RESUMEN
Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.
Asunto(s)
Aurodox , Sistemas de Secreción Tipo III , Sistemas de Secreción Tipo III/metabolismo , Aurodox/farmacología , Antibacterianos/farmacología , Antibacterianos/química , Bacterias Gramnegativas/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
Spiro compounds have been considered key scaffolds for pharmaceutical applications. Although many synthetic methods exist for monospirocycles, fewer approaches are known for dispirocycles. Here, we report a highly cis-selective method for constructing a 5/6/5-dispirocyclic structure containing pyrrolidine and γ-lactam rings with various substituents from a series of N-arylpropiolamides. The high cis-selectivity would result from isomerization under thermodynamic control. Cis- and trans-diastereomers can be in equilibrium, favoring cis-adducts.
RESUMEN
Cytotrienin A, an ansamycin-class antibiotic, exhibits potent apoptosis-inducing activity and has attracted much attention as a lead compound for anticancer drugs. Herein, we report a new asymmetric synthetic route to cytotrienin A, employing an unexplored approach involving the late-stage installation of a C11 side chain onto the macrolactam core. In this strategy, we utilized the redox properties of hydroquinone and installed a side chain on the sterically hindered C11 hydroxy group by the traceless Staudinger reaction. This study also demonstrated that the boron-Wittig/iterative Suzuki-Miyaura cross-coupling sequence was effective for the concise and selective construction of the (E,E,E)-conjugated triene moiety. The developed route opens new opportunities for the structure-activity relationship studies of the side chains of these ansamycin antibiotics and the preparation of other synthetic analogs and chemical probes for further biological studies.
Asunto(s)
Rifamicinas , Lactamas Macrocíclicas/farmacología , Rifamicinas/farmacología , Relación Estructura-Actividad , Oxidación-ReducciónRESUMEN
Trivalent PROTACs having a functionalization site with controlled orientation were designed, synthesized, and evaluated. Based on the X-ray structure of BRD protein degrader MZ1 (1) in complex with human VHL and BRD4BD2, we expected that the 1,2-disubstituted ethyl group near the JQ-1 moiety in MZ1 (1) could be replaced by a planar benzene tether as a platform for further functionalization. To test this hypothesis, we first designed six divalent MZ1 derivatives, 2a-c and 3a-c, by combining three variations of substitution patterns on the benzene ring (1,2-, 1,3-, and 1,4-substitution) and two variations in the number of ethylene glycol units (2 or 1). We then tested the synthesized compounds for the BRD4 degradation activity of each. As expected, we found that 1,2D-EG2-MZ1 (2a), an MZ1 derivative with 1,2-disubstituted benzene possessing two ethylene glycol units, had an activity profile similar to that of MZ1 (1). Based on the structure of 2a, we then synthesized and evaluated four isomeric trivalent MZ1 derivatives, 15a-15d, having a tert-butyl ester unit on the benzene ring as a handle for further functionalization. Among the four isomers, 1,2,5T-EG2-MZ1 (15c) retained a level of BRD4 depletion activity similar to that of 2a without inducing a measurable Hook effect, and its BRD4 depletion kinetics was the same as that of MZ1 (1). Other isomers were also shown to retain BRD4 depletion activity. Thus, the trivalent PROTACs we synthesized here may serve as efficient platforms for further applications.
Asunto(s)
Proteínas NuclearesRESUMEN
With the increasing importance of fluorine to medicinal chemistry and other areas, methods to access various fluorinated compounds are needed. Herein, we report the synthesis of difluoropropargyl vinyl ethers from ketones and aldehydes using difluoropropargyl bromide dicobalt complexes. We applied difluoropropargyl vinyl ethers to the synthesis of difluorodienone or difluoroallene under thermal conditions and trifluoro-pyran under acid-catalyzed conditions.
RESUMEN
A highly modular synthetic strategy for the heronamide C-type polyene macrolactams was established by synthesizing 8-deoxyheronamide C (2). The developed strategy enabled not only the total synthesis of 8-deoxyheronamide C (2) but also the unified synthesis of four heronamide-like molecules named "heronamidoids" (5-8). Conformational and reactivity analysis of the heronamidoids clarified that (1) the C19 stereochemistry mainly affected the conformation of the amide linkage, resulting in the change of alignment of two polyene units and reactivity toward photochemical [6π + 6π] cycloaddition, and (2) the C8,C9-diol moiety is important for the conversion to the heronamide A-type skeleton from the heronamide C skeleton.
Asunto(s)
Polienos , Reacción de Cicloadición , Lactamas Macrocíclicas , Conformación MolecularRESUMEN
16,17-Dihydroheronamide C (8) and ent-heronamide C (ent-1) were designed as probes for the mode-of-action analysis of heronamide C (1). These molecules were synthesized by utilizing a highly modular strategy developed in the preceding paper. The evaluation of the antifungal activity of these compounds revealed the exceptional importance of the C16-C17 double bond for the antifungal activity of heronamide C and the existence of chiral recognition between heronamide C (1) and cell membrane components.
Asunto(s)
Antifúngicos , Antifúngicos/farmacología , Lactamas Macrocíclicas , Relación Estructura-ActividadRESUMEN
Despite the potential of α-fluoroethers in medicinal chemistry, their synthetic methods, especially etherification of aliphatic alcohols, have been limited. Herein, we developed two- and three-step gem-difluoropropargylation of aliphatic alcohols including amino acid derivatives and naturally occurring bioactive molecules. Highly chemoselective etherification proceeded by using the gem-difluoropropargyl bromide dicobalt complex in the presence of silver triflate and triethylamine. Decomplexation of dicobalt complexes was achieved by using cerium ammonium nitrate or N,N,N'-trimethylethylenediamine. The thus obtained gem-difluoropropargyl ethers were converted to various α-difluoroethers which are expected to be useful for medicinal chemistry.
RESUMEN
A benchtop-stable reagent for the catalytic Nicholas reaction was developed. By combining a propargyl dicobalt hexacarbonyl cluster with an ortho-alkynylbenzoate unit and a fluorous tag, introduction of a propargyl hexacarbonyl complex on various aromatic compounds having acid- or base-sensitive functional groups becomes possible by using a gold(i) catalyst. In addition, the presence of a fluorous tag facilitates convenient separation of the target products from byproducts.
RESUMEN
GfsF is a multifunctional P450 monooxygenase that catalyzes epoxidation and subsequent hydroxylation in the biosynthesis of macrolide polyketide FD-891. Here, we describe the biochemical and structural analysis of GfsF. To obtain the structural basis of a dual-function reaction, we determined the crystal structure of ligand-free GfsF, which revealed GfsF to have a predominantly hydrophobic substrate binding pocket. The docking models, in conjunction with the results of the enzymatic assay with substrate analogues and site-directed mutagenesis suggested two distinct substrate binding modes for epoxidation and hydroxylation reactions, which explained how GfsF regulates the order of two oxidative reactions. These findings provide new insights into the reaction mechanism of multifunctional P450 monooxygenases.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Macrólidos/metabolismo , Sistema Enzimático del Citocromo P-450/análisis , Sistema Enzimático del Citocromo P-450/genética , Interacciones Hidrofóbicas e Hidrofílicas , Macrólidos/química , Conformación Molecular , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Especificidad por SustratoRESUMEN
A two-step method for introducing a propargyl group in aromatic bioactive small molecules has been developed. This method features propargylation of aromatic groups using a cationic propargyl hexacarbonyl complex in the presence of cesium carbonate, and decomplexation of the resultant cobalt complexes using 2,2,6,6-tetramethylpiperidine-1-oxoammonium tetrafluoroborate. These reactions proceed under mild conditions, and thus are applicable for acid- and/or oxidant-sensitive aromatic bioactive small molecules.
Asunto(s)
Propanoles/química , Bibliotecas de Moléculas Pequeñas/química , Transporte de ElectrónRESUMEN
A concise, protecting-group-free synthesis of the antipsychotic agent (+)-nemonapride has been achieved featuring a europium(III) trifluoromethanesulfonate (Eu(OTf)3)-catalyzed C4 selective aminolysis of a 3,4-epoxy alcohol by benzylamine and an expedient use of the resulting 4-benzylamino-1,3-diol product for constructing the pyrrolidine skeleton.
Asunto(s)
Alcoholes/química , Aminas/química , Benzamidas/síntesis química , Compuestos Epoxi/química , Mesilatos/química , Benzamidas/química , Catálisis , Estructura MolecularRESUMEN
Covering: up to the end of 2015A photo-cross-linked small-molecule affinity matrix is a unique platform for identifying targets for bioactive small molecules. It utilises a photogenerated carbene species to immobilise a variety of bioactive small molecules on an affinity matrix in a chemo- and site-nonselective manner. Although this platform would seem to run counter to the more typical approach of small-molecule immobilisation on an affinity matrix (i.e., selective coupling), it has been successfully utilised in the past decade to screen protein targets for many bioactive small molecules. This review describes the status of the photo-cross-linking methodology while providing a useful tutorial for academic and industrial chemical biologists who are involved or interested in drug target identification.
Asunto(s)
Productos Biológicos/farmacología , Metano/análogos & derivados , Metano/farmacología , Estructura Molecular , Proteínas/química , Bibliotecas de Moléculas PequeñasRESUMEN
Heronamides are biosynthetically related metabolites isolated from marine-derived actinomycetes. Heronamideâ C shows potent antifungal activity by targeting membrane phospholipids possessing saturated hydrocarbon chains with as-yet-unrevealed modes of action. In spite of their curious hypothesized biosynthesis and fascinating biological activities, there have been conflicts in regard to the reported stereochemistries of heronamides. Here, we describe the asymmetric total synthesis of the originally proposed and revised structures of heronamideâ C, which unambiguously confirmed the chemical structure of this molecule. We also demonstrated nonenzymatic synthesis of heronamidesâ A and B from heronamideâ C, which not only proved the postulated biosynthesis, but also confirmed the correct structures of heronamidesâ A and B. Investigation of the structure-activity relationship of synthetic and natural heronamides revealed the importance of both long-range stereochemical communication and the 20-membered macrolactam ring for the biological activity of these compounds.
Asunto(s)
Lactamas Macrocíclicas/síntesis química , Actinobacteria/química , Actinobacteria/metabolismo , Antifúngicos/síntesis química , Antifúngicos/química , Antifúngicos/farmacología , Catálisis , Dicroismo Circular , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacología , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente , Schizosaccharomyces/efectos de los fármacos , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Extracellular administration of water-soluble and membrane-permeant analogs of phosphatidylinositol phosphates (PIPs) is a useful strategy for understanding the cellular roles of PIPs as well as the mode of action of drugs whose biological activity is associated with PIPs. We herein established the synthetic route to the dioctanoyl analogue of phosphatidylinositol 3,5-bisphosphate (di-C8-PI(3,5)P2) and its penta(acetoxymethyl) ester (di-C8-PI(3,5)P2/5AM).
Asunto(s)
Diseño de Fármacos , Ésteres/química , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/síntesis químicaRESUMEN
C5-curcuminoids [a.k.a. bis(arylmethylidene)acetones] are curcumin analogues bearing a reactive cross-conjugated dienone structure essential for eliciting cytotoxicity. To gain insight into the mode of action of C5-curcuminoids, we investigated the reversibility of the thia-Michael reaction of 1,5-bis(3,5-bis(methoxymethoxy)phenyl)-1,4-pentadiene-3-one, named GO-Y030 which is the most potent cytotoxic C5-curcuminoid, using spectroscopic methods. A panel of GO-Y030-bis-thiol-adducts were synthesized and the structure-reactivity relationship regarding the retro thia-Michael reaction as well as the cell growth inhibitory activity against human colon cancer HCT116 were evaluated. Some C5-curcuminoid thiol-adducts exhibited comparable cytotoxicity with GO-Y030, demonstrating their potential use as prodrugs. These results imply that C5-curcuminoids elicit cytotoxicity by covalently interacting with various biothiols via a reversible thia-Michael reaction.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Derivados del Benceno/química , Derivados del Benceno/farmacología , Curcumina/análogos & derivados , Curcumina/farmacología , Cetonas/química , Cetonas/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Células HCT116 , Humanos , Profármacos/química , Profármacos/farmacología , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity.
Asunto(s)
Aminoglicósidos/farmacología , Antibacterianos/farmacología , Endosomas/efectos de los fármacos , Lactamas/farmacología , Macrólidos/farmacología , Fusión de Membrana/efectos de los fármacos , Vacuolas/efectos de los fármacos , Aminopiridinas/farmacología , Animales , Línea Celular , Colesterol/metabolismo , Endocitosis/fisiología , Endosomas/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Flavoproteínas/genética , Flavoproteínas/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Liposomas/química , Liposomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Transporte de Proteínas , Ratas , Transducción de Señal , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab5/genética , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Numerous cytochrome P450s are involved in secondary metabolite biosynthesis. The biosynthetic gene cluster for reveromycin A (RM-A), which is a promising lead compound with anti-osteoclastic activity, also includes a P450 gene, revI. To understand the roles of P450revI, we comprehensively characterized the enzyme by genetic, kinetic, and structural studies. The revI gene disruptants (ΔrevI) resulted in accumulation of reveromycin T (RM-T), and revI gene complementation restored RM-A production, indicating that the physiological substrate of P450revI is RM-T. Indeed, the purified P450revI catalyzed the C18-hydroxylation of RM-T more efficiently than the other RM derivatives tested. Moreover, the 1.4 Å resolution co-crystal structure of P450revI with RM-T revealed that the substrate binds the enzyme with a folded compact conformation for C18-hydroxylation. To address the structure-enzyme activity relationship, site-directed mutagenesis was performed in P450revI. R190A and R81A mutations, which abolished salt bridge formation with C1 and C24 carboxyl groups of RM-T, respectively, resulted in significant loss of enzyme activity. The interaction between Arg(190) and the C1 carboxyl group of RM-T elucidated why P450revI was unable to catalyze both RM-T 1-methyl ester and RM-T 1-ethyl ester. Moreover, the accumulation of RM-T in ΔrevI mutants enabled us to characterize its biological activity. Our results show that RM-T had stronger anticancer activity and isoleucyl-tRNA synthetase inhibition than RM-A. However, RM-T showed much less anti-osteoclastic activity than RM-A, indicating that hemisuccinate moiety is important for the activity. Structure-based P450revI engineering for novel hydroxylation and subsequent hemisuccinylation will help facilitate the development of RM derivatives with anti-osteoclast activity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Piranos/metabolismo , Compuestos de Espiro/metabolismo , Animales , Biocatálisis , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Electroforesis en Gel de Poliacrilamida , Células HeLa , Humanos , Hidroxilación , Isoleucina-ARNt Ligasa/antagonistas & inhibidores , Isoleucina-ARNt Ligasa/metabolismo , Células K562 , Cinética , Viabilidad Microbiana/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Mutación , Osteoclastos/citología , Osteoclastos/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Piranos/química , Piranos/farmacología , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Relación Estructura-Actividad , Especificidad por Sustrato , Espectrometría de Masas en TándemRESUMEN
The third generation of photoactivatable beads designed to capture bioactive small molecules in a chemo- and site-nonselective manner upon irradiation at 365 nm of UV light and release them as coumarin conjugates after exposure to UV light of 302 nm is described. These photoactivatable and photocleavable beads enable quantification of the amount and distribution of immobilized small molecules prior to the pull-down experiments to identify target protein(s) for the immobilized small molecules. The newly developed system was then used to analyze the functional group compatibility of the photo-cross-linking technology as well as the preferable nature of small molecules to be immobilized. As a result, compounds having a hydroxyl group, carboxylic acid, or aromatic ring were shown to give multiple conjugates, indicating that these compounds are well compatible with the photoactivatable beads system.