A proteome-wide atlas of lysine-reactive chemistry

Abstract

Recent advances in chemical proteomics have begun to characterize the reactivity and ligandability of lysines on a global scale. Yet, only a limited diversity of aminophilic electrophiles have been evaluated for interactions with the lysine proteome. Here, we report an in-depth profiling of >30 uncharted aminophilic chemotypes that greatly expands the content of ligandable lysines in human proteins. Aminophilic electrophiles showed disparate proteomic reactivities that range from selective interactions with a handful of lysines to, for a set of dicarboxaldehyde fragments, remarkably broad engagement of the covalent small-molecule-lysine interactions captured by the entire library. We used these latter 'scout' electrophiles to efficiently map ligandable lysines in primary human immune cells under stimulatory conditions. Finally, we show that aminophilic compounds perturb diverse biochemical functions through site-selective modification of lysines in proteins, including protein-RNA interactions implicated in innate immune responses. These findings support the broad potential of covalent chemistry for targeting functional lysines in the human proteome.

Extended Data Fig. 1.. Properties of aminophilic compound library for mapping small molecule-lysine interactions in the proteome.

cLogP versus molecular weight plot showing aminophilic compounds that follow Lipinski’s drug-likeness “rule-of-five” (Lipinski space Ro5, light gray box) and Congreve’s fragment-based lead-likeness “rule-of-three” (Lead-like space Ro3, dark gray box)^,.

Extended Data Fig. 2.. Features of aminophilic compound-lysine interaction map in human cancer cell proteomes.

a, Histogram showing number of quantified lysines across all isoTOP-ABPP datasets. b, Number of aminophilic compound hits per liganded lysine (left) and the number liganded lysines per protein (right). The results shown are average ratios from three experiments (n = 3 biologically independent experiments).

Extended Data Fig. 3.. Reactivity profiles of representative aminophilic compounds with a model amine nucleophile.

a, Aminophilic compounds (125 µM) were incubated at room temperature with the amine nucleophile Nα-acetyl-L-lysine-OMe (2 M, 1 h, at pH 10 (0.05 M NaHCO₃)). All samples contained 5 µM Nα-acetyl-L-methionine-OH as an internal standard. Samples were neutralized with formic acid and 20 µL of the resulting solution was inject on to an Agilent 6100 series single quadrupole LC/MS system. Samples were run with the following gradient of Buffer A (95/5 Water/MeCN with 0.1% formic acid) and Buffer B (5/95 Water/MeCN with 0.1% formic acid): 100% A from 0–1 min, 100% A → 100% B from 1–11 min, 100% B from 11–13 min, and 100% A from 13–15 min. Peaks corresponding to the amine nucleophile adducts were quantified using Agilent Open Lab software. b, Correlation plot comparing amine nucleophile adduct formation to liganded lysines for each compound (also see Supplementary Table 1). Representative aminophilic compound chemotypes are color-coded. For a and b, data represent average values ± SD; n = 2 per group (n = 2 independent experiments).

Extended Data Fig. 4.. Relating aminophilic compound-lysine interaction map to compound properties and representative features of liganded lysines.

a, cLogP versus molecular weight plot showing aminophilic compounds that follow Lipinski’s “rule of five” (Lipinski space Ro5) and lead-likeness “rule of three” (Lead-like space Ro3). The size of each bubble represents the number of liganded lysines per compound. b, Distribution of compounds (top, left) by the number of hydrogen-bond donors (HBDs, orange line), hydrogen-bond acceptors (HBAs, blue line) and rotatable bonds (RBs, black line). Correlation between the compound distribution and the number of liganded lysine interactions (gray bars, right y-axis) as relates to the number of HBDs (top, right), HBAs (bottom, left) and RBs (bottom, right). c, Heatmap (top) and extracted MS1 chromatograms (bottom) of representative liganded lysines that show broad reactivity with aminophilic compounds (also see Supplementary Dataset 3). d, isoTOP-ABPP ratio plot for the sulfonyl fluoride 17r containing a kinase-directed recognition element. Red points represent liganded active-site lysines in kinases and their corresponding extracted MS1 chromatograms. The dashed line marks the R value of 4 used to define a lysine liganding event (also see Supplementary Dataset 3). e, Comparison of reactivity of aminophilic compounds toward kinase lysines as a function of selectivity toward kinase lysines across the proteome (right panel). The kinase reactivity of individual compounds was defined by the total number of liganded kinase lysines. The selectivity of individual compounds toward kinase lysines was defined by the fraction of liganded kinase to non-kinase lysines. f-h, Location of liganded lysines that are also missense mutated in human disease (orange) in protein crystal structures (gray) of PMVK (K69) (f, PDB ID: 3CH4), CPOX (K404) (g, PDB ID: 2AEX), and RPL10 (K78) (h, PDB ID: 6OLE). Also shown highlighted in blue are active site residues or protein-RNA interaction regions of the proteins where the indicated lysines reside. Note the proximity of K404 in CPOX and K78 in RPL10 to the active site and RNA-interaction region of these proteins, respectively. K69 of PMVK is distant from the active site of the enzyme, but the missense mutation of this lysine causes substantial catalytic defects^,, pointing to an allosteric regulatory function.

Extended Data Fig. 5.. Functional impact of aminophilic compound-lysine interactions for representative proteins.

a, The location of liganded lysine K117 (orange) in the RIDA crystal structure (gray, PDB ID: 1ONI). Also shown is bound pyruvate (teal) in each of the three active sites at the interfaces of adjacent monomers. b, SAR for aminophilic compound engagement of K117 in RIDA, as determined by competitive isoTOP-ABPP is recapitulated by gel-ABPP of recombinant protein (also see Supplementary Datasets 3 and 4). Top, HEK293T cells recombinantly expressing WT-RIDA and the corresponding K117R mutant as Flag epitope-tagged proteins were treated with the indicated aminophilic compounds (50 µM, 1 h) followed by treatment with probe P2 and analyzed by gel-ABPP (top panel) and western blotting (bottom panel). Bottom, Extracted MS1 chromatograms depicting R values for the indicated aminophilic compound-RIDA-K117 interactions mapped by competitive isoTOP-ABPP (also see Supplementary Dataset 3). c, Top, gel-ABPP data showing concentration-dependent blockade of P2 labeling of recombinantly expressed WT-RIDA by 28h and 26l in HEK293T cell lysates. Bottom, structures of 28h and 26l with extracted MS1 chromatograms depicting R values for their respective engagement of K117 or RIDA determined by competitive isoTOP-ABPP (also see Supplementary Dataset 3). d, Corresponding fitted IC₅₀ curves for blockade of probe 2 labeling of WT-RIDA. Data represent average values ± SD; n = 3 per group. CI, confidence interval. e, Representative isoTOP-ABPP ratio plot showing proteome-wide lysine reactivity profile for 26l (50 μM). Among ~3,000 quantified lysines, only two - K117 of RIDA and K1070 of VCL - were liganded. The dashed line marks the R value of 4 used to define a liganded lysine event (also see Supplementary Dataset 3). f, g, Fitted IC₅₀ curves for the concentration-dependent inhibition of the deaminase activity of recombinantly expressed WT- and K117R and K117I mutants of RIDA in HEK293T cell lysates by 28h (f) and 26l (g). Data represent average values ± SD; n = 3 per group. CI, confidence interval. h, Catalytic activity (upper panel) and gel-ABPP analysis of P2 labeling (lower panel) of WT- and indicated K117 mutants. i, Presumed reversible-covalent and irreversible adducts formed between 26l with K117 and R117. Data represent average values ± SD; n = 3 per group. P values were 0.00081 and 0.000066. For western blot and gel-ABPP data in b, c, and h, experiments were conducted three times (n = 3 biologically independent experiments) with similar results. Statistical significance was calculated for changes >25% in magnitude in comparison to DMSO-treated samples with unpaired two-tailed Student’s t-tests: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Extended Data Fig. 6.. Compound 11e does not block preformed Ku70-Ku80 complex.

a, Western blot showing recombinantly co-expressed HA-tagged WT Ku80 with Flag-tagged WT and K351R mutant forms of Ku70 in HEK293T cells. b, Lysates of HEK293T cells co-expressing WT Ku80 with WT (left panel) and K351R (right panel) mutant forms of Ku70 were co-immunoprecipitated with anti-Flag antibody (1 h, 4 °C), treated with DMSO or 11e at the indicated concentrations (1 h, 23 °C), washed, and analyzed by Western blotting. Western blots in a and b are representative of four independent experiments.

Extended Data Fig. 7.. Dicarboxaldehyde scout fragments and their functional effects on LPCAT1.

a, Ternary plot showing the proportional lysine reactivity of 27c, 28o and 32i for each lysine. Each point represents a different composition of the three scout fragments based on their individual lysine reactivity ratio (R) values, with the maximum proportion (100%) of each fragment in each corner of the triangle and the minimum proportion (0%) at the opposite line. Extracted MS1 chromatograms of representative competed lysines targeted by scout fragments with differential R values (also see Supplementary Dataset 3). b, Percent identity matrix of human LPCAT1–4 and AGPAT1–4 (https://www.ebi.ac.uk/Tools/msa/clustalo). c, Conservation of K221 of LPCAT1 across species (https://www.ncbi.nlm.nih.gov/homologene). d, 28o produces concentration-depended blockades of WT-LPCAT1 activity. Data represent average values ± SD; n = 3 per group from three biologically independent experiments. P values were 0.00074, 0.000028, 0.000065, 0.0050, and 0.0000062. Statistical significance was calculated for changes >25% in magnitude in comparison to DMSO-treated samples with unpaired two-tailed Student’s t-tests: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. e, SAR for aminophilic compound blockade of lyso-PC hydrolysis activity of recombinantly expressed LPCAT1 in HEK293T cell lysates. Data represent average values ± SD; n = 3 per group. f, Compounds 28o and 28m produced greater blockade of LPCAT1 enzymatic activity compared to structural analogs 28p or 28l. g, Compound 28f produced greater blockade of LPCAT1 enzymatic activity compared to structural analog 28k. h, 28f produced concentration-depended blockades of WT-LPCAT1 activity. Data represent average values ± SD; n = 3 per group from three biologically independent experiments. Statistical significance was calculated for changes >25% in magnitude in comparison to DMSO-treated samples with unpaired two-tailed Student’s t-tests: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. i, j, HA-tagged ubiquitin and FLAG-tagged WT or K221R LPCAT1 were co-expressed in HEK293T cells in the presence of proteasome inhibitor MG132 (10 µM) for 14 h (i) or 2 h (j), after which cell lysates were subjected to anti-FLAG immunoprecipitation, and the affinity-enriched precipitates analyzed by anti-HA immunoblotting. For i, mock-transfected cells and LPCAT1-WT cells not treated with MG132 were used as controls. For j, FLAG-tagged GFP-transfected cells were used as a control. k, FLAG-tagged WT or K221R LPCAT1, or GFP-transfected HEK293T cells were treated with MG132 (10 µM, 2 h), followed by anti-FLAG immunoprecipitation and the affinity-enriched precipitates were analyzed by anti-ubiquitin immunoblotting. Western blots in i-k are representative of four independent biological experiments.

Extended Data Fig. 8.. Example of differential lysine ligandability event in stimulated immune cells.

The location of liganded lysine K252 (orange) mapped onto the crystal structure of ALAD (gray, PDB ID: 1PV8). K252 showed substantially weaker interactions with 27c (100 µM, 23 °C, 1 h) in LPS-stimulated (R = 1.5) vs quiescent PBMCs (R = 9.3), whereas the reactivity of K159 (green) remained largely unchanged by LPS treatment (R = 1.7). K252 is an active-site residue responsible for reversible Schiff-base formation with substrate (blue).

Extended Data Fig. 9.. Characterization of aminophilic compounds that selectively inhibit IFIT family of RNA-binding proteins.

a-b, Multiple sequence alignment (a) and percent identity matrix (b) of human IFIT paralogs (https://www.ebi.ac.uk/Tools/msa/clustalo). The red highlight marks a conserved and liganded lysine. c, Aggregate spectral counts for quantified lysine-containing peptides for IFIT proteins in human PBMCs ± LPS treatment. Data represent average values ± SD; n = 3 per group from three biologically independent experiments. d, Location of liganded lysines (orange) mapped onto the aligned crystal structures of N-terminal domains in IFIT5 (gray, PDB ID: 4HOT) and IFIT1 (yellow, PDB ID: 4HOU) displaying 5’-PPP-RNA (blue) in the nucleotide binding cleft.

Extended Data Fig. 10.. Characterization of aminophilic compounds that inhibit the IFIT family of antiviral RNA-binding proteins.

a, Extracted MS1 chromatograms with corresponding isoTOP-ABPP ratios (top) and Western blot analysis (bottom) from biotinylated RNA pulldown experiments of WT-IFIT1 and the K151R-IFIT1 mutant from HEK293T cell lysates treated with the indicated concentrations of aminophilic compounds. Western blot is representative of three independent experiments). Also see Supplementary Dataset 4. b, Western blot analysis from biotinylated RNA pulldown experiments of WT-IFIT1 and IFIT5 from HEK293T cell lysates showing concentration-dependent blockade of RNA binding by indicated aminophilic compounds. Western blot is representative of three independent experiments). Also see Supplementary Dataset 4. c, Concentration-dependent blockade (upper panel) and fitted IC₅₀ curve (lower panel) of RNA binding of WT-IFIT5 by 7a after 1 versus 4 h of pre-incubation (n = 2 biologically independent experiments). d, Structures of 7e containing an alkyne moiety on “staying group” and 7f with an alkyne moiety on “leaving group”. Highlighted in red are “staying groups” in both compounds. e, Representative competition gel showing concentration-dependent blockade of probe 3 labeling by 7a, 7e, and 7f of recombinant WT-IFIT5 in HEK293T cell lysates. f, Concentration-dependent labeling of recombinantly expressed WT-IFIT5 and the K150R mutant in HEK293T cell lysates by the clickable probes 7e and 7f. gel-ABPP data in e and f are representative of three independent experiments. g, Fitted in situ IC₅₀ curve for the concentration-dependent blockade of the 7e-WT-IFIT5 interaction by 7a in transfected HEK293T cells (n = 4 biologically independent experiments). h, Average ratio values for lysines quantified by isoTOP-ABPP in IFIT5-transfected HEK293T cells treated in situ with 7a (1 μM, 2 h) (n = 2 independent experiments; also see Supplementary Dataset 3). i, R values for quantified lysines in IFIT5 of experiment described in part h.

Figure 1.. An aminophilic compound library for mapping small molecule-lysine interactions in the proteome.

a, Mechanism-based categorization of aminophilic chemotypes by their predicted modes of reactivity. B, Structural composition and diversity of representative aminophilic chemotypes clustered by reactivity modes (presumed electrophilic centers are highlighted by blue circles). See Supplementary Dataset 1 for a complete list of compound structures for each chemotype. C, Distribution of aminophilic compounds by the number of hydrogen-bond donors (HBDs, orange line), hydrogen-bond acceptors (HBAs, blue line) and rotatable bonds (RBs, black line). Plot highlights compounds that follow Lipinski’s “rule-of-five” (drug-like space, light-grey box) and Congreve’s “rule-of-three” (fragment-based lead-like space, dark-grey box). D-f, Qualitative assessment of apparent amino acid reactivity of representative compounds 32a-I from the heterocyclic aldehyde chemotype, as measured by competitive gel-ABPP with the lysine-directed probe Alexa-Fluor® 488 (P3) (d), cysteine-directed probe iodoacetamide-rhodamine (P7) (e) and serine hydrolase-directed probe fluorophosphonate-rhodamine (P8) (f) in proteomic lysate of the MDA-MB-231 human breast cancer cell line. Competitive profiling experiments were generally performed as follows: soluble proteome from MDA-MB-231 cells was treated with the indicated compounds (100 µM, 1 h, 23 °C), followed by labeling with the indicated fluorogenic probe (2 µM, 1 h, 23 °C) and analysis by SDS-PAGE and in-gel fluorescence scanning. Red asterisks mark representative compound-competed proteins. This experiment was conducted twice (n = 2) with similar results.

Figure 2.. A global map of aminophilic compound-lysine interactions in the human proteome.

a, General schematic for competitive isoTOP-ABPP experiments and experimental workflow to identify lysines liganded by aminophilic compounds. b, Fraction of total quantified lysines (left) and proteins (right) liganded by aminophilic compounds. c, Plot comparing the number of liganded lysines for each aminophilic chemotype, with blue and black designating lysines that were engaged by a single or multiple chemotypes, respectively. d, Top, Heatmap showing R values of representative lysines preferentially liganded by a single chemotype. Middle, Extracted MS1 chromatograms with corresponding R values for K153 in ST13 showing a highly restricted SAR across individual members of the squarate chemotype (also see Supplementary Dataset 3). Bottom, corresponding recapitulation by gel-based ABPP of the recombinant ST13 (also see Supplementary Dataset 4). e, Overlap of proteins with liganded lysines targeted by chemotypes evaluated in this study and by activated esters evaluated in a previous study. f, Functional class distribution of liganded DrugBank (left) and non-DrugBank (right) proteins. g, Overlap of proteins harboring liganded lysines and liganded cysteines in Ramos and MDA-MB-231 proteomes. h, Categorization of liganded lysines based on the indicated functional categories. i, Distribution of liganded lysines in proteins that have human disease-relevance (as assessed by pathogenic mutations that lead to monogenic disorders defined in the OMIM database) and functional consequences of mutations of the liganded lysine residues themselves (in cases, where these mutations are associated with disease). For panels e-i, lysines and proteins exclusively liganded by compounds 27c, 28o, and 32i were excluded from the analyses, as we revisit these preferred targets of these “scout” compounds in Figure 5.

Figure 3.. Confirmation and SAR analysis of aminophilic compound-lysine interactions with recombinantly expressed proteins.

a, Structure of sulfonyl fluoride 17b and R values for quantified lysines in CPOX, identifying K404 as a liganded lysine in this protein. Each point represents a distinct aminophilic compound-lysine interaction quantified by isoTOP-ABPP. The dashed line marks the R value of 4 used to define a liganding event. The results shown are average ratios from three experiments (n = 3 biologically independent experiments). b, c, Site-specific aminophilic compound-lysine interactions are preserved in recombinant proteins. Right panels: Lysates from HEK293T cells recombinantly expressing representative liganded proteins and their corresponding lysine-to-arginine mutants as Flag epitope-tagged proteins were treated with the indicated aminophilic compounds (50 µM, 1 h) followed by treatment with the indicated lysine-reactive probes and analyzed by gel-ABPP (top) and western blotting (middle). Below the blots are shown extracted MS1 chromatograms depicting R values for the indicated aminophilic compound-lysine interactions mapped for endogenous proteins by competitive isoTOP-ABPP (also see Supplementary Datasets 3 and 4). Left images: Location of liganded lysines (orange) in protein crystal structures (gray) of CPOX (b, PDB ID: 2AEX) and GSTT2B in complex with glutathione (blue) (c, PDB ID: 4MPG). d, Top, Representative gel-ABPP data showing concentration-dependent blockade of probe P1 labeling of recombinant GSTT2B in HEK293T cell lysates by ammoniumsulfonyl carbamate 22b. Middle, Structure of 22b. Bottom, IC₅₀ curve for blockade of P1 labeling by 22b. Data represent average values ± SD; n = 3 per group. CI, confidence interval. e-f, Structure-activity relationships (SARs) determined for aminophilic compound interactions with the conserved lysine in endogenous SIN3A (e) and SIN3B (f) by competitive isoTOP-ABPP recapitulated by gel-ABPP of recombinant proteins. Top, HEK293T cells recombinantly expressing representative liganded proteins and their corresponding lysine-to-arginine mutants as Flag epitope-tagged proteins were treated with the indicated aminophilic compounds (50 µM, 1 h) followed by treatment with the indicated lysine-reactive probes and analyzed by gel-ABPP (top panel) and western blotting (bottom panel). Middle, Extracted MS1 chromatograms depicting R values for the indicated aminophilic compound-lysine interactions identified by competitive isoTOP-ABPP (also see Supplementary Datasets 3 and 4). Bottom, Liganded lysines (orange) mapped onto protein crystal structures (gray) of SIN3A (e, PDB ID: 2RMR) and SIN3B (f, PDB ID: 2CZY) in complex with neural repressor NRSF/REST (blue). g, Top, Representative gel-ABPP data showing concentration-dependent blockade of probe 5 labeling by N-hydroxyphthalimide 12a (middle) of recombinant SIN3A and SIN3B in HEK293T cell lysates. Bottom, Corresponding fitted IC₅₀ curves. Data represent average values ± SD; n = 3 per group. CI, confidence interval. For gel-ABPP data in b, c, d, and f, experiments were conducted three times (n = 3 biologically independent experiments) with similar results.

Figure 4.. Functional impact of aminophilic compound-lysine interactions for representative proteins.

a, Upper panel, R values for quantified lysines in XCRR6 (or Ku70), identifying K351 as the only observed liganded lysine in this protein. Each point represents a distinct aminophilic compound-lysine interaction quantified by isoTOP-ABPP. The dashed line marks the R value of 4 used to define a liganded lysine event (also see Supplementary Dataset 3). The results shown are average ratios from three experiments (n = 3 biologically independent experiments). Lower panel, Structures of fragments 33e and 11e with extracted MS1 chromatograms depicting R values for their respective engagement of K351 in XRCC6 mapped by competitive isoTOP-ABPP. b, The location of liganded lysine K351 of Ku70 (orange) mapped onto the crystal structure of the Ku heterodimer (PDB ID: 1JEY) consisting of Ku70 (gray) and Ku80 (yellow) bound to double-stranded DNA (teal). c-e, Aminophilic compounds engaging K351 of Ku70 block formation of the Ku70-Ku80 heterodimer. c, Western blot analysis showing recombinantly expressed Flag-tagged WT and K351R mutant forms of Ku70, as well as HA-tagged WT-Ku80 in HEK293T cells. d, Lysates of cells expressing Ku70 protein variants were treated with DMSO, 33e (left panel), or 11a (right panel) at the indicated concentrations (1 h, 23 °C) and then mixed with lysates expressing Ku80 protein (1 h, 23 °C), followed by co-immunoprecipitation with anti-Flag antibody (1 h, 4 °C) and western blot analysis. Also see Supplementary Dataset 4. e, Quantification of western blotting data for 33e and 11e from three biological replicates. Data represent average values ± SD; n = 3 per group from three biologically independent experiments.

Figure 5.. Dicarboxaldehyde scout fragments and their application for profiling lysine ligandability in human immune cells.

a, Overlap of proteins with liganded lysines targeted by scout fragments (27c, 28o, 32i), other chemotypes from this study, and activated esters from a previous study. b, Structures of scout fragments 27c, 28o, and 32i. c, Scout fragments 27c, 28o and 32i engage a much larger number of lysines in human cancer cell proteomes compared to other aminophilic compounds. d, Fraction of proteins harboring liganded lysines and subset of these liganded proteins that are immune-relevant. e, Top-20 enriched clusters of biological processes from gene ontology (GO)-term enrichment analysis of liganded proteins. Red bold font highlights immune-relevant biological processes. f, Fraction of liganded, immune-relevant proteins quantified in LPS-stimulated and quiescent PBMCs. g, Treatment with scout fragments inhibits the lyso-PC hydrolysis activity of membrane lysates of HEK293T cells recombinantly expressing WT-LPCAT1. Lysates expressing a K221R mutant of LPCAT1 also show impaired lyso-PC hydrolysis activity compared to lysates expressing WT-LPCAT1. The results shown are average ratios from three experiments (n = 3 biologically independent experiments). h, Fitted IC₅₀ curves for the concentration-dependent inhibition of lyso-PC hydrolysis activity of recombinantly expressed WT LPCAT1 in HEK293T cell lysates by 28o and 28f. Data represent average values ± SD; n = 3 per group from three biologically independent experiments. CI, confidence interval. P values were 0.0036 and 0.000027. Statistical significance was calculated for changes >25% in magnitude in comparison to DMSO-treated samples with unpaired two-tailed Student’s t-tests: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 6.. Identification of aminophilic compounds that inhibit the IFIT family of antiviral RNA-binding proteins.

a, Lower panel, Number of proteins with lysines liganded by scout fragments within each functional module of the immune system (modules defined in a previous study). Enrichment network of module 4 (inset, upper panel) showing protein clusters associated with RNA-related functions. Nodes are individual annotation terms, edges represent protein overlap between terms, node size represent annotation enrichment, and fill color represent clusters. Extracted MS1 chromatograms are shown for representative liganded lysines in RNA-binding proteins involved in RNA metabolism, splicing, localization and response to viral infection. b, Location of ligandable lysine K150 (orange) mapped onto the crystal structures of IFIT5 (gray, PDB ID: 4HOT) bound to 5’-PPP-RNA (blue) in the nucleotide binding cleft. c, SAR for aminophilic compound engagement of K150 of IFIT5, as determined by competitive isoTOP-ABPP is recapitulated by gel-ABPP of recombinant IFIT5. Top, HEK293T cells recombinantly expressing WT-IFIT5 and the corresponding K150R mutant as Flag epitope-tagged proteins were treated with the indicated aminophilic compounds (50 µM, 1 h) followed by treatment with probe 3 and analyzed by gel-ABPP (top panel) and western blotting (bottom panel). Bottom, extracted MS1 chromatograms depicting R values for the indicated aminophilic compound-IFIT5 (K150) interactions mapped by competitive isoTOP-ABPP (also see Supplementary Datasets 3 and 4). d, Extracted MS1 chromatograms with corresponding isoTOP-ABPP ratios (top) and Western blot analysis (bottom) from biotinylated RNA pulldown experiments of WT-IFIT5 and the K150R-IFIT5 mutant from HEK293T cell lysates treated with the indicated concentrations of aminophilic compounds. Also see Supplementary Dataset 4. e, f, Gel-ABPP data (e) and corresponding fitted IC₅₀ curves (f) for the concentration-dependent blockade of probe labeling of IFIT1, IFIT3, and IFIT5 by 32i and 7a. Data represent average values ± SD; n = 3 per group. CI, confidence interval. g, Concentration-dependent in situ labeling of WT-IFIT5, but not the K150R mutant of IFIT5, by an alkyne probe 7e in transfected HEK293T cells. Also see Supplementary Dataset 4. h, Representative gel-ABPP for the concentration-dependent blockade of the 7e-WT-IFIT5 interaction by 7a in transfected HEK293T cells. Data represent average values ± SD; n = 2 per group. CI, confidence interval. For gel-ABPP and Western blotting data in c-e, g, and h, experiments were conducted three times (n = 3 biologically independent experiments) with similar results.