Fig. 2: RMC-113 is a selective kinase inhibitor that stably binds PIKfyve and PIP4K2C.

a Kinase abundance ratio between RMC-113 (0.1, 1.0, and 10 µM)- and DMSO-treated SUM159 cell lysates measured by multiplexed inhibitor beads kinome profiling and mass spectrometry (MIB/MS). Shown are the Log2 fold change mean values ± SD of 2 replicates of a subset of the screen panel (see Supplementary Fig. 2a). b and c In vitro dose response of PIKfyve activity (b) and PIP4K2C binding (c) to RMC-113. d Biochemical parameters for RMC-113 and SRN2-002. ND = Not determined. e Chemical structure of SRN2-002. f Dose response of SARS-CoV-2 infection (MOI = 0.05, black) and cell viability (blue) to SRN2-002 in Calu-3 cells via plaque and alamarBlue assays at 24 hpi, respectively (n = 2 independent experiments). g Kinase expression in SARS-CoV-2-infected A549-ACE2 lysates following incubation with SRN2-002 individually or combined with RMC-113, UV irradiation, and streptavidin pull-down measured by Western blotting at 24 hpi. Representative membranes of n = 2 experiments are shown. Lanes: 1: SRN2-002, no UV; 2: SRN2-002, no pull-down; 3: SRN2-002 (1 µM); 4: SRN2-002 (5 µM); 5: SRN2-002 (1 µM) competitively inhibited by RMC-113 (10 µM); 6: SRN2-002 (5 µM) competitively inhibited by RMC-113 (50 µM). h, Putative binding mode of RMC-113 into the kinase ATP-binding pockets based on microsecond timescale MD simulations. A representative snapshot with key interactions is shown (see i–l for details). Binding pocket residues with >10% interaction frequencies to RMC-113 are shown. Positively charged residues are highlighted in blue; negative charge, red; hydrophobic, green; polar, cyan. RMC-113 is shown in yellow (carbon) stick model. H-bonds are illustrated with purple dashed lines, π–π interactions with green. i and k, Summary of main protein-ligand interactions ( > 10% frequency) in the MD simulations of PIKfyve (i) and PIP4K2C (k). j and l Aggregate of protein-ligand interactions (residues with >10%) in the simulations of PIKfyve (j) and PIP4K2C (l). Means ± SD are shown (f). Data in (i and j) consist of 72 μs, and (k and l) consist of 20 μs, both analyzed each 1 ns. Source data are provided as a Source Data file.