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

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: Figure 1. Purification of recombinant nitroreductase (NTR) and quantification of enzymatic activity. (A) Recombinant C-terminally His10-tagged NTR was expressed in E. coli BL21 Star (DE3) and purified using Ni-NTA agarose. In the lanes denoted as Marker, Lysate, Flowthrough, Wash1 and Wash2, a total of 10 μg protein were loaded. For Elution and Concentrated lanes, 2 μg were loaded. The estimated protein size is 26.7 kDa. (B) Scheme of the enzymatic reaction catalyzed by NTR reducing NCOU1 to umbelliferone (λex/em = 315/455 nm). (C) The fluorescent spectra of umbelliferone. Time-dependent increases in the fluorescent spectra were recorded for 60 min at room temperature in NTR assay buffer (30 mM Tris−HCl, pH 8.0, 0.25% Triton X-100) containing 5 μg/mL NTR, 20 μM NCOU1, and 50 μM NADH (λex/em = 315/350−600 nm over 0.5 nm intervals, recorded every 5 min). For better visualization, only the spectra from time points at 0, 10, 20, 30, 45, and 60 min are shown (gray: 0 min, orange: 10 min, yellow: 20 min, light blue: 30 min, blue: 45 min, purple: 60 min). Fluorescence intensity (FI) over time (λex/em = 315/455 nm) is plotted in the inset. NCOU1: nitrobenzyl-umbelliferone 1, NAD+: oxidized nicotinamide adenine dinucleotide, NADH: reduced nicotinamide adenine dinucleotide, a.u.: arbitrary unit.

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

Techniques: Purification, Recombinant, Activity Assay, Marker, Fluorescence

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: Figure 3. Nitroreductase (NTR) assay using near-infrared probe (NIR-P). (A) Scheme of enzymatic reaction of NTR and NIR-Pox producing the fluorescent NIR-Pred (λex/em = 615/670 nm). (B) Time-dependent fluorescent spectra of NIR-Pred produced by NTR. The enzymatic reaction catalyzed by NTR was monitored using 50 μM NIR-Pox and 50 μM NADH in 30 mM Tris-HCl, pH 8.0, buffer containing 0.5 μg/mL NTR. Fluorescent signals (λex/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. 1C). (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 NIRred signal (λex/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