RESUMEN
We have identified an acyl-carrier protein, Rv0100, that is up-regulated in a dormancy model. This protein plays a critical role in the fatty acid biosynthesis pathway, which is important for energy storage and cell wall synthesis in Mycobacterium tuberculosis (MTB). Knocking out the Rv0100 gene resulted in a significant reduction of growth compared to wild-type MTB in the Wayne model of non-replicating persistence. We have also shown that Rv0100 is essential for the growth and survival of this pathogen during infection in mice and a macrophage model. Furthermore, knocking out Rv0100 disrupted the synthesis of phthiocerol dimycocerosates, the virulence-enhancing lipids produced by MTB and Mycobacterium bovis. We hypothesize that this essential gene contributes to MTB virulence in the state of latent infection. Therefore, inhibitors targeting this gene could prove to be potent antibacterial agents against this pathogen.
Asunto(s)
Proteína Transportadora de Acilo , Proteínas Bacterianas , Mycobacterium tuberculosis , Animales , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidad , Ratones , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteína Transportadora de Acilo/metabolismo , Proteína Transportadora de Acilo/genética , Macrófagos/microbiología , Macrófagos/metabolismo , Virulencia , Regulación Bacteriana de la Expresión Génica , Tuberculosis/microbiología , Lípidos/químicaRESUMEN
Class II Fructose-1,6-bisphosphatases (FBPaseII) (EC: 3.1.3.11) are highly conserved essential enzymes in the gluconeogenic pathway of microorganisms. Previous crystallographic studies of FBPasesII provided insights into various inactivated states of the enzyme in different species. Presented here is the first crystal structure of FBPaseII in an active state, solved for the enzyme from Francisella tularensis (FtFBPaseII), containing native metal cofactor Mn2+ and complexed with catalytic product fructose-6-phosphate (F6P). Another crystal structure of the same enzyme complex is presented in the inactivated state due to the structural changes introduced by crystal packing. Analysis of the interatomic distances among the substrate, product, and divalent metal cations in the catalytic centers of the enzyme led to a revision of the catalytic mechanism suggested previously for class II FBPases. We propose that phosphate-1 is cleaved from the substrate fructose-1,6-bisphosphate (F1,6BP) by T89 in a proximal α-helix backbone (G88-T89-T90-I91-T92-S93-K94) in which the substrate transition state is stabilized by the positive dipole of the ã-helix backbone. Once cleaved a water molecule found in the active site liberates the inorganic phosphate from T89 completing the catalytic mechanism. Additionally, a crystal structure of Mycobacterium tuberculosis FBPaseII (MtFBPaseII) containing a bound F1,6BP is presented to further support the substrate binding and novel catalytic mechanism suggested for this class of enzymes.
Asunto(s)
Francisella tularensis , Fructosa-Bifosfatasa , Fructosa-Bifosfatasa/metabolismo , Francisella tularensis/metabolismo , Catálisis , Dominio Catalítico , Fructosa/metabolismo , Cristalografía por Rayos XRESUMEN
Three critical aspects that define high concentration antibody products (HCAPs) are as follows: 1) formulation composition, 2) dosage form, and 3) primary packaging configuration. HCAPs have become successful in the therapeutic sector due to their unique advantage of allowing subcutaneous self-administration. Technical challenges, such as physical and chemical instability, viscosity, delivery volume limitations, and product immunogenicity, can hinder successful development and commercialization of HCAPs. Such challenges can be overcome by robust formulation and process development strategies, as well as rational selection of excipients and packaging components. We compiled and analyzed data from US Food and Drug Administration-approved and marketed HCAPs that are ≥100 mg/mL to identify trends in formulation composition and quality target product profile. This review presents our findings and discusses novel formulation and processing technologies that enable the development of improved HCAPs at ≥200 mg/mL. The observed trends can be used as a guide for further advancements in the development of HCAPs as more complex antibody-based modalities enter biologics product development.
Asunto(s)
Embalaje de Medicamentos , Excipientes , Preparaciones Farmacéuticas , ViscosidadRESUMEN
The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4â Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02â Å, ß = 90.003°) with four chains in the asymmetric unit. Chain A had two coordinated Mg2+ ions in its active center, which is distinct from previous findings, and is presumably deactivated by their presence. The structure revealed an approximate 222 (D2) symmetry homotetramer analogous to that previously described for MtFBPaseII, which is formed by a crystallographic dyad and which differs from the exact tetramer found in EcFBPaseII at a 222 symmetry site in the crystal. Instead, the approximate homotetramer is very similar to that found in the dual phosphatase from Synechocystis, even though no allosteric effector was found in FtFBPase. The amino-acid sequence and folding of the active site of FtFBPaseII result in structural characteristics that are more similar to those of the previously published EcFBPaseII than to those of MtFBPaseII. The kinetic parameters of native FtFBPaseII were found to be in agreement with published studies. Kinetic analyses of the Thr89Ser and Thr89Ala mutations in the active site of the enzyme are consistent with the previously proposed mechanism for other class II bisphosphatases. The Thr89Ala variant enzyme was inactive but the Thr89Ser variant was partially active, with an approximately fourfold lower Km and Vmax than the native enzyme. The structural and functional insights derived from the structure of FtFBPaseII will provide valuable information for the design of specific inhibitors.
Asunto(s)
Francisella tularensis/enzimología , Fructosa-Bifosfatasa/química , Fructosa-Bifosfatasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Escherichia coli/enzimología , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/aislamiento & purificación , Modelos Moleculares , Mycobacterium tuberculosis/enzimología , Conformación Proteica , Estructura Cuaternaria de Proteína , Synechocystis/enzimologíaRESUMEN
The true identity of the protein found in the crystals reported by Bondoc et al. [(2019), Acta Cryst. F75, 646-651] is given.
RESUMEN
Acyl carrier proteins (ACPs) are important components in fatty-acid biosynthesis in prokaryotes. Rv0100 is predicted to be an essential ACP in Mycobacterium tuberculosis, the pathogen that is the causative agent of tuberculosis, and therefore has the potential to be a novel antituberculosis drug target. Here, the successful cloning and purification of Rv0100 using Mycobacterium smegmatis as a host is reported. Crystals of the purified protein were obtained that diffracted to a resolution of 1.9â Å. Overall, this work lays the foundation for the future pursuit of drug discovery and development against this potentially novel drug target.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteínas Bacterianas/química , Cristalización , Mycobacterium tuberculosis/química , Proteína Transportadora de Acilo/genética , Proteína Transportadora de Acilo/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cristalografía por Rayos X , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Leading regulatory agencies recommend biosimilar assessment to proceed in a stepwise fashion, starting with a detailed analytical comparison of the structural and functional properties of the proposed biosimilar and reference product. The degree of analytical similarity determines the degree of residual uncertainty that must be addressed through downstream in vivo studies. Substantive evidence of similarity from comprehensive analytical testing may justify a targeted clinical development plan, and thus enable a shorter path to licensing. The importance of a careful design of the analytical similarity study program therefore should not be underestimated. Designing a state-of-the-art analytical similarity study meeting current regulatory requirements in regions such as the USA and EU requires a methodical approach, consisting of specific steps that far precede the work on the actual analytical study protocol. This white paper discusses scientific and methodological considerations on the process of attribute and test method selection, criticality assessment, and subsequent assignment of analytical measures to US FDA's three tiers of analytical similarity assessment. Case examples of selection of critical quality attributes and analytical methods for similarity exercises are provided to illustrate the practical implementation of the principles discussed.
Asunto(s)
Biosimilares Farmacéuticos/farmacología , Investigación Farmacéutica/métodos , Control de Calidad , United States Food and Drug Administration/normas , Biosimilares Farmacéuticos/química , Química Farmacéutica/métodos , Desarrollo de Medicamentos/métodos , Desarrollo de Medicamentos/normas , Estructura Molecular , Investigación Farmacéutica/normas , Proyectos de Investigación , Relación Estructura-Actividad , Tecnología Farmacéutica/métodos , Tecnología Farmacéutica/normas , Estados UnidosRESUMEN
The crystal structures of native class II fructose-1,6-bisphosphatase (FBPaseII) from Mycobacterium tuberculosis at 2.6â Å resolution and two active-site protein variants are presented. The variants were complexed with the reaction product fructose 6-phosphate (F6P). The Thr84Ala mutant is inactive, while the Thr84Ser mutant has a lower catalytic activity. The structures reveal the presence of a 222 tetramer, similar to those described for fructose-1,6/sedoheptulose-1,7-bisphosphatase from Synechocystis (strain 6803) as well as the equivalent enzyme from Thermosynechococcus elongatus. This homotetramer corresponds to a homologous oligomer that is present but not described in the crystal structure of FBPaseII from Escherichia coli and is probably conserved in all FBPaseIIs. The constellation of amino-acid residues in the active site of FBPaseII from M. tuberculosis (MtFBPaseII) is conserved and is analogous to that described previously for the E. coli enzyme. Moreover, the structure of the active site of the partially active (Thr84Ser) variant and the analysis of the kinetics are consistent with the previously proposed catalytic mechanism. The presence of metabolites in the crystallization medium (for example citrate and malonate) and in the corresponding crystal structures of MtFBPaseII, combined with their observed inhibitory effect, could suggest the existence of an uncharacterized inhibition of this class of enzymes besides the allosteric inhibition by adenosine monophosphate observed for the Synechocystis enzyme. The structural and functional insights derived from the structure of MtFBPaseII will provide critical information for the design of lead inhibitors, which will be used to validate this target for future chemical intervention.
Asunto(s)
Regulación Alostérica , Citratos/antagonistas & inhibidores , Fructosa-Bifosfatasa/química , Mycobacterium tuberculosis/enzimología , Catálisis , Dominio Catalítico , Inhibidores Enzimáticos , Proteínas de Escherichia coli , Fructosa-Bifosfatasa/genética , Cinética , Proteínas Mutantes/química , Mutación , Multimerización de Proteína , Synechocystis/químicaRESUMEN
The glpX gene from Francisella tularensis encodes for the class II fructose 1,6-bisphosphatase (FBPaseII) enzyme. The glpX gene has been verified to be essential in F. tularensis, and the inactivation of this gene leads to impaired bacterial growth on gluconeogenic substrates. In the present work, we have complemented a ∆glpX mutant of Escherichia coli with the glpX gene of F. tularensis (FTF1631c). Our complementation work independently verifies that the glpX gene (FTF1631c) in F. tularensis is indeed an FBPase and supports the growth of the ΔglpX E. coli mutant on glycerol-containing media. We have performed heterologous expression and purification of the glpX encoded FBPaseII in F. tularensis. We have confirmed the function of glpX as an FBPase and optimized the conditions for enzymatic activity. Mn2+ was found to be an absolute requirement for activity, with no other metal substitutions rendering the enzyme active. The kinetic parameters for this enzyme were found as follows: Km 11 µM, Vmax 2.0 units/mg, kcat 1.2 s-1, kcat/Km 120 mM-1 s-1, and a specific activity of 2.0 units/mg. Size exclusion data suggested an abundance of a tetrameric species in solution. Our findings on the enzyme's properties will facilitate the initial stages of a structure-based drug design program targeting this essential gene of F. tularensis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Francisella tularensis/enzimología , Francisella tularensis/patogenicidad , Fructosa-Bifosfatasa/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Francisella tularensis/genética , Fructosa-Bifosfatasa/química , Fructosa-Bifosfatasa/genética , Prueba de Complementación GenéticaRESUMEN
Protein therapeutics have unique critical quality attributes (CQAs) that define their purity, potency, and safety. The analytical methods used to assess CQAs must be able to distinguish clinically meaningful differences in comparator products, and the most important CQAs should be evaluated with the most statistical rigor. High-risk CQA measurements assess the most important attributes that directly impact the clinical mechanism of action or have known implications for safety, while the moderate- to low-risk characteristics may have a lower direct impact and thereby may have a broader range to establish similarity. Statistical equivalence testing is applied for high-risk CQA measurements to establish the degree of similarity (e.g., highly similar fingerprint, highly similar, or similar) of selected attributes. Notably, some high-risk CQAs (e.g., primary sequence or disulfide bonding) are qualitative (e.g., the same as the originator or not the same) and therefore not amenable to equivalence testing. For biosimilars, an important step is the acquisition of a sufficient number of unique originator drug product lots to measure the variability in the originator drug manufacturing process and provide sufficient statistical power for the analytical data comparisons. Together, these analytical evaluations, along with PK/PD and safety data (immunogenicity), provide the data necessary to determine if the totality of the evidence warrants a designation of biosimilarity and subsequent licensure for marketing in the USA. In this paper, a case study approach is used to provide examples of analytical similarity exercises and the appropriateness of statistical approaches for the example data.
Asunto(s)
Biosimilares Farmacéuticos/normas , Evaluación de Medicamentos/estadística & datos numéricos , Industria Farmacéutica/normas , Aprobación de Drogas , Industria Farmacéutica/tendencias , Control de Calidad , Estados Unidos , United States Food and Drug AdministrationRESUMEN
Several enzymes involved in central carbon metabolism and gluconeogenesis play a critical role in survival and pathogenesis of Mycobacterium tuberculosis (Mtb). The only known functional fructose 1,6-bisphosphatase (FBPase) in Mtb is encoded by the glpX gene and belongs to the Class II sub-family of FBPase. We describe herein the generation of a ΔglpX strain using homologous recombination. Although the growth profile of ΔglpX is comparable to that of wild type Mtb when grown on the standard enrichment media, its growth is dysgonic with individual gluconeogenic substrates such as oleic acid, glycerol and acetate. In mice lung CFU titers of ΔglpX were 2-3 log10 lower than the wild-type Mtb strain. The results indicate that glpX gene encodes a functional FBPase and is essential for both in vitro and in vivo growth and survival of Mtb. Loss of glpX results in significant reduction of FBPase activity but not complete abolition. These findings verify that the glpX encoded FBPase II in Mtb can be a potential target for drug discovery.
Asunto(s)
Proteínas Bacterianas/genética , Fructosa-Bifosfatasa/genética , Genes Bacterianos , Gluconeogénesis , Viabilidad Microbiana , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/crecimiento & desarrollo , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Carbono/farmacología , Ácidos Grasos/farmacología , Fructosa-Bifosfatasa/metabolismo , Eliminación de Gen , Gluconeogénesis/efectos de los fármacos , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Nitroimidazoles/farmacología , Estrés Fisiológico/efectos de los fármacos , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level.
RESUMEN
Fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11), which is a key enzyme in gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. The present investigation reports the crystallization and preliminary crystallographic studies of the glpX-encoded class II FBPase from Mycobacterium tuberculosis H37Rv. The recombinant protein, which was cloned using an Escherichia coli expression system, was purified and crystallized using the hanging-drop vapor-diffusion method. The crystals diffracted to a resolution of 2.7â Å and belonged to the hexagonal space group P6(1)22, with unit-cell parameters a = b = 131.3, c = 143.2â Å. The structure has been solved by molecular replacement and is currently undergoing refinement.
Asunto(s)
Fructosadifosfatos/química , Mycobacterium tuberculosis/enzimología , Cristalización , Cristalografía por Rayos X , Fructosadifosfatos/aislamiento & purificaciónRESUMEN
The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 3.1.3.11); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis-Menten kinetics with an apparent km = 44 µM. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme.
Asunto(s)
Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/aislamiento & purificación , Fructosa-Bifosfatasa/metabolismo , Mycobacterium tuberculosis/enzimología , Calcio/metabolismo , Calcio/farmacología , Cationes/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Clonación Molecular , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Escherichia coli/genética , Fructosa-Bifosfatasa/antagonistas & inhibidores , Regulación Enzimológica de la Expresión Génica , Concentración de Iones de Hidrógeno , Litio , Magnesio/metabolismo , Magnesio/farmacología , Manganeso/metabolismo , Manganeso/farmacología , Metales/metabolismo , Peso Molecular , Mycobacterium tuberculosis/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The sequence of Mycobacterium tuberculosis, completed in 1998, facilitated both the development of genomic tools, and the creation of a number of mycobacterial mutants. These mutants have a wide range of phenotypes, from attenuated to hypervirulent strains. These phenotypes must be confirmed, to rule out possible secondary mutations that may arise during the generation of mutant strains. This may occur during the amplification of target genes or during the generation of the mutation, thus constructing a complementation strain, which expresses the wild-type copy of the gene in the mutant strain, becomes necessary. In this study we have introduced a two-step strategy to construct complementation strains using the Ag85 promoter. We have constitutively expressed dosR and have shown dosR expression is restored to wild-type level.