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European Journal of Medicinal Chemistry Nov 2021Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with...
Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with pemetrexed/carbo(cis)platin/pembrolizumab (PCP) chemo-immunotherapy in lung cancer. Despite the strong genetic support and therapeutic potential for a NRF2 transcriptional inhibitor, currently there are no known direct inhibitors of the NRF2 protein or its complexes with MAF and/or DNA. Herein we describe the design of a novel and high-confidence homology model to guide a medicinal chemistry effort that resulted in the discovery of a series of peptides that demonstrate high affinity, selective binding to the Antioxidant Response Element (ARE) DNA and thereby displace NRF2-MAFG from its promoter, which is an inhibitory mechanism that to our knowledge has not been previously described. In addition to their activity in electrophoretic mobility shift (EMSA) and TR-FRET-based assays, we show significant dose-dependent ternary complex disruption of NRF2-MAFG binding to DNA by SPR, as well as cellular target engagement by thermal destabilization of HiBiT-tagged NRF2 in the NCI-H1944 NSCLC cell line upon digitonin permeabilization, and SAR studies leading to improved cellular stability. We report the characterization and unique profile of lead peptide 18, which we believe to be a useful in vitro tool to probe NRF2 biology in cancer cell lines and models, while also serving as an excellent starting point for additional in vivo optimization toward inhibition of NRF2-driven transcription to address a significant unmet medical need in non-small cell lung cancer (NSCLC).
Topics: Antioxidant Response Elements; DNA; Drug Design; Drug Stability; Electrophoretic Mobility Shift Assay; Half-Life; HeLa Cells; Humans; MafG Transcription Factor; NF-E2-Related Factor 2; Neoplasms; Peptides; Structure-Activity Relationship
PubMed: 34303079
DOI: 10.1016/j.ejmech.2021.113686 -
Cell Reports Jun 2021The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP...
The sterol regulatory element-binding protein (SREBP) pathway monitors the cellular cholesterol level through sterol-regulated association between the SREBP cleavage-activating protein (Scap) and the insulin-induced gene (Insig). Despite structural determination of the Scap and Insig-2 complex bound to 25-hydroxycholesterol, the luminal domains of Scap remain unresolved. In this study, combining cryogenic electron microscopy (cryo-EM) analysis and artificial intelligence-facilitated structural prediction, we report the structure of the human Scap/Insig-2 complex purified in digitonin. The luminal domain loop 1 and a co-folded segment in loop 7 of Scap resemble those of the luminal/extracellular domain in NPC1 and related proteins, providing clues to the cholesterol-regulated interaction of loop 1 and loop 7. An additional luminal interface is observed between Scap and Insig. We also show that Scap(D428A), which inhibits SREBP activation even under sterol depletion, exhibits an identical conformation with the wild-type protein when complexed with Insig-2, and its constitutive suppression of the SREBP pathway may also involve a later step in protein trafficking.
Topics: Digitonin; HEK293 Cells; Humans; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Micelles; Models, Molecular; Protein Conformation; Protein Folding; Sterols; Structural Homology, Protein
PubMed: 34192549
DOI: 10.1016/j.celrep.2021.109299 -
Methods in Molecular Biology (Clifton,... 2021Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to...
Three-dimensional (3D) in vitro systems closely resemble tissue microenvironments and provide predictive models for studying cytotoxic drug responses. The ability to capture the kinetic profiles of such responses in a dynamic and noninvasive way can further advance the utility of 3D cell cultures. Here, we describe the use of a luminescent lactate dehydrogenase (LDH) toxicity assay for monitoring time- and dose-dependent effects of drug treatment in 3D cancer spheroids. HCT116 spheroids formed in 96-well ultralow attachment plates were treated with increasing drug concentrations. Medium samples were collected at different timepoints, frozen, stored, and analyzed at the end of experiments using the luminescent LDH-Glo™ Assay. High assay sensitivity and low volume sampling enabled drug-induced toxicity profiling in a time- and dose-dependent manner.
Topics: Antineoplastic Agents; Digitonin; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Indicators and Reagents; L-Lactate Dehydrogenase; Luminescent Measurements; Neoplasms; Spheroids, Cellular; Time Factors; Toxicity Tests; Tumor Cells, Cultured
PubMed: 34033096
DOI: 10.1007/978-1-0716-1162-3_8 -
MBio Apr 2021Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the...
Pyruvate is the final metabolite of glycolysis and can be converted into acetyl coenzyme A (acetyl-CoA) in mitochondria, where it is used as the substrate for the tricarboxylic acid cycle. Pyruvate availability in mitochondria depends on its active transport through the heterocomplex formed by the mitochondrial pyruvate carriers 1 and 2 (MPC1/MPC2). We report here studies on MPC1/MPC2 of , the etiologic agent of Chagas disease. Endogenous tagging of () and with 3× showed that both encoded proteins colocalize with MitoTracker to the mitochondria of epimastigotes. Individual knockout (KO) of and genes using CRISPR/Cas9 was confirmed by PCR and Southern blot analyses. Digitonin-permeabilized -KO and -KO epimastigotes showed reduced O consumption rates when pyruvate, but not succinate, was used as the mitochondrial substrate, while α-ketoglutarate increased their O consumption rates due to an increase in α-ketoglutarate dehydrogenase activity. Defective mitochondrial pyruvate import resulted in decreased Ca uptake. The inhibitors UK5099 and malonate impaired pyruvate-driven oxygen consumption in permeabilized control cells. Inhibition of succinate dehydrogenase by malonate indicated that pyruvate needs to be converted into succinate to increase respiration. -KO and -KO epimastigotes showed little growth differences in standard or low-glucose culture medium. However, the ability of trypomastigotes to infect tissue culture cells and replicate as intracellular amastigotes was decreased in -KOs. Overall, MPC1 and MPC2 are essential for cellular respiration in the presence of pyruvate, invasion of host cells, and replication of amastigotes. is the causative agent of Chagas disease. Pyruvate is the end product of glycolysis, and its transport into the mitochondrion is mediated by the mitochondrial pyruvate carrier (MPC) subunits. Using the CRISPR/Cas9 technique, we generated individual () and knockouts and demonstrated that they are essential for pyruvate-driven respiration. Interestingly, although glycolysis was reported as not an important source of energy for the infective stages, MPC was essential for normal host cell invasion and intracellular replication.
Topics: Anion Transport Proteins; Biological Transport; CRISPR-Cas Systems; DNA Replication; Gene Knockout Techniques; Mitochondrial Membrane Transport Proteins; Protozoan Proteins; Pyruvic Acid; Trypanosoma cruzi
PubMed: 33824204
DOI: 10.1128/mBio.00540-21 -
International Journal of Molecular... Mar 2021Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5'end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been...
Adenoviruses contain dsDNA covalently linked to a terminal protein (TP) at the 5'end. TP plays a pivotal role in replication and long-lasting infectivity. TP has been reported to contain a nuclear localisation signal (NLS) that facilitates its import into the nucleus. We studied the potential NLS motifs within TP using molecular and cellular biology techniques to identify the motifs needed for optimum nuclear import. We used confocal imaging microscopy to monitor the localisation and nuclear association of GFP fusion proteins. We identified two nuclear localisation signals, PV(R)6VP and MRRRR, that are essential for fully efficient TP nuclear entry in transfected cells. To study TP-host interactions further, we expressed TP in (). Nuclear uptake of purified protein was determined in digitonin-permeabilised cells. The data confirmed that nuclear uptake of TP requires active transport using energy and shuttling factors. This mechanism of nuclear transport was confirmed when expressed TP was microinjected into living cells. Finally, we uncovered the nature of TP binding to host nuclear shuttling proteins, revealing selective binding to Imp β, and a complex of Imp α/β but not Imp α alone. TP translocation to the nucleus could be inhibited using selective inhibitors of importins. Our results show that the bipartite NLS is required for fully efficient TP entry into the nucleus and suggest that this translocation can be carried out by binding to Imp β or Imp α/β. This work forms the biochemical foundation for future work determining the involvement of TP in nuclear delivery of adenovirus DNA.
Topics: Active Transport, Cell Nucleus; Adenoviridae; Cell Nucleus; Cytosol; DNA; Escherichia coli; Genome, Viral; Green Fluorescent Proteins; HEK293 Cells; HeLa Cells; Humans; Microscopy, Confocal; Nuclear Localization Signals; Protein Binding; Viral Proteins; alpha Karyopherins; beta Karyopherins
PubMed: 33804953
DOI: 10.3390/ijms22073310 -
International Journal of Molecular... Mar 2021This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and...
This study was focused on the molecular mechanisms of action of saponins and related compounds (sapogenins and alkaloids) on model lipid membranes. Steroids and triterpenes were tested. A systematic analysis of the effects of these chemicals on the physicochemical properties of the lipid bilayers and on the formation and functionality of the reconstituted ion channels induced by antimicrobial agents was performed. It was found that digitonin, tribulosin, and dioscin substantially reduced the boundary potential of the phosphatidylcholine membranes. We concluded that saponins might affect the membrane boundary potential by restructuring the membrane hydration layer. Moreover, an increase in the conductance and lifetime of gramicidin A channels in the presence of tribulosin was due to an alteration in the membrane dipole potential. Differential scanning microcalorimetry data indicated the key role of the sapogenin core structure (steroid or triterpenic) in affecting lipid melting and disordering. We showed that an alteration in pore forming activity of syringomycin E by dioscin might be due to amendments in the lipid packing. We also found that the ability of saponins to disengage the fluorescent marker calcein from lipid vesicles might be also determined by their ability to induce a positive curvature stress.
Topics: Cell Membrane; Cell Membrane Permeability; Dose-Response Relationship, Drug; Ion Channel Gating; Ion Channels; Lipid Bilayers; Membrane Lipids; Membrane Potentials; Molecular Structure; Phase Transition; Saponins
PubMed: 33804648
DOI: 10.3390/ijms22063167 -
Quarterly Reviews of Biophysics Mar 2021Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron...
Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.
Topics: Cryoelectron Microscopy; Electrons; Membrane Proteins; Surface-Active Agents
PubMed: 33785082
DOI: 10.1017/S0033583521000044 -
Journal of Fluorescence May 2021The varied applications of nanotechnology have paved way for several breakthroughs in the realm of biomedical technology. In this challenging era when illness...
The varied applications of nanotechnology have paved way for several breakthroughs in the realm of biomedical technology. In this challenging era when illness multiplies, timely and accurate disease diagnosis is very important. Thus, well founded novel approaches matter very much in areas like disease diagnosis and monitoring. Nanomedicine has tremendous implications in the given context. An elevated cholesterol concentration in blood is risky and is associated with cardiovascular diseases (CVD). CVD remains the No. 1 global cause of death and hence there is an urge to understand cholesterol level and take preventive measures. Highly fluorescent graphene quantum dots (GQs) are well known for their biocompatibility, non toxicity and aqueous solubility. Here in we report an easy and sensitive non enzymatic based cholesterol detection using digitonin conjugated graphene quantum dots (GDG). Selectivity studies and the cholesterol detection in human blood serum suggests the probe to be reliable and selective for blood cholesterol monitoring. Digitonin conjugated fluorescent graphene quantumdots, an efficient probe for cholesterol sensing.
Topics: Blood Chemical Analysis; Cholesterol; Graphite; Humans; Quantum Dots
PubMed: 33761068
DOI: 10.1007/s10895-021-02712-5 -
PLoS Neglected Tropical Diseases Feb 2021In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal,...
In Trypanosoma brucei, there are fourteen enzymatic biotransformations that collectively convert glucose into five essential nucleotide sugars: UDP-Glc, UDP-Gal, UDP-GlcNAc, GDP-Man and GDP-Fuc. These biotransformations are catalyzed by thirteen discrete enzymes, five of which possess putative peroxisome targeting sequences. Published experimental analyses using immunofluorescence microscopy and/or digitonin latency and/or subcellular fractionation and/or organelle proteomics have localized eight and six of these enzymes to the glycosomes of bloodstream form and procyclic form T. brucei, respectively. Here we increase these glycosome localizations to eleven in both lifecycle stages while noting that one, phospho-N-acetylglucosamine mutase, also localizes to the cytoplasm. In the course of these studies, the heterogeneity of glycosome contents was also noted. These data suggest that, unlike other eukaryotes, all of nucleotide sugar biosynthesis in T. brucei is compartmentalized to the glycosomes in both lifecycle stages. The implications are discussed.
Topics: Life Cycle Stages; Microbodies; Nucleotides; Sugars; Trypanosoma brucei brucei
PubMed: 33592041
DOI: 10.1371/journal.pntd.0009132 -
Environmental Science and Pollution... May 2021Pergularia tomentosa L. (P. tomentosa) has been largely used in Tunisian folk medicine as remedies against skin diseases, asthma, and bronchitis. The main objectives of...
Bioactivities and in silico study of Pergularia tomentosa L. phytochemicals as potent antimicrobial agents targeting type IIA topoisomerase, TyrRS, and Sap1 virulence proteins.
Pergularia tomentosa L. (P. tomentosa) has been largely used in Tunisian folk medicine as remedies against skin diseases, asthma, and bronchitis. The main objectives of this study were to identify phytochemical compounds that have antioxidant and antimicrobial properties from the stem, leaves, and fruit crude methanolic extracts of P. tomentosa, and to search for tyrosyl-tRNA synthetase (TyrRS), topoisomerase type IIA, and Candidapepsin-1 (SAP1) enzyme inhibitors through molecular docking study. Phytochemical quantification revealed that fruit and leaves extracts displayed the highest total flavonoids (582 mg QE/g Ex; 219 mg QE/g Ex) and tannins content (375 mg TAE/g Ex; 216 mg TAE/g Ex), also exhibiting significant scavenging activity to decrease free radicals for ABTS, DPPH, β-carotene, and FRAP assay with IC values (> 1 mg/mL). Additionally, promising antimicrobial activities towards different organs have been observed against several bacteria and Candida strains. From the liquid chromatography-mass spectrometry (LC-MS) analysis, five polyphenolic compounds, namely digitoxigenin, digitonin glycoside and calactina in the leaves, kaempferol in the fruit, and calotropagenin in the stems, were identified. They were also analyzed for their drug likeliness, based on computational methods. Molecular docking study affirmed that the binding affinity of calactin and actodigin to the active site of TyrRS, topoisomerase type IIA, and SAP1 target virulence proteins was the highest among the examined dominant compounds. Therefore, this study indicated that P. tomentosa methanolic extracts displayed great potential to become a potent antimicrobial agent and might be a promising source for therapeutic and nutritional functions. These phytocompounds could be further promoted as a candidate for drug discovery and development.
Topics: Anti-Infective Agents; Antioxidants; Bacteria; Candida; Caryophyllaceae; Fruit; Molecular Docking Simulation; Phytochemicals; Plant Extracts; Plant Leaves; Tyrosine-tRNA Ligase; Virulence; Virulence Factors
PubMed: 33454827
DOI: 10.1007/s11356-020-11946-y