Small molecules targeting RNA can be valuable chemical probes and potential therapeutics. The interactions between small molecules, particularly fragments, and RNA, however, can be difficult to detect due to their modest affinities and short residence times. Here, we describe the procedures for mapping the molecular fingerprints of small molecules in vitro and throughout the human transcriptome in live cells, identifying both the targets bound by the small molecule and the sites of binding therein. For complete details on the use and execution of this protocol, please refer to 1.
Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Here, we describe isotopically labeled desthiobiotin azide (isoDTB) tags that are easily synthesized, shorten the chemoproteomic workflow and allow an increased coverage of cysteines in bacterial systems. We quantify 59% of all cysteines in essential proteins in <i>Staphylococcus aureus</i> and discover 88 cysteines with high reactivity, which correlates with functional importance. Furthermore, we identify 268 cysteines that are engaged by covalent ligands. We verify inhibition of HMG-CoA synthase, which will allow addressing the bacterial mevalonate pathway through a new target. Overall, a comprehensive map of the bacterial cysteinome is obtained, which will facilitate the development of antibiotics with novel modes-of-action.
Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, that do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids
Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs. Editor's evaluation This manuscript describes an elegant chemical-genetic strategy to discover that human oxidoreductase HSD17B11 is a major contributor to the bioactivation of the dialkinylcarbinol class of cytotoxic natural products. Mechanistic work further revealed that the reactive metabolites generated by HSD17B11 modify lysine and cysteine side-chains on proteins, leading to an unfolded protein response and apoptotic cell death. This study thus provides a plausible mechanism to explain how dialkynylcarbinol compounds exert their cytotoxic properties and identifies enzyme targets for controlling this process in human cells. https://doi.org/10.7554/eLife.73913.sa0 Decision letter eLife's review process Introduction Nature is a rich source of bioactive compounds, some of which can be directly exploited to treat diseases. Some of them reveal sophisticated mechanisms of action which can be mimicked by designing synthetic molecules with specific features (Newman and Cragg, 2020). Marine sponges have attracted pharmaceutical interest since the discovery in the 1950s of C-nucleosides in Cryptotethia crypta that led to the development of cytosine arabinoside (ara-C or cytarabine) and analogues as anticancer treatments for acute myelogenous leukemia (Bergmann, 1950; Ellison et al., 1968). In a different structural series, several cytotoxic acetylenic lipids bearing a terminal alkenylalkynylcarbinol (AAC) pharmacophore have since been isolated from marine sponges, such as petrocortyne A (Figure 1—figure supplement 1), isolated from Petrosia sp. (Seo et al., 1998) and fulvinol isolated from Haliclona fulva (Ortega et al., 1996). The simplest cytotoxic AAC representative, (S)-eicos-(4E)-en-1-yn-3-ol ((S)–1, Figure 1—figure supplement 1), was isolated from the marine sponge Cribrochalina vasculum (Gunasekera and Faircloth, 1990). It demonstrated high cytotoxic activity selectively towards non-small cell lung carcinoma cells as compared to normal lung fibroblasts (Zovko et al., 2014). Starting from (S)–1, an extensive structure-activity relationship study in human cancer cell lines established that (Figure 1—figure supplement 1): (i) the non-natural enantiomer (R)–1 has higher cytotoxic activity, (ii) homologues with shorter lipidic tails are more cytotoxic, with an optimum total aliphatic backbone of 17 carbon atoms (e.g. (R)–2), and (iii) replacement of the internal C=C bond by a C≡C bond, giving rise to a terminal dialkynylcarbinol (DAC) pharmacophore, further increases cytotoxicity, to reach an IC50 down to 90 nM for the DAC (S)–3 (El Arfaoui et al., 2013; Listunov et al., 2015a; Listunov et al., 2018b). However, despite this significant level of activity, the mode of action of this family of molecules, including the natural compound (S)–1, remains elusive (Zovko et al., 2014). Here, we use functional genomics and chemoproteomics to decipher how cytotoxic DACs and related molecules mediate their biological effect. We discover that they behave as prodrugs enantiospecifically bioactivated by a member of the Short-chain Dehydrogenase/Reductase (SDR) family. Finally, we design new SDR-bioactivated DACs derivatives, establishing this family of lipidic alkynylcarbinols as a large and untapped reservoir of cytotoxic prodrugs. Results The SDR HSD17B11 governs (S)-DACs cytotoxicity To determine how cytotoxic DACs mediate their effect on human cells, we applied a genetic approach using the pseudo-haploid human cell line HAP-1 (Carette et al., 2009). Given that (S)–3 had the greatest cytotoxic activity of all the DACs previously tested (El Arfaoui et al., 2013; Listunov et al., 2015a; Listunov et al., 2018b), we screened for mutations that could render HAP-1 cells resistant to (S)–3. We first confirmed in HAP-1 that (S)–3, but not (R)–3 (Figure 1A), exhibits nanomolar cytotoxic activity (Figure 1B, IC50 62.4 nM), in agreement with previous results on HCT116 colon cancer cells (El Arfaoui et al., 2013). We used Ethyl-Methane Sulfonate (EMS) to generate a mutagenized HAP-1 population and selected resistant clones using a lethal 250 nM (S)–3 concentration. Ten individual (S)–3-resistant clones (DACR) were isolated, displaying a 38- to 62-fold resistance to (S)–3 (Figure 1C) but similar sensitivity as parental cells to two unrelated compounds, bortezomib and doxorubicin (Figure 1—figure supplement 2A,B). Based on previous work (Wacker et al., 2012; Bossaert et al., 2021), and considering that EMS induces mainly point mutations under these conditions (Forment et al., 2017), we selected four DACR clones for RNA-seq analysis, to identify mis- or non-sense mutations accounting for the resistance. Around nine mutated genes were identified per clone (Figure 1—figure supplement 2C), with KCTD5 and HSD17B11 being the only mutated genes shared by more than two clones (Figure 1—figure supplement 2D). KCTD5 encodes for an E3-ubiquitin ligase substrate adaptor identified in a genetic screen as a negative regulator of the Akt pathway (Brockmann et al., 2017). However, while KCTD5 mRNA was expressed in all DACR clones, HSD17B11 mRNA levels were strongly reduced in the only clone without HSD17B11 coding mutations (#A5, Figure 1—figure supplement 2E). This suggested that mutations or lack of expression of HSD17B11 were responsible for DACR clone resistance. To confirm this, we sequenced HSD17B11 cDNAs from six other DACR clones, and detected non-synonymous HSD17B11 mutations in five, and no HSD17B11 cDNA in the sixth, suggesting loss of expression (Figure 1D, E). These data strongly supported a role for HSD17B11 in mediating (S)–3 cytotoxicity. Figure 1 with 5 supplements see all Download asset Open asset HSD17B11 is necessary for DAC (S)–3 cytotoxic activity. (A) DAC (S)–3 and (R)–3 structures. (B) Cell viability analysis of HAP-1 or U2OS cells treated for 72 h with the indicated concentrations of (S)- or (R)–3. (C) Cell viability analysis of individual DAC-resistant clones or wild-type HAP-1 treated for 72 hr with the indicated concentrations of (S)–3. (D) List of mutations identified by RNA-seq or targeted sequencing of HSD17B11 in individual DAC-resistant clones. (E) Schematic representation of HSD17B11 functional domains. The positions of the identified mutations are indicated in red. The Y185, K189 (indicated in black), and S172 amino acids are critical for catalysis. (F) Analysis by immunoblotting of HSD17B11 levels in wild-type HAP-1 and DAC-resistant clones. Ku80 was used as a loading control. The black arrow indicates HSD17B11 position. (G) Analysis by immunoblotting of HSD17B11-GFP levels in individual clones of DAC-resistant clone A5 complemented with GFP, wild-type or S172L mutant HSD17B11-GFP. SAF-A and total H2AX were used as loading controls. (H) Cell viability analysis of individual clones of DAC-resistant clone A5 complemented with GFP, wild-type or S172L mutant HSD17B11-GFP treated for 72 h with the indicated concentrations of (S)–3. Figure 1—source data 1 Source data related to Figure 1F. The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains at the bottom an overlay with a picture of the membrane to locate the protein ladder positions. https://cdn.elifesciences.org/articles/73913/elife-73913-fig1-data1-v1.zip Download elife-73913-fig1-data1-v1.zip Figure 1—source data 2 Source data related to Figure 1G. The tiff files correspond to uncropped pictures of the chemiluminescent signal acquired on a BioRad Chemidoc. The regions used to generate the figure are highlighted by back squares in the jpg file, which also contains at the bottom an overlay with a picture of the membrane to locate the protein ladder positions. https://cdn.elifesciences.org/articles/73913/elife-73913-fig1-data2-v1.zip Download elife-73913-fig1-data2-v1.zip HSD17B11 encodes for the estradiol 17-beta-dehydrogenase 11, a member of the SDR super-family. HSD17B11, also called SDR16C2 [Persson et al., 2009], PAN1B, DHRS8, or retSDR2, localizes to the endoplasmic reticulum (ER) and lipid droplets (LD) via a N-terminal targeting domain (Figure 1E), where it uses NAD+ to catalyze oxidation of the C17 carbinol center of androstan-3-alpha,17-beta-diol to generate androsterone, a weak androgen (Brereton et al., 2001; Horiguchi et al., 2008a) (see Figure 2A). The HSD17B11 protein was barely detectable in all the DACR clones (Figure 1F, lower band), suggesting that the mutations result in protein instability. Using the DACR#A5 clone, in which HSD17B11 RNA was strongly down-regulated (~200 fold, Figure 1—figure supplement 2E), we performed complementation experiments with plasmids coding for GFP alone, or wild-type (WT) or S172L HSD17B11-GFP. This mutation was selected because the S172 residue is critical for catalysis (Filling et al., 2002; Gao et al., 2021), and the DACR#A4 clone, which carried S172L mutations, was the only one in which traces of full-length HSD17B11 could be detected (Figure 1F). Complemented DACR#A5 cells stably expressing WT and S172L HSD17B11-GFP at similar levels were successfully isolated (Figure 1G), and (S)–3 was ~50 times more active against cells expressing WT HSD17B11 compared to control GFP-complemented cells or cells expressing S172L HSD17B11 (Figure 1H). This supports that HSD17B11 catalytic activity is critical for (S)-DAC cytotoxicity. Notably, the DACR#A4 clone (S172L mutation) was also resistant to six other cytotoxic AACs: the naturally occurring AAC (S)–1 (Figure 1—figure supplement 3A), its synthetic enantiomer (R)–1, its shorter homologue (R)–2, the synthetic AAC (S)–4 with an internal C≡C bond and an external C=C bond (Figure 1—figure supplement 3B), the allenylalkynylcarbinol (AllAC) (R,Sa)–5 (Listunov et al., 2018a) and the more cytotoxic butadiynylalkynylcarbinol (BAC) (S)–6 (Bourkhis et al., 2018; Figure 1—figure supplement 3C). Thus, HSD17B11 functionality governs the enantiospecific cytotoxicity of the natural compound (S)–1 but also of all the more cytotoxic synthetic derivatives tested. In addition, HSD17B11 has been recently identified as mediating, through an unknown mechanism, the cytotoxic effect of dehydrofalcarinol, a polyacetylenic compound with a terminal butadiynylalkenylcarbinol motif isolated from several plants of the Asteraceae family (Grant et al., 2020). Figure 2 with 4 supplements see all Download asset Open asset DACones are protein reactive species. (A) Reaction catalyzed by HSD17B11. (B) Clickable DACs and DACones used in the study. (C) Viability analysis of U2OS cells treated in PBS for 1 h with (S)–3 or DACones and incubated for an additional 72 h after drug washout. (D) FBS or purified BSA were incubated 40 min at 30 °C with clickable DAC (S)–9 or clickable DACone 10. After reaction, CuAAC was used to ligate an azido-AlexaFluor647 to clickable molecules. Modified proteins were detected by scanning membrane fluorescence after SDS-PAGE and transfer. Ponceau S stains total proteins. (E) BSA or BLG were incubated with the indicated DACs or DACones, as in (D). After reaction, modified proteins were detected as in (D). Coomassie stains total proteins. (F) WT or S172L HSD17B11-GFP were immunoprecipitated from complemented U2OS KO HSD17B11 cells and incubated with clickable DAC 9 and BLG. After reaction, modified proteins were detected in the supernatant (BLG) or on the beads (HSD17B11-GFP) as in (D). GFP immunoblotting confirmed that equal amounts of WT and S172L proteins were used. (G) Analysis by direct-infusion mass spectrometry of purified BLG (mixture of isoform A and B) modified or not by DACone 10. Cyan and green arrows indicate the formation of a first and second adduct, respectively. (H) % of each amino acid detected as modified by DACones 10 or 11 in U2OS extracts as determined using an isoDTB-ABPP-based framework. (I) Proposed reactions of DACones with cysteine and lysine side chains in proteins. Figure 2—source data 1 Source data related to Figure 2D. The tiff files correspond to an uncropped picture of the AlexaFluor647 fluorescence signal, acquired on an Odyssey LI-COR, and of a scan of the membrane stained with Ponceau S. The jpg file combines both pictures and can be used to locate the protein ladders. https://cdn.elifesciences.org/articles/73913/elife-73913-fig2-data1-v1.zip Download elife-73913-fig2-data1-v1.zip Figure 2—source data 2 Source data related to Figure 2E. The tiff files correspond to uncropped pictures of the AlexaFluor647 fluorescence, acquired in gel on an Odyssey LI-COR, and of the gels after staining with Coomassie blue scanned with the BioRad Chemidoc. Two different gels were used (respectively labeled upper and lower). Each jpg file combines the two pictures used to generate upper and lower parts of the figure. https://cdn.elifesciences.org/articles/73913/elife-73913-fig2-data2-v1.zip Download elife-73913-fig2-data2-v1.zip Figure 2—source data 3 Source data related to Figure 2F. The tiff files correspond to uncropped pictures of the AlexaFluor647 fluorescence signal, acquired on an Odyssey LI-COR, of a scan of the membrane stained with Ponceau S, for the upper part (supernatant), and of the chemiluminescent signal acquired using autoradiographic films for the lower part (beads). The jpg file combines the pictures used to generate the figure and can be used to locate the protein ladders. https://cdn.elifesciences.org/articles/73913/elife-73913-fig2-data3-v1.zip Download elife-73913-fig2-data3-v1.zip We next tested the cytotoxic activity of (S)–3 on a panel of 15 cancer cell lines. This revealed that the osteosarcoma U2OS cell line was the most sensitive to (S)–3 while the breast cancer cell line T47D was highly resistant (Figure 1—figure supplement 4A). Accordingly, HSD17B11 protein was undetectable in T47D, while U2OS displayed the highest levels (Figure 1—figure supplement 4B), in agreement with reported mRNA levels (The Cancer Cell Line Encyclopedia dataset [Barretina et al., 2012]). In addition, (S)–3 was particularly cytotoxic toward four other osteosarcoma cell lines as compared to normal cell lines or primary osteoblasts (Figure 1—figure supplement 4C). CRISPR/Cas9-mediated inactivation of HSD17B11 also conferred significant (S)–3 resistance to U2OS cells, which was suppressed by wild-type HSD17B11-GFP but not by the S172L mutant or GFP alone (Figure 1—figure supplement 5A, B). In contrast, complementation with HSD17B11 carrying the L14P or V16D mutations, lying outside of the catalytic domain and identified in the DACR clones #B1 and #A1/#A2, respectively, restored (S)–3 cytotoxic activity, in agreement with these mutations affecting HSD17B11 protein stability and not its catalytic activity (Figure 1—figure supplement 5C, D). These data also support that the C-terminal FLAG-GFP tag and/or the CMV promoter-based overexpression partly overcome the impact of these mutations on HSD17B11 expression level. The role of HSD17B11 in (S)–3 cytotoxic activity was further confirmed using two different small-interfering RNAs (siRNA) to down-regulate HSD17B11 in U2OS (Figure 1—figure supplement 5E, F) and in the non-small cell lung carcinoma cell line A549, in which CRISPR/Cas9-mediated HSD17B11 inactivation also conferred (S)–3 resistance (Figure 1—figure supplement 5G,H). Altogether, these data establish that HSD17B11 is critical in multiple cell lines for (S)–3 cytotoxic activity, and suggest that (S)–3 behaves as an HSD17B11-bioactivated prodrug. In addition, the acute toxicity of (S)–3 towards osteosarcoma cell lines suggests that DACs could be developed into a targeted anticancer therapy, but this would need to be further investigated, especially in vivo. Dialkynylketones are protein-reactive species We next investigated the downstream mechanism of cytotoxic action of the DAC (S)–3. The C17 carbinol center of androstan-3-alpha,17-beta-diol, which is naturally oxidized by HSD17B11 (Figure 2; Brereton et al., 2001), has the same spatial orientation as the (S)–3 carbinol when its lipidic chain is superimposed with the C13(C18) side of the steroid skeleton (Figure 2B). This suggested that HSD17B11 enantiospecifically recognizes and oxidizes (S)–3 into a "dialkynylketone" 7 (DACone), a diynone that could be the cytotoxic species. However, when the DACone 7 was previously synthesized and tested, no cytotoxic activity was found (Listunov et al., 2015a). Given the high in vitro electrophilic reactivity of ynones as Michael acceptors of thiols and amines (Worch et al., 2021), we considered that medium components such as serum albumin may rapidly react with and inactivate DACones. To test this, we synthesized the DACone 7, as well as a homologue with a shorter alkyl chain 8, and treated U2OS cells in a protein-free medium (PBS containing CaCl2 and MgCl2 to maintain cellular adhesion). Both the DACones 7 and 8 were indeed cytotoxic in the absence of serum, with 8 (short chain) being even more active than (S)–3 (Figure 2C). While the cytotoxicity of (S)–3 was strongly reduced by inactivation of HSD17B11, the cytotoxicity of the DACones 7 and 8 was not affected, supporting the notion that the DACones are the cytotoxic products generated from DACs by HSD17B11. To further analyze the interaction between DACones and proteins, we synthesized 'clickable' analogues, that is bearing a terminal C≡CH tag, for each DAC enantiomer ((S)–9 and (R)–9), and for long and short DACones (10 and 11, Figure 2B), and used them to monitor the formation of covalent bonds between DACones and serum proteins by copper-catalyzed azide-alkyne cycloaddition (CuAAC 'click chemistry', [Tornøe et al., 2002; Rostovtsev et al., 2002]). The clickable DACone 10, or clickable DAC (S)–9 as control, were incubated with fetal bovine serum (FBS) or purified bovine serum albumin (BSA), followed by CuAAC-mediated ligation of an AlexaFluor647-azido fluorophore to the free C≡CH tag. The proteins were separated by SDS-PAGE and scanned for fluorescence (Speers and Cravatt, 2004). Covalent adducts were formed on BSA with DACone 10 but not with (S)–9 (Figure 2D). Moreover, the DACone 10 also reacted with several other model proteins, including the bovine beta-lactoglobuline (BLG) (Figure 2—figure supplement 1A). Using BSA and BLG, we established that DACone adducts are produced only when using the clickable DACones 10 or 11 (Figure 2E), suggesting that the terminal triple bond of the DACone pharmacophore is modified or masked after reaction. Finally, we could recapitulate the activation of (S)–9, but not of (R)–9, into protein-reactive species by immunopurified WT HSD17B11, but not by the S172L mutant (Figure 2F), supporting an enantiospecific bioactivation of (S)–9 into the BLG-reactive DACone 10 by HSD17B11. Considering that HSD17B11 known activity is the NAD+-dependent oxidation of a secondary carbinol into a ketone and that the only hydroxyl group on (S)–3 is the one occurring in the dialkynylcarbinol pharmacophore, this experiment strongly supports the notion that HSD17B11 enantiospecifically oxidizes (S)–3 into the DACone 7, which immediately reacts with nearby proteins, including HSD17B11-GFP itself as observed in Figure 2F. This high level of reactivity unfortunately precludes isolating the HSD17B11-produced DACones. To understand the basis for this enantiospecific bioactivation, we used AlphaFold2 (Jumper et al., 2021; Evans et al., 2021; Mirdita et al., 2021) to generate a structural model of HSD17B11 (Figure 2—figure supplement 2A) and performed molecular docking of (S)–3 and (R)–3 into its catalytic core. Both (S)–3 and (R)–3 docked into the catalytic cavity (Figure 2—figure supplement 2B,C), but (R)–3 had a lower computed affinity than (S)–3 (155 nM vs 15 nM) and only (S)–3 had its hydroxyl group properly positioned to engage hydrogen bonds with the S172 and Y185 catalytic amino acids (Figure 2—figure supplement 2D, E), which is critical for further carbinol oxidation via hydride transfer to NAD+ (Filling et al., 2002; Gao et al., 2021). Combining docking of (S)- and (R)–3 on AlphaFold2 models of the 12 other human 17β-hydroxysteroid dehydrogenase (17β-HSDs) SDRs, with filtering to select the most stringent interactions, was used to identify other SDRs that might be able to bioactivate (S)–3 (Figure 2—figure supplement 2F). This filtering revealed that, beyond HSD17B11, (S)–3 also docked onto the catalytic domains of only two 17β-HSDs, HSD17B13 and HSD17B3, while (R)–3 docked onto two different 17β-HSDs, HSD17B9 and HSD17B6. HSD17B3 (aka EDH17B3 or SDR12C2 [Persson et al., 2009]) is a reductive SDR involved in testosterone biosynthesisso it was not tested further. In contrast, since HSD17B13 (aka SCDR9 or SDR16C3 [Persson et al., 2009]), whose expression is restricted to the liver, is an oxidative SDR, the closest homologue of HSD17B11 and is also localized at the ER (Horiguchi et al., 2008b), we tested whether it could complement U2OS KO for HSD17B11. This revealed that HSD17B13 is also able to bioactivate (S)–3 into cytotoxic compounds, albeit in a less efficient manner as compared to HSD17B11 (IC50 of 30 nM vs 12 nM for HSD17B11 with similar complementation levels, Figure 2—figure supplement 2G, H). Reaction of DACones with proteins To further decipher the reaction of DACones with proteins, we used direct-infusion mass spectrometry to analyze BLG modified with the clickable DACone 10. Purified BLG contains two isoforms (A and B, differing by 86.0 Da) and, when incubated with DACone, both BLG isoforms were completely modified with the formation of one or two adducts of ~+242 Da (Figure 2G), which corresponds to the mass of the clickable DACone 10. Monitoring the absorbance spectra of modified BLG revealed that BLG gains an absorption band at ~323 nm upon modification by DACone (Figure 2—figure supplement 1B). Using this, we confirmed that both BLG and BSA are modified by the DACones 7 and 8 or their clickable analogues 10 and 11 (Figure 2—figure supplement 1B, C). The shorter DACone 8 proved to be even more reactive, in line with its greater cytotoxicity (Figure 2C, Figure 2—figure supplement 1B, C). Next, we assessed the selectivity of DACones towards amino acid residues in the whole proteome in an unbiased fashion. For this purpose, we incubated the DACones 10 and 11 with U2OS total cell extracts in PBS. We then used residue-specific chemoproteomics with isotopically labeled desthiobiotin azide (isoDTB) tags (Backus et al., 2016; Weerapana et al., 2010; Zanon et al., 2020) coupled to a novel MSFragger-based FragPipe computational platform (Zanon et al., 2021) to detect the modified amino acids on the enriched peptides. This revealed that both DACones reacted with cysteine and lysine side chains, with the expected modification being detected ( + 729.4498/ + 723.4408 Da (Heavy/Light) for DACone 10, + 631.3404/ + 625.3332 Da (Heavy/Light) for DACone 11, Figure 2H, Figure 2—figure supplement 1D, E, F, Supplementary file 1A, B). We also detected many unmodified peptides (Supplementary file 1D, E), most of them with at least one missed lysine trypsin cleavage site (~92% of sequences), suggesting that these were still modified during digest and the modification lost during the subsequent workflow, potentially during the final trifluoroacetic acid elution (0.1%, pH~2 [Zanon et al., 2021]). We cannot fully exclude that other amino acids were also modified by the probe to some degree and that this modification was also lost during the workflow, but this data points to the fact that lysines and cysteines are the main modification sites. We next confirmed the reactivity of DACones with cysteine and lysine side chains by monitoring the appearance of the ~323 nm absorbance band after reaction of the DACone 8 with isolated amino acids, using N-acetylated versions to prevent reactions with the N-terminal amino group. At neutral pH, DACones only reacted with N-acetyl-L-cysteine (NAC) but not with Nα-acetyl-L-lysine (NAK) (Figure 2—figure supplement 1G, left spectrum), whereas at higher pH they reacted with both NAC and NAK (Figure 2—figure supplement 1G, right spectrum), in agreement with the nucleophilic reactivity of the non-protonated ε−NH2 group of the lysine chain. No reaction was observed with N-acetyl glycine (NAG), supporting that the reaction involves the side chain. By monitoring the adducts absorbance, we confirmed that the DACone 8-NAK linkage is progressively lost when incubated in 0.1% TFA (38% reduction after 1 h incubation), while the DACone 8-NAC linkage remains unaffected in these conditions (Figure 2—figure supplement 1H, I). These data suggest that the reactivity with lysine side chains is underestimated by our isoDTB-ABPP experiment due to preferential loss of the lysine-DACone species. The reaction with lysine side chains is compatible with the pKa value for the lysine ε-amino group that can be as low as ~5 in hydrophobic domains in proteins (Isom et al., 2011). Accordingly, analysis of the sequence context of the amino acids identified as modified by the DACone 10 revealed an enrichment of hydrophobic amino acids around the modified lysines, which was not observed for the modified cysteines (Figure 2—figure supplement 3A, B, Supplementary file 2). Using nuclear magnetic resonance (NMR), we characterized the products of the reaction of the short DACone 8 with NAC (Figure 2—figure supplement 4A, B) or NAK (Figure 2—figure supplement 4C, D). This revealed that a covalent bond forms by addition of the thiol (NAC) or amino (NAK) group onto the terminal alkyne of the DACone head (Figure 2—figure supplement 4E). A similar reaction probably occurs with the cysteine and lysine residues of proteins, which is supported by their gain of a similar absorbance band upon treatment with DACone (Figure 2I). This additional absorption band can be accounted for by the donor-acceptor extension of π-electron delocalization in the enone adducts (S–CH=CH–C=O for NAC, N–CH=CH–C=O for NAK). Altogether, our data show that DACones are highly reactive with proteins in vitro. Bioactivated (S)-DACs lipoxidize multiple proteins in cells Protein modification by lipidic DACs equates to their lipoxidation (a term used to designate the covalent modification of a protein by a reactive lipid [Viedma-Poyatos et al., 2021]) by one or several C17 hydrophobic chain(s) (Figure 3—figure supplement 1). Considering that protein palmitoylation (addition of a C16 lipidic chain) can trigger membrane tethering of proteins, we hypothesized that lipoxidation by DACs could affect protein localization and/or function and account for the cytotoxicity of bioactivated DACs in cells as described for other reactive lipids (Viedma-Poyatos et al., 2021). To challenge this hypothesis in cells, we took advantage of the clickable DAC 9 (Figure 2B, [Listunov et al., 2015b]). As observed for the DAC 3, the cytotoxicity of the clickable DAC 9 was enantiospecific, biased toward (S)–9, and dependent on bioactivation by HSD17B11 (Figure 3—figure supplement 2A). Cells were treated with clickable (S)- or (R)–9 DACs, extracts prepared and click chemistry used to detect the covalent adducts of DACs onto proteins (Speers and Cravatt, 2004). Multiple modified proteins were detected in extracts from (S)–9-treated cells, while no adduct with (R)–9 was detected (Figure 3A). Figure 3 with 5 supplements see all Download asset Open asset (S)-DACs lipoxidize multiple cellular proteins, triggering their association with cellular membranes. (A) U2OS cells were incubated for 2 h with 2 µM (S)- or (R)–9, proteins were extracted and DAC-modified proteins were detected by CuAAC-mediated ligation of azido-AlexaFluor-647 to clickable molecules, separation by SDS-PAGE, transfer to a membrane which was scanned for fluorescence. (B) Landscape of proteins modified in U2OS cells by clickable DAC (S)–9 computed from three independent experiments. Fold enrichment (FC) as compared to the clickable (R)–9 is computed and color-co
Covalent inhibitors have recently seen a resurgence of interest in drug development. Nevertheless, compounds, which do not rely on an enzymatic activity, have almost exclusively been developed to target cysteines. Expanding the scope to other amino acids would be largely facilitated by the ability to globally monitor their engagement by covalent inhibitors. Here, we present the use of light-activatable 2,5-disubstituted tetrazoles that allow quantifying 8971 aspartates and glutamates in the bacterial proteome with excellent selectivity. Using these probes, we competitively map the binding sites of two isoxazolium salts and introduce hydrazonyl chlorides as a new class of carboxylic-acid-directed covalent protein ligands. As the probes are unreactive prior to activation, they allow global profiling even in living Gram-positive and Gram-negative bacteria. Taken together, this method to monitor aspartates and glutamates proteome-wide will lay the foundation to efficiently develop covalent inhibitors targeting these amino acids.
Abstract Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue‐specific chemoproteomic approaches enable proteome‐wide identification of binding sites for covalent inhibitors. Described here are easily synthesized isotopically labeled desthiobiotin azide (isoDTB) tags that shortened the chemoproteomic workflow and allowed an increased coverage of cysteines in bacterial systems. They were used to quantify 59 % of all cysteines in essential proteins in Staphylococcus aureus and enabled the discovery of 88 cysteines that showed high reactivity, which correlates with functional importance. Furthermore, 268 cysteines that are engaged by covalent ligands were identified. Inhibition of HMG‐CoA synthase was verified and will allow addressing the bacterial mevalonate pathway through a new target. Overall, a broad map of the bacterial cysteinome was obtained, which will facilitate the development of antibiotics with novel modes‐of‐action.
<p><a>Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome‑wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome‑wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the <i>N</i>‑terminus proteome‑wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.</a></p>
Various approaches have been developed to target RNA and modulate its function with modes of action including binding and cleavage. Herein, we explored how small molecule binding is correlated with cleavage induced by heterobifunctional ribonuclease targeting chimeras (RiboTACs), where RNase L is recruited to cleave the bound RNA target, in a transcriptome-wide, unbiased fashion. Only a fraction of bound targets was cleaved by RNase L, induced by RiboTAC binding. Global analysis suggested that (i) cleaved targets generally form a region of stable structure that encompasses the small molecule binding site; (ii) cleaved targets have preferred RNase L cleavage sites nearby small molecule binding sites; (iii) RiboTACs facilitate a cellular interaction between cleaved targets and RNase L; and (iv) the expression level of the target influences the extent of cleavage observed. In one example, we converted a binder of LGALS1 (galectin-1) mRNA into a RiboTAC. In MDA-MB-231 cells, the binder had no effect on galectin-1 protein levels, while the RiboTAC cleaved LGALS1 mRNA, reduced galectin-1 protein abundance, and affected galectin-1-associated oncogenic cellular phenotypes. Using LGALS1, we further assessed additional factors including the length of the linker that tethers the two components of the RiboTAC, cellular uptake, and the RNase L-recruiting module on RiboTAC potency. Collectively, these studies may facilitate triangulation of factors to enable the design of RiboTACs.
Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome-wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome-wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the N-terminus proteome-wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.
ABSTRACT Hundreds of cytotoxic natural or synthetic lipidic compounds contain chiral alkynylcarbinol motifs, but the mechanism of action of those potential therapeutic agents remains unknown. Using a genetic screen in haploid human cells, we discovered that the enantiospecific cytotoxicity of numerous terminal alkynylcarbinols, including the highly cytotoxic dialkynylcarbinols, involves a bioactivation by HSD17B11, a short-chain dehydrogenase/reductase (SDR) known to oxidize the C-17 carbinol center of androstan-3-alpha,17-beta-diol to the corresponding ketone. A similar oxidation of dialkynylcarbinols generates dialkynylketones, that we characterize as highly protein-reactive electrophiles. We established that, once bioactivated in cells, the dialkynylcarbinols covalently modify several proteins involved in protein-quality control mechanisms, resulting in their lipoxidation on cysteines and lysines through Michael addition. For some proteins, this triggers their association to cellular membranes and results in endoplasmic reticulum stress, unfolded protein response activation, ubiquitin-proteasome system inhibition and cell death by apoptosis. Finally, as a proof-of-concept, we show that generic lipidic alkynylcarbinols can be devised to be bioactivated by other SDRs, including human RDH11 and HPGD/15-PGDH. Given that the SDR superfamily is one of the largest and most ubiquitous, this unique cytotoxic mechanism-of-action could be widely exploited to treat diseases, in particular cancer, through the design of tailored prodrugs. Graphical abstract
