Journal: Oncotarget

Article Title: Host targeted antiviral (HTA): functional inhibitor compounds of scaffold protein RACK1 inhibit herpes simplex virus proliferation

doi: 10.18632/oncotarget.26907

Figure Lengend Snippet: ( A ) Shown are sample two receptor-based three-point pharmacophore models generated on the RACK1A phosphorylation site with exclusion spheres colored pink, geometric and distance constraints (flexible) shown as lines and filled white circles as centers. HP-hydrophobic; D-donor; A-acceptor. ( B ) Ligand-based pharmacophore model generated on SD-29 with pharmacophore constraints acceptor, donor, hydrophobic ring, and hydrophilic sites represented filled circles. Structures of compounds SD-29-12 and SD-29-14 are shown.

Article Snippet: Twenty five microgram of proteins were loaded on the BioRad’s 4-12% precast Bis-Tris polyacrylamide gel, transferred to a nitrocellulose membrane and then blocked with 5% Bovine Serum Albumin (BSA) for one hour, washed and incubated with the an antibody (1:100 dilution) to detect phosphorylated Y248 residue of RACK1A protein which was raised using the epitope: FSPNR{pTYR}WLCAATEH (Genscript, Piscataway, NJ, USA).

Techniques: Generated, Phospho-proteomics

Journal: Oncotarget

Article Title: Host targeted antiviral (HTA): functional inhibitor compounds of scaffold protein RACK1 inhibit herpes simplex virus proliferation

doi: 10.18632/oncotarget.26907

Figure Lengend Snippet: ( A ) Docked Model of RACK1A with SD-29 at the Y248 phosphorylation site. (left panel) Modeled structure of RACK1A with SD-29 (carbon in green) docked into it. The targeted binding pocket is highlighted in green. RACK1A is shown as ribbon model (white). (right panel) Detailed view of the SD-29 (carbon in green) interaction with RACK1A site pocket. The residues interacting with SD-29 are shown in a ball-and-stick model. Hydrogen bonds are shown as red broken lines. SD-29 binding site is surrounded by both hydrophobic (HP1) and hydrophilic residues (HP2). The structural model of ‘SD-29’ with RACK1A showing hydrogen bonds with Ser244, Trp249 and hydrophobic interactions with Tyr248, Phe243, Pro204, Leu 263and Trp249 residues. ( B ) RACK1 functional inhibitor compounds inhibit stress hormone induced RACK1A Y248 phosphorylation. One-week old Arabidopsis seedlings were treated with 10 μM of stress hormone Abscisic acid (ABA) in the presence/absence of the inhibitor compounds for 12 hours in a growth chamber (overnight) at 22 ° C. Lysates were probed with an antibody raised to detect phosphorylated Y248 residue of RACK1A protein in Arabidopsis . Lysates from a rack1a-1 knock-out mutant seedlings grown and treated similarly as the Wild Type seedlings were used as negative control. The compounds were dissolved in DMSO (D) and ABA was dissolved in methanol (M). The lower panel shows the same membrane stripped with stripping buffer and then probed with an Arabidopsis Actin antibody to show the loading control. ( C ) Salt stress-induced upregulation of RACK1 expression was inhibited by SD-29. The abundant leaf protein Rubisco large subunit (RbcL) was used as loading control for the blot. The 37kD RACK1 band was absent from the genetic knockout of RACK1 plants (double mutant- rack1ab lane). ( D ) Purified RACK1 protein on a SDS-PAGE gel. E. coli BL21(DE3) host strain was transformed with recombinant plasmid containing rice RACK1 (Chr05 Os05g47890) cDNA with a 3’ His tag. PMSF-induced bacterial lysate eluted from the glutathione-resin column was resolved by the SDS-PAGE electrophoresis for purity check. Lane M: Protein Marker; Lane S: Supernatant; Lane F: Flow through of supernatant; Lane W: Wash; and Lane E1~4: Elutions. ( E ) In the SPR assay, SD-29 (left panel) and SD-29-12 (right panel) bind directly to immobilized RACK1A on the surface of the chip via similar patterns, as evident in the sensogram. SD-29 (left panel) and SD-29-12 (right panel) were separately injected three times on the CM5 chip at 0, 1.56 μM, at 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM (top sensor) concentrations (left panel) and at 3.13 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM (top sensor) concentrations (right panel).

Article Snippet: Twenty five microgram of proteins were loaded on the BioRad’s 4-12% precast Bis-Tris polyacrylamide gel, transferred to a nitrocellulose membrane and then blocked with 5% Bovine Serum Albumin (BSA) for one hour, washed and incubated with the an antibody (1:100 dilution) to detect phosphorylated Y248 residue of RACK1A protein which was raised using the epitope: FSPNR{pTYR}WLCAATEH (Genscript, Piscataway, NJ, USA).

Techniques: Phospho-proteomics, Binding Assay, Functional Assay, Residue, Knock-Out, Mutagenesis, Negative Control, Membrane, Stripping Membranes, Control, Expressing, Purification, SDS Page, Transformation Assay, Recombinant, Plasmid Preparation, Electrophoresis, Marker, SPR Assay, Injection

Journal: bioRxiv

Article Title: Novel RNA m 6 A methyltransferase METTL16 inhibitors

doi: 10.1101/2023.03.27.534333

Figure Lengend Snippet: Identification of the METTL16 inhibitors. (a) The relative inhibitory effect Δm 6 A of compound 1 and 2 on the methylation of the probe RNA by full-length METTL16. The error bars represent SD of each data point calculated from 3 independent measurements. (b) Binding of compounds 1 and 2 to the recombinant His-tagged METTL16 (residues 1-291) protein measured by microscale thermophoresis (MST assay). The error bars represent SD of each data point calculated from 4 independent measurements. The confidence range for Kd (±) defines the range where it falls with a 68% of certainty.

Article Snippet: Recombinant METTL16 MTD (residues 1-291) protein was labelled via His-tag using Monolith His-Tag Labeling Kit RED-tris-NTA 2 nd Generation (NanoTemper Technologies GmbH, Germany).

Techniques: Methylation, Binding Assay, Recombinant, Microscale Thermophoresis

Journal: bioRxiv

Article Title: Novel RNA m 6 A methyltransferase METTL16 inhibitors

doi: 10.1101/2023.03.27.534333

Figure Lengend Snippet: Graphical schemes of binding interactions of inhibitor compounds to METTL16 MTD obtained by molecular docking: (a) compound 1 ; (b) compound 2 . The results of the molecular dynamics (MD) simulation of METTL16 MTD (pdb: 6GFN ) in complex with compound 1 . (c) The root mean square deviation (RMSD) of Cα atoms of METTL16 MTD in complex with compound 1 calculated by 10 ns MD simulation. (d) Desmond 2D profile data for the compound 1 binding to METTL16 MTD. (e) Normalized stacked bar chart representation of interactions and contacts between the compound 1 and METTL16 MTD over the course of the MD trajectory (values over 1.0 occur when amino acid residue has multiple contacts of the same subtype with the ligand). Results of the MD simulation of METTL16 MTD (pdb: 6GFN ) in complex with compound 2 . (f) The RMSD of Cα atoms of METTL16 MTD in complex with compound 2 calculated by 10 ns MD simulation. (g) Desmond 2D profile data for compound 2 binding to METTL16 MTD. (h) Normalized stacked bar chart representation of interactions and contacts between compound 2 and METTL16 MTD over the course of the MD trajectory (values over 1.0 occur when amino acid residue has multiple contacts of the same subtype with the ligand).

Article Snippet: Recombinant METTL16 MTD (residues 1-291) protein was labelled via His-tag using Monolith His-Tag Labeling Kit RED-tris-NTA 2 nd Generation (NanoTemper Technologies GmbH, Germany).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Novel RNA m 6 A methyltransferase METTL16 inhibitors

doi: 10.1101/2023.03.27.534333

Figure Lengend Snippet: Cytotoxicity of compounds 1 and 2 in HEK293T cells assessed by WST-1 assay after (a) 24h and (b) 48 h of the exposure. Data presented as mean ± SD of 4 independent measurements. The concentration dependence of the inhibition of HL-60 cells at different time points of inhibitors (c) 1 and (d) 2 assessed by trypan blue exclusion assay. Concentration dependence of the inhibition of the CCRF-CEM cells by inhibitors (e) 1 and (f) 2 at different time points assessed by trypan blue exclusion assay. Data presented as mean ± SD of 3 independent measurements. The cytotoxicity of compounds 1 and 2 in HL-60 cells assessed by WST-1 assay after (g) 24 h and (h) 48 h of exposure. Data presented as mean ± SD of 3 independent measurements. The percentage of the viable, early-stage apoptotic, late-stage apoptotic, and necrotic HL-60 cells after (i) 24 h and (j) 48 h of treatment by identified METTL16 inhibitors at 10 μM concentration. Data presented as mean ± SD of 2 independent measurements. (k) The effect of compound 1 on the ratio of N 6 -methyladenosine and adenosine (m 6 A/A) in HL-60 cells mRNA measured by LC/MS. Data presented as mean ± SD of 3 independent measurements: * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA test.

Article Snippet: Recombinant METTL16 MTD (residues 1-291) protein was labelled via His-tag using Monolith His-Tag Labeling Kit RED-tris-NTA 2 nd Generation (NanoTemper Technologies GmbH, Germany).

Techniques: WST-1 Assay, Concentration Assay, Inhibition, Trypan Blue Exclusion Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Paenipeptin Analogues Potentiate Clarithromycin and Rifampin against mcr-1 -Mediated Polymyxin-Resistant Escherichia coli In Vivo

doi: 10.1128/AAC.02045-19

Figure Lengend Snippet: Paenipeptin analogue 1 in combination with clarithromycin or rifampin was efficacious against Escherichia coli FDA-CDC AR 0494 in a murine thigh infection model. Treatment groups (antimicrobial injection time, hours postinfection): PBS, phosphate-buffered saline; PMXB_7.5, 7.5 mg/kg polymyxin B (2 h); CLR_60, 60 mg/kg clarithromycin (2 and 5 h); RIF_2.5, 2.5 mg/kg rifampin (2 and 5 h); RIF_5, 5 mg/kg rifampin (2 and 5 h); Paen_40: 40 mg/kg analogue 1 (2 h); Paen_60, 60 mg/kg analogue 1 (2 h); Paen_60+CLR_60, 60 mg/kg analogue 1 (2 h) plus 60 mg/kg clarithromycin (2 h and 5 h); Paen_40+RIF_5, 40 mg/kg analogue 1 (2 h) plus 5 mg/kg rifampin (2 h and 5 h); Paen_60+RIF_2.5, 60 mg/kg analogue 1 (2 h) plus 2.5 mg/kg rifampin (2 h and 5 h); Paen_60+RIF_5, 60 mg/kg analogue 1 (2 h) plus 5 mg/kg rifampin (2 h and 5 h). Statistical significance between the antimicrobial treatment groups and the PBS control group is indicated by an asterisk(s) (*, P < 0.05; ***, P < 0.001).

Article Snippet: The objective of this study was to evaluate the efficacy of the combination between paenipeptins and clarithromycin/rifampin against mcr-1 mediated, polymyxin-resistant E. coli in vitro and in vivo using a murine thigh infection model. Paenipeptin analogues 1 and 15 (>95% purity) are synthetic lipopeptides produced by a commercial peptide synthesis company (GenScript Inc., Piscataway, NJ).

Techniques: Infection, Injection, Saline, Control