RESUMEN
Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase that serves a critical function in numerous developmental, morphogenic, and proliferative signaling pathways. If dysregulated, MET has been shown to be involved in the development and survival of several cancers, including non-small cell lung cancer (NSCLC), renal cancer, and other epithelial tumors. Currently, the clinical efficacy of FDA approved MET inhibitors is limited by on-target acquired resistance, dose-limiting toxicities, and less than optimal efficacy against brain metastasis. Therefore, there is still an unmet medical need for the development of MET inhibitors to address these issues. Herein we report the application of structure-based design for the discovery and development of a novel class of brain-penetrant MET inhibitors with enhanced activity against clinically relevant mutations and improved selectivity. Compound 13 with a MET D1228N cell line IC50 value of 23 nM showed good efficacy in an intracranial tumor model and increased the median overall survival of the animals to 100% when dosed orally at 100 mg/kg daily for 21 days.
Asunto(s)
Antineoplásicos , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas c-met , Pirazoles , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-met/metabolismo , Humanos , Animales , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/uso terapéutico , Pirazoles/farmacología , Pirazoles/química , Pirazoles/síntesis química , Línea Celular Tumoral , Relación Estructura-Actividad , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Antineoplásicos/uso terapéutico , Descubrimiento de Drogas , Pirazinas/farmacología , Pirazinas/síntesis química , Pirazinas/química , Pirazinas/uso terapéutico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Ratones , Mutación , RatasRESUMEN
Hydrogen sulfide (H2S) production in the intestinal microbiota has many contributions to human health and disease. An important source of H2S in the human gut is anaerobic respiration of sulfite released from the abundant dietary and host-derived organic sulfonate substrate in the gut, taurine (2-aminoethanesulfonate). However, the enzymes that allow intestinal bacteria to access sulfite from taurine have not yet been identified. Here we decipher the complete taurine desulfonation pathway in Bilophila wadsworthia 3.1.6 using differential proteomics, in vitro reconstruction with heterologously produced enzymes, and identification of critical intermediates. An initial deamination of taurine to sulfoacetaldehyde by a known taurine:pyruvate aminotransferase is followed, unexpectedly, by reduction of sulfoacetaldehyde to isethionate (2-hydroxyethanesulfonate) by an NADH-dependent reductase. Isethionate is then cleaved to sulfite and acetaldehyde by a previously uncharacterized glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslA). The acetaldehyde produced is oxidized to acetyl-CoA by a dehydrogenase, and the sulfite is reduced to H2S by dissimilatory sulfite reductase. This unique GRE is also found in Desulfovibrio desulfuricans DSM642 and Desulfovibrio alaskensis G20, which use isethionate but not taurine; corresponding knockout mutants of D. alaskensis G20 did not grow with isethionate as the terminal electron acceptor. In conclusion, the novel radical-based C-S bond-cleavage reaction catalyzed by IslA diversifies the known repertoire of GRE superfamily enzymes and enables the energy metabolism of B. wadsworthia This GRE is widely distributed in gut bacterial genomes and may represent a novel target for control of intestinal H2S production.
Asunto(s)
Oxidorreductasas de Alcohol/genética , Bilophila/enzimología , Sulfuro de Hidrógeno/metabolismo , Proteómica , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Anaerobiosis/genética , Bilophila/química , Bilophila/metabolismo , Microbioma Gastrointestinal/genética , Humanos , Sulfuro de Hidrógeno/química , Oxidación-Reducción , Taurina/metabolismoRESUMEN
The C-terminal domain of the dehydrophos biosynthetic enzyme DhpH (DhpH-C) catalyzes the condensation of Leu-tRNALeu with (R)-1-aminoethylphosphonate, the aminophosphonate analog of alanine called Ala(P). The product of this reaction, Leu-Ala(P), is a phosphonodipeptide, a class of compounds that have previously been investigated for use as clinical antibiotics. In this study, we show that DhpH-C is highly substrate tolerant and can condense various aminophosphonates (Gly(P), Ser(P), Val(P), 1-amino-propylphosphonate, and phenylglycine(P)) to Leu. Moreover, the enzyme is also tolerant with respect to the amino acid attached to tRNALeu. Using a mutant of leucyl tRNA synthetase that is deficient in its proofreading ability allowed the preparation of a series of aminoacyl-tRNALeu derivatives (Ile, Ala, Val, Met, norvaline, and norleucine). DhpH-C accepted these aminoacyl-tRNA derivatives and condensed the amino acid with l-Ala(P) to form the corresponding phosphonodipeptides. A subset of these peptides displayed antimicrobial activities demonstrating that the enzyme is a versatile biocatalyst for the preparation of antimicrobial peptides. We also investigated another enzyme from the dehydrophos biosynthetic pathway, the 2-oxoglutarate dependent enzyme DhpA. This enzyme oxidizes 2-hydroxyethylphosphonate to 1,2-dihydroxyethylphosphonate en route to l-Ala(P), but longer incubation results in overoxidation to 1-oxo-2-hydroxyethylphosphonate. This α-ketophosphonate was converted by the pyridoxal phosphate dependent enzyme DhpD into l-Ser(P). Thus, the dehydrophos biosynthetic enzymes can generate not only l-Ala(P) but also l-Ser(P).
Asunto(s)
Alanina/análogos & derivados , Antibacterianos/biosíntesis , Leucina-ARNt Ligasa/metabolismo , Alanina/biosíntesis , Alanina/química , Antibacterianos/química , Biocatálisis , Conformación Molecular , Streptomyces/enzimologíaRESUMEN
Methylphosphonate synthase (MPnS) produces methylphosphonate, a metabolic precursor to methane in the upper ocean. Here, we determine a 2.35-angstrom resolution structure of MPnS and discover that it has an unusual 2-histidine-1-glutamine iron-coordinating triad. We further solve the structure of a related enzyme, hydroxyethylphosphonate dioxygenase from Streptomyces albus (SaHEPD), and find that it displays the same motif. SaHEPD can be converted into an MPnS by mutation of glutamine-adjacent residues, identifying the molecular requirements for methylphosphonate synthesis. Using these sequence markers, we find numerous putative MPnSs in marine microbiomes and confirm that MPnS is present in the abundant Pelagibacter ubique. The ubiquity of MPnS-containing microbes supports the proposal that methylphosphonate is a source of methane in the upper, aerobic ocean, where phosphorus-starved microbes catabolize methylphosphonate for its phosphorus.
Asunto(s)
Organismos Acuáticos/enzimología , Proteínas Bacterianas/química , Compuestos Organofosforados/metabolismo , Oxigenasas/química , Alphaproteobacteria/enzimología , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/ultraestructura , Dominio Catalítico , Glutamina/química , Histidina/química , Microbiota , Oxigenasas/clasificación , Oxigenasas/ultraestructura , Filogenia , Streptomyces/enzimologíaRESUMEN
Activation of O-H bonds by inorganic metal-oxo complexes has been documented, but no cognate enzymatic process is known. Our mechanistic analysis of 2-hydroxyethylphosphonate dioxygenase (HEPD), which cleaves the C1-C2 bond of its substrate to afford hydroxymethylphosphonate on the biosynthetic pathway to the commercial herbicide phosphinothricin, uncovered an example of such an O-H-bond-cleavage event. Stopped-flow UV-visible absorption and freeze-quench Mössbauer experiments identified a transient iron(IV)-oxo (ferryl) complex. Maximal accumulation of the intermediate required both the presence of deuterium in the substrate and, importantly, the use of 2H2O as solvent. The ferryl complex forms and decays rapidly enough to be on the catalytic pathway. To account for these unanticipated results, a new mechanism that involves activation of an O-H bond by the ferryl complex is proposed. This mechanism accommodates all available data on the HEPD reaction.
Asunto(s)
Dioxigenasas/metabolismo , Compuestos de Hierro/metabolismo , Organofosfonatos/metabolismo , Biocatálisis , Dioxigenasas/química , Compuestos de Hierro/química , Cinética , Conformación Molecular , Organofosfonatos/química , Espectrofotometría Ultravioleta , Espectroscopía de MossbauerRESUMEN
This review discusses the current mechanistic understanding of a group of mononuclear non-heme iron-dependent enzymes that catalyze four-electron oxidation of their organic substrates without the use of any cofactors or cosubstrates. One set of enzymes acts on α-ketoacid-containing substrates, coupling decarboxylation to oxygen activation. This group includes 4-hydroxyphenylpyruvate dioxygenase, 4-hydroxymandelate synthase, and CloR involved in clorobiocin biosynthesis. A second set of enzymes acts on substrates containing a thiol group that coordinates to the iron. This group is comprised of isopenicillin N synthase, thiol dioxygenases, and enzymes involved in the biosynthesis of ergothioneine and ovothiol. The final group of enzymes includes HEPD and MPnS that both carry out the oxidative cleavage of the carbon-carbon bond of 2-hydroxyethylphosphonate but generate different products. Commonalities amongst many of these enzymes are discussed and include the initial substrate oxidation by a ferric-superoxo-intermediate and a second oxidation by a ferryl species.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Hierro , Transporte de ElectrónRESUMEN
Nonheme iron oxygenases that carry out four-electron oxidations of substrate have been proposed to employ iron(III) superoxide species to initiate this reaction [Paria, S.; Que, L.; Paine, T. K. Angew. Chem. Int. Ed. 2011, 50, 11129]. Here we report experimental evidence in support of this proposal. (18)O KIEs were measured for two recently discovered mononuclear nonheme iron oxygenases: hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS). Competitive (18)O KIEs measured with deuterated substrates are larger than those measured with unlabeled substrates, which indicates that C-H cleavage must occur before an irreversible reductive step at molecular oxygen. A similar observation was previously used to implicate copper(II) superoxide in the H-abstraction reactions catalyzed by dopamine ß-monooxygenase [Tian, G. C.; Klinman, J. P. J. Am. Chem. Soc. 1993, 115, 8891] and peptidylglycine α-hydroxylating monooxygenase [Francisco, W. A.; Blackburn, N. J.; Klinman, J. P. Biochemistry 2003, 42, 1813].
Asunto(s)
Dioxigenasas/metabolismo , Hidrógeno/química , Hierro/química , Superóxidos/química , Transporte de Electrón , Cinética , Isótopos de Oxígeno/químicaRESUMEN
2-Hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS) are nonheme iron oxygenases that both catalyze the carbon-carbon bond cleavage of 2-hydroxyethylphosphonate but generate different products. Substrate labeling experiments led to a mechanistic hypothesis in which the fate of a common intermediate determined product identity. We report here the generation of a bifunctional mutant of HEPD (E176H) that exhibits the activity of both HEPD and MPnS. The product distribution of the mutant is sensitive to a substrate isotope effect, consistent with an isotope-sensitive branching mechanism involving a common intermediate. The X-ray structure of the mutant was determined and suggested that the introduced histidine does not coordinate the active site metal, unlike the iron-binding glutamate it replaced.
Asunto(s)
Dioxigenasas/química , Dioxigenasas/metabolismo , Oxigenasas/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Dioxigenasas/genética , Ácido Glutámico/genética , Ácido Glutámico/metabolismo , Histidina , Espectroscopía de Resonancia Magnética , Mutación , Organofosfonatos/metabolismo , Compuestos Organofosforados/metabolismo , Oxigenasas/química , Conformación ProteicaRESUMEN
Phosphonates (C-PO3²â») have applications as antibiotics, herbicides, and detergents. In some environments, these molecules represent the predominant source of phosphorus, and several microbes have evolved dedicated enzymatic machineries for phosphonate degradation. For example, most common naturally occurring phosphonates can be catabolized to either phosphonoacetaldehyde or phosphonoacetate, which can then be hydrolyzed to generate inorganic phosphate and acetaldehyde or acetate, respectively. The phosphonoacetaldehyde oxidase gene (phnY) links these two hydrolytic processes and provides a previously unknown catabolic mechanism for phosphonoacetate production in the microbial metabolome. Here, we present biochemical characterization of PhnY and high-resolution crystal structures of the apo state, as well as complexes with substrate, cofactor, and product. Kinetic analysis of active site mutants demonstrates how a highly conserved aldehyde dehydrogenase active site has been modified in nature to generate activity with a phosphonate substrate.
Asunto(s)
Acetaldehído/análogos & derivados , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Ácido Fosfonoacético/metabolismo , Acetaldehído/química , Acetaldehído/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Dominio Catalítico/genética , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Estructura Molecular , NAD/química , NAD/metabolismo , Oxidorreductasas/genética , Ácido Fosfonoacético/química , Sinorhizobium meliloti/enzimologíaRESUMEN
The iron-dependent epoxidase HppE converts (S)-2-hydroxypropyl-1-phosphonate (S-HPP) to the antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonate] in an unusual 1,3-dehydrogenation of a secondary alcohol to an epoxide. HppE has been classified as an oxidase, with proposed mechanisms differing primarily in the identity of the O2-derived iron complex that abstracts hydrogen (Hâ¢) from C1 of S-HPP to initiate epoxide ring closure. We show here that the preferred cosubstrate is actually H2O2 and that HppE therefore almost certainly uses an iron(IV)-oxo complex as the H⢠abstractor. Reaction with H2O2 is accelerated by bound substrate and produces fosfomycin catalytically with a stoichiometry of unity. The ability of catalase to suppress the HppE activity previously attributed to its direct utilization of O2 implies that reduction of O2 and utilization of the resultant H2O2 were actually operant.
Asunto(s)
Antibacterianos/biosíntesis , Fosfomicina/biosíntesis , Proteínas de Hierro no Heme/química , Oxidorreductasas/química , Peroxidasas/química , Peróxido de Hidrógeno/química , Proteínas de Hierro no Heme/clasificación , Oxidorreductasas/clasificación , Peroxidasas/clasificación , Yersinia pseudotuberculosis/enzimologíaRESUMEN
Natural product biosynthesis has proven a fertile ground for the discovery of novel chemistry. Herein we review the progress made in elucidating the biosynthetic pathways of phosphonate and phosphinate natural products such as the antibacterial compounds dehydrophos and fosfomycin, the herbicidal phosphinothricin-containing peptides, and the antimalarial compound FR-900098. In each case, investigation of the pathway has yielded unusual, and often unprecedented, biochemistry. Likewise, recent investigations have uncovered novel ways to cleave the CP bond to yield phosphate under phosphorus starvation conditions. These include the discovery of novel oxidative cleavage of the CP bond catalyzed by PhnY and PhnZ as well as phosphonohydrolases that liberate phosphate from phosphonoacetate. Perhaps the crown jewel of phosphonate catabolism has been the recent resolution of the longstanding problem of the C-P lyase responsible for reductively cleaving the CP bond of a number of different phosphonates to release phosphate. Taken together, the strides made on both metabolic and catabolic fronts illustrate an array of fascinating biochemistry.
Asunto(s)
Organofosfonatos/metabolismo , Biocatálisis , Vías Biosintéticas , Humanos , Compuestos Organofosforados/metabolismo , Oxidación-ReducciónRESUMEN
The P-C bonds in phosphonate and phosphinate natural products endow them with a high level of stability and the ability to mimic phosphate esters and carboxylates. As such, they have a diverse range of enzyme targets that act on substrates containing such functionalities. Recent years have seen a renewed interest in discovery efforts focused on this class of compounds as well as in understanding their biosynthetic pathways. This chapter focuses on current knowledge of these biosynthetic pathways as well as tools for phosphonate discovery.
Asunto(s)
Productos Biológicos/metabolismo , Organofosfonatos/metabolismo , Ácidos Fosfínicos/metabolismo , Streptomyces/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Vías Biosintéticas , Cromatografía Liquida , Descubrimiento de Drogas , Cinética , Espectroscopía de Resonancia Magnética , Organofosfonatos/química , Organofosfonatos/aislamiento & purificación , Ácidos Fosfínicos/química , Ácidos Fosfínicos/aislamiento & purificación , Espectroscopía Infrarroja por Transformada de Fourier , Streptomyces/enzimología , Streptomyces/genética , Especificidad por SustratoRESUMEN
Methylphosphonate synthase is a non-heme iron-dependent oxygenase that converts 2-hydroxyethylphosphonate (2-HEP) to methylphosphonate. On the basis of experiments with two enantiomers of a substrate analog, 2-hydroxypropylphosphonate, catalysis is proposed to commence with stereospecific abstraction of the pro-S hydrogen on C2 of the substrate. Experiments with isotopologues of 2-HEP indicate stereospecific hydrogen transfer of the pro-R hydrogen at C2 of the substrate to the methyl group of methylphosphonate. Kinetic studies with these substrate isotopologues reveal that neither hydrogen transfer is rate limiting under saturating substrate conditions. A mechanism is proposed that is consistent with the available data.
Asunto(s)
Hemo Oxigenasa (Desciclizante)/metabolismo , Hierro/metabolismo , Oxigenasas/metabolismo , Análisis de Fourier , Cinética , Espectrometría de Masas , Resonancia Magnética Nuclear BiomolecularRESUMEN
HEPD belongs to the superfamily of 2-His-1-carboxylate non-heme iron-dependent dioxygenases. It converts 2-hydroxyethylphosphonate (2-HEP) to hydroxymethylphosphonate (HMP) and formate. Previously postulated mechanisms for the reaction catalyzed by HEPD cannot explain its conversion of 1-HEP to acetylphosphate. Alternative mechanisms that involve either phosphite or methylphosphonate as intermediates, which potentially explain all experimental studies including isotope labeling experiments and use of substrate analogues, were investigated. The results of these studies reveal that these alternative mechanisms are not correct. Site-directed mutagenesis studies of Lys16, Arg90, and Tyr98 support roles of these residues in binding of 2-HEP. Mutation of Lys16 to Ala resulted in an inactive enzyme, whereas mutation of Arg90 to Ala or Tyr98 to Phe greatly decreased k(cat)/K(m,2-HEP). Furthermore, the latter mutants could not be saturated in O(2). These results suggest that proper binding of 2-HEP is important for O(2) activation and that the enzyme uses a compulsory binding order with 2-HEP binding before O(2). The Y98F mutant produces methylphosphonate as a minor side product providing indirect support for the proposal that the last step during catalysis involves a ferric hydroxide reacting with a methylphosphonate radical.
Asunto(s)
Dioxigenasas/química , Organofosfonatos/química , Relación Estructura-Actividad Cuantitativa , Streptomyces/enzimología , Aminobutiratos/química , Vías Biosintéticas/genética , Cristalografía por Rayos X , Dioxigenasas/biosíntesis , Dioxigenasas/genética , Herbicidas/química , Mutagénesis Sitio-Dirigida , Estereoisomerismo , Especificidad por Sustrato/genéticaRESUMEN
Stereochemical investigations have shown that the conversion of 2-hydroxyethylphosphonate to hydroxymethylphosphonate by the enzyme HEPD involves removal of the pro-S hydrogen at C2 and, surprisingly, the loss of stereochemical information at C1. As a result, the mechanisms previously proposed for HEPD must be re-evaluated.