Journal: ACS Infectious Diseases

Article Title: Accelerating Antimalarial Drug Discovery with a New High-Throughput Screen for Fast-Killing Compounds

doi: 10.1021/acsinfecdis.4c00328

Figure Lengend Snippet: Nitroreductase (NTR) assay using near-infrared probe (NIR-P). (A) Scheme of enzymatic reaction of NTR and NIR-P ox producing the fluorescent NIR-P red (λ ex/em = 615/670 nm). (B) Time-dependent fluorescent spectra of NIR-P red produced by NTR. The enzymatic reaction catalyzed by NTR was monitored using 50 μM NIR-P ox and 50 μM NADH in 30 mM Tris-HCl, pH 8.0, buffer containing 0.5 μg/mL NTR. Fluorescent signals (λ ex/em = 605/620–740 nm over 1 nm intervals) were measured over a 60 min period at room temperature (orange: 0 min, yellow: 2 min, light green: 3 min, blue: 5 min, purple: 10 min, red: 60 min). Fluorescence intensity (FI) over time is plotted in the inset. Note that the reduction of NIR-P by NTR is much faster than the reaction observed using the NCOU1 substrate ( Figure. C). (C) Parasitemia titration measured using the NTR-NIR assay. Each parasite culture (1% hematocrit (Hct)) was assayed in PfLDH buffer (100 mM Tris-HCl, pH8.0, 300 mM lithium L-lactate, and 0.25% Triton X-100) containing 500 μM APAD + , 200 μM NIR probe, and 0.05 U/mL NTR at room temperature. The quenching (“stop”) solution consisted of 0.9 M pyruvate. Data are presented as the mean ± standard deviation (SD) ( n = 4). (D) Correlation of %Inhibition between conventional SYBR green and NTR-NIR assays of PfLDH activity. The red line in the graph represents the line of equality ( y = x ). a.u.: arbitrary unit, NAD + : oxidized nicotinamide adenine dinucleotide, NADH: reduced nicotinamide adenine dinucleotide, RFU: relative fluorescence units.

Article Snippet: The centrifuged plates were incubated for 1 h at room temperature in a moist chamber, at which point the NIR red signal (λ ex/em = 615/690 nm) was measured using a PHERAstar Plus microplate reader (BMG LABTECH).

Techniques: Produced, Fluorescence, Titration, Standard Deviation, Inhibition, SYBR Green Assay, Activity Assay