Activity-based Protein Profiling Research Articles

Activity-based chemical proteomics reveals caffeic acid ameliorates pentylenetetrazol-induced seizures by covalently targeting aconitate decarboxylase 1

BackgroundEpilepsy is a neurological disorder characterized by recurrent seizures, tightly associated with neuroinflammation. Activation of inflammatory cells and molecules in damaged nervous tissues plays a pivotal role in epilepsy. Caffeic acid, one of the most abundant polyphenols in coffee, has shown potent protective effects as a phytomedicine in various neurological disorders. However, the direct protein targets and exact molecular mechanisms of caffeic acid in epilepsy, remain largely elusive.PurposeThis study aimed to explore the protective effects of caffeic acid in epilepsy and elucidate its underlying mechanism.MethodsIn this study, we established pentylenetetrazol-induced acute and kindling models of seizures. Additionally, a BV2 microglial cellular inflammation model was established by lipopolysaccharide stimulation. The potential direct protein targets of caffeic acid in BV2 cells were analyzed using an activity-based protein profiling (ABPP) with a caffeic acid probe. Various methods such as pull-down assay, immunofluorescence and cellular heat transfer assays were used for experimental validation. The anti-inflammatory effects of caffeic acid in LPS-activated BV2 cells was proved by knocking down the target protein.ResultsHere, we found that caffeic acid exhibits antiepileptic effects in pentylenetetrazol-induced epilepsy mice and exerts anti-neuroinflammation effect in vivo and in vitro. Besides, we discovered that caffeic acid directly binds to aconitate decarboxylase 1 and influenced its enzymatic activity. Moreover, we indicated that caffeic acid exhibits anti-neuroinflammation effect through aconitate decarboxylase 1 mediated PERK-NF-κB pathway in vitro.ConclusionIn summary, this study elucidates, for the first time, the potential antiepileptic targets and mechanism of action of caffeic acid using the ABPP strategy. Our study provides evidence supporting the utilization of caffeic acid as a promising therapeutic agent for treating epilepsy and neuroinflammation-related disorders.

Triticain alpha represents the major active papain-like cysteine protease in naturally senescing and ozone-treated leaves of wheat

Current background tropospheric ozone (O3) concentrations have significant adverse effects on wheat. O3 generally induces oxidative damages and premature leaf senescence leading to important yield losses. As leaf protein degradation and recycling is involved in both maintaining cell longevity during abiotic stresses and performing efficient nitrogen remobilization during senescence, we aimed to identify proteases involved in acidic endoproteolytic activities during natural and O3-induced leaf senescence in wheat. Field-grown plants of two winter wheat cultivars were exposed to ambient and semi-controlled chronic O3 concentrations, from pre-anthesis to grain harvest. Yield parameters were impacted by the most elevated O3 exposure for both cultivars. At the cellular level, our analysis revealed that both natural leaf senescence and O3 treatments induced a stimulation of acidic (pH 5.5) endoproteolytic activities, mostly due to papain-like cysteine proteases (PLCPs). Identification of active PLCPs using activity-based protein profiling (ABPP) revealed that Triticain α was the major active PLCP in senescing flag leaves and the only PLCP whose abundance increased with O3 stress, a result of positive transcriptional regulation. Our study provides novel insight into the implication of PLCP-mediated proteolysis in O3 sensitivity in a major crop.

Ailanthone induces triple-negative breast cancer cells death involving the inhibition of OTUB1-mediated ERRα deubiquitylation.

Triple-negative breast cancer (TNBC) remains the most aggressive subtype of breast cancer, and effective therapeutic strategies are needed. Estrogen-related receptor alpha (ERRα) is considered a promising target for managing TNBC. Here, we aimed to screen natural products to find downregulator of ERRα and elucidate its mechanism of action. TNBC cells (MDA-MB-231, MDA-MB-468, MDA-MB-453, and BT-549) were used for in vitro studies, and a subcutaneous MDA-MB-231 tumor model was created for in vivo studies. Immunofluorescence assessed protein distribution, while competitive activity-based protein profiling identified potential target proteins. Co-immunoprecipitation detected protein interactions and modifications, and a luciferase reporter assay evaluated ERRα transcriptional activity. The natural product Ailanthone (AIL) effectively induced cell death in TNBC cells by reducing the protein level of ERRα. The mechanism of action involved AIL promoting the degradation of ERRα through the ubiquitin-proteasome system, consequently reducing its transcriptional activity. The competitive-ABPP method mapped the profile of target proteins for AIL, and OTU domain-containing ubiquitin aldehyde-binding protein 1 (OTUB1) was identified as a pivotal target of AIL in regulating ERRα protein levels. OTUB1 was validated as a novel deubiquitinating enzyme for ERRα, with its C91 residue being crucial for this deubiquitination process. AIL was found to inhibit the enzyme activity of OTUB1 by interacting with the C91 residue and disrupt the interaction between OTUB1 and ERRα, ultimately leading to the inhibition of ERRα. AIL is a promising downregulator of ERRα, and the mechanism of this downregulation has been elucidated. Additionally, a new regulatory relationship between ERRα and OTUB1 is identified. The research presented in this article is anticipated to yield potential lead compounds for ERRα regulatory agents and to stimulate the development of novel therapeutic strategies designed to modulate ERRα activity for the treatment of TNBC.

Chemical tools to define and manipulate interferon-inducible Ubl protease USP18

Ubiquitin-specific protease 18 (USP18) is a multifunctional cysteine protease primarily responsible for deconjugating the interferon-inducible ubiquitin-like modifier ISG15 from protein substrates. Here, we report the design and synthesis of activity-based probes (ABPs) that incorporate unnatural amino acids into the C-terminal tail of ISG15, enabling the selective detection of USP18 activity over other ISG15 cross-reactive deubiquitinases (DUBs) such as USP5 and USP14. Combined with a ubiquitin-based DUB ABP, the USP18 ABP is employed in a chemoproteomics screening platform to identify and assess inhibitors of DUBs including USP18. We further demonstrate that USP18 ABPs can be utilized to profile differential activities of USP18 in lung cancer cell lines, providing a strategy that will help define the activity-related landscape of USP18 in different disease states and unravel important (de)ISGylation-dependent biological processes.

Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags.

Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ∼100 kinase active sites in a single LC-MS analysis. The complementary use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and pinpoint the exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC50 of 17 nM and EphB4 with an IC50 of 20 nM. Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 (DDR1) with an IC50 of 2.1 nM, suggesting that a DDR1-targeting regio-isomer of NVP-BHG712 was used. The promiscuity of XO44 toward ATP-binding pockets on non-kinase proteins facilitated the screening of additional off-target sites, revealing inosine-5'-monophosphate dehydrogenase 2 (IMPDH2) as a putative off-target. Expanding the search to other amino acids revealed that XO44, in addition to 745 lysines, also covalently linked 715 tyrosines, which significantly expands the competitive ABPP search space and highlights the added value of the site-specific method. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable new approach for competitive site-specific kinase inhibitor target profiling.

12/15-Lipoxygenase-Derived Electrophilic Lipid Modifications in Phagocytic Macrophages.

Macrophages remove apoptotic cells via phagocytosis, also known as efferocytosis, during inflammation to maintain tissue homeostasis. This process is accompanied by various metabolic changes in macrophages including the production of lipid metabolites by fatty acid oxygenases. Among these, highly reactive metabolites, called lipid-derived electrophiles (LDEs), modify cysteines and other nucleophilic amino acids in intracellular proteins. However, the landscape and functions of the modifications by these electrophilic metabolites have been poorly characterized. In this study, we used activity-based protein profiling to quantitatively profile the cysteine reactivity landscape and identify the potential targets of endogenous LDE modification during efferocytosis in mouse peritoneal macrophages. Using this methodology, we identified multiple cysteine sites that are highly likely to be modified by LDEs generated by 12/15-lipoxygenase (12/15-LOX), an efferocytosis-related fatty acid oxygenase that is highly expressed in peritoneal macrophages. Among these, actin-depolymerizing protein Cofilin-1 was found to be a target of 12/15-LOX-derived LDEs. In vitro Cofilin-1 activity was attenuated by 12/15-LOX-derived LDEs, and intracellular actin stabilization and efferocytosis were substantially enhanced by the LDE treatment of mouse peritoneal macrophages. These results highlighted the role of intracellular LDE modification during efferocytosis in macrophages.

Enantioselective OTUD7B fragment discovery through chemoproteomics screening and high-throughput optimisation

Deubiquitinating enzymes (DUBs) are key regulators of cellular homoeostasis, and their dysregulation is associated with several human diseases. The ovarian tumour protease (OTU) family of DUBs are biochemically well-characterised and of therapeutic interest, yet only a few tool compounds exist to study their cellular function and therapeutic potential. Here we present a chemoproteomics fragment screening platform for identifying novel DUB-specific hit matter, that combines activity-based protein profiling with high-throughput chemistry direct-to-biology optimisation to enable rapid elaboration of initial fragment hits against OTU DUBs. Applying these approaches, we identify an enantioselective covalent fragment for OTUD7B, and validate it using chemoproteomics and biochemical DUB activity assays.

Total syntheses of cyclohelminthol I-IV reveal a new cysteine-selective covalent reactive group.

Biocompatible covalent reactive groups (CRGs) play pivotal roles in several areas of chemical biology and the life sciences, including targeted covalent inhibitor design and preparation of advanced biologic drugs, such as antibody-drug conjugates. In this study, we present the discovery that the small, chlorinated polyketide natural product cyclohelminthiol II (CHM-II) acts as a new type of cysteine/thiol-targeting CRG incorporating both reversible and irreversible reactivity. We devise the first syntheses of four simple cyclohelminthols, (±)-cyclohelminthol I-IV, with selective chlorinations (at C2 and C5) and a Ni-catalyzed reductive cross coupling between an enone, a vinyl bromide and triethylsilyl chloride as the key steps. Unbiased biological profiling (cell painting) was used to discover a putative covalent mechanism for CHM-II in cells with subsequent validation experiments demonstrating mechanistic similarity to dimethyl fumarate (DMF) - a known (covalent) drug used in the treatment of multiple sclerosis. Focused biochemical experiments revealed divergent thiol-reactivity inherent to the CHM-II scaffold and through further chemical derivatization of CHM-II we applied activity-based protein profiling (ABPP)-workflows to show exclusive cysteine-labelling in cell lysate. Overall, this study provides both efficient synthetic access to the CHM-II chemotype - and neighboring chemical space - and proof-of-concept for several potential applications of this new privileged CRG-class within covalent chemical biology.

Identifying ENO1 as a protein target of chlorogenic acid to inhibit cellular senescence and prevent skin photoaging in mice.

Cellular senescence plays a critical role in repeated ultraviolet (UV) exposure-induced skin photoaging. Currently, from the perspective of regulating senescent cells, potent compounds or reliable protein targets that could effectively prevent skin photoaging have not yet been reported. Herein, we demonstrated that chlorogenic acid (CGA) significantly inhibited UVA-induced senescence of human dermis skin fibroblasts (HDF) cells by screening the natural product library. The activity-based protein profiling (ABPP) result revealed that Enolase 1 (ENO1) is one of the direct targets of CGA in HDF cells. Further mechanism research indicated that CGA covalently binds to ENO1, and prevented UVA-induced cellular senescence by suppressing the activity of ENO1 protein to block the glycolytic pathway. Importantly, we found that CGA dose-dependently reduced the skin wrinkle score, alleviated skin pathological features and inhibited senescent characteristics in a photoaging mouse model. The proteomic analysis revealed that CGA treatment effectively inhibited senescence-associated secretory phenotype (SASP) secretion and glycolysis in skin samples of mice. Collectively, our study not only demonstrated that inhibiting cell senescence is an effective anti-skin photoaging strategy, but also revealed that ENO1 is a promising protein target to prevent photoaging.

Activity-Based Proteome Profiling of Serum Serine Hydrolases: Application in Pediatric Abusive Head Trauma.

Traumatic brain injury (TBI), including pediatric abusive head trauma (AHT), is the leading cause of death and disability in children and young adults worldwide. The current understanding of trauma-induced molecular changes in the brain of human subjects with intracranial hemorrhage (ICH) remains inadequate and requires further investigation to improve the outcome and management of TBI in the clinic. Calcium-mediated damage at the site of brain injury has been shown to activate several catalytic enzymes. Serine hydrolases (SHs) are major catalytic enzymes involved in the biochemical pathways of blood coagulation, systemic inflammation, and neuronal signaling. Here, we investigated activity-based protein profiling (ABPP) coupled to liquid chromatography-mass spectrometry (LC-MS) by measuring the activity status of SH enzymes in the serum of infants with severe ICH as a consequence of AHT or atraumatic infants who died of sudden infant death syndrome (SIDS). Our proof-of-principle study revealed significantly reduced physiological activity of dozens of metabolic SHs in the serum of infants with severe AHT compared to the SIDS group, with some of the enzymes being related to neurodevelopment and basic brain metabolism. To our knowledge, this is the first study to investigate the ABPP of the SHs enzyme family to detect changes in their physiological activity in blood serum in severe TBI. We used antemortem (AM) serum from infants under the age of 2 years who were victims of AHT with a severe form of ICH. The analytical approach used in the proof-of-principle study shows reduced activities of serum serine lipases in AHT cases and could be further investigated in mild forms of AHT, which currently show 30% of misdiagnosed cases in clinics.

Kinact/KI Value Determination for Penicillin-Binding Proteins in Live Cells.

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are covalently inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts peptidoglycan biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC50 values are often employed as descriptors of enzyme inhibition and inhibitor selectivity, but can be misleading in the study of time-dependent, covalent inhibitors. Due to this disconnect, the second-order rate constant, kinact/KI, is a more appropriate metric of covalent-inhibitor potency. Despite being the gold standard measurement of potency, kinact/KI values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologues (such as the PBPs). Therefore, we developed a whole-cell kinact/KI assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent, activity-based probe, Bocillin-FL. Our results align with in vitro kinact/KI data and show a comparable relationship to previously established IC50 values. These results support the validity of our in vivo kinact/KI method as a means of obtaining β-lactam potency for a suite of PBPs to enable structure-activity relationship studies.

Haloacetonitriles Induce Structure-Related Cellular Toxicity Through Distinct Proteome Thiol Reaction Mechanisms.

Haloacetonitriles (HANs) are a class of toxic drinking water disinfection byproducts (DBPs). However, the toxicity mechanisms of HANs remain unclear. We herein investigated the structure-related in vitro toxicity of 6 representative HANs by utilizing complementary bioanalytical approaches. Dibromoacetonitrile (DBAN) displayed strong cytotoxicity and Nrf2 oxidative stress responses, followed by monohalogenated HANs (monoHANs) while other polyhalogenated HANs (polyHANs) exhibited little toxicity. Activity based protein profiling (ABPP) revealed that toxic HANs adduct to human proteome thiols, supporting thiol reactivity as the primary toxicity mechanism for HANs. By using glutathione (GSH) as a thiol surrogate, monoHANs reacted with GSH via SN2 while polyHANs reacted through ultrafast addition reactions. In contrast, DBAN generated an unexpected fully debrominated product and glutathione disulfide (GSSG). The unique reaction of DBAN with GSH was found to be mediated by radicals which was supported by electron paramagnetic resonance (EPR) spectroscopy and by radical trapping reagent reaction quenching. Shotgun proteomics further revealed that monoHANs and DBAN adducted to proteome thiols in live cells forming dehalogenated adducts. Multiple antioxidant proteins, SOD1, CSTB, and GAPDH, were adducted by toxic HANs at specific cysteine residues. This study highlights the structurally selective toxicity of HANs in human cells, which are attributed to their distinct reactions with proteome thiols.

Identification of TIGAR, a direct proteomic target associated with the hypoglycemic effect of Berberine

Diabetes mellitus is a global chronic metabolic disease and the prevalence of diabetes mellitus is increasing dramatically every year. Berberine (BBR) from Coptidis Rhizoma has potent hypoglycemic effects, however, the specific proteins targeted by berberine that contribute to its hypoglycemic action remain to be elucidated. In this work, TIGAR (TP53-induced glycolysis and apoptosis regulator) was identified as a direct target protein for berberine using activity-based protein profiling (ABPP) and other chemical proteomics techniques with active photoaffinity probes as chemical tools. In addition, the study revealed that berberine-targeted TIGAR attenuated the conversion of fructose-2, 6-bisphosphate to fructose-6-phosphate. This study demonstrated an innovative mechanism by which berberine directly targets TIGAR and its hypoglycemic effects. Therefore, TIGAR emerges as a novel target for the treatment of diabetes mellitus, with TIGAR inhibitors offering a new and promising therapeutic strategy for managing the disease.

Open-source Electrophilic Fragment Screening Platform to Identify Chemical Starting Points for UCHL1 Covalent Inhibitors

Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 <5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.

Precision Activity-Based α-Amylase Probes for Dissection and Annotation of Linear and Branched-Chain Starch-Degrading Enzymes.

α-Amylases are the workhorse enzymes of starch degradation. They are central to human health, including as targets for anti-diabetic compounds, but are also the key enzymes in the industrial processing of starch for biofuels, corn syrups, brewing and detergents. Dissection of the activity, specificity and stability of α-amylases is crucial to understanding their biology and allowing their exploitation. Yet, functional characterization lags behind DNA sequencing and genomics; and new tools are required for rapid analysis of α-amylase function. Here, we design, synthesize and apply new branched α-amylase activity-based probes. Using both α-1,6 branched and unbranched α-1,4 maltobiose activity-based probes we were able to explore the stability and substrate specificity of both a panel of human gut microbial α-amylases and a panel of industrially relevant α-amylases. We also demonstrate how we can detect and annotate the substrate specificity of α-amylases in the complex cell lysate of both a prominent gut microbe and a diverse compost sample by in-gel fluorescence and mass spectrometry. A toolbox of starch-active activity-based probes will enable rapid functional dissection of α-amylases. We envisage activity-based probes contributing to better selection and engineering of enzymes for industrial application as well as fundamental analysis of enzymes in human health.

Aggregation Induced Emission Based Luminogenic (AIEgenic) Probes for the Biomarker Detection.

Various biomarkers such as proteins play key roles in controlling crucial biochemical processes. The critical concentration of the biomarkers is important to maintain a healthy life. In fact, imbalance in concentration or irregular activity of these can lead to various diseases like Cancer, Alzheimer's etc. Therefore, the disease related biomarkers and their timely detection would be the key to control the illness. In the literature, a few fluorescent activity-based probes for the detection of such biomarkers are available.In this regard, several fluorescent probes are of great choice. Although these fluorescent probes face certain challenges such as aggregation caused quenching, which heavily affects the sensitivity and photostability is another major concern for many fluorescent probes. To overcome these challenges aggregation-induced emissive fluorescent probes could be an excellent alternative. Aggregation induced emissive luminogens (AIEgens) offer higher signal to noise ratios and found to possess better photostability during sensing and imaging. In the present review we have summarized the development of AIEgenic probes for sensing and imaging of disease related biomarkers.

Carmaphycin B-Based Proteasome Inhibitors to Treat Human African Trypanosomiasis: Structure-Activity Relationship and In Vivo Efficacy.

The proteasome is essential for eukaryotic cell proteostasis, and inhibitors of the 20S proteasome are progressing preclinically and clinically as antiparasitics. We screenedTrypanosoma brucei, the causative agent of human and animal African trypanosomiasis, in vitro with a set of 27 carmaphycin B analogs, irreversible epoxyketone inhibitors that were originally developed to inhibit thePlasmodium falciparum20S (Pf20S). The structure-activity relationship was distinct from that of the human c20S antitarget by the acceptance of d-amino acids at the P3 position of the peptidyl backbone to yield compounds with greatly decreased toxicity to human cells. For the three most selective compounds, binding to the Tb20S β5 catalytic subunit was confirmed by competition with a fluorescent activity-based probe. For one compound, J-80, with its P3 d-configuration, the differential binding to the parasite's β5 subunit was supported by both covalent and noncovalent docking analysis. Further, J-80 was equipotent against both Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense in vitro. In a mouse model of Stage 1 T. brucei infection, a single intraperitoneal (i.p.) dose of 40 mg/kg J-80 halted the growth of the parasite, and when given at 50 mg/kg i.p. twice daily for 5 days, parasitemia was decreased to below the detectable limit, with parasite recrudescence 48 h after the last dose. The in vivo proof of principle demonstrated by a potent, selective, and irreversible inhibitor of Tb20S reveals an alternative path to the development of kinetoplastid proteasome inhibitors that differs from the current focus on allosteric reversible inhibitors.

Production of Natural Penicillin-Binding Protein 2 and Development of a Signal-Amplified Fluorescence Polarization Assay for the Determination of 28 Beta-Lactam Antibiotics in Milk.

In this study, two magnetic activity-based protein profiling probes based on cephradine and amoxicillin were first synthesized that were used to produce the natural penicillin-binding protein 2 of Escherichia coli. After characterization by using LC-ESI-MS/MS, it was found that the obtained proteins by using the two probes were the same. The molecular docking for 28 beta-lactam antibiotics showed that the key amino acids were Ser330 and Ser387, the main intermolecular forces were hydrogen bond and hydrophobic interaction, and the main binding sites in their molecules were on the beta-lactam ring. Then this protein was combined with streptavidin-labeled tracer and biotinylated fluorescein isothiocyanate to establish a signal-amplified fluorescence polarization assay to determine the 28 drugs in milk. The limits of detection ranged from 0.07 to 2.21 ng/mL, and the sensitivities for the 28 drugs were improved 4 to 48-fold in comparison with the use of a fluorescein isothiocyanate-labeled fluorescent tracer. Therefore, this method could be used for rapid multiscreening of the 28 beta-lactam antibiotics in milk.

Covalent Inhibitors of KEAP1 with Exquisite Selectivity.

The NRF2-KEAP1 interaction is central for cytoprotection against stresses, giving it high clinical significance. Covalent modification of KEAP1 is an efficient approach, but the covalent inhibitors used in the clinic carry undesired side effects originating in their moderate selectivity. Starting with a phenotypic screen, we identified a new covalent inhibitor chemotype that was optimized to deliver a series of potent and highly selective KEAP1 binders. While the developed compounds showed both cellular and in vivo activity, upregulating antioxidant response element-dependent target genes, they showed no genotoxicity in vitro. The lead compound exhibited broad selectivity in activity-based protein profiling and showed no significant interaction with a panel of commonly studied receptors nor with a broad panel of kinases. The nature of its interaction with KEAP1 and the origin of its selectivity were revealed by X-ray crystallography.

Systematic Optimization of Activity-Based Protein Profiling for Identification of Polysorbate-Degradative Enzymes in Biotherapeutic Drug Substance down to 10 ppb.

The identification and control of high-risk host cell proteins (HCPs) in biotherapeutics development are crucial for ensuring product quality and shelf life. Specifically, HCPs with hydrolase activity can cause the degradation of excipient polysorbates (PS), leading to a decrease in the shelf life of the drug product. In this study, we systematically optimized every step of an activity-based protein profiling (ABPP) workflow to identify trace amounts of active polysorbate-degradative enzymes (PSDEs) in biotherapeutic process intermediates. Evaluation of various parameters during sample preparation pinpointed the optimal pH level and fluorophosphonate (FP)-biotin concentration. Moreover, the combined use of a short liquid chromatography gradient and the fast-scanning parallel accumulation-serial fragmentation (PASEF) methodology increased sample throughput without compromising identification coverage. Tuning the trapped ion mobility spectrometry (TIMS) parameters further enhanced sensitivity. In addition, we evaluated various data acquisition modes, including PASEF combined with data-dependent acquisition (DDA PASEF), data-independent acquisition (diaPASEF), or parallel reaction monitoring (prm-PASEF). By employing the newly optimized ABPP workflow, we successfully identified PSDEs at a concentration as low as 10 ppb in a drug substance sample. Finally, the new workflow enabled us to detect a PSDE that could not be detected with the original workflow during a PS degradation root-cause investigation.