scholarly journals Towards Selective Mycobacterial ClpP1P2 Inhibitors with Reduced Activity against the Human Proteasome

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Wilfried Moreira ◽  
Sridhar Santhanakrishnan ◽  
Grace J. Y. Ngan ◽  
Choon Bing Low ◽  
Kanda Sangthongpitag ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Aqueous Solubility ◽  
Structural Data ◽  
Proteasome Inhibition ◽  
Selective Inhibition ◽  
Lead Target ◽  
Content Type ◽  
Bacterial Agent ◽  
Human Liver Cell ◽  
Oral Pharmacokinetics

ABSTRACT Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti-M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti-M. tuberculosis activity.

scholarly journals Coxiella burnetii Alters Cyclic AMP-Dependent Protein Kinase Signaling during Growth in Macrophages

2012 ◽  
Vol 80 (6) ◽  
pp. 1980-1986 ◽  
Author(s):  
Laura J. MacDonald ◽  
Richard C. Kurten ◽  
Daniel E. Voth
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Coxiella Burnetii ◽  
Alveolar Macrophages ◽  
Q Fever ◽  
Parasitophorous Vacuole ◽  
Dependent Protein Kinase ◽  
Content Type ◽  
Bacterial Agent ◽  
Dependent Protein

ABSTRACTCoxiella burnetiiis the bacterial agent of human Q fever, an acute, flu-like illness that can present as chronic endocarditis in immunocompromised individuals. Following aerosol-mediated transmission,C. burnetiireplicates in alveolar macrophages in a unique phagolysosome-like parasitophorous vacuole (PV) required for survival. The mechanisms ofC. burnetiiintracellular survival are poorly defined and a recent Q fever outbreak in the Netherlands emphasizes the need for better understanding this unique host-pathogen interaction. We recently demonstrated that inhibition of host cyclic AMP-dependent protein kinase (PKA) activity negatively impacts PV formation. In the current study, we confirmed PKA involvement in PV biogenesis and probed the role of PKA signaling duringC. burnetiiinfection of macrophages. Using PKA-specific inhibitors, we found the kinase was needed for biogenesis of prototypical PV andC. burnetiireplication. PKA and downstream targets were differentially phosphorylated throughout infection, suggesting prolonged regulation of the pathway. Importantly, the pathogen actively triggered PKA activation, which was also required for PV formation by virulentC. burnetiiisolates during infection of primary human alveolar macrophages. A subset of PKA-specific substrates were differentially phosphorylated duringC. burnetiiinfection, suggesting the pathogen uses PKA signaling to control distinct host cell responses. Collectively, the current results suggest a versatile role for PKA inC. burnetiiinfection and indicate virulent organisms usurp host kinase cascades for efficient intracellular growth.

scholarly journals Complete Genome Sequences of Three Bacillus amyloliquefaciens Strains That Inhibit the Growth of Listeria monocytogenes In Vitro

2018 ◽  
Vol 6 (25) ◽  
Author(s):  
Thao D. Tran ◽  
Steven Huynh ◽  
Craig T. Parker ◽  
Robert Hnasko ◽  
Lisa Gorski ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Secondary Metabolites ◽  
Growth Inhibition ◽  
Complete Genome ◽  
Bacillus Amyloliquefaciens ◽  
Gene Clusters ◽  
Genome Sequences ◽  
Multiple Gene ◽  
Content Type

ABSTRACT Here, we report the complete genome sequences of three Bacillus amyloliquefaciens strains isolated from alfalfa, almond drupes, and grapes that inhibited the growth of Listeria monocytogenes strain 2011L-2857 in vitro. We also report multiple gene clusters encoding secondary metabolites that may be responsible for the growth inhibition of L. monocytogenes.

scholarly journals Genetic Evidence for SecY Translocon-Mediated Import of Two Contact-Dependent Growth Inhibition (CDI) Toxins

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Allison M. Jones ◽  
Petra Virtanen ◽  
Disa Hammarlöf ◽  
William J. Allen ◽  
Ian Collinson ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Target Cell ◽  
Bacterial Species ◽  
Genetic Evidence ◽  
Target Cells ◽  
Content Type ◽  
Sec Translocon ◽  
Dependent Growth ◽  
Cell Envelopes ◽  
Contact Dependent Growth Inhibition

ABSTRACT The C-terminal (CT) toxin domains of contact-dependent growth inhibition (CDI) CdiA proteins target Gram-negative bacteria and must breach both the outer and inner membranes of target cells to exert growth inhibitory activity. Here, we examine two CdiA-CT toxins that exploit the bacterial general protein secretion machinery after delivery into the periplasm. A Ser281Phe amino acid substitution in transmembrane segment 7 of SecY, the universally conserved channel-forming subunit of the Sec translocon, decreases the cytotoxicity of the membrane depolarizing orphan10 toxin from enterohemorrhagic Escherichia coli EC869. Target cells expressing secYS281F and lacking either PpiD or YfgM, two SecY auxiliary factors, are fully protected from CDI-mediated inhibition either by CdiA-CTo10EC869 or by CdiA-CTGN05224, the latter being an EndoU RNase CdiA toxin from Klebsiella aerogenes GN05224 that has a related cytoplasm entry domain. RNase activity of CdiA-CTGN05224 was reduced in secYS281F target cells and absent in secYS281F ΔppiD or secYS281F ΔyfgM target cells during competition co-cultures. Importantly, an allele-specific mutation in secY (secYG313W) renders ΔppiD or ΔyfgM target cells specifically resistant to CdiA-CTGN05224 but not to CdiA-CTo10EC869, further suggesting a direct interaction between SecY and the CDI toxins. Our results provide genetic evidence of a unique confluence between the primary cellular export route for unfolded polypeptides and the import pathways of two CDI toxins. IMPORTANCE Many bacterial species interact via direct cell-to-cell contact using CDI systems, which provide a mechanism to inject toxins that inhibit bacterial growth into one another. Here, we find that two CDI toxins, one that depolarizes membranes and another that degrades RNA, exploit the universally conserved SecY translocon machinery used to export proteins for target cell entry. Mutations in genes coding for members of the Sec translocon render cells resistant to these CDI toxins by blocking their movement into and through target cell membranes. This work lays the foundation for understanding how CDI toxins interact with the protein export machinery and has direct relevance to development of new antibiotics that can penetrate bacterial cell envelopes.

scholarly journals In vitro synergy of 5-nitrofurans, vancomycin and sodium deoxycholate against Gram-negative pathogens

2021 ◽  
Author(s):  
Catrina Olivera ◽  
Vuong Van Hung Le ◽  
Catherine Davenport ◽  
Jasna Rakonjac
Keyword(s):  
Growth Inhibition ◽  
Type Species ◽  
Sodium Deoxycholate ◽  
Gram Positive ◽  
Bacterial Killing ◽  
Gram Negative ◽  
Content Type ◽  
Link Type ◽  
Time Kill

Introduction. There is an urgent need for effective therapies against bacterial infections, especially those caused by antibiotic-resistant Gram-negative pathogens. Hypothesis. Synergistic combinations of existing antimicrobials show promise due to their enhanced efficacies and reduced dosages which can mitigate adverse effects, and therefore can be used as potential antibacterial therapy. Aim. In this study, we sought to characterize the in vitro interaction of 5-nitrofurans, vancomycin and sodium deoxycholate (NVD) against pathogenic bacteria. Methodology. The synergy of the NVD combination was investigated in terms of growth inhibition and bacterial killing using checkerboard and time-kill assays, respectively. Results. Using a three-dimensional checkerboard assay, we showed that 5-nitrofurans, sodium deoxycholate and vancomycin interact synergistically in the growth inhibition of 15 out of 20 Gram-negative strains tested, including clinically significant pathogens such as carbapenemase-producing Escherichia coli , Klebsiella pneumoniae and Acinetobacter baumannii , and interact indifferently against the Gram-positive strains tested. The time-kill assay further confirmed that the triple combination was bactericidal in a synergistic manner. Conclusion. This study demonstrates the synergistic effect of 5-nitrofurans, sodium deoxycholate and vancomycin against Gram-negative pathogens and highlights the potential of the combination as a treatment for Gram-negative and Gram-positive infections.

Magnitude, timing, and rate of slip along the Atacama fault system, northern Chile: Implications for Early Cretaceous slip partitioning and plate convergence

2021 ◽  
pp. jgs2020-142
Author(s):  
N.M. Seymour ◽  
J.S. Singleton ◽  
R. Gomila ◽  
S.P. Mavor ◽  
G. Heuser ◽  
...  
Keyword(s):  
Convergence Rates ◽  
Slip Rate ◽  
Structural Data ◽  
Fault System ◽  
Content Type ◽  
Plate Convergence ◽  
Oblique Convergence ◽  
Slip History ◽  
Supplementary Material ◽  
Atacama Fault System

Displacement estimates along the Atacama fault system (AFS), a crustal-scale sinistral structure that accommodated oblique convergence in the Mesozoic Coastal Cordillera arc, vary widely due to a lack of piercing points. We mapped the distribution of plutons and mylonitic deformation along the northern ∼70 km of the El Salado segment and use U-Pb geochronology to establish the slip history of the AFS. Along the eastern branch, mylonitic fabrics associated with the synkinematic ∼134–132 Ma Cerro del Pingo Complex are separated by 34–38 km, and mylonites associated with a synkinematic ∼120–119 Ma tonalite are separated by 20.5–25 km. We interpret leucocratic intrusions to be separated across the western branch by ∼16–20 km, giving a total slip magnitude of ∼54 ± 6 km across the El Salado segment. Kinematic indicators consistently record sinistral shear and zircon (U-Th)/He data suggest dip-slip motion was insignificant. Displacement occurred between ∼133–110 Ma at a slip rate of ∼2.1–2.6 km/Myr. This slip rate is low compared to modern intra-arc strike-slip faults, suggesting (1) the majority of lateral slip was accommodated along the slab interface or distributed through the forearc or (2) plate convergence rates/obliquity were significantly lower than previously modeled.Supplementary material including full U-Pb, (U-Th)/He, petrographic, and structural data with locations is available at https://doi.org/10.6084/m9.figshare.c.5262177.

scholarly journals Imidazole Derivatives as Promising Agents for the Treatment of Chagas Disease

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Julianna Siciliano de Araújo ◽  
Alfonso García-Rubia ◽  
Victor Sebastián-Pérez ◽  
Titilola D. Kalejaiye ◽  
Patrícia Bernardino da Silva ◽  
...  
Keyword(s):  
Chagas Disease ◽  
Latin American ◽  
Protozoan Parasite ◽  
Aqueous Solubility ◽  
Content Type ◽  
Cellular Effects ◽  
Transmission Electron ◽  
Phenotypic Screen ◽  
Current Therapies ◽  
Potential Inhibitors

ABSTRACT More than 100 years after being first described, Chagas disease remains endemic in 21 Latin American countries and has spread to other continents. Indeed, this disease, which is caused by the protozoan parasite Trypanosoma cruzi, is no longer just a problem for the American continents but has become a global health threat. Current therapies, i.e., nifurtimox and benznidazole (Bz), are far from being adequate, due to their undesirable effects and their lack of efficacy in the chronic phases of the disease. In this work, we present an in-depth phenotypic evaluation in T. cruzi of a new class of imidazole compounds, which were discovered in a previous phenotypic screen against different trypanosomatids and were designed as potential inhibitors of cAMP phosphodiesterases (PDEs). The confirmation of several activities similar or superior to that of Bz prompted a synthesis program of hit optimization and extended structure-activity relationship aimed at improving drug-like properties such as aqueous solubility, which resulted in additional hits with 50% inhibitory concentration (IC50) values similar to that of Bz. The cellular effects of one representative hit, compound 9, on bloodstream trypomastigotes were further investigated. Transmission electron microscopy revealed cellular changes, after just 2 h of incubation with the IC50 concentration, that were consistent with induced autophagy and osmotic stress, mechanisms previously linked to cAMP signaling. Compound 9 induced highly significant increases in both cellular and medium cAMP levels, confirming that inhibition of T. cruzi PDE(s) is part of its mechanism of action. The potent and selective activity of this imidazole-based PDE inhibitor class against T. cruzi constitutes a successful repurposing of research into inhibitors of mammalian PDEs.

scholarly journals Liposomal delivery of methylphosphonate antisense oligodeoxynucleotides in chronic myelogenous leukemia [see comments]

Blood ◽  
1994 ◽  
Vol 84 (2) ◽  
pp. 601-607 ◽  
Author(s):  
AM Tari ◽  
SD Tucker ◽  
A Deisseroth ◽  
G Lopez-Berestein
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Chronic Myelogenous Leukemia ◽  
Fusion Gene ◽  
Hematologic Malignancy ◽  
Control Cell ◽  
Selective Inhibition ◽  
Inhibitory Effect ◽  
Myelogenous Leukemia ◽  
Ph Chromosome

Abstract Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by the presence of the Philadelphia (Ph) chromosome. Bcr- abl, the fusion gene associated with the Ph chromosome, expresses a p210bcr-abl protein that promotes a selective expansion of mature myeloid progenitor cells. Methylphosphonate (MP) oligodeoxynucleotides complementary to specific regions of the bcr-abl mRNA were incorporated in liposomes. We studied the effects of liposomal MP (L-MP) on the growth inhibition of CML-like cell lines. L-MP targeted to the breakpoint junctions of the bcr-abl mRNA inhibited the growth of CML cells. Fifty percent inhibition was achieved at approximately 1 mumol/L of L-MP oligonucleotide concentrations. The inhibitory effect was selective because growth inhibition was observed only with CML but not with control cell lines. Moreover, CML cell growth inhibition was dependent on the sequence of the MP oligodeoxynucleotides incorporated in the liposomes. The growth inhibition of CML cells by L-MP resulted from selective inhibition of the expression of the p210bcr-abl protein.

scholarly journals Selective Inhibition of Coxiella burnetii Replication by the Steroid Hormone Progesterone

2020 ◽  
Vol 88 (12) ◽  
Author(s):  
Zachary P. Howard ◽  
Anders Omsland
Keyword(s):  
Coxiella Burnetii ◽  
Efflux Pump ◽  
Q Fever ◽  
Steroid Hormone ◽  
Natural Infection ◽  
Placental Tissue ◽  
Selective Inhibition ◽  
Inhibitory Effect ◽  
Content Type ◽  
Direct Inhibitory Effect

ABSTRACT Coxiella burnetii is a zoonotic bacterial obligate intracellular parasite and the cause of query (Q) fever. During natural infection of female animals, C. burnetii shows tropism for the placenta and is associated with late-term abortion, at which time the pathogen titer in placental tissue can exceed one billion bacteria per gram. During later stages of pregnancy, placental trophoblasts serve as the major source of progesterone, a steroid hormone known to affect the replication of some pathogens. During infection of placenta-derived JEG-3 cells, C. burnetii showed sensitivity to progesterone but not the immediate precursor pregnenolone or estrogen, another major mammalian steroid hormone. Using host cell-free culture, progesterone was determined to have a direct inhibitory effect on C. burnetii replication. Synergy between the inhibitory effect of progesterone and the efflux pump inhibitors verapamil and 1-(1-naphthylmethyl)-piperazine is consistent with a role for efflux pumps in preventing progesterone-mediated inhibition of C. burnetii activity. The sensitivity of C. burnetii to progesterone, but not structurally related molecules, is consistent with the ability of progesterone to influence pathogen replication in progesterone-producing tissues.

scholarly journals Outcrop-scale manifestations of reactivation during multiple superimposed rifting and basin inversion events: the Devonian Orcadian Basin, northern Scotland

2020 ◽  
Vol 178 (1) ◽  
pp. jgs2020-089 ◽  
Author(s):  
A.M. Dichiarante ◽  
R.E. Holdsworth ◽  
E.D. Dempsey ◽  
K.J.W. McCaffrey ◽  
T.A.G. Utley
Keyword(s):  
Structural Complexity ◽  
Structural Data ◽  
Dominant Group ◽  
Content Type ◽  
Basin Inversion ◽  
Supplementary Material ◽  
Group 2 ◽  
Orcadian Basin ◽  
East West ◽  
Group 1

The Devonian Orcadian Basin in Scotland hosts extensional fault systems assumed to be related to the initial formation of the basin, with only limited post-Devonian inversion and reactivation. However, a recent detailed structural study across Caithness, underpinned by published Re–Os geochronology, shows that three phases of deformation are present. North–south- and NW–SE-trending Group 1 faults are related to Devonian ENE–WSW transtension associated with sinistral shear along the Great Glen Fault during the formation of the Orcadian Basin. Metre- to kilometre-scale north–south-trending Group 2 folds and thrusts are developed close to earlier sub-basin-bounding faults and reflect late Carboniferous–early Permian east–west inversion associated with dextral reactivation of the Great Glen Fault. The dominant Group 3 structures are dextral oblique NE–SW-trending and sinistral east–west-trending faults with widespread syndeformational carbonate mineralization (± pyrite and bitumen) and are dated using Re–Os geochronology as Permian (c. 267 Ma). Regional Permian NW–SE extension related to the development of the offshore West Orkney Basin was superimposed over pre-existing fault networks, leading to local oblique reactivation of Group 1 faults in complex localized zones of transtensional folding, faulting and inversion. The structural complexity in surface outcrops onshore therefore reflects both the local reactivation of pre-existing faults and the superimposition of obliquely oriented rifting episodes during basin development in the adjacent offshore areas.Supplementary material: Stereographic projections of compiled structural data from individual fieldwork localities are available at https://doi.org/10.6084/m9.figshare.c.5115228

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