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System-wide methods to monitor protein activity are still underused in drug discovery. This Review discusses the potential of proteomics and chemoproteomics approaches for target identification, validation and identification of safety hazards. Proteins are the main targets of most drugs; however, system-wide methods to monitor protein activity and function are still underused in drug discovery. Novel biochemical approaches, in combination with recent developments in mass spectrometry-based proteomics instrumentation and data analysis pipelines, have now enabled the dissection of disease phenotypes and their modulation by bioactive molecules at unprecedented resolution and dimensionality. In this Review, we describe proteomics and chemoproteomics approaches for target identification and validation, as well as for identification of safety hazards. We discuss innovative strategies in early-stage drug discovery in which proteomics approaches generate unique insights, such as targeted protein degradation and the use of reactive fragments, and provide guidance for experimental strategies crucial for success.

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing four different cell types derived from 14 NGLY1 deficient patients and 17 controls. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared with parents, residual and likely non-functional NGLY1 protein was detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosome biogenesis and mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cysteine ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human “mammalian target of rapamycin” (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kβ) and cyclic guanosine monophosphate–dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kβ in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kβ. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kβ and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.

Focal adhesion kinase (FAK) is a key mediator of tumour progression and metastasis. To date, clinical trials of FAK inhibitors have reported disappointing efficacy for oncology indications. We report the design and characterisation of GSK215, a potent, selective, FAK-degrading Proteolysis Targeting Chimera (PROTAC) based on a binder for the VHL E3 ligase and the known FAK inhibitor VS-4718. X-ray crystallography revealed the molecular basis of the highly cooperative FAK-GSK215-VHL ternary complex, and GSK215 showed differentiated in-vitro pharmacology compared to VS-4718. In mice, a single dose of GSK215 induced rapid and prolonged FAK degradation, giving a long-lasting effect on FAK levels (≈96 h) and a marked PK/PD disconnect. This tool PROTAC molecule is expected to be useful for the study of FAK-degradation biology in vivo, and our results indicate that FAK degradation may be a differentiated clinical strategy versus FAK inhibition for the treatment of cancer.

We present a one-step Ugi reaction protocol for the expedient synthesis of photoaffinity probes for live-cell MS-based proteomics. The reaction couples an amine affinity function with commonly used photoreactive groups, and a variety of handle functionalities. Using this technology, a series of pan-BET (BET: bromodomain and extra-terminal domain) selective bromodomain photoaffinity probes were obtained by parallel synthesis. Studies on the effects of photoreactive group, linker length and irradiation wavelength on photocrosslinking efficiency provide valuable insights into photoaffinity probe design. Optimal probes were progressed to MS-based proteomics to capture the BET family of proteins from live cells and reveal their potential on- and off-target profiles.

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. However, current data analysis strategies use preset thresholds that can lead to suboptimal sensitivity/specificity tradeoffs and limited comparability across datasets. Here, we present a method based on statistical hypothesis testing on curves, which provides control of the false discovery rate. We apply it to several datasets probing epigenetic drugs and a metabolite. This leads us to detect off-target drug engagement, including the finding that the HDAC8 inhibitor PCI-34051 and its analog BRD-3811 bind to and inhibit leucine aminopeptidase 3. An implementation is available as an R package from Bioconductor (https://bioconductor.org/packages/TPP2D). We hope that our method will facilitate prioritizing targets from thermal profiling experiments., 2D-thermal proteome profiling (2D-TPP) is a powerful assay for probing interactions of proteins with small molecules in their native context. Here the authors provide a statistical method for false discovery rate controlled analysis for 2D-TPP applications.

Reversible protein phosphorylation is an important mechanism for regulating (dis)assembly of biomolecular condensates. However, condensate-specific phosphosites remain largely unknown, thereby limiting our understanding of the underlying mechanisms. Here, we combine solubility proteome profiling with phosphoproteomics to quantitatively map several hundred phosphosites enriched in either soluble or condensate-bound protein subpopulations, including a subset of phosphosites modulating protein–RNA interactions. We show that multi-phosphorylation of the C-terminal disordered segment of heteronuclear ribonucleoprotein A1 (HNRNPA1), a key RNA-splicing factor, reduces its ability to locate to nuclear clusters. For nucleophosmin 1 (NPM1), an essential nucleolar protein, we show that phosphorylation of S254 and S260 is crucial for lowering its partitioning to the nucleolus and additional phosphorylation of distal sites enhances its retention in the nucleoplasm. These phosphorylation events decrease RNA and protein interactions of NPM1 to regulate its condensation. Our dataset is a rich resource for systematically uncovering the phosphoregulation of biomolecular condensates.

N-Glycanase 1 (NGLY1) deficiency is an ultra-rare, complex and devastating neuromuscular disease. Patients display multi-organ symptoms including developmental delays, movement disorders, seizures, constipation and lack of tear production. NGLY1 is a deglycosylating protein involved in the degradation of misfolded proteins retrotranslocated from the endoplasmic reticulum (ER). NGLY1-deficient cells have been reported to exhibit decreased deglycosylation activity and an increased sensitivity to proteasome inhibitors. We show that the loss of NGLY1 causes substantial changes in the RNA and protein landscape of K562 cells and results in downregulation of proteasomal subunits, consistent with its processing of the transcription factor NFE2L1. We employed the CMap database to predict compounds that can modulate NGLY1 activity. Utilizing our robust K562 screening system, we demonstrate that the compound NVP-BEZ235 (Dactosilib) promotes degradation of NGLY1-dependent substrates, concurrent with increased autophagic flux, suggesting that stimulating autophagy may assist in clearing aberrant substrates during NGLY1 deficiency.

Proteolysis-Targeting Chimeras (PROTACs) are heterobifunctional small-molecules that can promote the rapid and selective proteasome-mediated degradation of intracellular proteins through the recruitment of E3 ligase complexes to non-native protein substrates. The catalytic mechanism of action of PROTACs represents an exciting new modality in drug discovery that offers several potential advantages over traditional small-molecule inhibitors, including the potential to deliver pharmacodynamic (PD) efficacy which extends beyond the detectable pharmacokinetic (PK) presence of the PROTAC, driven by the synthesis rate of the protein. Herein we report the identification and development of PROTACs that selectively degrade Receptor-Interacting Serine/Threonine Protein Kinase 2 (RIPK2) and demonstrate in vivo degradation of endogenous RIPK2 in rats at low doses and extended PD that persists in the absence of detectable compound. This disconnect between PK and PD, when coupled with low nanomolar potency, offers the potential for low human doses and infrequent dosing regimens with PROTAC medicines., Mares et al. develop Proteolysis-Targeting Chimeras (PROTACs) that degrade its target RIPK2 in vivo at low doses for a prolonged period of time. This study suggests that PROTAC has a therapeutic potential that is superior to traditional RIPK2 small-molecule inhibitors.

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.

Optimization of a lead series of PI3Kδ inhibitors based on a dihydroisobenzofuran core led to the identification of potent, orally bioavailable compound 19. Selectivity profiling of compound 19 showed similar potency for class III PI3K, Vps34, and PI3Kδ, and compound 19 was not well-tolerated in a 7-day rat toxicity study. Structure-based design led to an improvement in selectivity for PI3Kδ over Vps34 and, a focus on oral phramacokinetics properties resulted in the discovery of compound 41, which showed improved toxicological outcomes at similar exposure levels to compound 19.

N-Glycanase 1 (NGLY1) deficiency is a rare and complex genetic disorder. Although recent studies have shed light on the molecular underpinnings of NGLY1 deficiency, a systematic characterization of gene and protein expression changes in patient-derived cells has been lacking. Here, we performed RNA-sequencing and mass spectrometry to determine the transcriptomes and proteomes of 66 cell lines representing 4 different cell types derived from 14 NGLY1 deficient patients and 17 controls. While gene and protein expression levels agreed well with each other, expression differences were more pronounced at the protein level. Although NGLY1 protein levels were up to 9.5-fold downregulated in patients compared to parent controls, depending on the genotype, NGLY1 protein was still detectable in all patient-derived lymphoblastoid cell lines. Consistent with the role of NGLY1 as a regulator of the transcription factor Nrf1, we observed a cell type-independent downregulation of proteasomal genes in NGLY1 deficient cells. In contrast, genes involved in ribosomal mRNA processing were upregulated in multiple cell types. In addition, we observed cell type-specific effects. For example, genes and proteins involved in glutathione synthesis, such as the glutamate-cystein ligase subunits GCLC and GCLM, were downregulated specifically in lymphoblastoid cells. We provide a web application that enables access to all results generated in this study at https://apps.embl.de/ngly1browser. This resource will guide future studies of NGLY1 deficiency in directions that are most relevant to patients.

In order to understand the full mechanism of action of candidate drug molecules, it is critical to thoroughly characterize their interactions with endogenously expressed pharmacological targets and potentially undesired off-targets. Here we describe a chemoproteomics approach that is based on functionalized analogs of the compound of interest to affinity enrich target proteins from cell or tissue extracts. Experiments are designed as competition binding assays where free parental compound is spiked at a range of concentrations into the extracts to compete specific binders off the immobilized compound matrix. Quantification of matrix-bound proteins enables generation of dose-response curves and half-binding concentrations. In addition, the influence of the affinity matrix on the equilibrium is determined in rebinding experiments. TMT10 isobaric mass tags enable analyzing repeat binding and dose-dependent competition samples in a single mass spectrometry analysis run, thus enabling the efficient identification of targets, apparent dissociation constants, and selectivity of small molecules in a single experiment. The workflow is exemplified with the kinase inhibitor sunitinib.

Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys and gut. Angiotensin converting enzyme (ACE) 2, the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines, but did not affect NF-κB-regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB-signaling could potentially have clinical application for severe COVID-19.

[Image: see text] The serine/threonine protein kinase TBK1 (Tank-binding Kinase-1) is a noncanonical member of the IkB kinase (IKK) family. This kinase regulates signaling pathways in innate immunity, oncogenesis, energy homeostasis, autophagy, and neuroinflammation. Herein, we report the discovery and characterization of a novel potent and highly selective TBK1 inhibitor, GSK8612. In cellular assays, this small molecule inhibited toll-like receptor (TLR)3-induced interferon regulatory factor (IRF)3 phosphorylation in Ramos cells and type I interferon (IFN) secretion in primary human mononuclear cells. In THP1 cells, GSK8612 was able to inhibit secretion of interferon beta (IFNβ) in response to dsDNA and cGAMP, the natural ligand for STING. GSK8612 is a TBK1 small molecule inhibitor displaying an excellent selectivity profile and therefore represents an ideal probe to further dissect the biology of TBK1 in models of immunity, neuroinflammation, obesity, or cancer.

Mass spectrometry-based secretomics approaches frequently utilize serum-free culture conditions to circumvent serum-induced interference and to increase analytical depth. However, this can negatively affect a wide range of cellular functions and cell viability. These effects become particularly apparent when investigating transcriptionally regulated secretion events and feedback-loops in response to perturbations that require 48 h or more to fully manifest. We present an "interval-based" secretomics workflow, which determines protein secretion rates in short serum-free time windows. Relative quantification using tandem mass tags enables precise monitoring of time-dependent changes. We applied this approach to determine temporal profiles of protein secretion in the hepatocyte model cell lines HepG2 and HepaRG after stimulation of the acute-phase response (APR) by the cytokines IL1b and IL6. While the popular hepatocarcinoma cell line HepG2 showed an incomplete APR, secretion patterns derived from differentiated HepaRG cells recapitulated the expected APR more comprehensively. For several APR response proteins, substantial secretion was only observed after 72 h, a time window at which cell fitness is substantially impaired under serum-free cell culture conditions. The interval-based secretomics approach enabled the first comprehensive analysis of time-dependent secretion of liver cell models in response to these proinflammatory cytokines. The extended time range facilitated the observation of distinct chronological phases and cytokine-dependent secretion phenotypes of the APR. IL1b directed the APR toward pathogen defense over three distinct phases-chemotaxis, effector, clearance-while IL6 directed the APR toward regeneration. Protein shedding on the cell surface was pronounced upon IL1b stimulation, and small molecule inhibition of ADAM and matrix metalloproteases identified induced as well as constitutive shedding events. Inhibition of ADAM proteases with TAPI-0 resulted in reduced shedding of the sorting receptor SORT1, and an attenuated cytokine response suggesting a direct link between cell surface shedding and cytokine secretion rates.

Multiplexed quantitative proteomics enabled complex workflows to study the mechanisms by which small molecule drugs interact with the proteome such as thermal proteome profiling (TPP) or multiplexed proteome dynamics profiling (mPDP). TPP measures changes in protein thermal stability in response to drug treatment and thus informs on direct targets and downstream regulation events, while the mPDP approach enables the discovery of regulated protein synthesis and degradation events caused by small molecules and other perturbations. The isobaric mass tags available for multiplexed proteomics have thus far limited the efficiency and sensitivity by which such experiments could be performed. Here we evaluate a recent generation of 16-plex isobaric mass tags and demonstrate the sensitive and time efficient identification of Staurosporine targets in HepG2 cell extracts by recording full thermal denaturation/aggregation profiles of vehicle and compound treated samples in a single mass spectrometry experiment. In 2D-TPP experiments, isothermal titration over seven concentrations per temperature enabled comprehensive selectivity profiling of Staurosporine with EC50 values for kinase targets tightly matching to the kinobeads gold standard assay. Finally, we demonstrate time and condition-based multiplexing of dynamic SILAC labeling experiments to delineate proteome-wide effects of the molecular glue Indisulam on synthesis and degradation rates.

In order to understand the full mechanism of action of candidate drug molecules, it is critical to thoroughly characterize their interactions with endogenously expressed pharmacological targets and potentially undesired off-targets. Here we describe a chemoproteomics approach that is based on functionalized analogs of the compound of interest to affinity enrich target proteins from cell or tissue extracts. Experiments are designed as competition binding assays where free parental compound is spiked at a range of concentrations into the extracts to compete specific binders off the immobilized compound matrix. Quantification of matrix-bound proteins enables generation of dose-response curves and half-binding concentrations. In addition, the influence of the affinity matrix on the equilibrium is determined in rebinding experiments. TMT10 isobaric mass tags enable analyzing repeat binding and dose-dependent competition samples in a single mass spectrometry analysis run, thus enabling the efficient identification of targets, apparent dissociation constants, and selectivity of small molecules in a single experiment. The workflow is exemplified with the kinase inhibitor sunitinib.

The thermal stability of proteins can be altered when they interact with small molecules. Thus monitoring the thermal stability of proteins under various cellular perturbations can provide insights into protein function, as well as potentially determine drug targets and off-targets. Thermal proteome profiling is a multiplexed mass-spectrommetry method for monitoring the melting behaviour of thousands of proteins in a single experiment. In essence, thermal proteome profiling assumes that proteins denature upon heating and hence become insoluble. Thus, by tracking the relative solubility of proteins at sequentially increasing temperatures, one can report on protein thermal stability. Standard thermodynamics predicts a sigmoidal relationship between temperature and relative solubility and this is the basis of current statistical procedures. However, current methods do not model deviations from this behaviour and they do not quantify uncertainty in the melting profiles. To overcome these challenges, we propose Bayesian functional data analysis tools which allow complex temperature-solubility behaviours. Our methods have improved sensitivity over the state-of-the art, identify new drug-protein associations and have less restrictive assumptions than current approaches. Our methods allows for comprehensive analysis of proteins that deviate from the predicted sigmoid behaviour and we uncover potentially biphasic phenomena with a series of published datasets.

Lysine methylation of BRD4 selectively determines the recruitment of E2F1 to specific target genes to regulate mRNA translation., The transcriptional coactivator BRD4 has a fundamental role in transcription regulation and thus became a promising epigenetic therapeutic candidate to target diverse pathologies. However, the regulation of BRD4 by posttranslational modifications has been largely unexplored. Here, we show that BRD4 is methylated on chromatin at lysine-99 by the protein lysine methyltransferase SETD6. BRD4 methylation negatively regulates the expression of genes that are involved in translation and inhibits total mRNA translation in cells. Mechanistically, we provide evidence that supports a model where BRD4 methylation by SETD6 does not have a direct role in the association with acetylated histone H4 at chromatin. However, this methylation specifically determines the recruitment of the transcription factor E2F1 to selected target genes that are involved in mRNA translation. Together, our findings reveal a previously unknown molecular mechanism for BRD4 methylation–dependent gene-specific targeting, which may serve as a new direction for the development of therapeutic applications.

In malaria parasites, all cGMP-dependent signalling is mediated through a single cGMP-dependent protein kinase (PKG), a major function of which is to control essential calcium signals. However, how PKG transmits these signals in the absence of known second messenger-dependent calcium channels or scaffolding proteins is unknown. Here we identify a polytopic membrane protein, ICM1, with homology to transporters and calcium channels that is tightly-associated with PKG in both Plasmodium falciparum asexual blood stages and P. berghei gametocytes. Phosphoproteomic analyses in both Plasmodium species reveal multiple ICM1 phosphorylation events dependent upon PKG activity. Stage-specific depletion of P. berghei ICM1 blocks gametogenesis due to the inability of mutant parasites to mobilise intracellular calcium upon PKG activation, whilst conditional loss of P. falciparum ICM1 results in reduced calcium mobilisation, defective egress and lack of invasion. Our findings provide new insights into atypical calcium homeostasis in malaria parasites essential for pathology and disease transmission.

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. We present a statistical analysis method with reliable control of the false discovery rate and apply it to several datasets probing epigenetic drugs. We detect off-target drug engagement in unrelated protein families.

Myotonic dystrophy type 1 (DM1) is an RNA-based disease with no current treatment. It is caused by a transcribed CTG repeat expansion within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Mutant repeat expansion transcripts remain in the nuclei of patients' cells, forming distinct microscopically detectable foci that contribute substantially to the pathophysiology of the condition. Here, we report small-molecule inhibitors that remove nuclear foci and have beneficial effects in the HSALR mouse model, reducing transgene expression, leading to improvements in myotonia, splicing, and centralized nuclei. Using chemoproteomics in combination with cell-based assays, we identify cyclin-dependent kinase 12 (CDK12) as a druggable target for this condition. CDK12 is a protein elevated in DM1 cell lines and patient muscle biopsies, and our results showed that its inhibition led to reduced expression of repeat expansion RNA. Some of the inhibitors identified in this study are currently the subject of clinical trials for other indications and provide valuable starting points for a drug development program in DM1.

Bromodomain inhibitors revisited Bromodomain and extraterminal domain (BET) proteins contribute to the pathogenesis of cancer and immune diseases through their effects on transcriptional regulation. BET proteins contain two nearly identical bromodomains, BD1 and BD2, structural modules that have attracted great interest as targets for drug development. First-generation drugs that inhibited both BD1 and BD2 showed promising therapeutic activity in preclinical models but proved to be less efficacious in clinical trials. Gilan et al. took a different approach and designed drugs that selectively inhibited BD1 or BD2 (see the Perspective by Filippakopoulos and Knapp). They found that BD1 and BD2 inhibitors altered gene expression in different ways and that BD2 inhibitors had greater therapeutic activity than BD1 inhibitors in preclinical models of inflammation and autoimmune disease. Science , this issue p. 387 ; see also p. 367

Numerous drugs and endogenous ligands bind to cell surface receptors leading to modulation of downstream signaling cascades and frequently to adaptation of the plasma membrane proteome. In-depth analysis of dynamic processes at the cell surface is challenging due to biochemical properties and low abundances of plasma membrane proteins. Here we introduce cell surface thermal proteome profiling for the comprehensive characterization of ligand-induced changes in protein abundances and thermal stabilities at the plasma membrane. We demonstrate drug binding to extracellular receptors and transporters, discover stimulation-dependent remodeling of T cell receptor complexes and describe a competition-based approach to measure target engagement of G-protein-coupled receptor antagonists. Remodeling of the plasma membrane proteome in response to treatment with the TGFB receptor inhibitor SB431542 leads to partial internalization of the monocarboxylate transporters MCT1/3 explaining the antimetastatic effects of the drug. Cell surface thermal proteome profiling allows characterization of ligand-induced changes in protein abundances and thermal stabilities at the plasma membrane.

Plants are essential for life and are extremely diverse organisms with unique molecular capabilities(1). Here we present a quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana. Our analysis provides initial answers to how many genes exist as proteins (more than 18,000), where they are expressed, in which approximate quantities (a dynamic range of more than six orders of magnitude) and to what extent they are phosphorylated (over 43,000 sites). We present examples of how the data may be used, such as to discover proteins that are translated from short open-reading frames, to uncover sequence motifs that are involved in the regulation of protein production, and to identify tissue-specific protein complexes or phosphorylation-mediated signalling events. Interactive access to this resource for the plant community is provided by the ProteomicsDB and ATHENA databases, which include powerful bioinformatics tools to explore and characterize Arabidopsis proteins, their modifications and interactions.A quantitative atlas of the transcriptomes, proteomes and phosphoproteomes of 30 tissues of the model plant Arabidopsis thaliana provides a valuable resource for plant research.

Kinobeads are a set of promiscuous kinase inhibitors immobilized on sepharose beads for the comprehensive enrichment of endogenously expressed protein kinases from cell lines and tissues. These beads enable chemoproteomics profiling of kinase inhibitors of interest in dose-dependent competition studies in combination with quantitative mass spectrometry. We present improved bead matrices that capture more than 350 protein kinases and 15 lipid kinases from human cell lysates, respectively. A multiplexing strategy is suggested that enables determination of apparent dissociation constants in a single mass spectrometry experiment. Miniaturization of the procedure enabled determining the target selectivity of the clinical BCR-ABL inhibitor dasatinib in peripheral blood mononuclear cell (PBMC) lysates from individual donors. Profiling of a set of Jak kinase inhibitors revealed kinase off-targets from nearly all kinase families underpinning the need to profile kinase inhibitors against the kinome. Potently bound off-targets of clinical inhibitors suggest polypharmacology, e.g. through MRCK alpha and beta, which bind to decernotinib with nanomolar affinity.

The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of TB research. There are multiple different experimental approaches that can be employed in the discovery of anti-TB agents. Notably, inhibitors of MmpL3 are numerous and structurally diverse in Mtb and have been discovered through the generation of spontaneous resistant mutants and subsequent whole genome sequencing studies. However, this approach is not always reliable and can lead to incorrect target assignment and requires orthogonal confirmatory approaches. In fact, many of these inhibitors have also been shown to act as multi-target agents, with secondary targets in Mtb, as well as in other non-MmpL3-containing pathogens. Herein, we have investigated further the cellular targets of the MmpL3-inhibitor BM212 and a number of BM212 analogues. To determine the alternative targets of BM212, which may have been masked by MmpL3 mutations, we have applied a combination of chemo-proteomic profiling using bead-immobilised BM212 derivatives and protein extracts, along with whole-cell and biochemical assays. The study identified EthR2 (Rv0078) as a protein that binds BM212 analogues. We further demonstrated binding of BM212 to EthR2 through an in vitro tryptophan fluorescence assay, which showed significant quenching of tryptophan fluorescence upon addition of BM212. Our studies have demonstrated the value of revisiting drugs with ambiguous targets, such as MmpL3, in an attempt to find alternative targets and the study of off-target effects to understand more precisely target engagement of new hits emerging from drug screening campaigns.

The availability of high quality probes for specific protein targets is fundamental to the investigation of their function and their validation as therapeutic targets. We report the utilization of a dedicated chemoproteomic assay platform combining affinity enrichment technology with high-resolution protein mass spectrometry to the discovery of a novel nicotinamide isoster, the tetrazoloquinoxaline 41, a highly potent and selective tankyrase inhibitor. We also describe the use of 41 to investigate the biology of tankyrase, revealing the compound induced growth inhibition of a number of tumor derived cell lines, demonstrating the potential of tankyrase inhibitors in oncology.

The plasma membrane proteome plays a crucial role in inter- and intracellular signaling, cell survival, and cell identity. As such, it is a prominent target for pharmacological intervention. The relatively low abundance of this subproteome in conjunction with challenging extractability and solubility still hampers its comprehensive analysis. Here, we combined a chemical glycoprotein-tagging strategy with mass spectrometry to enable comprehensive analysis of the cell-surface glycoproteome. To benchmark this workflow and to provide guidance for cell line selection for functional experiments, we generated an inventory of the N-linked cell-surface glycoproteomes of 15 standard laboratory human cell lines and three primary lymphocytic cell types. On average, about 900 plasma membrane and secreted proteins were identified per experiment, including more than 300 transporters and ion channels. Primary cells displayed distinct expression of surface markers and transporters underpinning the importance of carefully validating model cell lines selected for the study of cell surface-mediated processes. To monitor dynamic changes of the cell-surface proteome in a highly multiplexed experiment, we employed an isobaric mass tag-based chemical labeling strategy. This enabled the time-resolved analysis of plasma membrane protein presentation during differentiation of the monocytic suspension cell line THP-1 into macrophage-like adherent cells. Time-dependent changes observed in membrane protein presentation reflect functional remodeling during the phenotypic transition in three distinct phases: rapid surface presentation and secretion of proteins from intracellular pools concurrent with rapid internalization of no longer needed proteins and finally delayed presentation of newly synthesized macrophage markers. Perturbation of this process using marketed receptor tyrosine kinase inhibitors revealed dasatinib to severely compromise macrophage differentiation due to an off-target activity. This finding suggests that dynamic processes can be highly vulnerable to drug treatment and should be monitored more rigorously to identify adverse drug effects.

Gene knockouts (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While DNA editing efficiency is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here, we devised an experimental strategy combining RNA-seq and triple-stage mass spectrometry (SPS-MS3) to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins, or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9 generated KO lines is necessary for phenotype interpretation.

We have used a mass spectrometry-based proteomic approach to compile an atlas of the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans and covering melting temperatures of 30–90 °C. Protein sequence, composition and size affect thermal stability in prokaryotes and eukaryotic proteins show a nonlinear relationship between the degree of disordered protein structure and thermal stability. The data indicate that evolutionary conservation of protein complexes is reflected by similar thermal stability of their proteins, and we show examples in which genomic alterations can affect thermal stability. Proteins of the respiratory chain were found to be very stable in many organisms, and human mitochondria showed close to normal respiration at 46 °C. We also noted cell-type-specific effects that can affect protein stability or the efficacy of drugs. This meltome atlas broadly defines the proteome amenable to thermal profiling in biology and drug discovery and can be explored online at http://meltomeatlas.proteomics.wzw.tum.de:5003/ and http://www.proteomicsdb.org. The meltome atlas compiles the thermal stability of 48,000 proteins across 13 species ranging from archaea to humans, providing a resource for analyzing protein stability in the context of function and interactions.

Plant growth and development are regulated by a tightly controlled interplay between cell division, cell expansion and cell differentiation during the entire plant life cycle from seed germination to maturity and seed propagation. To explore some of the underlying molecular mechanisms in more detail, we selected different aerial tissue types of the model plant Arabidopsis thaliana, namely rosette leaf, flower and silique/seed and performed proteomic, phosphoproteomic and transcriptomic analyses of sequential growth stages using tandem mass tag-based mass spectrometry and RNA sequencing. With this exploratory multi-omics dataset, development dynamics of photosynthetic tissues can be investigated from different angles. As expected, we found progressive global expression changes between growth stages for all three omics types and often but not always corresponding expression patterns for individual genes on transcript, protein and phosphorylation site level. The biggest difference between proteomic- and transcriptomic-based expression information could be observed for seed samples. Proteomic and transcriptomic data is available via ProteomeXchange and ArrayExpress with the respective identifiers PXD018814 and E-MTAB-7978., Measurement(s) transcriptome • Proteome • Phosphoproteome Technology Type(s) RNA sequencing • liquid chromatography-tandem mass spectrometry Factor Type(s) organism part • growth stage Sample Characteristic - Organism Arabidopsis thaliana Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12998993