96 well black bottom plates

NHEK conditioned medium was added at 50 μl to 96-well black-bottom plates (Corning) followed by addition of 150 μl of the peptide Boc- Val-Pro-Arg-AMC (trypsin-like substrate; Bachem) at a final concentration of 200 μM in 1× digestion buffer [10 mM tris-HCl (pH7.8)] and incubated at 37°C for 24 hours. Relative fluorescence intensity (excitation, 354 nm; emission, 435 nm) was analyzed with a SpectraMax Gemini EM fluorometer (Thermo Fisher Scientific). For murine skin trypsin activity analysis, 0.5-cm² full-thickness skin was bead-beat (2.0-mm zirconia beads, 2 × 30 s with 5 min after each) in 1 ml of 1 M acetic acid, followed by an overnight rotation at 4°C. Samples were centrifuged (10 min, 13,000 rpm, 4°C) and then added to a new microcentrifuge tube followed by protein concentration using a SpeedVac to remove all remaining acetic acid. Proteins were resuspended in molecular-grade water (500 μl) and rotated overnight at 4°C followed by another centrifugation. Clear protein lysates were added to a new tube, and bicinchoninic acid (Thermo Fisher Scientific) analysis was used to determine protein concentration. Last, 10 μg of total protein was added to a 96-well plate followed by analysis with the trypsin substrate as above.

Inhibitor residence times were determined by monitoring the rate of association of a fluorescent probe to the enzyme-inhibitor complexes using a Penefsky column-based method. Fifty μL of a solution containing 10 μM purified ecLpxC and 20 μM of LpxC inhibitor (CHIR-090, PT810 or PT805) in 25 mM NaH₂PO₄, pH 8.0 buffer containing 300 mM KCl and 0.1 mg/mL BSA, was incubated at 25 °C for 16 h. Then, 5 μL of the incubated mixture was rapidly diluted into 1 mL of the reaction buffer containing 1 μM fluorescence competitor PT900 at 25 °C. Subsequently, 100 μL aliquots of the diluted mixture were collected at different time points and loaded onto the spin-column (PD SpinTrap™ G-25, Cytiva), which was then centrifuged in a swinging bucket rotor (Eppendorf 5810R, 15-amp version) at 800xg for 2 min. One hundred μL aliquots of the eluate were dispensed into 96 well black bottom plates (Corning, NY) in duplicate, and the fluorescence intensity was quantified using a plate reader (BioTek, Gen5, 3.09) at λ_ex 315 nm and λ_em 420 nm. The change in fluorescence as a function of time was fit to a one-phase association equation in GraphPad Prism 9.0. The rate of PT900 association to ecLpxC was taken to be equivalent to the overall off rate for dissociation of the inhibitor from ecLpxC (k_off), and residence times (t_R) were calculated by taking the reciprocal of k_off (t_R = 1/k_off).