Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: Introduction to the tetracycline destructase family of FMO enzymes and structure of the first inhibitor, anhydrotetracycline (5). A. Phylogenetic tree [aligned with Clustal Omega and viewed using iTOL software]. B. X-ray crystal structure of chlortetracycline bound to Tet(X) (PDB ID 2y6r). C. X-ray crystal structure of chlortetracycline bound to Tet(50) (PDB ID 5tui).

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: Software

Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: Michaelis-Menten kinetics of tetracycline destructase degradation of first generation tetracyclines. A. Representative Michaelis-Menten plot of tetracycline destructase degradation of oxytetracycline. B. Representative optical absorbance kinetic plots for the degradation of oxytetracycline by tetracycline destructase enzymes [as observed at 400 nm for Tet(50) and Tet(X)_3; 380 nm for Tet(X)]. C. Apparent Km, kapp, and catalytic efficiencies for the tetracycline destructase-mediated degradation of tetracycline, chlortetracycline, demeclocycline, and oxytetracycline. Error bars represent standard deviation for two independent trials.

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: Standard Deviation

Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: in vitro aTc inhibition of tetracycline destructase degradation of first-generation tetracycline antibiotics as observed via an optical absorbance kinetic assay. A. aTc inhibition of Tet(50) degradation of tetracyclines. B. aTc inhibition of Tet(X) degradation of tetracyclines. C. aTc inhibition of Tet(X)_3 degradation of tetracyclines. D. Apparent IC50 for aTc inhibition (denoted for each substrate and enzyme). Error bars represent standard deviation for three independent trials. All data points possess error bars, though some are not visible at the plotted scale.

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: In Vitro, Inhibition, Kinetic Assay, Standard Deviation

Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: In vitro inhibition of tetracycline destructase degradation of first-generation tetracycline antibiotics as observed via optical absorbance kinetic assay. A. Inhibitory activity of aTc library against Tet(50) degradation of tetracycline. B. Inhibitory activity of aTc library against Tet(X) degradation of tetracycline. C. Inhibitory activity of aTc library against Tet(X)_3 degradation of tetracycline. D. Inhibitory activity of aTc library against Tet(50) degradation of chlortetracycline. E. Inhibitory activity of aTc library against Tet(X) degradation of chlortetracycline. F. Inhibitory activity of aTc library against Tet(X)_3 degradation of chlortetracycline. Error bars represent standard deviation for three independent trials. All data points possess error bars, though some are not visible at the plotted scale.

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: In Vitro, Inhibition, Kinetic Assay, Activity Assay, Standard Deviation

Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: aDem inhibition of tetracycline destructase-mediated degradation of first generation tetracyclines. A. aDem inhibition of tetracycline destructase degradation of tetracycline. B. aDem inhibition of tetracycline destructase degradation of demeclocycline. C. Apparent IC50 for the in vitro inhibition of tetracycline and demeclocycline degradation by tetracycline destructase enzymes with aTc and aDem. D. Michaelis-Menten plot of dose-dependent aDem acceleration of Tet(50) consumption of NADPH, apparent Km, and calculated catalytic efficiency. Error bars represent standard deviation for two to three independent trials. All data points possess error bars, though some are not visible at the plotted scale.

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: Inhibition, In Vitro, Standard Deviation

Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: A. Whole cell inhibition of E. coli expressing tetracycline destructase enzymes including calculated FICI and observed fold change enhancements. B. Working model of the inhibition of tetracycline destructase enzymes by aTc-like small molecules (competitive inhibitor versus sacrificial substrate).

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: Inhibition, Expressing

Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: Michaelis-Menten kinetics of tetracycline destructase-mediated degradation of last generation tetracyclines. A. Michaelis-Menten plot and apparent kinetic parameters (Km, kapp, and catalytic efficiencies) for the Tet(X)-mediated degradation of tigecycline. C. Last generation tetracycline antibiotics tigecycline and eravacycline. Error bars represent standard deviation for three independent trials. All data points possess error bars, though some are not visible at the plotted scale.

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: Standard Deviation

Journal: ACS infectious diseases

Article Title: Semisynthetic Analogues of Anhydrotetracycline as Inhibitors of Tetracycline Destructase Enzymes

doi: 10.1021/acsinfecdis.8b00349

Figure Lengend Snippet: Toward extended library synthesis of aTc-like inhibitors of tetracycline destructase enzymes. A. An adjuvant approach to combat enzymatic inactivation of tetracycline antibiotics and summary of preliminary structure-activity information. B. Surface view of X-ray crystal structure of Tet(X) bound to tigecycline (PDB ID 4a6n) highlights importance of open active site to accommodate bulky D-ring substituents, suggesting further use of glycylcycline antibiotics may drive selective pressure of tetracycline destructase-involved resistance mechanisms.

Article Snippet: The plates were inoculated with ~1 μL of tetracycline destructase expressing E. coli MegaX (Invitrogen) diluted to OD 600 0.1 using a sterile 96-pin replicator (Scinomix).

Techniques: Adjuvant, Activity Assay