Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered ‘undruggable’1,2. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries1,3. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes4,5,6,7,8,9,10, including those that can access regions of proteins that are difficult to target through binding affinity alone5,10,11. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand–protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.
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This work was supported by the National Institutes of Health (CA087660 (B.F.C.), GM090294 (B.F.C.), GM108208 (K.M.B.), GM069832 (S.F., A.J.O.)). We thank J. Cisar, K. Mowen, C. Wang, M. Suciu, M. Dix, G. Simon, M. Carrillo, and J. Hulce for experimental assistance, M. Lenardo, L. Zheng and R. Siegel for helpful suggestions, the Marletta and Vogt laboratories at The Scripps Research Institute for sharing instrumentation, and Iterative Threading ASSEmbly Refinement (I-TASSER) for the structural modelling of IMPDH2.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Composition of fragment electrophile library and structures of additional tool compounds, click probes and fragments.
Extended Data Figure 2 Analysis of proteomic reactivities of fragment electrophiles in human cell lysates.
a, Initial analysis of the proteomic reactivity of fragments using an IA-rhodamine probe 16. Soluble proteome from Ramos cells was treated with the indicated fragments (500 μM each) for 1 h, followed by labelling with IA-rhodamine (1 μM, 1 h) and analysis by SDS–PAGE and in-gel fluorescence scanning. Several proteins were identified that show impaired reactivity with IA-rhodamine in the presence of one or more fragments (asterisks). Fluorescent gel shown in greyscale. b, Frequency of quantification of all cysteines across the complete set of competitive isoTOP-ABPP experiments performed with fragment electrophiles. Note that cysteines were required to have been quantified in at least three isoTOP-ABPP data sets for interpretation. c, Rank order of proteomic reactivity values (or liganded cysteine rates) of fragments calculated as the percentage of all quantified cysteines with R values ≥ 4 for each fragment. The majority of fragments were evaluated in 2–4 replicate experiments in MDA-MB-231 and/or Ramos cell lysates, and their proteomic reactivity values are reported as mean ± s.e.m. values for the replicates. d, Comparison of the proteomic reactivities of representative fragments screened at 500 versus 25 μM in cell lysates. e, Comparison of proteomic reactivity values for fragments tested in both Ramos and MDA-MB-231 lysates. f, Proteomic reactivity values of representative fragments. g, Relative GSH reactivity for representative fragment electrophiles. Consumption of GSH (125 μM) was measured using Ellman’s reagent (5 mM) after 1 h incubation with the indicated fragments (500 μM). h, Proteomic reactivity values for fragments electrophiles (500 μM) possessing different electrophilic groups attached to a common binding element. i, Concentration-dependent labelling of MDA-MB-231 soluble proteomes with acrylamide 18 and chloroacetamide 19 click probes detected by CuACC with a rhodamine-azide tag and analysis by SDS–PAGE and in-gel fluorescence scanning. For f and g, data represent mean values ± s.e.m. for at least three independent experiments.
Extended Data Figure 3 Analysis of cysteines liganded by fragment electrophiles in competitive isoTOP-ABPP experiments.
a, Representative MS1 ion chromatograms for peptides containing C481 of BTK and C131 of MAP2K7, two cysteines known to be targeted by the anti-cancer drug ibrutinib. Ramos cells were treated with ibrutinib (1 μM, 1 h, red trace) or DMSO (blue trace) and evaluated by isoTOP-ABPP. b, Total number of liganded cysteines found in the active sites and non-active sites of enzymes for which X-ray and/or NMR structures have been reported (or reported for a close homologue of the enzyme). c, Functional categorization of liganded and unliganded cysteines based on residue annotations from the UniProt database. d, Number of liganded and quantified cysteines per protein measured by isoTOP-ABPP. Respective average values of one and three for liganded and quantified cysteines per protein were measured by isoTOP-ABPP. e, R values for six cysteines in XPO1 quantified by isoTOP-ABPP, identifying C528 as the most liganded cysteine in this protein. Each point represents a distinct fragment–cysteine interaction quantified by isoTOP-ABPP. f–h, Histograms depicting the percentage of fragments that are hits (R ≥ 4) for all 768 liganded cysteines (f), for liganded cysteines found in enzymes for which X-ray and/or NMR structures have been reported (or reported for a close homologue of the enzyme) (g), or for active- and non-active site cysteines in kinases (h). i, Percentage of liganded cysteines targeted only by group A (red) or B (blue) fragments or both group A and B fragments (black). Shown for all liganded cysteines, liganded cysteines in enzyme active and non-active sites, and liganded cysteines in transcription factors/regulators. For g, i, active-site cysteines were defined as those that reside <10 Å from established active-site residues and/or bound substrates/inhibitors in enzyme structures. j, The percentage of liganded cysteines in kinases that were targeted by only group A, only group B, or both group A and B compounds. k, Heat map showing representative fragment interactions for liganded cysteines found in the active sites and non-active sites of kinases. l, Heat map showing representative fragment interactions for liganded cysteines found in transcription factors/regulators. m, The fraction of liganded (62%; 341 of 553 quantified cysteines) and unliganded (20%; 561 of 2,870 quantified cysteines) cysteines that are sensitive to heat denaturation measured by IA-alkyne labelling (R > 3 native/heat denatured). n, Percentage of proteins identified by isoTOP-ABPP as liganded by fragments 3 and 14 and enriched by their corresponding click probes 19 and 18 that are sensitive to heat denaturation (64% (85 of 133 quantified protein targets) and 73% (19 of 26 quantified protein targets), respectively). Protein enrichment by 18 and 19 was measured by whole-protein capture of isotopically SILAC-labelled MDA-MB-231 cells using quantitative (SILAC) proteomics. o, The fraction of cysteines predicted to be ligandable or unligandable by reactive docking that were quantified in isoTOP-ABPP experiments. p, The fraction of cysteines predicted to be ligandable or unligandable by reactive docking that show heat-sensitive labelling by the IA-alkyne probe (R > 3 native/heat denatured).
Extended Data Figure 4 Confirmation and functional analysis of fragment–cysteine interactions in PRMT1 and MLTK.
a, Representative MS1 chromatograms for the indicated Cys-containing peptides from PRMT1 quantified in competitive isoTOP-ABPP experiments of MDA-MB-231 cell lysates, showing blockade of IA-alkyne 1 labelling of C109 by fragment 11, but not control fragment 3. b, 11, but not 3, blocked IA-rhodamine (2 μM) labelling of recombinant, purified wild-type PRMT1 (1 μM protein doped into HEK293T cell lysates). Note that a C109S PRMT1 mutant did not react with IA-rhodamine. c, Apparent IC50 curve for blockade of 16 labelling of PRMT1 by 11. CI, 95% confidence intervals. d, Effect of 11 and control fragment 3 on methylation of recombinant histone 4 by recombinant PRMT1. Shown is one representative experiment of three independent experiments that yielded similar results. e, Representative MS1 ion chromatograms for the MLTK tryptic peptide containing liganded cysteine C22 quantified by isoTOP-ABPP in MDA-MB-231 lysates treated with fragment 4 or control fragment 3 (500 μM each). f, 60, but not control fragment 3 (50 μM of each fragment), blocked labelling of recombinant MLTK kinase by a previously reported ibrutinib-derived activity probe 59 (top)20. A C22A-MLTK mutant did not react with the ibrutinib probe. Anti-Flag blotting confirmed similar expression of wild-type and C22A-MLTK proteins, which were expressed as Flag-fusion proteins in HEK293T cells (bottom). g, Lysates from HEK293T cells expressing wild type or C22A MLTK treated with the indicated fragments and then an ibrutinib-derived activity probe 59 (ref. 20) at 10 μM. MLTK labelling by 59 was detected by CuAAC conjugation to a rhodamine-azide tag and analysis by SDS–PAGE and in-gel fluorescence scanning. h, Apparent IC50 curve for blockade of ibrutinib probe-labelling of MLTK by 60. i, 60, but not control fragment 3 (100 μM of each fragment), inhibited the kinase activity of wild-type, but not C22A-MLTK. For c, h and i, data represent mean values ± s.e.m. for at least three independent experiments. Statistical significance was calculated with unpaired Student’s t-tests comparing DMSO- to fragment-treated samples; **P < 0.1.
Extended Data Figure 5 Confirmation and functional analysis of fragment-cysteine interactions in IMPDH2 and TIGAR.
a, Representative MS1 ion chromatograms for IMPDH2 tryptic peptides containing the catalytic cysteine, C331, and Bateman domain cysteine, C140, quantified by isoTOP-ABPP in cell lysates treated with the indicated fragments (500 μM each). b, Structure of human IMPDH2 (PDB accession 1NF7) (light grey) and its structurally unresolved Bateman domain modelled by I-TASSER43 (dark grey) showing the positions of C331 (red spheres), ribavirin monophosphate and C2-mycophenolic adenine dinucleotide (blue), and C140 (yellow spheres). c, Click probe 18 (25 μM) labelled wild-type IMPDH2 and C331S IMPDH2, but not C140S IMPDH2 (or C140S/C331S IMPDH2). Labelling was detected by CuAAC conjugation to a rhodamine-azide reporter tag and analysis by SDS–PAGE and in-gel fluorescence scanning. Recombinant IMPDH2 wild type and mutants were expressed and purified from Escherichia coli and added to Jurkat lysates to a final concentration of 1 μM protein. d, Fragment reactivity with recombinant, purified IMPDH2 added to Jurkat lysates to a final concentration of 1 μM protein, where reactivity was detected in competition assays using the click probe 18 (25 μM). Note that 18 reacted with wild-type and C331S IMPDH2, but not C140S or C140S/C331S IMPDH2. e, Nucleotide competition of 18 (25 μM) labelling of wild-type IMPDH2 added to MDA-MB-231 lysates to a final concentration of 1 μM protein. f, Nucleotide competition profile for 18 labelling of recombinant wild-type IMPDH2 (500 μM of each nucleotide). g, Apparent IC50 curve for blockade of 18 labelling of IMPDH2 by ATP. h, Representative MS1 chromatograms for TIGAR tryptic peptides containing C114 and C161 quantified by isoTOP-ABPP in cell lysates treated with the indicated fragments (500 μM each). i, Crystal structure of TIGAR (PDB accession 3DCY) showing C114 (red spheres), C161 (yellow spheres), and inorganic phosphate (blue). j, Labelling of recombinant, purified TIGAR and mutant proteins by the IA-rhodamine (2 μM) probe. TIGAR proteins were added to MDA-MB-231 lysates, to a final concentration of 2 μM protein. k, 5, but not control fragment 3, blocked IA-rhodamine (2 μM) labelling of recombinant, purified C161S TIGAR (2 μM protein doped into Ramos cell lysates). l, Apparent IC50 curve for blockade of IA-rhodamine labelling of C161S TIGAR by 5. m, 5, but not control fragment 3 (100 μM of each fragment) inhibited the catalytic activity of wild-type TIGAR, C161S TIGAR, but not C114S TIGAR or C114S/C161S TIGAR. n, Concentration-dependent inhibition of wild-type TIGAR by 5. Note that the C140S-TIGAR mutant was not inhibited by 5. Data represent mean values ± s.e.m. for four replicate experiments at each concentration. For f, g and l–n, data represent mean values ± s.e.m. for at least three independent experiments. Statistical significance was calculated with unpaired Student’s t-tests comparing DMSO- to fragment-treated samples; **P < 0.01, ****P < 0.0001.
Extended Data Figure 6 IDH1-related and general in situ activity of fragment electrophiles.
a, X-ray crystal structure of IDH1 (PDB accession 3MAS) showing the position of C269 and the frequently mutated residue in cancer, R132. b, Blockade of 16 labelling of wild-type IDH1 by representative fragment electrophiles. Recombinant, purified wild-type IDH1 was added to MDA-MB-231 lysates at a final concentration of 2 μM, treated with fragments at the indicated concentrations, followed by IA-rhodamine probe 16 (2 μM) and analysis by SDS–PAGE and in-gel fluorescence scanning. Note that a C269S mutant of IDH1 did not label with IA-rhodamine 16. c, d, Reactivity of 20 and control fragment 2 with recombinant, purified wild-type IDH1 (b) or R132H IDH1 (c) added to MDA-MB-231 lysates to a final concentration of 2 or 4 μM protein, respectively. Fragment reactivity was detected in competition assays using the IA-rhodamine probe (2 μM) e, f, Apparent IC50 curve for blockade of IA-rhodamine labelling of IDH1 (e) and R132H IDH1 (f) by 20. Note that the control fragment 2 showed much lower activity. g, Representative MS1 ion chromatograms for the IDH1 tryptic peptides containing liganded cysteine C269 and an unliganded cysteine C379 quantified by isoTOP-ABPP in MDA-MB-231 lysates treated with fragment 20 (25 μM). h, 20, but not 2, inhibited IDH1-catalysed oxidation of isocitrate to α-ketoglutarate (α-KG) as measured by an increase in NADPH production (340 nm absorbance). 20 did not inhibit the C269S-IDH1 mutant. i, 20 inhibited oncometabolite 2-hydroxyglutarate (2-HG) production by R132H IDH1. MUM2C cells stably overexpressing the oncogenic R132H-IDH1 mutant or control green fluorescent protein (GFP)-expressing MUM2C cells were treated with the indicated fragments (2 h, in situ). Cells were harvested, lysed and IDH1-dependent production of 2-HG from α-KG and NADPH was measured by LC-MS and from which 2-HG production of GFP-expressing MUM2C cells was subtracted (GFP-expressing MUM2C cells produced <10% of the 2-HG generated by R132H-IDH1-expressing MUM2C cells). j, Western blot of MUM2C cells stably overexpressing GFP (mock) or R132H-IDH1 proteins. k, Representative MS1 chromatograms for the IDH1 tryptic peptides containing liganded cysteine C269 and an unliganded cysteine C379 quantified by isoTOP-ABPP in R132H-IDH-expressing MUM2C lysates treated with 20 or control fragment 2 (50 μM, 2 h, in situ). l, Proteomic reactivity values for individual fragments are comparable in vitro and in situ. One fragment (11) marked in red showed notably lower reactivity in situ versus in vitro. Reactivity values were calculated as in Fig. 1c. Dashed line mark 90% prediction intervals for the comparison of in vitro and in situ proteomic reactivity values for fragment electrophiles. Blue and red circles mark fragments that fall above (or just at) or below these prediction intervals, respectively. m, Fraction of cysteines liganded in vitro that are also liganded in situ. Shown are liganded cysteine numbers for individual fragments determined in vitro and the fraction of these cysteines that were liganded by the corresponding fragments in situ. n, Representative cysteines that were selectively targeted by fragments in situ, but not in vitro. For in situ-restricted fragment–cysteine interactions, a second cysteine in the parent protein was detected with an unchanging ratio (R ≈ 1), thus controlling for potential fragment-induced changes in protein expression. For e, f, h and i, data represent mean values ± s.e.m. for at least three independent experiments. Statistical significance was calculated with unpaired Student’s t-tests comparing DMSO- to fragment-treated samples; ****P < 0.0001.
Extended Data Figure 7 Fragment electrophiles that target pro-CASP8.
a, Representative MS1 chromatograms for CASP8 tryptic peptide containing the catalytic cysteine C360 quantified by isoTOP-ABPP in cell lysates or cells treated with fragment 4 (250 μM, in vitro; 100 μM, in situ) and control fragment 21 (500 μM, in vitro; 200 μM, in situ). b, Neither 7 nor control fragment 62 (100 μM each) inhibited recombinant, purified active CASP3 and CASP8 assayed using N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC) and Ac-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin (IETD-AFC) fluorogenic substrates, respectively. DEVD-CHO (20 μM) inhibited both caspases. c, Fragment reactivity with recombinant, purified active CASP8 added to cell lysates, where reactivity was detected in competition assays using the caspase activity probe Rho-DEVD-AOMK probe (2 μM, 1 h). d, Western blot of proteomes from MDA-MB-231, Jurkat, and CASP8-null Jurkat proteomes showing that CASP8 was only found in the pro-enzyme form in these cells. e, Fragment reactivity with recombinant, purified pro-CASP8 (D374A, D384A, C409S) added to cell lysates to a final concentration of 1 μM protein, where reactivity was detected in competition assays with the IA-rhodamine probe (2 μM). Note that mutation of both Cys360 and Cys409 to Ser prevented labelling of pro-CASP8 by IA-rhodamine. f, Inactive control fragment 62 did not compete IA-rhodamine labelling of C360 of pro-CASP8. g, Apparent IC50 curve for blockade of IA-rhodamine labelling of pro-CASP8 (C409S) by 7. h, 7 (50 μM) fully competed IA-alkyne labelling of C360 of endogenous CASP8 in cell lysates as measured by isoTOP-ABPP. Representative MS1 chromatograms are shown for the C360-containing peptide of CASP8. i, Concentration-dependent reactivity of click probe 61, with recombinant, purified pro-CASP8 (D374A, D384A) added to cell lysates to a final concentration of 1 μM protein. Note that pre-treatment with 7 blocked 61 reactivity with pro-CASP8 and mutation of C360 to Ser prevented labelling of pro-CASP8 by 61 (25 μM).). j, 7 (30 μM) blocked IA-alkyne labelling of C360 of pro-CASP8, but not active CASP8, as measured by isoTOP-ABPP. Recombinant pro- and active CASP8 were added to Ramos lysates at 1 μM and then treated with 7 (30 μM) followed by isoTOP-ABPP. k, Fragments 7 and 62 did not block labelling by Rho-DEVD-AOMK (2 μM) of recombinant, purified active CASP8 and active CASP3 added to MDA-MB-231 cell lysates to a final concentration of 1 μM protein. l, 7 does not inhibit active caspases. Recombinant, active caspases were added to MDA-MD-231 lysate to a final concentration of 200 nM (CASP2, 3, 6, 7) or 1 μM (CASP8, 10), treated with z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-FMK) (25 μM) or 7 (50 μM), followed by labelling with the Rho-DEVD-AOMK probe (2 μM). m, Representative MS1 chromatograms for tryptic peptides containing the catalytic cysteines of CASP8 (C360), CASP2 (C320), and CASP7 (C186) quantified by isoTOP-ABPP in Jurkat cell lysates treated with 7 or 62 (50 μM, 1 h). n, 7, but not control fragment 62, blocked extrinsic, but not intrinsic apoptosis. Jurkat cells (1.5 million cells per ml) were incubated with 7 or 62 (30 μM) or the pan-caspase inhibitor VAD-FMK (100 μM) for 30 min before addition of staurosporine (2 μM) or SuperFasLigand (100 ng ml−1). Cells were incubated for 4 h and viability was quantified with CellTiter-Glo. RLU, relative light unit. o, For cells treated as described in n, cleavage of PARP (96 kDa), CASP8 (p43/p41, p18), and CASP3 (p17) was visualized by western blot. p, 7 protects Jurkat cells from extrinsic, but not intrinsic apoptosis. Cleavage of PARP, CASP8 and CASP3 detected by western blotting as shown in o was quantified for three (STS) or two (FasL) independent experiments. Cleavage products (PARP (96 kDa), CASP8 (p43/p41), CASP3 (p17)) were quantified for compound treatment and the percentage cleavage relative to DMSO-treated samples was calculated. For b, g and n, data represent mean values ± s.e.m. for at least three independent experiments. For p, STS data represent mean values ± s.e.m. for three independent experiments, and FasL data represent mean values ± s.d. for two independent experiments. Statistical significance was calculated with unpaired Student’s t-tests comparing active compounds (VAD-FMK and 7) to control compound 62; **P < 0.01, ***P < 0.001, ****P < 0.0001.
Extended Data Figure 8 CASP10 is involved in intrinsic apoptosis in primary human T cells.
a, Representative MS1 peptide signals showing R values for caspases detected by quantitative proteomics using probe 61. ABPP-SILAC experiments. Jurkat cells (10 million cells) were treated with either DMSO (heavy cells) or the indicated compounds (light cells) for 2 h followed by probe 61 (10 μM, 1 h). b, 7 blocked 61 labelling of pro-CASP8 and CASP10, whereas 63-R selectively blocked probe labelling of pro-CASP8. c, 7, but not 63-R blocked probe labelling of pro-CASP10. Recombinant pro-CASP10 was added to MDA-MB-231 lysates to a final concentration of 300 nM, treated with the indicated compounds, and labelled with probe 61. Mutation of the catalytic cysteine C401A fully prevented labelling by 61. d, Apparent IC50 curve for blockade of 61 labelling of pro-CASP10 by 7, 63-R or 63-S. e, Neither 7 nor 63-R (25 μM each) inhibited the activity of recombinant, purified active CASP10 (500 nM), which was assayed after addition of the protein to MDA-MB-231 lysate using fluorometric Ac-Ala-Glu-Val-Asp-7-amino-4-methylcoumarin (AEVD-AMC) substrate. DEVD-CHO (20 μM) inhibited the activity of CASP10. f, Apparent IC50 curve for blockade of 61 labelling of pro-CASP8 and pro-CASP10 by 63-R. g, 63-R shows increased potency against pro-CASP8. Recombinant pro-CASP8 was added to MDA-MB-231 lysates to a final concentration of 300 nM, treated with the indicated compounds and labelled with probe 61. h, Apparent IC50 curve for blockade of 61 labelling of pro-CASP8 by 63-R compared with 63-S. The structure of 63-S is shown. i, CASP10 is more highly expressed in primary human T cells compared to Jurkat cells. Western blot analysis of full-length CASP10, CASP8 and GAPDH expression levels in Jurkat and T-cell lysates (2 mg ml−1). j, Jurkat cells (150,000 cells per well) were incubated with 7 or 63-R at the indicated concentrations for 30 min before addition of staurosporine (2 μM) or SuperFasLigand (100 ng ml−1). Cells were incubated for 4 h and viability was quantified with CellTiter-Glo (CTG). k, Jurkat cells treated as in j, but with 63-R or 63-S. l, HeLa cells (20,000 cells per well) were seeded and 24 h later treated with the indicated compounds for 30 min before the addition of SuperFasLigand (100 ng ml−1) and cycloheximide (CHX; 2.5 ng ml−1). Cells were incubated for 6 h and viability was quantified with CTG. m, For T cells treated as in Fig. 4d cleavage of CASP10 (p22), CASP8 (p18), CASP3 (p17) and RIPK (33 kDa) was visualized by western blotting. For d–f, h and j–k, data represent mean values ± s.e.m. for at least three independent experiments. Statistical significance was calculated with unpaired Student’s t-tests comparing DMSO- to fragment-treated samples; **P < 0.01, ****P < 0.0001.
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Backus, K., Correia, B., Lum, K. et al. Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574 (2016). https://doi.org/10.1038/nature18002
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