Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Inhibition of prolyl-tRNA synthetase and efflux pumps as a dual-targeting strategy against multidrug-resistant bacteria

doi: 10.1080/14756366.2026.2640718

Figure Lengend Snippet: Structure-based design of HF analogs against KpProRS. Protein sequence alignment (A) and superposition of the HsProRS cyt (grey), SaProRS (pink) and PfProRS (green) crystal structures, in complex with HF (yellow, from PDB 4HVC), with the HsProRs mit (blue) structural model, showing amino acid polymorphisms in the piperidine (B) and HF quinazolinone pockets (C, left and right panels) between eukaryotic and prokaryotic ProRS. (D) Structural alignment of the complexes of HsProRS cyt (PDB 7Y1W) and SaProRS, bound respectively to bersiporocin (cyan) and HF (yellow). In the sequence alignment, HF-interacting residues in human (Hs) and P. falciparum (Pf) ProRS are shown in red, while interacting residues in SaProRS (Sa) are shown in blue. Additional polymorphism in the piperidine and quinazolinone pockets are marked in orange. In panel C, right panel, H331 of PfProRS is not shown as it is positioned away from the other residues in the structure. Hs cyt , human cytosolic ProRS (PDB 4HVC); Pf , P. falciparum (PDB 4YDQ); Sa, S. aureus (PDB 5ZNJ); Ba, Bacillus anthracis (Uniprot C3P5M1 ); Bc, Burkholderia cenocepacia (Uniprot B4E5W6 ); Ab, Acinetobacter baumannii (Uniprot B2HY33 ); Pa, Pseudomonas aeruginosa (PDB 5UCM); Kp, K. pneumoniae (Uniprot A6T4Z7 ) and Ec, E. coli (UniProt P16659 ).

Article Snippet: Phenotypic assays for MIC determination of compounds on the growth of K. pneumoniae (ATCC BAA-1705 and ATCC BAA-1706), E. coli (ATCC 25922 and ATCC 35218), B. cenocepacia (ATCC BAA-245) and S. aureus (NCTC 12493) were performed using broth microdilution method in sterile 96-well flat bottoms microplates.

Techniques: Sequencing

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Inhibition of prolyl-tRNA synthetase and efflux pumps as a dual-targeting strategy against multidrug-resistant bacteria

doi: 10.1080/14756366.2026.2640718

Figure Lengend Snippet: Checkerboard analysis of the effect of the efflux pump inhibitor PAβN on Cpd-6 activity against bacterial pathogens. The colour grade is relative to absorbance values of bacterial growth cultures, from white (minimum or no growth) to dark green (maximum growth). Absorbance values are the means of at least four independent measurements. Cpd-6 concentration representing MIC values in µg/mL are indicated in grey. PAβN was used at 32 µg/mL for all bacterial pathogens, except B. cenocepacia where it was used at the maximum tolerable concentration of 16 µg/mL.

Article Snippet: Phenotypic assays for MIC determination of compounds on the growth of K. pneumoniae (ATCC BAA-1705 and ATCC BAA-1706), E. coli (ATCC 25922 and ATCC 35218), B. cenocepacia (ATCC BAA-245) and S. aureus (NCTC 12493) were performed using broth microdilution method in sterile 96-well flat bottoms microplates.

Techniques: Activity Assay, Concentration Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Inhibition of prolyl-tRNA synthetase and efflux pumps as a dual-targeting strategy against multidrug-resistant bacteria

doi: 10.1080/14756366.2026.2640718

Figure Lengend Snippet: Structure-based design of HF analogs against KpProRS. Protein sequence alignment (A) and superposition of the HsProRS cyt (grey), SaProRS (pink) and PfProRS (green) crystal structures, in complex with HF (yellow, from PDB 4HVC), with the HsProRs mit (blue) structural model, showing amino acid polymorphisms in the piperidine (B) and HF quinazolinone pockets (C, left and right panels) between eukaryotic and prokaryotic ProRS. (D) Structural alignment of the complexes of HsProRS cyt (PDB 7Y1W) and SaProRS, bound respectively to bersiporocin (cyan) and HF (yellow). In the sequence alignment, HF-interacting residues in human (Hs) and P. falciparum (Pf) ProRS are shown in red, while interacting residues in SaProRS (Sa) are shown in blue. Additional polymorphism in the piperidine and quinazolinone pockets are marked in orange. In panel C, right panel, H331 of PfProRS is not shown as it is positioned away from the other residues in the structure. Hs cyt , human cytosolic ProRS (PDB 4HVC); Pf , P. falciparum (PDB 4YDQ); Sa, S. aureus (PDB 5ZNJ); Ba, Bacillus anthracis (Uniprot C3P5M1 ); Bc, Burkholderia cenocepacia (Uniprot B4E5W6 ); Ab, Acinetobacter baumannii (Uniprot B2HY33 ); Pa, Pseudomonas aeruginosa (PDB 5UCM); Kp, K. pneumoniae (Uniprot A6T4Z7 ) and Ec, E. coli (UniProt P16659 ).

Article Snippet: Thus, the antimicrobial activity of the HF derivatives was tested against several human bacterial pathogens, including K. pneumoniae , reference strains carbapenemase (KPC) producer (ATCC BAA-1705) and carbapenem-sensitive (ATCC BAA-1706), E. coli , reference strains bla - (ATCC 25922) and bla + (ATCC 35218), MDR B. cenocepacia (ATCC BAA-245) and methicillin-resistant (MRSA) S. aureus (NCTC 12493).

Techniques: Sequencing

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Inhibition of prolyl-tRNA synthetase and efflux pumps as a dual-targeting strategy against multidrug-resistant bacteria

doi: 10.1080/14756366.2026.2640718

Figure Lengend Snippet: Checkerboard analysis of the effect of the efflux pump inhibitor PAβN on Cpd-6 activity against bacterial pathogens. The colour grade is relative to absorbance values of bacterial growth cultures, from white (minimum or no growth) to dark green (maximum growth). Absorbance values are the means of at least four independent measurements. Cpd-6 concentration representing MIC values in µg/mL are indicated in grey. PAβN was used at 32 µg/mL for all bacterial pathogens, except B. cenocepacia where it was used at the maximum tolerable concentration of 16 µg/mL.

Article Snippet: Thus, the antimicrobial activity of the HF derivatives was tested against several human bacterial pathogens, including K. pneumoniae , reference strains carbapenemase (KPC) producer (ATCC BAA-1705) and carbapenem-sensitive (ATCC BAA-1706), E. coli , reference strains bla - (ATCC 25922) and bla + (ATCC 35218), MDR B. cenocepacia (ATCC BAA-245) and methicillin-resistant (MRSA) S. aureus (NCTC 12493).

Techniques: Activity Assay, Concentration Assay

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Aurora B phosphorylates serine 16 and serine 39 of BubR1 upon nocodazole treatment, which is required for K250 acetylation. ( A ) Identification of Aurora B-mediated phosphorylation sites of BubR1 in response to nocodazole treatment. (Top) Schematic illustration of BubR1 marked with AurkB-binding region and phosphorylation sites. Potential phosphorylation sites (Ser16 and Ser39) by AurkB and K250 acetylation site are marked with red. N-terminus of BubR1 (1–150) binds to AurkB (results from Fig. ). (Bottom) IP-WB analysis to identify phosphorylation sites that crosstalk with K250 acetylation. mCherry-tagged BubR1 expression constructs were mutagenized in vitro to substitute the phosphorylation sites with alanine, based on proteome analysis . They were then transfected into HeLa cells and treated with nocodazole (200 ng/ml) for 20 h. Mitotic cells were collected by shake-off, and attached cells (Attach) were employed as a control. Mitotic cell lysates, along with lysates from attached cells (WT, Attach), were subjected to immunoprecipitation with anti-mCherry antibody, followed by WB with anti-AcK250 mAb. The same blot was reprobed with anti-BubR1 and anti-mCherry antibodies for normalization. Total cell lysates were subjected to WB with anti-phospho-H3 and anti-cyclin B antibodies to assess the mitosis stage. WB with anti-mCherry and anti-β-actin antibodies in TCL were used as loading controls. Relative band intensities (AcK250/BubR1) were measured using a densitometer and are indicated. ( B ) Identification of the Aurora B-binding region in BubR1. IP with 9E10 (anti-Myc) and WB with anti-AurkB (Aurora B kinase) were performed. All BubR1-expressing constructs were Myc-tagged. The same blot was reprobed with 9E10 for normalization. Two percent of TCL was subjected to WB with the indicated antibodies. ( C ) Effect of Ser16 or Ser39 phosphorylation in K250 acetylation. Immunofluorescence assay in cells expressing the indicated mCherry-tagged BubR1 variants. Nocodazole-treated cells were formaldehyde-fixed and subjected to immunostaining with anti-AcK250, anti-ACA (CREST), and anti-mCherry antibodies. ( D ) Graph showing the intensities of anti-AcK250 immunofluorescence from panel (C). Anti-AcK250 immunofluorescence signals were normalized to anti-mCherry. The results are from two independent experiments. Number of cells: WT, n = 30; K250R, n = 20; K250Q, n = 24; S16A, n = 28; S39A, n = 29 (mean ± s.e.m.). ( E ) WB analysis showing the effects of mitotic kinase inhibitors on phosphorylation of BubR1 (1–150) in unattached kinetochore (nocodazole treatment). HeLa cells were subjected to nocodazole treatment and mitotic shake-off. The mitotic lysates were then resuspended in in vitro phosphorylation buffer containing 100 μM ATP (see the “Materials and methods” section). MBP-tagged recombinant BubR1 (amino acids 1–150) was employed as the substrate. The mitotic lysate and recombinant BubR1 were incubated in the buffer for 2 h with/without indicated inhibitors (10 nM BI 2536, 2 μM reversine, 200 nM hesperadin, and 2 μM ZM447439). In parallel, the lysates were subjected to WB with anti-AurkB-pT232, -AurkB, and -β-actin. ( F ) In vitro phosphorylation assay showing that phosphorylation of S39 and S16 is AurkB-dependent. Recombinant BubR1 (amino acids 1–150) was incubated with anti-AurkB immunoprecipitate of nocodazole-arrested HeLa cells. Reactions were carried out for 2 h in the presence or absence of the AurkB inhibitor ZM447439 (2 μM). [γ- 32 P]-ATP label detects the phosphorylation of the substrate BubR1 (1–150). WB with anti-MBP and anti-AurkB in the same lysate was performed for control.

Article Snippet: Commercially sourced antibodies used in this study include anti-BubR1 (612 503; BD Biosciences), anti-α-tubulin-FITC (F2168; Upstate), anti-Centromere protein antibody (15–235; Antibodies Inc.), anti-HDAC2 (ab12169; Abcam), anti-HDAC3 (ab7030; Abcam), anti-CDC20 (A15656; ABclonal), anti-MAD2 (A11469; ABclonal), anti-Cyclin B (sc-245; Santa Cruz Biotechnology), anti-β-actin (A700-057; Bethyl), anti-CDC27 (BD 610455; BD Biosciences), anti-FLAG (F1804; Sigma–Aldrich), anti-GAPDH (#2118; Cell Signaling Technology), anti-CENP-A (ab13939;Abcam), anti-ZW10 (sc-81430; Santa Cruz Biotechnology), anti-Myc (sc-40; Santa Cruz Biotechnology), anti-AIM-1 (BD611082; BD Biosciences), anti-Vinculin (sc-73614; Santa Cruz Biotechnology), anti-AurB-pT232 (636 102; BioLegend), anti-PLK1 (ab17057; Abcam), anti-mCherry (ab125096; Abcam), anti-Bub3 (611 730; BD Biosciences), ani-CENP-E (A15263; ABclonal), Cyclin B (sc-245; Santa Cruz Biotechnology), anti-pH3 (06–570; EMD Millipore), anti-pan-Phospho-serine (AP0932; ABclonal), and anti-Cdc25C (A12234, ABclonal).

Techniques: Phospho-proteomics, Binding Assay, Expressing, Construct, In Vitro, Transfection, Control, Immunoprecipitation, Immunofluorescence, Immunostaining, Recombinant, Incubation

Journal: Nucleic Acids Research

Article Title: Linking kinetochore attachment to checkpoint control: the role of Aurora B in BubR1 acetylation

doi: 10.1093/nar/gkaf1517

Figure Lengend Snippet: Aurora B phosphorylates serine 16 and serine 39 of BubR1 upon nocodazole treatment, which is required for K250 acetylation. ( A ) Identification of Aurora B-mediated phosphorylation sites of BubR1 in response to nocodazole treatment. (Top) Schematic illustration of BubR1 marked with AurkB-binding region and phosphorylation sites. Potential phosphorylation sites (Ser16 and Ser39) by AurkB and K250 acetylation site are marked with red. N-terminus of BubR1 (1–150) binds to AurkB (results from Fig. ). (Bottom) IP-WB analysis to identify phosphorylation sites that crosstalk with K250 acetylation. mCherry-tagged BubR1 expression constructs were mutagenized in vitro to substitute the phosphorylation sites with alanine, based on proteome analysis . They were then transfected into HeLa cells and treated with nocodazole (200 ng/ml) for 20 h. Mitotic cells were collected by shake-off, and attached cells (Attach) were employed as a control. Mitotic cell lysates, along with lysates from attached cells (WT, Attach), were subjected to immunoprecipitation with anti-mCherry antibody, followed by WB with anti-AcK250 mAb. The same blot was reprobed with anti-BubR1 and anti-mCherry antibodies for normalization. Total cell lysates were subjected to WB with anti-phospho-H3 and anti-cyclin B antibodies to assess the mitosis stage. WB with anti-mCherry and anti-β-actin antibodies in TCL were used as loading controls. Relative band intensities (AcK250/BubR1) were measured using a densitometer and are indicated. ( B ) Identification of the Aurora B-binding region in BubR1. IP with 9E10 (anti-Myc) and WB with anti-AurkB (Aurora B kinase) were performed. All BubR1-expressing constructs were Myc-tagged. The same blot was reprobed with 9E10 for normalization. Two percent of TCL was subjected to WB with the indicated antibodies. ( C ) Effect of Ser16 or Ser39 phosphorylation in K250 acetylation. Immunofluorescence assay in cells expressing the indicated mCherry-tagged BubR1 variants. Nocodazole-treated cells were formaldehyde-fixed and subjected to immunostaining with anti-AcK250, anti-ACA (CREST), and anti-mCherry antibodies. ( D ) Graph showing the intensities of anti-AcK250 immunofluorescence from panel (C). Anti-AcK250 immunofluorescence signals were normalized to anti-mCherry. The results are from two independent experiments. Number of cells: WT, n = 30; K250R, n = 20; K250Q, n = 24; S16A, n = 28; S39A, n = 29 (mean ± s.e.m.). ( E ) WB analysis showing the effects of mitotic kinase inhibitors on phosphorylation of BubR1 (1–150) in unattached kinetochore (nocodazole treatment). HeLa cells were subjected to nocodazole treatment and mitotic shake-off. The mitotic lysates were then resuspended in in vitro phosphorylation buffer containing 100 μM ATP (see the “Materials and methods” section). MBP-tagged recombinant BubR1 (amino acids 1–150) was employed as the substrate. The mitotic lysate and recombinant BubR1 were incubated in the buffer for 2 h with/without indicated inhibitors (10 nM BI 2536, 2 μM reversine, 200 nM hesperadin, and 2 μM ZM447439). In parallel, the lysates were subjected to WB with anti-AurkB-pT232, -AurkB, and -β-actin. ( F ) In vitro phosphorylation assay showing that phosphorylation of S39 and S16 is AurkB-dependent. Recombinant BubR1 (amino acids 1–150) was incubated with anti-AurkB immunoprecipitate of nocodazole-arrested HeLa cells. Reactions were carried out for 2 h in the presence or absence of the AurkB inhibitor ZM447439 (2 μM). [γ- 32 P]-ATP label detects the phosphorylation of the substrate BubR1 (1–150). WB with anti-MBP and anti-AurkB in the same lysate was performed for control.

Article Snippet: Commercially sourced antibodies used in this study include anti-BubR1 (612 503; BD Biosciences), anti-α-tubulin-FITC (F2168; Upstate), anti-Centromere protein antibody (15–235; Antibodies Inc.), anti-HDAC2 (ab12169; Abcam), anti-HDAC3 (ab7030; Abcam), anti-CDC20 (A15656; ABclonal), anti-MAD2 (A11469; ABclonal), anti-Cyclin B (sc-245; Santa Cruz Biotechnology), anti-β-actin (A700-057; Bethyl), anti-CDC27 (BD 610455; BD Biosciences), anti-FLAG (F1804; Sigma–Aldrich), anti-GAPDH (#2118; Cell Signaling Technology), anti-CENP-A (ab13939;Abcam), anti-ZW10 (sc-81430; Santa Cruz Biotechnology), anti-Myc (sc-40; Santa Cruz Biotechnology), anti-AIM-1 (BD611082; BD Biosciences), anti-Vinculin (sc-73614; Santa Cruz Biotechnology), anti-AurB-pT232 (636 102; BioLegend), anti-PLK1 (ab17057; Abcam), anti-mCherry (ab125096; Abcam), anti-Bub3 (611 730; BD Biosciences), ani-CENP-E (A15263; ABclonal), Cyclin B (sc-245; Santa Cruz Biotechnology), anti-pH3 (06–570; EMD Millipore), anti-pan-Phospho-serine (AP0932; ABclonal), and anti-Cdc25C (A12234, ABclonal).

Techniques: Phospho-proteomics, Binding Assay, Expressing, Construct, In Vitro, Transfection, Control, Immunoprecipitation, Immunofluorescence, Immunostaining, Recombinant, Incubation