RESUMEN

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Asunto(s)

Adenosina Trifosfato/metabolismo , Antituberculosos/uso terapéutico , Mycobacterium tuberculosis/efectos de los fármacos , Piridinas/uso terapéutico , Quinina/análogos & derivados , Tuberculosis/tratamiento farmacológico , Animales , Antituberculosos/química , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Éteres/química , Éteres/farmacocinética , Éteres/farmacología , Éteres/uso terapéutico , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Piridinas/química , Piridinas/farmacocinética , Piridinas/farmacología , Quinina/química , Quinina/farmacocinética , Quinina/farmacología , Quinina/uso terapéutico , Tuberculosis/metabolismo

RESUMEN

The transfer of DNA between Enterococcus faecium strains has been characterized both by the movement of well-defined genetic elements and by the large-scale transfer of genomic DNA fragments. In this work, we report on the whole-genome analysis of transconjugants resulting from mating events between the vancomycin-resistant E. faecium C68 strain and the vancomycin-susceptible D344RRF strain to discern the mechanism by which the transferred regions enter the recipient chromosome. Vancomycin-resistant transconjugants from five independent matings were analyzed by whole-genome sequencing. In all cases but one, the penicillin binding protein 5 (pbp5) gene and the Tn5382 vancomycin resistance transposon were transferred together and replaced the corresponding pbp5 region of D344RRF. In one instance, Tn5382 inserted independently downstream of the D344RRF pbp5 gene. Single nucleotide variant (SNV) analysis suggested that entry of donor DNA into the recipient chromosome occurred by recombination across regions of homology between donor and recipient chromosomes, rather than through insertion sequence-mediated transposition. The transfer of genomic DNA was also associated with the transfer of C68 plasmid pLRM23 and another putative plasmid. Our data are consistent with the initiation of transfer by cointegration of a transferable plasmid with the donor chromosome, with subsequent circularization of the plasmid-chromosome cointegrant in the donor prior to transfer. Entry into the recipient chromosome most commonly occurred across regions of homology between donor and recipient chromosomes.

Asunto(s)

Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Resistencia a la Vancomicina/genética , Resistencia betalactámica/genética , Proteínas Bacterianas/genética , Cromosomas Bacterianos , Conjugación Genética , Elementos Transponibles de ADN , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Recombinación Homóloga , Operón , Proteínas de Unión a las Penicilinas/genética , Plásmidos , Resistencia a la Vancomicina/efectos de los fármacos , Resistencia betalactámica/efectos de los fármacos

RESUMEN

OBJECTIVES: The objectives of this study were to characterize contemporary MRSA isolates and understand the prevalence and impact of sequence variability in PBP2a on ceftaroline susceptibility. METHODS: A total of 184 MRSA isolates collected from 28 countries were collected and characterized. RESULTS: WT PBP2a proteins were found in MRSA distributed evenly over the ceftaroline MIC range of 0.5-2 mg/L (n=56). PBP2a variations found in 124 isolates fell into two categories: (i) 12 isolates contained a substitution in the transpeptidase pocket located in the penicillin-binding domain and exhibited significantly decreased ceftaroline susceptibility (typically 8 mg/L); and (ii) isolates with substitutions in the non-penicillin-binding domain (nPBD) in a region proposed to be functionally important for cell wall biogenesis. The majority (71%) of isolates containing only nPBD variations were inhibited by 2 mg/L ceftaroline, 23% by ≤1 mg/L and 6% by 4 mg/L. These data suggest that the WT MRSA distribution extends beyond the current EUCAST and CLSI susceptible breakpoints and includes isolates inhibited by 2 mg/L ceftaroline. SCCmec type IV was the predominant type in the ceftaroline-susceptible population (68%), whereas it only represented 6% of the non-susceptible population. The variations of MLST lineages were fewer among the non-susceptible group. CONCLUSIONS: This study suggests that MRSA populations with a WT PBP2a and those with nPBD variations overlap significantly and that PBP2a sequence-independent factors contribute to ceftaroline susceptibility. Whereas characterization of isolates with a ceftaroline MIC of 2 mg/L enriched for isolates with nPBD variations, it was not a discrete population. In contrast, the rare isolates containing a substitution in the transpeptidase-binding pocket were readily differentiated.

Asunto(s)

Antibacterianos/farmacología , Proteínas Bacterianas/genética , Cefalosporinas/farmacología , Variación Genética , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Staphylococcus aureus Resistente a Meticilina/genética , Proteínas de Unión a las Penicilinas/genética , Genotipo , Humanos , Staphylococcus aureus Resistente a Meticilina/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/microbiología , Ceftarolina

RESUMEN

OBJECTIVES: Pseudomonas aeruginosa is an important nosocomial pathogen that can cause a wide range of infections resulting in significant morbidity and mortality. Avibactam, a novel non-ß-lactam ß-lactamase inhibitor, is being developed in combination with ceftazidime and has the potential to be a valuable addition to the treatment options for the infectious diseases practitioner. We compared the frequency of resistance development to ceftazidime/avibactam in three P. aeruginosa strains that carried derepressed ampC alleles. METHODS: The strains were incubated in the presence of increasing concentrations of ceftazidime with a fixed concentration (4 mg/L) of avibactam to calculate the frequency of spontaneous resistance. The mutants were characterized by WGS to identify the underlying mechanism of resistance. A representative mutant protein was characterized biochemically. RESULTS: The resistance frequency was very low in all strains. The resistant variants isolated exhibited ceftazidime/avibactam MIC values that ranged from 64 to 256 mg/L. All of the mutants exhibited changes in the chromosomal ampC gene, the majority of which were deletions of various sizes in the Ω-loop region of AmpC. The mutant enzyme that carried the smallest Ω-loop deletion, which formed a part of the avibactam-binding pocket, was characterized biochemically and found to be less effectively inhibited by avibactam as well as exhibiting increased hydrolysis of ceftazidime. CONCLUSIONS: The development of high-level resistance to ceftazidime/avibactam appears to occur at low frequency, but structural modifications in AmpC can occur that impact the ability of avibactam to inhibit the enzyme and thereby protect ceftazidime from hydrolysis.

Asunto(s)

Antibacterianos/farmacología , Compuestos de Azabiciclo/farmacología , Proteínas Bacterianas/biosíntesis , Ceftazidima/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/enzimología , Selección Genética , Resistencia betalactámica , beta-Lactamasas/biosíntesis , Proteínas Bacterianas/genética , Combinación de Medicamentos , Humanos , Pruebas de Sensibilidad Microbiana , Tasa de Mutación , Pseudomonas aeruginosa/genética , beta-Lactamasas/genética

RESUMEN

In Gram-negative bacteria, lipoproteins are transported to the outer membrane by the Lol system. In this process, lipoproteins are released from the inner membrane by the ABC transporter LolCDE and passed to LolA, a diffusible periplasmic molecular chaperone. Lipoproteins are then transferred to the outer membrane receptor protein, LolB, for insertion in the outer membrane. Here we describe the discovery and characterization of novel pyridineimidazole compounds that inhibit this process. Escherichia coli mutants resistant to the pyridineimidazoles show no cross-resistance to other classes of antibiotics and map to either the LolC or LolE protein of the LolCDE transporter complex. The pyridineimidazoles were shown to inhibit the LolA-dependent release of the lipoprotein Lpp from E. coli spheroplasts. These results combined with bacterial cytological profiling are consistent with LolCDE-mediated disruption of lipoprotein targeting to the outer membrane as the mode of action of these pyridineimidazoles. The pyridineimidazoles are the first reported inhibitors of the LolCDE complex, a target which has never been exploited for therapeutic intervention. These compounds open the door to further interrogation of the outer membrane lipoprotein transport pathway as a target for antimicrobial therapy.

Asunto(s)

Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/metabolismo , Imidazoles/farmacología , Lipoproteínas/metabolismo , Transporte de Proteínas/efectos de los fármacos , Antibacterianos/química , Antifúngicos/química , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Bacterias Gramnegativas/genética , Imidazoles/química , Estructura Molecular , Mutación , Fenotipo

RESUMEN

The unmet medical need for novel intervention strategies to treat Neisseria gonorrhoeae infections is significant and increasing, as rapidly emerging resistance in this pathogen is threatening to eliminate the currently available treatment options. AZD0914 is a novel bacterial gyrase inhibitor that possesses potent in vitro activities against isolates with high-level resistance to ciprofloxacin and extended-spectrum cephalosporins, and it is currently in clinical development for the treatment of N. gonorrhoeae infections. The propensity to develop resistance against AZD0914 was examined in N. gonorrhoeae and found to be extremely low, a finding supported by similar studies with Staphylococcus aureus. The genetic characterization of both first-step and second-step mutants that exhibited decreased susceptibilities to AZD0914 identified substitutions in the conserved GyrB TOPRIM domain, confirming DNA gyrase as the primary target of AZD0914 and providing differentiation from fluoroquinolones. The analysis of available bacterial gyrase and topoisomerase IV structures, including those bound to fluoroquinolone and nonfluoroquinolone inhibitors, has allowed the rationalization of the lack of cross-resistance that AZD0914 shares with fluoroquinolones. Microbiological susceptibility data also indicate that the topoisomerase inhibition mechanisms are subtly different between N. gonorrhoeae and other bacterial species. Taken together, these data support the progression of AZD0914 as a novel treatment option for the oral treatment of N. gonorrhoeae infections.

Asunto(s)

Barbitúricos/farmacología , Neisseria gonorrhoeae/efectos de los fármacos , Compuestos de Espiro/farmacología , Inhibidores de Topoisomerasa II/farmacología , Girasa de ADN/química , Girasa de ADN/genética , Farmacorresistencia Bacteriana , Isoxazoles , Pruebas de Sensibilidad Microbiana , Morfolinas , Mutación , Neisseria gonorrhoeae/genética , Oxazolidinonas

RESUMEN

Many clinical isolates of Pseudomonas aeruginosa cause infections that are difficult to eradicate due to their resistance to a wide variety of antibiotics. Key genetic determinants of resistance were identified through genome sequences of 390 clinical isolates of P. aeruginosa, obtained from diverse geographic locations collected between 2003 and 2012 and were related to microbiological susceptibility data for meropenem, levofloxacin, and amikacin. ß-Lactamases and integron cassette arrangements were enriched in the established multidrug-resistant lineages of sequence types ST111 (predominantly O12) and ST235 (O11). This study demonstrates the utility of next-generation sequencing (NGS) in defining relevant resistance elements and highlights the diversity of resistance determinants within P. aeruginosa. This information is valuable in furthering the design of diagnostics and therapeutics for the treatment of P. aeruginosa infections.

Asunto(s)

Amicacina/farmacología , Antibacterianos/farmacología , Levofloxacino/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Tienamicinas/farmacología , Amicacina/uso terapéutico , Antibacterianos/uso terapéutico , Técnicas de Tipificación Bacteriana , Secuencia de Bases , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano/genética , Humanos , Levofloxacino/uso terapéutico , Meropenem , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/aislamiento & purificación , Análisis de Secuencia de ADN , Tienamicinas/uso terapéutico , beta-Lactamasas/genética

RESUMEN

In Gram-negative bacteria, the cell wall is surrounded by an outer membrane, the outer leaflet of which is comprised of charged lipopolysaccharide (LPS) molecules. Lipid A, a component of LPS, anchors this molecule to the outer membrane. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent metalloamidase that catalyzes the first committed step of biosynthesis of Lipid A, making it a promising target for antibiotic therapy. Formation of soluble aggregates of Pseudomonas aeruginosa LpxC protein when overexpressed in Escherichia coli has limited the availability of high quality protein for X-ray crystallography. Expression of LpxC in the presence of an inhibitor dramatically increased protein solubility, shortened crystallization time and led to a high-resolution crystal structure of LpxC bound to the inhibitor. However, this approach required large amounts of compound, restricting its use. To reduce the amount of compound needed, an overexpression strain of E. coli was created lacking acrB, a critical component of the major efflux pump. By overexpressing LpxC in the efflux deficient strain in the presence of LpxC inhibitors, several structures of P. aeruginosa LpxC in complex with different compounds were solved to accelerate structure-based drug design.

Asunto(s)

Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Pseudomonas aeruginosa/enzimología , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/genética , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/genética , Catálisis , Cromatografía Liquida , Cristalografía por Rayos X , Escherichia coli , Expresión Génica , Espectrometría de Masas , Conformación Proteica , Zinc/química , Zinc/metabolismo

RESUMEN

Diarylthiazole (DAT), a hit from diversity screening, was found to have potent antimycobacterial activity against Mycobacterium tuberculosis (Mtb). In a systematic medicinal chemistry exploration, we demonstrated chemical opportunities to optimize the potency and physicochemical properties. The effort led to more than 10 compounds with submicromolar MICs and desirable physicochemical properties. The potent antimycobacterial activity, in conjunction with low molecular weight, made the series an attractive lead (antibacterial ligand efficiency (ALE)>0.4). The series exhibited excellent bactericidal activity and was active against drug-sensitive and resistant Mtb. Mutational analysis showed that mutations in prrB impart resistance to DAT compounds but not to reference drugs tested. The sensor kinase PrrB belongs to the PrrBA two component system and is potentially the target for DAT. PrrBA is a conserved, essential regulatory mechanism in Mtb and has been shown to have a role in virulence and metabolic adaptation to stress. Hence, DATs provide an opportunity to understand a completely new target system for antimycobacterial drug discovery.

Asunto(s)

Antituberculosos/química , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/efectos de los fármacos , Proteínas Quinasas/metabolismo , Tiazoles/química , Animales , Antituberculosos/síntesis química , Antituberculosos/farmacología , Proteínas Bacterianas/genética , Farmacorresistencia Bacteriana , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Pruebas de Sensibilidad Microbiana , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Polimorfismo de Nucleótido Simple , Proteínas Quinasas/genética , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/farmacología

RESUMEN

In a previous report, we described the discovery of 1,4-azaindoles, a chemical series with excellent in vitro and in vivo antimycobacterial potency through noncovalent inhibition of decaprenylphosphoryl-ß-d-ribose-2'-epimerase (DprE1). Nevertheless, high mouse metabolic turnover and phosphodiesterase 6 (PDE6) off-target activity limited its advancement. Herein, we report lead optimization of this series, culminating in potent, metabolically stable compounds that have a robust pharmacokinetic profile without any PDE6 liability. Furthermore, we demonstrate efficacy for 1,4-azaindoles in a rat chronic TB infection model. We believe that compounds from the 1,4-azaindole series are suitable for in vivo combination and safety studies.

Asunto(s)

Antituberculosos/síntesis química , Indoles/síntesis química , Oxidorreductasas de Alcohol , Animales , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/antagonistas & inhibidores , Modelos Animales de Enfermedad , Humanos , Indoles/farmacocinética , Ratones , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Ratas , Relación Estructura-Actividad

RESUMEN

4-Aminoquinolone piperidine amides (AQs) were identified as a novel scaffold starting from a whole cell screen, with potent cidality on Mycobacterium tuberculosis (Mtb). Evaluation of the minimum inhibitory concentrations, followed by whole genome sequencing of mutants raised against AQs, identified decaprenylphosphoryl-ß-d-ribose 2'-epimerase (DprE1) as the primary target responsible for the antitubercular activity. Mass spectrometry and enzyme kinetic studies indicated that AQs are noncovalent, reversible inhibitors of DprE1 with slow on rates and long residence times of ∼100 min on the enzyme. In general, AQs have excellent leadlike properties and good in vitro secondary pharmacology profile. Although the scaffold started off as a single active compound with moderate potency from the whole cell screen, structure-activity relationship optimization of the scaffold led to compounds with potent DprE1 inhibition (IC50 < 10 nM) along with potent cellular activity (MIC = 60 nM) against Mtb.

Asunto(s)

Amidas/química , Antituberculosos/química , Proteínas Bacterianas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Piperidinas/química , Quinolonas/química , Oxidorreductasas de Alcohol , Amidas/farmacocinética , Amidas/farmacología , Animales , Antituberculosos/farmacocinética , Antituberculosos/farmacología , Dominio Catalítico , Línea Celular Tumoral , Farmacorresistencia Bacteriana , Genoma Bacteriano , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mutación , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Piperidinas/farmacocinética , Piperidinas/farmacología , Unión Proteica , Quinolonas/farmacocinética , Quinolonas/farmacología , Ratas Wistar , Estereoisomerismo , Relación Estructura-Actividad

RESUMEN

We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-ß-D-ribose2'-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.

Asunto(s)

Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Indoles/síntesis química , Mycobacterium tuberculosis/efectos de los fármacos , Oxidorreductasas/antagonistas & inhibidores , Oxidorreductasas de Alcohol , Animales , Antituberculosos/farmacocinética , Antituberculosos/uso terapéutico , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Indoles/farmacocinética , Indoles/farmacología , Indoles/uso terapéutico , Ratones , Ratas , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológico

RESUMEN

A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

Asunto(s)

Deshidrogenasas de Carbohidratos/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Adenosina Difosfato/metabolismo , Antibacterianos/química , Deshidrogenasas de Carbohidratos/análisis , Pared Celular , Descubrimiento de Drogas/métodos , Escherichia coli/metabolismo , Fluorescencia , Peptidoglicano/biosíntesis , Polirribonucleótido Nucleotidiltransferasa/metabolismo

RESUMEN

Methionine aminopeptidase (MAP) (E.C. 3.4.11.18) is a metallopeptidase that cleaves the N-terminal methionine (Met) residue from some proteins. MAP is essential for growth of several bacterial pathogens, making it a target for antibacterial drug discovery. MAP enzymes are also present in eukaryotic cells, and one is a target for antiangiogenic cancer therapy. To screen large compound libraries for MAP inhibitors as the starting point for drug discovery, a high-throughput-compatible assay is valuable. Here the authors describe a novel assay, which detects the Met product of MAP-catalyzed peptide cleavage by coupling it to adenosine triphosphate (ATP)-dependent production of S-adenosyl-L-methionine (SAM) and inorganic phosphate (P(i)) by SAM synthetase (MetK) combined with inorganic pyrophosphatase. The three P(i) ions produced for each Met consumed are detected using Malachite Green/molybdate reagent. This assay can use any unmodified peptide MAP substrate with an N-terminal Met. The assay was used to measure kinetic constants for Escherichia coli MAP using Mn(2+) as the activator and the peptide Met-Gly-Met-Met as the substrate, as well as to measure the potency of a MAP inhibitor. A Mn(2+) buffer is described that can be used to prevent free Mn(2+) depletion by chelating compounds from interfering in screens for MAP inhibitors.

Asunto(s)

Aminopeptidasas/metabolismo , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Ligasas/metabolismo , Metionina/metabolismo , S-Adenosilmetionina/biosíntesis , Aminopeptidasas/antagonistas & inhibidores , Cationes Bivalentes/farmacología , Ácido Edético/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Escherichia coli/enzimología , Escherichia coli/metabolismo , Ensayos Analíticos de Alto Rendimiento/normas , Cinética , Manganeso/farmacología , Metionil Aminopeptidasas , Estándares de Referencia , S-Adenosilmetionina/metabolismo