MassIVE MSV000092363

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Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential Eur J Med Chem. 2023 Feb 5;247:115035. doi: 10.1016/j.ejmech.2022.115035. Epub 2022 Dec 22. Slavish, Jake et al. The enzymatic activity of PAN endonuclease in the presence of inhibitors was measured using a MALDI-FTICR-MS-based functional assay. An ECHO 650 acoustic dispenser plate reformat protocol (Labcyte) was used to make 16-point dose responses of each compound (67.5 nL, from 45 uM to 0.8 pM) in 384-well plate format (Greiner Bio-One). The compounds were pre-incubated for 1 h with 10 uL of the enzyme assay mix; 300 nM PAN endonuclease, 5 mM manganese chloride (Aldrich), 20 mM 2-mercaptoethanol (Calbiochem) in 10 mM Tris pH 8.0 (Fisher Scientific). The reaction was initiated by the addition of 10 uL of RNA (Integrated DNA Technologies) to a final concentration of 50 uM. The plate was incubated at room temperature for 90 min before being quenched with the addition of 10% formic acid (EMD Millipore Corp.). Two microliters of each reaction were mixed with 8 uL of 40 mg/mL 3-hydroxypicolinic acid (Sigma-Aldrich) in 70% acetonitrile (Fisher Scientific) containing 10 mg/ml ammonium citrate (Fisher Scientific), manually spotted on a 384 Anchorchip MALDI target (Bruker Daltonics), and let dry at room temperature. The experiments were performed on a 7-T SolariX XR FT-ICR Mass Spectrometer (Bruker Daltonics) in MALDI negative mode using ftmsControl v2.3. The spectrometer instrument method was developed specifically to improve its dynamic range for substrate/product RNA with minimal sample preparation. Thus, with a mass range of 150 to 5000 m/z, signal from a minimum 10 transients were accumulated and each transient analyzing ions produced from 300 MALDI laser shots at a 500 Hz frequency. Other FTMS settings were as follows: sweep excitation power of 44%, excitation waveform calculation version 2, Time of Flight of 0.002 s, Q1 mass of 300 m/z, ion accumulation time of 0.025 s, laser power 40 lp. The readout for this assay was the disappearance of a single-stranded, 17-mer RNA substrate (5'-UCUCUAGCAGUGGCGCC -3'; m/z = 2687.3 for doubly-charged species) and appearance of the 7-mer hydrolyzed product (5'-UCUCUAG-3' m/z = 2139.6 for singly-charged species). Five assay replicates across three days were performed with two instrument replicates per assay. The highest 2500 local maxima with an absolute intensity threshold of 100,000 were extracted, and those within +/- 0.1 m/z of target m/z were used to calculate product and substrate intensity. Enzyme turnover (T) was calculated as a ratio of product intensity (I) over total intensity of substrate plus product (TRdRp = I2139.6/I2687.3 + I2139.6). Peak extraction and data analysis were performed in R using the RStudio IDE [59,60]. Z-prime was calculated for each individual FTMS target and targets with a resulting Z-prime below 0 were eliminated. FTMS replicates were then combined and Z-prime for each assay replicate was calculated (Table 6). Turnover values were normalized to negative controls and dose-response curves were fit to a four-parameter log-logistic model using non-linear least-squares optimization. Resulting fits were plotted using the ggplot2 visualization package. R code is available on request. [doi:10.25345/C52B8VN8B] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Biochemical ; High-Throughput ; RNA ; MALDI ; RdRp ; Drug Screen

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Publications

Slavish PJ, Cuypers MG, Rimmer MA, Abdolvahabi A, Jeevan T, Kumar G, Jarusiewicz JA, Vaithiyalingam S, Jones JC, Bowling JJ, Price JE, DuBois RM, Min J, Webby RJ, Rankovic Z, White SW.
Chemical scaffold recycling: Structure-guided conversion of an HIV integrase inhibitor into a potent influenza virus RNA-dependent RNA polymerase inhibitor designed to minimize resistance potential.
Eur J Med Chem. Epub 2022 Dec 22.