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1.
Proc Natl Acad Sci U S A ; 119(32): e2204473119, 2022 08 09.
Artículo en Inglés | MEDLINE | ID: mdl-35921442

RESUMEN

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped ß-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.


Asunto(s)
Anticuerpos Monoclonales , Cadherinas , Adhesión Celular , Anticuerpos Monoclonales/química , Cadherinas/química , Cadherinas/inmunología , Cristalografía por Rayos X , Humanos , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Dominios Proteicos
2.
Biochemistry ; 62(17): 2587-2596, 2023 09 05.
Artículo en Inglés | MEDLINE | ID: mdl-37552766

RESUMEN

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.


Asunto(s)
Transferasas Intramoleculares , Ribonucleótidos , Humanos , Ribonucleótidos/química , Escherichia coli/metabolismo , Transferasas Intramoleculares/metabolismo , Nucleótidos de Purina/metabolismo
3.
Antimicrob Agents Chemother ; 67(4): e0160722, 2023 04 18.
Artículo en Inglés | MEDLINE | ID: mdl-36920188

RESUMEN

Mycobacterium fortuitum represents one of the most clinically relevant rapid-growing mycobacterial species. Treatments are complex due to antibiotic resistance and to severe side effects of effective drugs, prolonged time of treatment, and co-infection with other pathogens. Herein, we explored the activity of NITD-916, a direct inhibitor of the enoyl-ACP reductase InhA of the type II fatty acid synthase in Mycobacterium tuberculosis. We found that this compound displayed very low MIC values against a panel of M. fortuitum clinical strains and exerted potent antimicrobial activity against M. fortuitum in macrophages. Remarkably, the compound was also highly efficacious in a zebrafish model of infection. Short duration treatments were sufficient to significantly protect the infected larvae from M. fortuitum-induced killing, which correlated with reduced bacterial burdens and abscesses. Biochemical analyses demonstrated an inhibition of de novo synthesis of mycolic acids. Resolving the crystal structure of the InhAMFO in complex with NAD and NITD-916 confirmed that NITD-916 is a direct inhibitor of InhAMFO. Importantly, single nucleotide polymorphism leading to a G96S substitution in InhAMFO conferred high resistance levels to NITD-916, thus resolving its target in M. fortuitum. Overall, these findings indicate that NITD-916 is highly active against M. fortuitum both in vitro and in vivo and should be considered in future preclinical evaluations for the treatment of M. fortuitum pulmonary diseases.


Asunto(s)
Mycobacterium fortuitum , Mycobacterium tuberculosis , Animales , Pez Cebra , Ácidos Micólicos/farmacología , Oxidorreductasas
4.
PLoS Pathog ; 17(9): e1009887, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34525130

RESUMEN

Brucellosis is one of the most widespread bacterial zoonoses worldwide. Here, our aim was to identify the effector mechanisms controlling the early stages of intranasal infection with Brucella in C57BL/6 mice. During the first 48 hours of infection, alveolar macrophages (AMs) are the main cells infected in the lungs. Using RNA sequencing, we identified the aconitate decarboxylase 1 gene (Acod1; also known as Immune responsive gene 1), as one of the genes most upregulated in murine AMs in response to B. melitensis infection at 24 hours post-infection. Upregulation of Acod1 was confirmed by RT-qPCR in lungs infected with B. melitensis and B. abortus. We observed that Acod1-/- C57BL/6 mice display a higher bacterial load in their lungs than wild-type (wt) mice following B. melitensis or B. abortus infection, demonstrating that Acod1 participates in the control of pulmonary Brucella infection. The ACOD1 enzyme is mostly produced in mitochondria of macrophages, and converts cis-aconitate, a metabolite in the Krebs cycle, into itaconate. Dimethyl itaconate (DMI), a chemically-modified membrane permeable form of itaconate, has a dose-dependent inhibitory effect on Brucella growth in vitro. Interestingly, structural analysis suggests the binding of itaconate into the binding site of B. abortus isocitrate lyase. DMI does not inhibit multiplication of the isocitrate lyase deletion mutant ΔaceA B. abortus in vitro. Finally, we observed that, unlike the wt strain, the ΔaceA B. abortus strain multiplies similarly in wt and Acod1-/- C57BL/6 mice. These data suggest that bacterial isocitrate lyase might be a target of itaconate in AMs.


Asunto(s)
Brucelosis/inmunología , Carboxiliasas/inmunología , Enfermedades Pulmonares/inmunología , Macrófagos Alveolares/inmunología , Animales , Isocitratoliasa/metabolismo , Ratones , Ratones Endogámicos C57BL
5.
Proc Natl Acad Sci U S A ; 116(14): 7015-7020, 2019 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-30894487

RESUMEN

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.


Asunto(s)
Criptosporidiosis , Cryptosporidium parvum/enzimología , Inhibidores Enzimáticos/farmacología , Lisina-ARNt Ligasa/antagonistas & inhibidores , Malaria Falciparum , Plasmodium falciparum/enzimología , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Criptosporidiosis/tratamiento farmacológico , Criptosporidiosis/enzimología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/química , Humanos , Lisina-ARNt Ligasa/metabolismo , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Ratones SCID , Proteínas Protozoarias/metabolismo
6.
Biochemistry ; 59(36): 3285-3289, 2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32841567

RESUMEN

Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, ß-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ1-pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD+ and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231-491 M-1 s-1. The reaction is reversible, and nuclear magnetic resonance characterization (1H, 13C, and 31P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD+. We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP+-TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P)+. Because NAD(P)+-dependent oxidoreductases are widespread in nature, our results may be broadly relevant.


Asunto(s)
Burkholderia/enzimología , Ditiotreitol/química , NAD/metabolismo , Fosfinas/química , Sustancias Reductoras/química , Deshidrogenasas-Reductasas de Cadena Corta/química , Deshidrogenasas-Reductasas de Cadena Corta/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Disulfuros/química , Oxidación-Reducción , Conformación Proteica , Dominios Proteicos
7.
Nucleic Acids Res ; 45(10): 6217-6227, 2017 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-28369538

RESUMEN

Deoxyribonuclease II (DNase II) is also known as acid deoxyribonuclease because it has optimal activity at the low pH environment of lysosomes where it is typically found in higher eukaryotes. Interestingly, DNase II has also been identified in a few genera of bacteria and is believed to have arisen via horizontal transfer. Here, we demonstrate that recombinant Burkholderia thailandensis DNase II is highly active at low pH in the absence of divalent metal ions, similar to eukaryotic DNase II. The crystal structure of B. thailandensis DNase II shows a dimeric quaternary structure which appears capable of binding double-stranded DNA. Each monomer of B. thailandensis DNase II exhibits a similar overall fold as phospholipase D (PLD), phosphatidylserine synthase (PSS) and tyrosyl-DNA phosphodiesterase (TDP), and conserved catalytic residues imply a similar mechanism. The structural and biochemical data presented here provide insights into the atomic structure and catalytic mechanism of DNase II.


Asunto(s)
Proteínas Bacterianas/química , Burkholderia/enzimología , Endodesoxirribonucleasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cobre/farmacología , Cristalografía por Rayos X , ADN Bacteriano/metabolismo , Endodesoxirribonucleasas/antagonistas & inhibidores , Endodesoxirribonucleasas/metabolismo , Células Eucariotas/enzimología , Evolución Molecular , Transferencia de Gen Horizontal , Concentración de Iones de Hidrógeno , Modelos Moleculares , Simulación del Acoplamiento Molecular , Filogenia , Células Procariotas/enzimología , Conformación Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido
8.
J Biol Chem ; 292(42): 17449-17460, 2017 10 20.
Artículo en Inglés | MEDLINE | ID: mdl-28878017

RESUMEN

The neonatal Fc receptor FcRn plays a critical role in the trafficking of IgGs across tissue barriers and in retaining high circulating concentrations of both IgG and albumin. Although generally beneficial from an immunological perspective in maintaining IgG populations, FcRn can contribute to the pathogenesis of autoimmune disorders when an abnormal immune response targets normal biological components. We previously described a monoclonal antibody (DX-2507) that binds to FcRn with high affinity at both neutral and acidic pH, prevents the simultaneous binding of IgG, and reduces circulating IgG levels in preclinical animal models. Here, we report a 2.5 Å resolution X-ray crystal structure of an FcRn-DX-2507 Fab complex, revealing a nearly complete overlap of the IgG-Fc binding site in FcRn by complementarity-determining regions in DX-2507. This overlap explains how DX-2507 blocks IgG binding to FcRn and thereby shortens IgG half-life by preventing IgGs from recycling back into circulation. Moreover, the complex structure explains how the DX-2507 interaction is pH-insensitive unlike normal Fc interactions and how serum albumin levels are unaffected by DX-2507 binding. These structural studies could inform antibody-based therapeutic approaches for limiting the effects of IgG-mediated autoimmune disease.


Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Antígenos de Histocompatibilidad Clase I/química , Inmunoglobulina G/química , Receptores Fc/antagonistas & inhibidores , Receptores Fc/química , Animales , Cristalografía por Rayos X , Células HEK293 , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Ratones , Estructura Cuaternaria de Proteína , Ratas , Receptores Fc/genética
9.
Bioorg Med Chem Lett ; 28(8): 1376-1380, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29551481

RESUMEN

Methionine aminopeptidase (MetAP) is a dinuclear metalloprotease responsible for the cleavage of methionine initiator residues from nascent proteins. MetAP activity is necessary for bacterial proliferation and is therefore a projected novel antibacterial target. A compound library consisting of 294 members containing metal-binding functional groups was screened against Rickettsia prowazekii MetAP to determine potential inhibitory motifs. The compounds were first screened against the target at a concentration of 10 µM and potential hits were determined to be those exhibiting greater than 50% inhibition of enzymatic activity. These hit compounds were then rescreened against the target in 8-point dose-response curves and 11 compounds were found to inhibit enzymatic activity with IC50 values of less than 10 µM. Finally, compounds (1-5) were docked against RpMetAP with AutoDock to determine potential binding mechanisms and the results were compared with crystal structures deposited within the PDB.


Asunto(s)
Antibacterianos/química , Metaloproteasas/antagonistas & inhibidores , Metionil Aminopeptidasas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Bibliotecas de Moléculas Pequeñas/química , Dominio Catalítico , Pruebas de Enzimas , Metaloproteasas/química , Metionil Aminopeptidasas/química , Simulación del Acoplamiento Molecular , Rickettsia prowazekii/enzimología
10.
Biometals ; 31(6): 1081-1089, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-30311019

RESUMEN

There are several options available for intravenous application of iron supplements, but they all have a similar structure:-an iron core surrounded by a carbohydrate coating. These nanoparticles require processing by the reticuloendothelial system to release iron, which is subsequently picked up by the iron-binding protein transferrin and distributed throughout the body, with most of the iron supplied to the bone marrow. This process risks exposing cells and tissues to free iron, which is potentially toxic due to its high redox activity. A new parenteral iron formation, ferric pyrophosphate citrate (FPC), has a novel structure that differs from conventional intravenous iron formulations, consisting of an iron atom complexed to one pyrophosphate and two citrate anions. In this study, we show that FPC can directly transfer iron to apo-transferrin. Kinetic analyses reveal that FPC donates iron to apo-transferrin with fast binding kinetics. In addition, the crystal structure of transferrin bound to FPC shows that FPC can donate iron to both iron-binding sites found within the transferrin structure. Examination of the iron-binding sites demonstrates that the iron atoms in both sites are fully encapsulated, forming bonds with amino acid side chains in the protein as well as pyrophosphate and carbonate anions. Taken together, these data demonstrate that, unlike intravenous iron formulations, FPC can directly and rapidly donate iron to transferrin in a manner that does not expose cells and tissues to the damaging effects of free, redox-active iron.


Asunto(s)
Ácido Cítrico/química , Difosfatos/química , Hierro/química , Transferrina/química , Sitios de Unión , Ácido Cítrico/metabolismo , Cristalografía por Rayos X , Difosfatos/metabolismo , Humanos , Hierro/metabolismo , Cinética , Modelos Moleculares , Transferrina/metabolismo
11.
PLoS Pathog ; 11(11): e1005248, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26544981

RESUMEN

Anaplasma phagocytophilum, the causative agent of Human Granulocytic Anaplasmosis (HGA), is an obligately intracellular α-proteobacterium that is transmitted by Ixodes spp ticks. However, the pathogen is not transovarially transmitted between tick generations and therefore needs to survive in both a mammalian host and the arthropod vector to complete its life cycle. To adapt to different environments, pathogens rely on differential gene expression as well as the modification of proteins and other molecules. Random transposon mutagenesis of A. phagocytophilum resulted in an insertion within the coding region of an o-methyltransferase (omt) family 3 gene. In wild-type bacteria, expression of omt was up-regulated during binding to tick cells (ISE6) at 2 hr post-inoculation, but nearly absent by 4 hr p.i. Gene disruption reduced bacterial binding to ISE6 cells, and the mutant bacteria that were able to enter the cells were arrested in their replication and development. Analyses of the proteomes of wild-type versus mutant bacteria during binding to ISE6 cells identified Major Surface Protein 4 (Msp4), but also hypothetical protein APH_0406, as the most differentially methylated. Importantly, two glutamic acid residues (the targets of the OMT) were methyl-modified in wild-type Msp4, whereas a single asparagine (not a target of the OMT) was methylated in APH_0406. In vitro methylation assays demonstrated that recombinant OMT specifically methylated Msp4. Towards a greater understanding of the overall structure and catalytic activity of the OMT, we solved the apo (PDB_ID:4OA8), the S-adenosine homocystein-bound (PDB_ID:4OA5), the SAH-Mn2+ bound (PDB_ID:4PCA), and SAM- Mn2+ bound (PDB_ID:4PCL) X-ray crystal structures of the enzyme. Here, we characterized a mutation in A. phagocytophilum that affected the ability of the bacteria to productively infect cells from its natural vector. Nevertheless, due to the lack of complementation, we cannot rule out secondary mutations.


Asunto(s)
Anaplasma phagocytophilum/enzimología , Ehrlichiosis/microbiología , Ixodes/microbiología , Metiltransferasas/metabolismo , Garrapatas/microbiología , Animales , Ehrlichiosis/genética , Ixodes/inmunología , Metiltransferasas/genética , Activación Transcripcional , Regulación hacia Arriba
12.
Bioorg Med Chem Lett ; 27(8): 1840-1847, 2017 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-28274633

RESUMEN

A series of 2'-fluorinated C-nucleosides were prepared and tested for anti-HCV activity. Among them, the triphosphate of 2'-fluoro-2'-C-methyl adenosine C-nucleoside (15) was a potent and selective inhibitor of the NS5B polymerase and maintained activity against the S282T resistance mutant. A number of phosphoramidate prodrugs were then prepared and evaluated leading to the identification of the 1-aminocyclobutane-1-carboxylic acid isopropyl ester variant (53) with favorable pharmacokinetic properties including efficient liver delivery in animals.


Asunto(s)
Antivirales/química , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Nucleósidos/química , Nucleósidos/farmacología , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas/química , Amidas/farmacocinética , Amidas/farmacología , Animales , Antivirales/farmacocinética , Células CACO-2 , Línea Celular , Cricetinae , Descubrimiento de Drogas , Farmacorresistencia Viral , Halogenación , Hepacivirus/genética , Hepacivirus/fisiología , Hepatitis C/tratamiento farmacológico , Humanos , Metilación , Simulación del Acoplamiento Molecular , Nucleósidos/farmacocinética , Ácidos Fosfóricos/química , Ácidos Fosfóricos/farmacocinética , Ácidos Fosfóricos/farmacología , Mutación Puntual , Profármacos/química , Profármacos/farmacocinética , Profármacos/farmacología , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos
13.
BMC Infect Dis ; 17(1): 231, 2017 03 24.
Artículo en Inglés | MEDLINE | ID: mdl-28340562

RESUMEN

BACKGROUND: We describe drug-induced liver injury (DILI) secondary to antituberculous treatment (ATT) in a large tuberculosis (TB) centre in London; we identify the proportion who had risk factors for DILI and the timing and outcome of DILI. METHODS: We identified consecutive patients who developed DILI whilst on treatment for active TB; patients with active TB without DILI were selected as controls. Comprehensive demographic and clinical data, management and outcome were recorded. RESULTS: There were 105 (6.9%) cases of ATT-associated DILI amongst 1529 patients diagnosed with active TB between April 2010 and May 2014. Risk factors for DILI were: low patient weight, HIV-1 co-infection, higher baseline ALP, and alcohol intake. Only 25.7% of patients had British or American Thoracic Society defined criteria for liver test (LT) monitoring. Half (53%) of the cases occurred within 2 weeks of starting ATT and 87.6% occurred within 8 weeks. Five (4.8%) of seven deaths were attributable to DILI. CONCLUSIONS: Only a quarter of patients who developed DILI had British or American Thoracic Society defined criteria for pre-emptive LT monitoring, suggesting that all patients on ATT should be considered for universal liver monitoring particularly during the first 8 weeks of treatment.


Asunto(s)
Antituberculosos , Enfermedad Hepática Inducida por Sustancias y Drogas , Tuberculosis , Adolescente , Adulto , Antituberculosos/efectos adversos , Antituberculosos/uso terapéutico , Enfermedad Hepática Inducida por Sustancias y Drogas/epidemiología , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Femenino , Humanos , Londres/epidemiología , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tuberculosis/complicaciones , Tuberculosis/tratamiento farmacológico , Tuberculosis/epidemiología , Reino Unido , Adulto Joven
14.
Bioorg Med Chem ; 25(3): 813-824, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28089350

RESUMEN

Methionine aminopeptidase (MetAP) is a class of ubiquitous enzymes essential for the survival of numerous bacterial species. These enzymes are responsible for the cleavage of N-terminal formyl-methionine initiators from nascent proteins to initiate post-translational modifications that are often essential to proper protein function. Thus, inhibition of MetAP activity has been implicated as a novel antibacterial target. We tested this idea in the present study by targeting the MetAP enzyme in the obligate intracellular pathogen Rickettsia prowazekii. We first identified potent RpMetAP inhibitory species by employing an in vitro enzymatic activity assay. The molecular docking program AutoDock was then utilized to compare published crystal structures of inhibited MetAP species to docked poses of RpMetAP. Based on these in silico and in vitro screens, a subset of 17 compounds was tested for inhibition of R. prowazekii growth in a pulmonary vascular endothelial cell (EC) culture infection model system. All compounds were tested over concentration ranges that were determined to be non-toxic to the ECs and 8 of the 17 compounds displayed substantial inhibition of R. prowazekii growth. These data highlight the therapeutic potential for inhibiting RpMetAP as a novel antimicrobial strategy and set the stage for future studies in pre-clinical animal models of infection.


Asunto(s)
Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Metionil Aminopeptidasas/antagonistas & inhibidores , Rickettsia prowazekii/efectos de los fármacos , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Metionil Aminopeptidasas/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Arteria Pulmonar/efectos de los fármacos , Ratas , Rickettsia prowazekii/enzimología , Relación Estructura-Actividad
15.
Biochemistry ; 55(51): 7099-7111, 2016 Dec 27.
Artículo en Inglés | MEDLINE | ID: mdl-27936614

RESUMEN

The complex life cycle of Mycobacterium tuberculosis requires diverse energy mobilization and utilization strategies facilitated by a battery of lipid metabolism enzymes. Among lipid metabolism enzymes, the Lip family of mycobacterial serine hydrolases is essential to lipid scavenging, metabolic cycles, and reactivation from dormancy. On the basis of the homologous rescue strategy for mycobacterial drug targets, we have characterized the three-dimensional structure of full length LipW from Mycobacterium marinum, the first structure of a catalytically active Lip family member. LipW contains a deep, expansive substrate-binding pocket with only a narrow, restrictive active site, suggesting tight substrate selectivity for short, unbranched esters. Structural alignment reinforced this strict substrate selectivity of LipW, as the binding pocket of LipW aligned most closely with the bacterial acyl esterase superfamily. Detailed kinetic analysis of two different LipW homologues confirmed this strict substrate selectivity, as each homologue selected for unbranched propionyl ester substrates, irrespective of the alcohol portion of the ester. Using comprehensive substitutional analysis across the binding pocket, the strict substrate selectivity of LipW for propionyl esters was assigned to a narrow funnel in the acyl-binding pocket capped by a key hydrophobic valine residue. The polar, negatively charged alcohol-binding pocket also contributed to substrate orientation and stabilization of rotameric states in the catalytic serine. Together, the structural, enzymatic, and substitutional analyses of LipW provide a connection between the structure and metabolic properties of a Lip family hydrolase that refines its biological function in active and dormant tuberculosis infection.


Asunto(s)
Proteínas Bacterianas/metabolismo , Ésteres/metabolismo , Hidrolasas/metabolismo , Mycobacterium marinum/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Estabilidad de Enzimas , Ésteres/química , Hidrolasas/química , Hidrolasas/genética , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Estructura Molecular , Mutación , Mycobacterium marinum/genética , Unión Proteica , Dominios Proteicos , Serina/química , Serina/genética , Serina/metabolismo , Especificidad por Sustrato , Temperatura , Valina/química , Valina/genética , Valina/metabolismo
16.
J Infect Dis ; 212 Suppl 2: S167-71, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-25957961

RESUMEN

Ebolaviruses cause severe hemorrhagic fever. Central to the Ebola life cycle is the matrix protein VP40, which oligomerizes and drives viral budding. Here we present the crystal structure of the Sudan virus (SUDV) matrix protein. This structure is higher resolution (1.6 Å) than previously achievable. Despite differences in the protein purification, we find that it still forms a stable dimer in solution, as was noted for other Ebola VP40s. Although the N-terminal domain interface by which VP40 dimerizes is conserved between Ebola virus and SUDV, the C-terminal domain interface by which VP40 dimers may further assemble is significantly smaller in this SUDV assembly.


Asunto(s)
Ebolavirus/química , Proteínas de la Matriz Viral/química , Ebolavirus/metabolismo , Escherichia coli/metabolismo , Multimerización de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Soluciones/química , Sudán , Proteínas de la Matriz Viral/metabolismo
17.
J Struct Funct Genomics ; 16(2): 91-9, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25832174

RESUMEN

The methylmalonyl Co-A mutase-associated GTPase MeaB from Methylobacterium extorquens is involved in glyoxylate regulation and required for growth. In humans, mutations in the homolog methylmalonic aciduria associated protein (MMAA) cause methylmalonic aciduria, which is often fatal. The central role of MeaB from bacteria to humans suggests that MeaB is also important in other, pathogenic bacteria such as Mycobacterium tuberculosis. However, the identity of the mycobacterial MeaB homolog is presently unclear. Here, we identify the M. tuberculosis protein Rv1496 and its homologs in M. smegmatis and M. thermoresistibile as MeaB. The crystal structures of all three homologs are highly similar to MeaB and MMAA structures and reveal a characteristic three-domain homodimer with GDP bound in the G domain active site. A structure of Rv1496 obtained from a crystal grown in the presence of GTP exhibited electron density for GDP, suggesting GTPase activity. These structures identify the mycobacterial MeaB and provide a structural framework for therapeutic targeting of M. tuberculosis MeaB.


Asunto(s)
Proteínas Bacterianas/química , GTP Fosfohidrolasas/química , Mycobacterium tuberculosis/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Mycobacterium tuberculosis/genética
18.
J Biol Chem ; 289(4): 2139-47, 2014 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-24311791

RESUMEN

Ribonucleases (RNases) maintain the cellular RNA pool by RNA processing and degradation. In many bacteria, including the human pathogen Mycobacterium tuberculosis (Mtb), the enzymes mediating several central RNA processing functions are still unknown. Here, we identify the hypothetical Mtb protein Rv2179c as a highly divergent exoribonuclease. Although the primary sequence of Rv2179c has no detectable similarity to any known RNase, the Rv2179c crystal structure reveals an RNase fold. Active site residues are equivalent to those in the DEDD family of RNases, and Rv2179c has close structural homology to Escherichia coli RNase T. Consistent with the DEDD fold, Rv2179c has exoribonuclease activity, cleaving the 3' single-strand overhangs of duplex RNA. Functional orthologs of Rv2179c are prevalent in actinobacteria and found in bacteria as phylogenetically distant as proteobacteria. Thus, Rv2179c is the founding member of a new, large RNase family with hundreds of members across the bacterial kingdom.


Asunto(s)
Proteínas Bacterianas/química , Exorribonucleasas/química , Mycobacterium tuberculosis/enzimología , Filogenia , Factores de Virulencia/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Humanos , Mycobacterium tuberculosis/genética , Homología Estructural de Proteína , Factores de Virulencia/genética , Factores de Virulencia/metabolismo
19.
J Biol Chem ; 289(12): 8720-34, 2014 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-24415767

RESUMEN

IL-6 is a secreted cytokine that functions through binding two cell surface receptors, IL-6Rα and gp130. Because of its involvement in the progression of several chronic inflammatory diseases, IL-6 is a target of pharmacologic interest. We have recently identified a novel class of ligands called SOMAmers (S low Off-rate Modified Aptamers) that bind IL-6 and inhibit its biologic activity. SOMAmers exploit the chemical diversity of protein-like side chains assembled on flexible nucleic acid scaffolds, resulting in an expanded repertoire of intra- and intermolecular interactions not achievable with conventional aptamers. Here, we report the co-crystal structure of a high affinity SOMAmer (Kd = 0.20 nm) modified at the 5-position of deoxyuridine in a complex with IL-6. The SOMAmer, comprised of a G-quartet domain and a stem-loop domain, engages IL-6 in a clamp-like manner over an extended surface exhibiting close shape complementarity with the protein. The interface is characterized by substantial hydrophobic interactions overlapping the binding surfaces of the IL-6Rα and gp130 receptors. The G-quartet domain retains considerable binding activity as a disconnected autonomous fragment (Kd = 270 nm). A single substitution from our diversely modified nucleotide library leads to a 37-fold enhancement in binding affinity of the G-quartet fragment (Kd = 7.4 nm). The ability to probe ligand surfaces in this manner is a powerful tool in the development of new therapeutic reagents with improved pharmacologic properties. The SOMAmer·IL-6 structure also expands our understanding of the diverse structural motifs achievable with modified nucleic acid libraries and elucidates the nature with which these unique ligands interact with their protein targets.


Asunto(s)
Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Interleucina-6/química , Interleucina-6/metabolismo , Cristalografía por Rayos X , Descubrimiento de Drogas , Humanos , Ligandos , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Técnica SELEX de Producción de Aptámeros
20.
J Biol Chem ; 289(34): 23596-608, 2014 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-24970892

RESUMEN

Plasma kallikrein (pKal) proteolytically cleaves high molecular weight kininogen to generate the potent vasodilator and the pro-inflammatory peptide, bradykinin. pKal activity is tightly regulated in healthy individuals by the serpin C1-inhibitor, but individuals with hereditary angioedema (HAE) are deficient in C1-inhibitor and consequently exhibit excessive bradykinin generation that in turn causes debilitating and potentially fatal swelling attacks. To develop a potential therapeutic agent for HAE and other pKal-mediated disorders, we used phage display to discover a fully human IgG1 monoclonal antibody (DX-2930) against pKal. In vitro experiments demonstrated that DX-2930 potently inhibits active pKal (Ki = 0.120 ± 0.005 nM) but does not target either the zymogen (prekallikrein) or any other serine protease tested. These findings are supported by a 2.1-Å resolution crystal structure of pKal complexed to a DX-2930 Fab construct, which establishes that the pKal active site is fully occluded by the antibody. DX-2930 injected subcutaneously into cynomolgus monkeys exhibited a long half-life (t½ ∼ 12.5 days) and blocked high molecular weight kininogen proteolysis in activated plasma in a dose- and time-dependent manner. Furthermore, subcutaneous DX-2930 reduced carrageenan-induced paw edema in rats. A potent and long acting inhibitor of pKal activity could be an effective treatment option for pKal-mediated diseases, such as HAE.


Asunto(s)
Anticuerpos/inmunología , Calicreínas/inmunología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Humanos , Calicreínas/sangre , Datos de Secuencia Molecular , Ratas , Ratas Sprague-Dawley , Resonancia por Plasmón de Superficie
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