Mass Spectrometry-Based Solutions for Drugging the Undruggable

p. 1 Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable Author: Francois Autelitano, VP Translational Proteomics and Metabolomics For further information: Francois Autelitano VP Translational Proteomics and Metabolomics Francois.Autelitano@evotec.com Considering that current drugs mainly target proteins, proteomic technologies are revolutionizing the analytical toolbox available to support preclinical drug discovery. Through recent advances in workflows and instrumentation, mass spectrometry (MS)-based quantitative proteomics has been successfully applied for comprehensive proteome-scale profiling to support the discovery of drugs that target proteins that were assumed to be undruggable in the past. Therefore, both pharmaceutical and biotech industries have sparked a growing interest in molecular glues, targeted protein degraders (TPD), targeted protein stabilizer (TPS), and targeted covalent ligands. ScreenPep™ – Proteomics without Compromise Evotec operates one of the largest high-end mass spectrometry facilities worldwide for Next-Generation Proteomics research. With more than 25 years’ experience in mass spectrometry, chemical proteomics, and state-of-the-art data analytics, Evotec offers a broad range of cutting-edge mass spectrometry-based proteomic technologies and services to address key issues in drug and biomarker discovery. We continuously advance our capabilities in mass spectrometry-based proteomics to ensure unrivalled comprehensiveness and data quality when analyzing cells, animal models and patient samples. Evotec’s ScreenPep™ platform drives the paradigm shift towards high-throughput proteomics profiling without making any compromise on coverage and precision. Implementing fast automation and parallelization for processing of samples across many instruments, we can scale up our throughput for largescale discovery proteomics to 300,000 samples per year or 800 samples per day. ScreenPep™ is a single-shot Data Independent Acquisition (DIA) based protein quantification workflow (1) offering ultra-deep proteome coverage with reproducible and precise quantification of 10,000+ proteins per sample. The ScreenPep™ workflow (Figure 1) is also extendable to post-translational modifications including ubiquitination, phosphorylation, acetylation, methylation, and S-nitrosylation.ScreenPep™ provides a scalable, tractable, fully automated proteome-based pipeline in human cell lines for screening small molecules that bind to or alter the abundance of endogenous proteins, such as targeted protein degraders and stabilizers. Discovery of Covalent Drugs using HighThroughput Activity-Based Protein Profiling Irreversible inhibitors are a powerful modality in drug discovery. By forming a covalent bond with the protein target, these inhibitors can exhibit great potency and prolonged duration of action. In addition, irreversible inhibitors offer other advantages, such as targeting therapeutically relevant mutations or addressing difficult-to-drug targets. Despite these advantages, pharma has historically avoided them because of the potential for unpredictable off-target toxicities or immune responses. In the cases when pharma have proceeded with the development of covalent compounds, compromises between reactivity, selectivity and potency have produced safe and effective drugs. The approval of multiple covalent drugs by the FDA in the last years has prompted a resurgence of interest in this class of compound. Key examples include covalent tyrosine kinase inhibitors (TKIs) for cancer treatment, including the Bruton’s tyrosine kinase (BTK) inhibitor Ibrutinib (2 – 4), the epidermal growth factor receptor (EGFR) inhibitor Osimertinib (5 – 7), and Sotorasib, a RAS GTPase family inhibitor developed for the treatment of solid tumours with KRAS G12C mutations (8 – 10). In the discovery of novel covalent inhibitors, covalent fragments (typical MW of less than 300 Da) provide a vital toolbox. These fragments have several advantages that can be summarized as follows: (i) excellent target occupancy via covalent bond formation; (ii) selectivity towards distinct protein target residues (cysteine, lysine, serine, …) using chemically diverse electrophilic warheads (11 – 12); (iii) possibility to establish target engagement and on-target mechanism of action using mass spectrometry-based methods in parallel with target residue mutagenesis; (iv) ability to pharmacologically target pockets within disordered regions and cryptic binding sites beyond the substrate pockets, thus allowing the discovery of new allosteric drugs or ligands for targets that lack well-defined binding pockets; (v) discovery of potentially unknown binding sites. At Evotec, several strategies have been developed to discover, develop, and validate covalent fragments as a promising path to covalent ligand discovery. Intact protein liquid chromatography mass spectrometry (LC-MS) is a widely used target-based covalent fragment screening strategy (13 – 15). Intact protein LC-MS can provide information on the accurate mass of a protein (and proteoforms) alone or conjugated with a covalent fragment, and the relative abundance of the different species across different conditions. Intact protein LC-MS provides stoichiometry of covalent protein–fragment complex formation by comparing the calculated mass difference across vehicle control to fragment-treated samples. We use high-throughput intact protein LC-MS to screen covalent fragment libraries against pure recombinant protein constructs and identify the fragments hits and quantify their labeling. Subsequent steps to the primary screen can include biochemical assays, surface-plasmon resonance (SPR) (16), and intact protein LC-MS to deconvolute the reversible and irreversible components of the interactions and the kinetic rate constants (kon and koff and thus the KD, k inact/KI) of each step. These parameters are particularly useful to rank order compound hits by potency and help medicinal chemistry teams better understand and optimize structure-activity relationship (SAR). To localize the site of modification, Peptide Mass-Fingerprinting (PMF) using LC-MS/MS is applied. Finally, this data can be paired with crystal structures that fully characterize the modification sites and guide the subsequent optimization to therapeutic leads (15 – 17). Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 2 Cell Treatment Human cells Lyse Red/Alk Digest Clean-up Compounds Sample Preparation DIA-MS Target Identification and Quantification Figure 1: Overview of the ScreenPep™ platformFigure 2: Overview of the HT-ABPP platform Treat Lyse Combine Probe Red/Alk Digest Clean-up Enrich DIA-MS Target Identification and Quantification Human cells Covalent library % occupancy Compounds A more powerful approach is the application of bottom-up mass spectrometry-based proteomics to peptide mapping on a larger scale. The basic workflow involves enzymatic digestion of a target protein modified by a covalent fragment, followed by the analysis of the resulting peptides via tandem mass spectrometry (LC-MS/ MS) and peptide fragment data analysis (18 – 19). This approach can resolve each amino acid of the peptide sequence allowing unbiased identification of the targeted residue with site occupancy information. Although target-based electrophilic library screens using intact protein LC-MS is effective and relatively easy to implement, it requires production and isolation of stable recombinant target proteins. For many targets of interest like multi-pass membrane proteins (e.g., G-proteincoupled receptors and multi-subunit receptors or ion channels), this is either not feasible on a scale needed for these experiments or impossible given intrinsic instability of the target. Moreover, in many cases, selectivity against isolated protein constructs does not directly translate into global chemoselectivity in a cellular environment. To address this, we have established a state-of-art mass-spectrometry based chemoproteomic screening platform with two main objectives: 1) discover previously unknown target sites within disordered regions, cryptic or shallow binding pockets, and allosteric pockets on the surface of proteins 2) identify small molecule fragments that interact in a highly selective manner with those pockets directly in complex biological systems (cell/tissue lysates, live cells, or live organisms). Evotec’s proteome-wide chemoproteomic screening Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 3 platform provides selectivity and target engagement measurements that delivers a map of ligandable sites across the entire proteome. This serves as a starting point for understanding the druggability of potential therapeutic targets and for the development of small molecules against any class of protein in their native cellular context. Our platform leverages residue-based probes (RBPs) that possess broadly reactive covalent warheads developed to modify side chains on the targeted amino acid residue, including cysteine (20 – 21), lysine (22 – 23), tyrosine (24 – 25), and aspartate/glutamate residues (26). RBPs also bear a reporter group, such as a fluorophore (i.e. TAMRA) or an affinity tag (i.e. desthiobiotin) for identification and enrichment purposes. Alternatively, a bioorthogonal chemical handle such as an alkyne or azide can be installed on the probe, allowing the attachment of reporter or affinity tags after the protein target conjugation step for subsequent click chemistry-enabled applications. Our High-Throughput Activity-Based Protein Profiling (HT-ABPP) platform combines a robotic liquid handling platform for enrichment and peptide clean-up, stable isotope labeling by amino acids in cell culture (SILAC), and data-independent acquisition (DIA)-mass spectrometry (MS)-based proteomic analysis (Figure 2). The platform allows the consistent mapping of 60,000+ reactive cysteine sites on 13,000+ unique proteins, and 12,000+ distinct reactive lysine sites on 3,500+ unique proteins, across many different human cell lines and primary cells with a throughput of up to 100 samples per day. Mapping of reactive tyrosine and Asp/Glu sites in various cell lines is currently in progress.We have used this platform to screen diverse covalent reactive libraries in live cells to identify new chemical matter targeting proteins or protein families of interest. This strategy enables the assessment of cysteine reactivity and ligandability directly in a cellular context. Using dose-dependent competition assays in combination with quantitative MS, we can determine IC50 values and apparent dissociation constants (Ki app) of the electrophilic covalent fragments against all specifically captured proteins in a single experiment (27 – 29). This information enables rapid identification of covalent ligands against reactive hotspots, along with providing selectivity information on each ligand in various cellular states. When paired with a parallel phenotypic screen against a desired outcome, this methodology facilitates the identification of functional covalent ligands and their corresponding protein targets in a high-throughput manner. Discovery of Reversible Drugs using High-Throughput PhotoAffinity Labeling Mass Spectrometry The HT-ABPP approach can identify protein targets featuring suitably reactive nucleophilic (primarily cysteine or lysine) residues. Since this significantly narrows the target space, we developed an integrated photoaffinity labeling mass spectrometry screening platform to test Fully Functionalized Fragment (FFF) libraries in the presence of purified protein (30) or live cells (31 – 32). Each FFF probe contains a variable small-molecule fragment conjugated to (i) a photoactivatable diazirine group, which is embedded within its structure to permit UV light-induced cross-linking to interacting proteins, and (ii) a click chemistry derivatizable handle allowing the attachment of reporter or affinity tags for Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 4 subsequent visualization, enrichment, and identification of interacting proteins. Using our High-Throughput PhotoAffinity Labeling Mass Spectrometry (HT-PALMS) platform in combination with FFF libraries, we can globally map thousands of reversible small-molecule fragment-protein interactions directly in human cells (Figure 3). In follow-up studies, we can map the sites of protein binding for FFF hits. Furthermore, FFF hits can immediately be used as reporters in competition assays to screen focused libraries of structurally elaborated underivatized fragment analogs (where the constant photoreactive/clickable component is replaced with a propanamide group) to establish SAR and identify more potent and selective cell-active binders targeting protein(s) of interest. Competition studies can then eloquently discern phenotypically relevant target(s) of FFF hits from proteins that interact with the compounds but do not contribute to the phenotype. From the elaborated competitor hits, a second-generation library of FFF probes can be generated, expanded, and screened in live cells, as described previously, to identify more potent FFF probes in a site-specific manner. Using our HT-PALMS platform to screen FFF libraries in the presence of a purified protein in a biochemical setting improves the throughput and enables targeted screening against proteins of interest. FFF hits can be detected by intact protein LC-MS, enabling identification of known and new chemotypes. FFF hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel starting points in the development of therapeutics. Treat UV Lyse Click Red/Alk Enrich Digest Clean-up Elaborated fragment library FFF probe DIA-MS Target Identification and Quantification Figure 3: Overview of the HT-PALMS platformEvotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 5 Evotec’s Proprietary Covalent Libraries – Covering diverse warheads and Chemical Space Evotec created an extensive collection of 5,300 acrylamide derivatives as cysteine modifiers (Figure 4). First focus was on acrylamides because of (i) the growing interest in selective covalent modification of cysteine residues, (ii) the increasing number of acrylamide-derived drugs reaching advanced clinical phases and the market [Ibrutinib (2 - 4), Osimertinib (5 – 7), Sotorasib (8 – 10)], (iii) our internal expertise built over years showing developability of such molecules and (iv) their long-term stability (over years), as demonstrated by their storage in our sample management facilities. This collection covers fragment and lead-like chemical spaces and was designed using a thorough molecular design workflow based on well-thought molecular descriptors, Evotec list of structural alerts, and diversity selection. The outcome of the workflow was supervised by skilled medicinal chemists experienced in covalent drug discovery. Figure 4: Evotec’s acrylamide-focused electrophilic fragment library. Selection workflow (A) and main descriptors (B) of the Evotec acrylamide screening collection. (A) (B) Finely-tuned acrylamide library Long term storage, rapid compound progression, proven development potential  Curation using an in-house database of frequent hitters and structural alerts  Relevant chemical property distribution  Visual inspection by seasoned Medicinal chemists Representative examples Distribution of molecular properties ~50k set Final 5k set Pooling of commercially available covalent compounds from trusted vendors Evotec Structural Alerts Molecular descriptors Confirmation of availability Retrosynthetic Tractability Scoring Diverse Subset Selection Eye-check of remaining structures Evotec is now extending the collection towards other protein residues that are susceptible to chemical modification, such as lysine, serine/threonine, tyrosine, aspartic/glutamic acids, methionine, arginine, and histidine. We pay particular attention to stability when creating new library sets, which has particularly high importance when dealing with reactive molecules. Hence our decision to test mid-term stability of islets of relevant chemical modifiers of various protein residues in regular storage conditions. Warheads that appear to be suitable for mid-term storage will be extended to create the “beyond cysteine” Evotec covalent collection. Evotec also uses the libraries of covalent compounds and FFFs commercially available from different suppliers (e.g. Enamine, Life Chemicals, Molport) as well as the libraries built up by his partners.Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 6 Conclusion We have established new drug discovery platforms that leverage groundbreaking scientific approaches in MS-based proteomics to uncover and pharmacologically target previously undruggable proteins targets, making them accessible to therapeutic intervention by small-molecule drugs. With the SreenPep™ platform, Evotec operates MS-based proteomics at industrial scale, without compromise on sensitivity, precision, or proteome coverage, to screen extensive small-molecule libraries for TPD of pathogenic proteins previously considered undruggable. Significant advances in fragment-based screening, including the emergence of FFFs and innovations in covalent fragment screening provide a new paradigm for ligand and target discovery. Evotec’s HT-PALMS platform has the potential to fill major gaps in smallmolecule probe development by enabling the discovery of reversible ligands, and the sites of ligand binding, for many proteins in parallel, directly in human cells. Screening covalent fragments using our HT-ABPP platform enables exploration of novel druggable pockets through irreversible fragment-amino acid residue interactions, complementing their fully functionalized counterparts. FFFs and covalent fragments are not only useful for identifying functional modulators, but also play important roles in the emerging induced proximity modalities, including molecular glues and rationally designed bifunctional agents. HT-PALMS and HT-ABPP platforms facilitate the expansion of TPD and TPS approaches by enabling the discovery of new E3 ubiquitin ligase or deubiquitinase recruiters. With ScreenPep™, intact protein LC-MS, HT-PALMS, and HT-ABPP platforms, Evotec provides the screening community with multiple unbiased and complementary routes for discovering disease-focused targets and new chemical entities toward first-in-class therapeutics.Case study 1: Discovery of selenocysteineselective covalent inhibitors of a SelenoTarget protein Rational Selectively targeting tumors with drugs that induce reactive oxygen species (ROS) production is a promising and well-studied approach for cancer treatment, with the promise to succeed in a clinical setting. Selenium (Se) is one of the trace elements that is essential for human survival. Its essential benefits to human health are mainly due to its presence in proteins as selenocysteine (33). The main role of Se is participating in the redox catalytic process (34). Many selenoproteins have been found to be associated with the occurrence and poor prognosis of cancer (35 –36). In this study, we were interested in an Se-related enzyme whose role in cancer is established that we here call SelenoTarget (SeTarget). SeTarget inhibition rapidly induces high ROS-levels in cancer cells, resulting in cell death. Inhibition of SeTarget may thus contribute to cancer therapy and improving chemotherapeutic agents. Mammalian SeTarget contains a highly reactive, surface-exposed selenocysteine residue (Se-Cys or U) in its active site. Selenocysteine is easily targeted by electrophilic compounds that inhibit SeTarget activity. Herein, we decribe a strategy to discover novel chemical matter that inhibit the enzymatic activity of SeTarget through covalent modification of the C-terminal selenocysteine (Figure 5). Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 7 Electrophile-focused library We compiled a library of 1,047 electrophilic fragments featuring mildly reactive cysteine-targeting warheads, including chloroacetamide- (733) and acrylamide-based (229) warheads, with average molecular weights of 241 and 233 Da, respectively, as well as heterocyclic electrophiles (85) with various types of warheads (e.g. haloalkyl, vinyl sulfone, aldehyde ketone, and cyano groups) and an average molecular weight of 413 Da. To this collection, we added 95 electrophiles (average molecular weight of 413 Da and armed with α,β-unsaturated amides or ketones or cyano groups) selected by target-based virtual screening of over 100,000 cysteine-focused covalent compounds against two X-ray structures of the SeTarget of interest, targeting its catalytic selenocysteine residue. Intact protein LC-MS screening Human SeTarget protein construct was incubated with each fragment in singleton at 230 μM for 2 h at room temperature. Following incubation, intact protein liquid chromatography–mass spectrometry (LC–MS) was applied to identify the fragment hits and quantify the percentage of SeTarget labeling. In total, 96 compounds covalently bound to SeTarget protein with a Drug-Antibody Ratio (DAR* is defined as the average number of compounds adducts attached to a single SeTarget protein) between 1 (mono-adduct) and 2 (bis-adduct) were selected for a counter-screen on the mutated SeTarget construct (U -> S). Of these, 59 hits were shown to form a single covalent adduct with SeTarget selenocysteine over other cysteines on the protein with high selectivity (ΔDAR** ≥ 0.95). Enzymatic activity and thiol reactivity Among the selected hits, 8 and 23 were confirmed to inhibit enzymatic activity of the purified enzyme by an endpoint enzymatic assay with IC50 values between 2–10 µM and 10–30 µM, respectively. Selected substances were tested for glutathione (GSH) reactivity using glutathione reactivity assay (37–38). While there was variability in the intrinsic reactivity of the fragments toward thiols, it was sufficiently small to allow the identification of hits sufficiently mild to ensure that the main driver of protein labeling is recognition rather than reactivity. Hit Expansion phase Next, six clusters were selected based on enzymatic activity (IC50 < 10 µM), lipophilicity (cLogD7.4 ≤ 3.5), and GSH reactivity (t1/2 > 1 h), and advanced for hit expansion via medicinal chemistry. In total, 273 analogs were synthesized and tested for their enzymatic activity and thiol reactivity. Several compounds exhibiting low single digit micromolar Figure 5: High-throughput screening cascade for the discovery of SeTarget covalent inhibitors targeting the catalytic selenocysteine residue. Summary of electrophile screening cascade and hit validation showing number of primary hits from the highthroughput screen and how these were prioritized and filtered. 1,142 electrophiles from a pre-plated cysteinefocused fragment library 95 ligands selected by target-based virtual screening of >100K cysteinefocused covalent compounds 96 hits 1 < DAR < 2 Intact protein LC-MS primary screen on SeTarget wild-type Intact protein LC-MS counterscreen on SeTarget (U->S) mutant Enzymatic assay Glutathione reactivity assay Hit expansion phase 273 analogs synthesized In-cell ABPP 59 hits ΔDAR ≥ 0.95 6 clusters IC50 < 30 µM; t1/2 > 1 h 31 hits IC50 < 30 µM 16 hits IC50 < 10 µM; cLogD (7.4) ≤ 3.5; t1/2 > 1 h 14 hits engaged SeTarget Se-Cys at ≥ 94%SeTarget enzymatic potency were discovered. Among these hits, 16 compounds with IC50 values below 10 µM, t1/2 > 1 h, and cLogD7.4 ≤ 3.5 with molecular weight (Lipinsky) below 400 Da were assessed for target engagement and selectivity using in-cell Activity-Based Protein Profiling (ABPP) (19–20). Activity-based protein profiling in human cells Ligandable cysteine and selenocysteine sites were profiled using a generalized desthiobiotin iodoacetamide probe (DB-IAA) to differentiate conjugated cysteine- and selenocysteine-containing peptides not bound by electrophiles. NCI-H23 cells were incubated with DMSO or 100 µM electrophilic compound hits for 2 h at 37°C in culture medium. After treatment, cysteines and Se-Cys that did not get covalently labeled with the compounds were then alkylated with the DB-IAA probe. Each compound was analyzed in three biological replicates. Cells were lyzed in the presence of urea and the free cysteine and Se-Cys sites reduced with DTT then alkylated with iodoacetamide (IAA). After digestion with trypsin/LysC cocktail, LC-MS/MS was used to quantify the peptides containing cysteines and Se-Cys modified with DB-IAA. The final output is displayed as a fold change (FC, compound/DMSO) for each electrophile, where an FC ≤ -2 (with an adjusted p-value < 5%) represents > 50% attenuation of cysteine/Se-Cys probe alkylation at that site. Starting with 100 μg of sample input per condition, in-cell ABPP quantified > 24,500 cysteine sites including 8 Se-Cys for > 6,600 unique proteins, with 15,900 - 21,800 ligandable sites per Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 8 Figure 6: Target engagement and selectivity profiling of SeTarget inhibitors in NCI-H23 cells using competition ABPP. Competition ABPP of NCI-H23 cells was performed with compounds (20 and 100 µM) for 2 h using single-shot label-free proteomics by data-independent acquisition (DIA)-MS. Data is presented as the mean of biological replicated experiments (n = 3). The final output is displayed as a fold change (FC, compound/DMSO) for each compound, where an FC ≤ -2 (with an adjusted p-value < 5%) represents > 50% attenuation of cysteine/selenocysteine DBIAA probe alkylation at that site. (A) Diagram showing the percentage of occupancy of SeTarget selenocysteine (U) as well as the total number of cysteine/ selenocysteine sites significantly engaged for each compound (A–L). (B) Volcano plot showing Log2[Fold Change] and significance (-Log10[p-value]) between cysteine/selenocysteine sites enriched by DBIAA probe from cells treated with compound K versus vehicle. Compound K (100 µM) significantly engaged SeTarget selenocysteine (U) as well as 10 other reactive cysteines and 1 selenocysteine from 11 different proteins. (A) (B) condition. Testing of 16 hits at high concentration (100 µM) in NCI-H23 cells confirmed that all but two compounds engage the C-terminal Se-Cys of SeTarget protein at ≥ 94% among other cysteine and Se-Cys sites (12 to > 4,300) from other proteins, presumably due to "overdosing" in this experiment (Figure 6A). Two compounds (C and H) tested at a lower dose (20 µM) showed as dose-dependent behavior with a better selectivity towards SeTarget catalytic Se-Cys residue. Finally, the most interesting hit compound (K), when used at the highest concentration engaged SeTarget selenocysteine > 98% with an excellent selectivity, with only 10 other cysteines and 1 selenocysteine from 11 different proteins liganded between 77% and 95% (Figure 6B). In conclusion, this study highlights the potential of electrophile-fragment screening as a practical and efficient tool for covalent-ligand discovery. HT-ABPP platform enables screening of electrophile libraries, and present comprehensive reactive cysteine maps for > 6,600 proteins and ligandability information across electrophiles in cells at low micromolar concentrations. *DAR = ∑((0 adduct * v%) + (1 adducts * w%) + (2 adducts * x%) + (3 adduct * y%) + (4 adduct * z%) / ∑(v% + w%+ x% + y% + z%) where v, w, x, y and z is the % of 0, 1, 2, 3 and 4 adducts **ΔDAR = DAR (wt form) – DAR (mutant form)Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 9 Case study 2: Discovery of small molecule binders of the E3 ubiquitin ligase Trim 47 using photoaffinity-labeling combined with mass spectrometry-based proteomics. Rational Tripartite Motif 47 (TRIM47) functions as a RING-finger E3 ubiquitin ligase. Here, we report a holistic photoaffinity-labeling mass spectrometry screening approach to identify novel binders for TRIM47 as warheads for future Trim47 PROteolysis TArgeting Chimeras (PROTACs) and molecular glues development. In the present study, we used 3 Fully Functionalized Fragment (FFF) probes with each member possessing a variable small-molecule fragment conjugated to a constant tag bearing an alkyne and photoactivatable diazirine group (39) (Figure 7A). For FFF1 and FFF2 probes, the variable fragment groups had an average molecular weight of approximately 180 Da. The variable fragment of FFF1 probe is a benzylpiperazine group found in many biologically active natural products and clinically approved drugs, like Imatinib (Gleevec) and Meclizine (Bonine) to name just a few. The variable fragment structure of FFF2 probe was not disclosed for confidentiality reasons. Finally, a “fragmentless” photoreactive probe bearing a methyl group, was used as a negative probe (NP). Gel-based profiling of FFF probes in human cells We initially assessed the FFF probes using gel-based profiling by treating HEK293T cells with each fragment probe (20 or 200 µM, 30 min), followed by exposure to UV light (20 min, 4°C), cell lysis, coupling to a tetramethylrhodamine (TAMRA)-azide tag using copper-catalyzed azide alkyne cycloaddition (CuAAC) chemistry (40), and separation and visualization of fragment-modified proteins by SDS-PAGE coupled with in-gel fluorescence scanning. Despite the structural simplicity and small size of the variable fragment groups, FFF1 and FFF2 probes produced marked and differential concentration-dependent protein labeling in HEK293T cells (Figure 7B). Negligible protein labeling was observed in the absence of UV light (data not shown), indicating that the fragment-protein interactions correspond to reversible binding events that were converted to covalent adducts upon photolysis. Protein labeling with NP probe was almost undetectable and comparable to that obtained in the absence of probe, indicating that the variable group of FFF probes is critical for protein binding. MS-based profiling of FFF probes in human cells We next set out to globally map fragment-binding proteins in HEK293T cells by quantitative chemical proteomics following the general protocol shown in Figure 3. We compared FFF probes at 200 µM (30 min incubation) to control probe NP in pairwise experiments using single-shot label-free proteomics by data-independent acquisition (DIA)-MS, where proteins enriched by the test FFF probe over NP with a fold change (FC) > 2 and adj p-value < 5% were designated as test probe targets. In aggregate, 2,295 protein targets were identified for FFF1 or FFF2 probes, with both probes individually displaying a broad range of protein enrichments (820 and 2,227 proteins, respectively). When tested at lower concentrations (20 µM), FFF probes enriched significantly less protein targets (data not shown), confirming that the extent of proteome engagement depends on probe concentration. The probe-versuscontrol comparisons showed that 752 target proteins (33%) were significantly enriched with both FFF probes, whereas a subset of target proteins exhibited exclusive interactions with either FFF1 (68) or FFF2 (1,475) probes. TRIM47 was significantly enriched with FFF2 probe over NP probe (FC = 2.43, adj p-value = 4.23E-10) but not with FFF1 probe (Figure 7C). Reversible small molecule interactome mapping using PALMS in human cells To confirm engagement of Trim47 by FFF2 probe in cells, we performed competition photoaffinity displacement assay using two structurally elaborated fragment analogues, CP1 and CP2, lacking the photoreactive warhead. HEK293T cells were treated for 30 min with 10 µM FFF2 or NP as a control. For competitive profiling, a 10 min-pretreatment with 160 µM competitor was followed by FFF2 probe treatment. After photolysis, FFF-modified proteins were enriched and identified as previously described. Candidates are designated as proteins enriched by FFF2 probe over NP with an FC >2 and adj p-value < 5% and competed off by the competitor with a FC > 2 and adj p-value < 5% over FFF2 probe. A total of 10 and 28 competed protein targets of FFF2 were mapped for CP1 and CP2, respectively (Figure 7D). The overlap between CP1 and CP2 fragment targets remained modest, with only three common proteins including Trim47. We found Trim47 to be a primary target of FFF2 and the two competitors, as it is significantly enriched over NP (FC = 2.94 and adj p-value = 3.71E-7) and this binding could be blocked upon co-incubation with CP1 (FC = 2.98 and adj p-value = 2.59E-6) or CP2 (FC = 3.16 and adj p-value = 7.70E-7).Evotec’s Industrial-Scale Mass Spectrometry-based Proteomics Solutions for Drugging the Undruggable p. 10 Figure 7: Small molecule interactome mapping by photoaffinity labeling mass spectrometry identifies reversible binders for Trim 47 in human cells (A) Structures of FFF probes. The “constant” (containing the diazirine photoreactive group and clickable alkyne group) and “variable” (consisting of small-molecule fragments) regions of FFF probes are shown in red and blue, respectively. (B) FFF probe-protein interactions in cells. HEK293T cells were treated with indicated FFF probes (20 or 200 µM) for 30 min, followed by photocrosslinking, lysis, CuAAC conjugation to a tetramethylrhodamine (TAMRA)-azide tag, separation by SDS-PAGE, and visualization by in-gel fluorescence scanning and Coomassie Blue staining. (C) Volcano plots showing relative protein enrichment values of FFF probes (200 µM) versus NP in HEK293T cells. FFF probes at 200 µM (30 min incubation) were compared to NP in pairwise experiments using single-shot label-free proteomics by dataindependent acquisition (DIA)-MS. Proteins enriched by the test FFF probe over NP with a fold change (FC) > 2 and adj pvalue < 5% were designated as test probe targets (red circles). Data is presented as the mean of biological replicated experiments (n = 3). (D) Chemical proteomic target engagement of CP1 and CP2 in HEK293T cells. The x-axis shows protein enrichment by active probe FFF2 over NP (10 μM), while the y-axis shows protein competition in cells treated with FFF2 (10 μM) and DMSO or the designated competitor analog (160 μM). Proteins that were significantly enriched by FFF2 over NP are plotted as gray circles. Proteins that were significantly competed off by competitor are plotted as pink circles. Candidates (highlighted in red circles) are designated as proteins enriched by FFF2 probe over NP with an FC >2 and adj p-value <5% and competed off by the competitor with a FC > 2 and adj p-value < 5% over FFF2 probe. Data is presented as the mean of biological replicated experiments (n = 3). (A) (D) (B) (B) (C) In this work, we have shown that HT-PALMS platform has the potential to map small-molecule fragmentprotein interactions directly in human cells. 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