Engineering a Cell-Free Murein Biosynthetic Pathway:  Combinatorial Enzymology in Drug Discovery

1998 ◽  
Vol 120 (51) ◽  
pp. 13527-13528 ◽  
Author(s):  
Kenny K. Wong ◽  
David W. Kuo ◽  
Renee M. Chabin ◽  
Carole Fournier ◽  
Laura D. Gegnas ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Biosynthetic Pathway ◽  
A Cell

scholarly journals The synthesis of murine ferrochelatase in vitro and in vivo

1988 ◽  
Vol 254 (3) ◽  
pp. 799-803 ◽  
Author(s):  
S R Karr ◽  
H A Dailey
Keyword(s):  
Membrane Protein ◽  
Biosynthetic Pathway ◽  
Integral Membrane Protein ◽  
Mitochondrial Proteins ◽  
Isolated Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Carbonyl Cyanide ◽  
A Cell

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.

scholarly journals Nod2-Nodosome in a Cell-Free System: Implications in Pathogenesis and Drug Discovery for Blau Syndrome and Early-Onset Sarcoidosis

2016 ◽  
Vol 2016 ◽  
pp. 1-7 ◽  
Author(s):  
Tomoyuki Iwasaki ◽  
Naoe Kaneko ◽  
Yuki Ito ◽  
Hiroyuki Takeda ◽  
Tatsuya Sawasaki ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Early Onset ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Blau Syndrome ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Early Onset Sarcoidosis

Nucleotide-binding oligomerization domain-containing protein (Nod) 2 is an intracellular pattern recognition receptor, which recognizes muramyl dipeptide (N-Acetylmuramyl-L-Alanyl-D-Isoglutamine: MDP), a bacterial peptidoglycan component, and makes a NF-κB-activating complex called nodosome with adaptor protein RICK (RIP2/RIPK2). Nod2 mutants are associated with the autoinflammatory diseases, Blau syndrome (BS)/early-onset sarcoidosis (EOS). For drug discovery of BS/EOS, we tried to develop Nod2-nodosome in a cell-free system. FLAG-tagged RICK, biotinylated-Nod2, and BS/EOS-associated Nod2 mutants were synthesized, and proximity signals between FLAG-tagged and biotinylated proteins were detected by amplified luminescent proximity homogeneous assay (ALPHA). Upon incubation with MDP, the ALPHA signal of interaction between Nod2-WT and RICK was increased in a dose-dependent manner. The ALPHA signal of interaction between RICK and the BS/EOS-associated Nod2 mutants was more significantly increased than Nod2-WT. Notably, the ALPHA signal between Nod2-WT and RICK was increased upon incubation with MDP, but not when incubated with the same concentrations, L-alanine, D-isoglutamic acid, or the MDP-D-isoform. Thus, we successfully developed Nod2-nodosome in a cell-free system reflecting its function in vivo, and it can be useful for screening Nod2-nodosome-targeted therapeutic molecules for BS/EOS and granulomatous inflammatory diseases.

scholarly journals Fungal phospholipid metabolism for antifungal drug discovery

2010 ◽  
Vol 31 (2) ◽  
pp. 93
Author(s):  
Tania C Sorrrell ◽  
Julianne T Djordjevic ◽  
Sharon CA Chen ◽  
Katrina A Jolliffe
Keyword(s):  
Drug Discovery ◽  
Mammalian Cells ◽  
Biosynthetic Pathway ◽  
Fungicidal Activity ◽  
Fungal Infections ◽  
Toxicity Testing ◽  
Phospholipid Metabolism ◽  
Antifungal Drugs ◽  
Major Mode ◽  
Novel Targets

Invasive fungal infections often respond poorly to antifungal drugs. The fungal invasin phospholipase B (PLB) and/or its biosynthetic pathway are novel targets for drug development. Compounds with structural similarities to phosphatidylcholine, which is a preferred substrate of cryptococcal PLB1, were purchased or synthesised. For many, there was a correlation between antifungal and anti-PLB activity but not all demonstrated selectivity for fungal compared with mammalian phospholipase, and some were toxic to mammalian cells in culture. The most promising, a bis-pyridinium compound, is undergoing toxicity testing in mice. Miltefosine (MI), a stable phospholipid analogue used in the treatment of leishmaniasis also has broad spectrum fungicidal activity, but inhibition of PLB is not its major mode of action. To improve antifungal potency and reduce toxicity of MI, analogues of this alkyl phospholipid have been synthesised and are under investigation.

scholarly journals A Single-Cell Platform for Monitoring Viral Proteolytic Cleavage in Different Cellular Compartments

2015 ◽  
Vol 8s2 ◽  
pp. BCI.S30379
Author(s):  
Darin Abbadessa ◽  
Cameron A. Smurthwaite ◽  
Connor W. Reed ◽  
Roland Wolkowicz
Keyword(s):  
Drug Discovery ◽  
Biomedical Research ◽  
Envelope Protein ◽  
Viral Infectivity ◽  
Host Proteins ◽  
Subcellular Compartment ◽  
A Cell ◽  
The Individual ◽  
Cellular Compartments ◽  
Hiv 1

Infectious diseases affect human health despite advances in biomedical research and drug discovery. Among these, viruses are especially difficult to tackle due to the sudden transfer from animals to humans, high mutational rates, resistance to current treatments, and the intricacies of their molecular interactions with the host. As an example of these interactions, we describe a cell-based approach to monitor specific proteolytic events executed by either the viral-encoded protease or by host proteins on the virus. We then emphasize the significance of examining proteolysis within the subcellular compartment where cleavage occurs naturally. We show the power of stable expression, highlighting the usefulness of the cell-based multiplexed approach, which we have adapted to two independent assays previously developed to monitor (a) the activity of the HIV-1-encoded protease or (b) the cleavage of the HIV-1-encoded envelope protein by the host. Multiplexing was achieved by mixing cells each carrying a different assay or, alternatively, by engineering cells expressing two assays. Multiplexing relies on the robustness of the individual assays and their clear discrimination, further enhancing screening capabilities in an attempt to block proteolytic events required for viral infectivity and spread.

scholarly journals Deletion of the mmpL4b gene in the Mycobacterium abscessus glycopeptidolipid biosynthetic pathway results in loss of surface colonization capability, but enhanced ability to replicate in human macrophages and stimulate their innate immune response

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1187-1195 ◽  
Author(s):  
Rachid Nessar ◽  
Jean-Marc Reyrat ◽  
Lisa B. Davidson ◽  
Thomas F. Byrd
Keyword(s):  
Pathogenic Bacteria ◽  
Biosynthetic Pathway ◽  
Gene Knockout ◽  
Cellular Level ◽  
Mycobacterium Abscessus ◽  
Bacterial Gene ◽  
Human Macrophages ◽  
Knockout Mutants ◽  
A Cell ◽  
Toll Like Receptor 2

Mycobacterium abscessus is considered to be the most virulent of the rapidly growing mycobacteria. Generation of bacterial gene knockout mutants has been a useful tool for studying factors that contribute to virulence of pathogenic bacteria. Until recently, the optimal genetic approach to generation of M. abscessus gene knockout mutants was not clear. Based on the recent identification of genetic recombineering as the preferred approach, a M. abscessus mutant was generated in which the gene mmpL4b, critical to glycopeptidolipid synthesis, was deleted. Compared to the previously well-characterized parental strain 390S, the mmpL4B deletion mutant had lost sliding motility and the ability to form biofilm, but acquired the ability to replicate in human macrophages and stimulate macrophage Toll-like receptor 2. This study demonstrates that deletion of a gene associated with expression of a cell-wall lipid can result in acquisition of an immunostimulatory, invasive bacterial phenotype and has important implications for the study of M. abscessus pathogenesis at the cellular level.

scholarly journals Screening for Mevalonate Biosynthetic Pathway Inhibitors Using Sensitized Bacterial Strains

2011 ◽  
Vol 16 (6) ◽  
pp. 637-646 ◽  
Author(s):  
Sandrine Ferrand ◽  
Jianshi Tao ◽  
Xiaoyu Shen ◽  
Dorothy McGuire ◽  
Andres Schmid ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Mevalonate Pathway ◽  
Inducible Promoter ◽  
High Density ◽  
Bacterial Strains ◽  
Differential Growth ◽  
Low Concentrations ◽  
Mevalonate Diphosphate Decarboxylase ◽  
A Cell ◽  
Dependent Pathway

A simple, optical density-based assay for inhibitors of the mevalonate-dependent pathway for isoprenoid biosynthesis was developed. The assay uses pathway-sensitized Staphylococcus aureus strains and is fully compatible with high-density screening in a 1536-well format. S. aureus strains were constructed in which genes required for mevalonate-dependent isopentenyl pyrophosphate (IPP) synthesis were regulated by an isopropyl-β-D-thiogalactopyranoside (IPTG)–inducible promoter. Inhibitors of the target enzymes displayed greater antibacterial potency in media containing low concentrations of IPTG, and therefore less induction of mevalonate pathway genes, than in media with high IPTG conditions. This differential growth phenotype was exploited to bias the cell-based screening hits toward specific inhibitors of mevalonate-dependent IPP biosynthesis. Screens were run against strains engineered for regulation of the enzymes HMG-CoA synthase (MvaS) and mevalonate kinase (mvaK1), mevalonate diphosphate decarboxylase (mvaD), and phosphomevalonate kinase (mvaK2). The latter three enzymes are regulated as an operon. These assays resulted in the discovery of potent antibacterial hits that were progressed to an active hit-to-lead program. The example presented here demonstrates that a cell sensitization strategy can be successfully applied to a 1.3-million compound high-throughput screen in a high-density 1536-well format.

scholarly journals Identification of a Direct Biosynthetic Pathway for UDP–N-Acetylgalactosamine from Glucosamine-6-Phosphate in Thermophilic Crenarchaeon Sulfolobus tokodaii

2018 ◽  
Vol 200 (10) ◽  
pp. e00048-18
Author(s):  
Mohammad Dadashipour ◽  
Mariko Iwamoto ◽  
Mohammad Murad Hossain ◽  
Jun-ichi Akutsu ◽  
Zilian Zhang ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Cell Extract ◽  
Biosynthetic Pathways ◽  
The Novel ◽  
Nucleotide Sugar ◽  
Content Type ◽  
Sulfolobus Tokodaii ◽  
Archaeal Species ◽  
A Cell ◽  
Bacterial Type

ABSTRACT Most organisms, from Bacteria to Eukarya, synthesize UDP–N-acetylglucosamine (UDP-GlcNAc) from fructose-6-phosphate via a four-step reaction, and UDP–N-acetylgalactosamine (UDP-GalNAc) can only be synthesized from UDP-GlcNAc by UDP-GlcNAc 4-epimerase. In Archaea, the bacterial-type UDP-GlcNAc biosynthetic pathway was reported for Methanococcales. However, the complete biosynthetic pathways for UDP-GlcNAc and UDP-GalNAc present in one archaeal species are unidentified. Previous experimental analyses on enzymatic activities of the ST0452 protein, identified from the thermophilic crenarchaeon Sulfolobus tokodaii, predicted the presence of both a bacterial-type UDP-GlcNAc and an independent UDP-GalNAc biosynthetic pathway in this archaeon. In the present work, functional analyses revealed that the recombinant ST2186 protein possessed an glutamine:fructose-6-phosphate amidotransferase activity and that the recombinant ST0242 protein possessed a phosphoglucosamine-mutase activity. Along with the acetyltransferase and uridyltransferase activities of the ST0452 protein, the activities of the ST2186 and ST0242 proteins confirmed the presence of a bacterial-type UDP-GlcNAc biosynthetic pathway in S. tokodaii. In contrast, the UDP-GlcNAc 4-epimerase homologue gene was not detected within the genomic data. Thus, it was expected that galactosamine-1-phosphate or galactosamine-6-phosphate (GalN-6-P) was provided by conversion of glucosamine-1-phosphate or glucosamine-6-phosphate (GlcN-6-P). A novel epimerase converting GlcN-6-P to GalN-6-P was detected in a cell extract of S. tokodaii, and the N-terminal sequence of the purified protein indicated that the novel epimerase was encoded by the ST2245 gene. Along with the ST0242 phosphogalactosamine-mutase activity, this observation confirmed the presence of a novel UDP-GalNAc biosynthetic pathway from GlcN-6-P in S. tokodaii. Discovery of the novel pathway provides a new insight into the evolution of nucleotide sugar metabolic pathways. IMPORTANCE In this work, a novel protein capable of directly converting glucosamine-6-phosphate to galactosamine-6-phosphate was successfully purified from a cell extract of the thermophilic crenarchaeon Sulfolobus tokodaii. Confirmation of this novel activity using the recombinant protein indicates that S. tokodaii possesses a novel UDP-GalNAc biosynthetic pathway derived from glucosamine-6-phosphate. The distributions of this and related genes indicate the presence of three different types of UDP-GalNAc biosynthetic pathways: a direct pathway using a novel enzyme and two conversion pathways from UDP-GlcNAc using known enzymes. Additionally, Crenarchaeota species lacking all three pathways were found, predicting the presence of one more unknown pathway. Identification of these novel proteins and pathways provides important insights into the evolution of nucleotide sugar biosynthesis, as well as being potentially important industrially.

scholarly journals A cell-free approach to accelerate the study of protein–protein interactions in vitro

2013 ◽  
Vol 3 (5) ◽  
pp. 20130018 ◽  
Author(s):  
E. Sierecki ◽  
N. Giles ◽  
M. Polinkovsky ◽  
M. Moustaqil ◽  
K. Alexandrov ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Interaction Networks ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Single Molecule Fluorescence Spectroscopy ◽  
A Cell

Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery.

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