhibitor with high membrane and blood-brain barrier permeability, recently received accelerated approval for treatment of ALK-rearranged non-small-cell lung cancer (NSCLC), and its further clinical development is ongoing. We previously found that the efflux transporter P-glycoprotein (MDR1/ABCB1) restricts lorlatinib brain accumulation and that the drug-metabolizing enzyme cytochrome P450-3A (CYP3A) limits its oral availability. Using genetically modified mouse models, we investigated the impact of targeted pharmacological inhibitors on lorlatinib pharmacokinetics and bioavailability. Upon oral administration of lorlatinib, the plasma AUC0-8h in CYP3A4-humanized mice was â¼1.8-fold lower than in wild-type and Cyp3a-/- mice. Oral coadministration of the CYP3A inhibitor ritonavir caused reversion to the AUC0-8h levels seen in wild-type and Cyp3a-/- mice, without altering the relative tissue distribution of lorlatinib. Moreover, simultaneous pharmacological inhibition of P-glycoprotein and CYP3A4 with oral elacridar and ritonavir in CYP3A4-humanized mice profoundly increased lorlatinib brain concentrations, but not its oral availability or other relative tissue distribution. Oral lorlatinib pharmacokinetics was not significantly affected by absence of the multispecific Oatp1a/1b drug uptake transporters. The absolute oral bioavailability of lorlatinib over 8â¯h in wild-type, Cyp3a-/-, and CYP3A4-humanized mice was 81.6%, 72.9%, and 58.5%, respectively. Lorlatinib thus has good oral bioavailability, which is markedly restricted by human CYP3A4 but not by mouse Cyp3a. Pharmacological inhibition of CYP3A4 reversed these effects, and simultaneous P-gp inhibition with elacridar boosted absolute brain levels of lorlatinib by 16-fold without obvious toxicity. These insights may help to optimize the clinical application of lorlatinib.
Asunto(s)
Acridinas/metabolismo , Quinasa de Linfoma Anaplásico/metabolismo , Encéfalo/metabolismo , Lactamas Macrocíclicas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ritonavir/metabolismo , Tetrahidroisoquinolinas/metabolismo , Acridinas/administración & dosificación , Administración Intravenosa , Administración Oral , Aminopiridinas , Quinasa de Linfoma Anaplásico/antagonistas & inhibidores , Animales , Disponibilidad Biológica , Encéfalo/efectos de los fármacos , Inhibidores del Citocromo P-450 CYP3A/administración & dosificación , Inhibidores del Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas/fisiología , Sinergismo Farmacológico , Lactamas , Lactamas Macrocíclicas/administración & dosificación , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Pirazoles , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Ritonavir/administración & dosificación , Tetrahidroisoquinolinas/administración & dosificaciónRESUMEN
P-Glycoprotein is a well-known membrane transporter responsible for the efflux of an ample spectrum of anticancer drugs. Its relevance in the management of cancer chemotherapy is increased in view of its high expression in cancer stem cells, a population of cancer cells with strong tumor-promoting ability. In the present study, a series of compounds were synthesized through structure modulation of [4'-(6,7-dimethoxy-3,4-dihydro-1 H-isoquinolin-2-ylmethyl)biphenyl-4-ol] (MC70), modifying the phenolic group of the lead compound. Among them, compound 5b emerged for its activity against the transporter (EC50 = 15 nM) and was capable of restoring doxorubicin antiproliferative activity at nontoxic concentration. Its behavior was rationalized through a molecular modeling study consisting of a well-tempered metadynamics simulation, which allowed one to identify the most favorable binding pose, and of a subsequent molecular dynamics run, which indicated a peculiar effect of the compound on the motion pattern of the transporter.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Antineoplásicos/farmacología , Diseño de Fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Células Madre Neoplásicas/efectos de los fármacos , Tetrahidroisoquinolinas/química , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/metabolismo , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Perros , Doxorrubicina/farmacología , Edición Génica , Humanos , Ligandos , Células de Riñón Canino Madin Darby , Simulación de Dinámica Molecular , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Permeabilidad/efectos de los fármacos , Relación Estructura-Actividad , Tetrahidroisoquinolinas/metabolismo , Tetrahidroisoquinolinas/farmacologíaRESUMEN
Alkaloids are a class of organic compounds with a wide range of biological properties, including anti-HIV activity. The 1,2,3,4-tetrahydroisoquinoline is a ubiquitous structural motif of many alkaloids. Using a short and an efficient route for synthesis, a series of 1,2,3,4-tetrahydroisoquinolines/isoquinolines was developed. These compounds have been analysed for their ability to inhibit an important interaction between HIV-1 integrase enzyme (IN) and human LEDGF/p75 protein (p75) which assists in the viral integration into the active genes. A lead compound 6d is found to inhibit the LEDGF/p75-IN interaction in vitro with an IC50 of ~10 µm. Molecular docking analysis of the isoquinoline 6d reveals its interactions with the LEDGF/p75-binding residues of IN. Based on an order of addition experiment, the binding of 6d or LEDGF/p75 to IN is shown to be mutually exclusive. Also, the activity of 6d in vitro is found to be unaffected by the presence of a non-specific DNA. As reported earlier for the inhibitors of LEDGF/p75-IN interaction, 6d exhibits a potent inhibition of both the early and late stages of HIV-1 replication. Compound 6d differing from the known inhibitors in the chemical moieties and interactions with CCD could potentially be explored further for developing small molecule inhibitors of LEDGF/p75-IN interaction having a higher potency.