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High throughput in vitro assays for screening chemical hazards focus primarily on specific receptors that are linked with certain adverse outcome pathways, neglecting potential novel endpoints or pathways induced by emerging pollutants. Identifying target proteins that interact with pollutants contributes to finding potential molecular initiating events under the adverse outcome pathways framework. Mass spectrometry-based thermal proteome profiling (TPP) assays have permitted uncovering binding targets of pollutants across the whole proteome. Based on the principle that proteins are thermally stabilized after binding with chemicals, TPP differentiates protein targets by determining the soluble fraction of proteins that remain stable after heat stress. Thus, TPP facilitates qualitative and quantitative measurements of chemical-protein interactions (CPIs) without modifications on chemical structures or immobilization of target proteins. In this mini-review, we introduced the principles, development and procedures of TPP, and summarized its applications in identifying protein targets and speculating toxicity pathways for emerging pollutants in environmental toxicological studies. Additionally, since measurements of CPIs using TPP for multiple chemicals could be labor- and cost-intensive, machine learning-based modeling is a feasible alternative to dissect CPIs due to its capability to mine intrinsic properties determining CPIs. Therefore, the recent development of machine learning models for CPI prediction was reviewed. Lastly, we envisioned prospects of combining TPP data with machine learning for CPI prediction, and the possibility of applying TPP to interpret toxicity pathways and phenotypes generated from multi-omics data, to inform future environmental toxicological research on forecasting targets and outcomes for emerging pollutants. [ABSTRACT FROM AUTHOR]

Advances in high-throughput high-resolution mass spectrometry and the development of thermal proteome profiling approach (TPP) have made it possible to accelerate a drug target search. Since its introduction in 2014, TPP quickly became a method of choice in chemical proteomics for identifying drug-to-protein interactions on a proteome-wide scale and mapping the pathways of these interactions, thus further elucidating the unknown mechanisms of action of a drug under study. However, the current TPP implementations based on tandem mass spectrometry (MS/MS), associated with employing lengthy peptide separation protocols and expensive labeling techniques for sample multiplexing, limit the scaling of this approach for the ever growing variety of drug-to-proteomes. A variety of ultrafast proteomics methods have been developed in the last couple of years. Among them, DirectMS1 provides MS/MS-free quantitative proteome-wide analysis in 5-min time scale, thus opening the way for sample-hungry applications, such as TPP. In this work, we demonstrate the first implementation of the TPP approach using the ultrafast proteome-wide analysis based on DirectMS1. Using a drug topotecan, which is a known topoisomerase I (TOP1) inhibitor, the feasibility of the method for identifying drug targets at the whole proteome level was demonstrated for an ovarian cancer cell line. [ABSTRACT FROM AUTHOR]

The accumulation of amyloid beta (Aβ1–42) results in neurotoxicity and is strongly related to neurodegenerative disorders, especially Alzheimer's disease (AD), but the underlying molecular mechanism is still poorly understood. Therefore, there is an urgent need for researchers to discover the proteins that interact with Aβ1–42 to determine the molecular basis. Previously, we developed peptide-ligand-induced changes in the abundance of proTeinS (PACTS)-assisted thermal proteome profiling (TPP) to identify proteins that interact with peptide ligands. In the present study, we applied this technique to analyze clinical samples to identify Aβ1–42-interacting proteins. We detected 115 proteins that interact with Aβ1–42 in human frontal lobe tissue. Pathway enrichment analysis revealed that the differentially expressed proteins were involved mainly in neurodegenerative diseases. Further orthogonal validation revealed that Aβ1–42 interacted with the AD-associated protein mitogen-activated protein kinase 3 (MAPK3), and knockdown of the Aβ1–42 amyloid precursor protein (APP) inhibited the MAPK signaling pathway, suggesting potential functional roles for Aβ1–42 in interacting with MAPK3. Overall, this study demonstrated the application of the PACTS-TPP in clinical samples and provided a valuable data source for research on neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Abstract Complex disease phenotypes often span multiple molecular processes. Functional characterization of these processes can shed light on disease mechanisms and drug effects. Thermal Proteome Profiling (TPP) is a mass-spectrometry (MS) based technique assessing changes in thermal protein stability that can serve as proxies of functional protein changes. These unique insights of TPP can complement those obtained by other omics technologies. Here, we show how TPP can be integrated with phosphoproteomics and transcriptomics in a network-based approach using COSMOS, a multi-omics integration framework, to provide an integrated view of transcription factors, kinases and proteins with altered thermal stability. This allowed us to recover consequences of Poly (ADP-ribose) polymerase (PARP) inhibition in ovarian cancer cells on cell cycle and DNA damage response as well as interferon and hippo signaling. We found that TPP offers a complementary perspective to other omics data modalities, and that its integration allowed us to obtain a more complete molecular overview of PARP inhibition. We anticipate that this strategy can be used to integrate functional proteomics with other omics to study molecular processes.

Complex disease phenotypes often span multiple molecular processes. Functional characterization of these processes can shed light on disease mechanisms and drug effects. Thermal Proteome Profiling (TPP) is a mass-spectrometry (MS) based technique assessing changes in thermal protein stability that can serve as proxies of functional protein changes. These unique insights of TPP can complement those obtained by other omics technologies. Here, we show how TPP can be integrated with phosphoproteomics and transcriptomics in a network-based approach using COSMOS, a multi-omics integration framework, to provide an integrated view of transcription factors, kinases and proteins with altered thermal stability. This allowed us to recover consequences of Poly (ADP-ribose) polymerase (PARP) inhibition in ovarian cancer cells on cell cycle and DNA damage response as well as interferon and hippo signaling. We found that TPP offers a complementary perspective to other omics data modalities, and that its integration allowed us to obtain a more complete molecular overview of PARP inhibition. We anticipate that this strategy can be used to integrate functional proteomics with other omics to study molecular processes. Synopsis: COSMOS, a network-based framework, can integrate functional proteomics data, such as Thermal Proteome Profiling (TPP), with other omics data modalities. It is used to generate multi-omics mechanistic hypotheses, as showcased in this study for PARP inhibition. Functional proteomics (TPP data) can be integrated with phosphoproteomics and transcriptomics in network models. TPP offers a complementary perspective to other omics data modalities. This approach provides a more complete and integrative overview of molecular processes related to PARP inhibition. COSMOS, a network-based framework, can integrate functional proteomics data, such as Thermal Proteome Profiling (TPP), with other omics data modalities. It is used to generate multi-omics mechanistic hypotheses, as showcased in this study for PARP inhibition. [ABSTRACT FROM AUTHOR]

Infections and diseases caused by parasitic nematodes have a major adverse impact on the health and productivity of animals and humans worldwide. The control of these parasites often relies heavily on the treatment with commercially available chemical compounds (anthelmintics). However, the excessive or uncontrolled use of these compounds in livestock animals has led to major challenges linked to drug resistance in nematodes. Therefore, there is a need to develop new anthelmintics with novel mechanism(s) of action. Recently, we identified a small molecule, designated UMW-9729, with nematocidal activity against the free-living model organism Caenorhabditis elegans. Here, we evaluated UMW-9729's potential as an anthelmintic in a structure-activity relationship (SAR) study in C. elegans and the highly pathogenic, blood-feeding Haemonchus contortus (barber's pole worm), and explored the compound-target relationship using thermal proteome profiling (TPP). First, we synthesised and tested 25 analogues of UMW-9729 for their nematocidal activity in both H. contortus (larvae and adults) and C. elegans (young adults), establishing a preliminary nematocidal pharmacophore for both species. We identified several compounds with marked activity against either H. contortus or C. elegans which had greater efficacy than UMW-9729, and found a significant divergence in compound bioactivity between these two nematode species. We also identified a UMW-9729 analogue, designated 25, that moderately inhibited the motility of adult female H. contortus in vitro. Subsequently, we inferred three H. contortus proteins (HCON_00134350, HCON_00021470 and HCON_00099760) and five C. elegans proteins (F30A10.9, F15B9.8, B0361.6, DNC-4 and UNC-11) that interacted directly with UMW-9729; however, no conserved protein target was shared between the two nematode species. Future work aims to extend the SAR investigation in these and other parasitic nematode species, and validate individual proteins identified here as possible targets of UMW-9729. Overall, the present study evaluates this anthelmintic candidate and highlights some challenges associated with early anthelmintic investigation.

Within the context of our anthelmintic discovery program, we recently identified and evaluated a quinoline derivative, called ABX464 or obefazimod, as a nematocidal candidate; synthesised a series of analogues which were assessed for activity against the free-living nematode Caenorhabditis elegans; and predicted compound-target relationships by thermal proteome profiling (TPP) and in silico docking. Here, we logically extended this work and critically evaluated the anthelmintic activity of ABX464 analogues on Haemonchus contortus (barber's pole worm) – a highly pathogenic nematode of ruminant livestock. First, we tested a series of 44 analogues on H. contortus (larvae and adults) to investigate the nematocidal pharmacophore of ABX464, and identified one compound with greater potency than the parent compound and showed moderate activity against a select number of other parasitic nematodes (including Ancylostoma, Heligmosomoides and Strongyloides species). Using TPP and in silico modelling studies, we predicted protein HCON_00074590 (a predicted aldo-keto reductase) as a target candidate for ABX464 in H. contortus. Future work aims to optimise this compound as a nematocidal candidate and investigate its pharmacokinetic properties. Overall, this study presents a first step toward the development of a new nematocide.

In recent years, cannabidiol (CBD), a non‐psychotropic cannabinoid, has garnered substantial interest in drug development due to its broad pharmacological activity and multi‐target effects. Diabetes is a chronic metabolic disease that can damage multiple organs in the body, leading to the development of complications such as abnormal kidney function, vision loss, neuropathy, and cardiovascular disease. CBD has demonstrated significant therapeutic potential in treating diabetes mellitus and its complications owing to its various pharmacological effects. This work summarizes the role of CBD in diabetes and its impact on complications such as cardiovascular dysfunction, nephropathy, retinopathy, and neuropathy. Strategies for discovering molecular targets for CBD in the treatment of diabetes and its complications are also proposed. Moreover, ways to optimize the structure of CBD based on known targets to generate new CBD analogues are explored. [ABSTRACT FROM AUTHOR]

SUMMARY: Identification of protein interactors is ideally suited for the functional characterization of small molecules. 3′,5′‐cAMP is an evolutionary ancient signaling metabolite largely uncharacterized in plants. To tap into the physiological roles of 3′,5′‐cAMP, we used a chemo‐proteomics approach, thermal proteome profiling (TPP), for the unbiased identification of 3′,5′‐cAMP protein targets. TPP measures shifts in the protein thermal stability upon ligand binding. Comprehensive proteomics analysis yielded a list of 51 proteins significantly altered in their thermal stability upon incubation with 3′,5′‐cAMP. The list contained metabolic enzymes, ribosomal subunits, translation initiation factors, and proteins associated with the regulation of plant growth such as CELL DIVISION CYCLE 48. To functionally validate obtained results, we focused on the role of 3′,5′‐cAMP in regulating the actin cytoskeleton suggested by the presence of actin among the 51 identified proteins. 3′,5′‐cAMP supplementation affected actin organization by inducing actin‐bundling. Consistent with these results, the increase in 3′,5′‐cAMP levels, obtained either by feeding or by chemical modulation of 3′,5′‐cAMP metabolism, was sufficient to partially rescue the short hypocotyl phenotype of the actin2 actin7 mutant, severely compromised in actin level. The observed rescue was specific to 3′,5′‐cAMP, as demonstrated using a positional isomer 2′,3′‐cAMP, and true for the nanomolar 3′,5′‐cAMP concentrations reported for plant cells. In vitro characterization of the 3′,5′‐cAMP–actin pairing argues against a direct interaction between actin and 3′,5′‐cAMP. Alternative mechanisms by which 3′,5′‐cAMP would affect actin dynamics, such as by interfering with calcium signaling, are discussed. In summary, our work provides a specific resource, 3′,5′‐cAMP interactome, as well as functional insight into 3′,5′‐cAMP‐mediated regulation in plants. Significance Statement: The present study provides the list of potential 3′,5′‐cAMP interactors pointing towards actin as one of them. We demonstrate that 3′,5′‐cAMP can regulate actin bundling in vivo. [ABSTRACT FROM AUTHOR]

Abstract In recent years, cannabidiol (CBD), a non‐psychotropic cannabinoid, has garnered substantial interest in drug development due to its broad pharmacological activity and multi‐target effects. Diabetes is a chronic metabolic disease that can damage multiple organs in the body, leading to the development of complications such as abnormal kidney function, vision loss, neuropathy, and cardiovascular disease. CBD has demonstrated significant therapeutic potential in treating diabetes mellitus and its complications owing to its various pharmacological effects. This work summarizes the role of CBD in diabetes and its impact on complications such as cardiovascular dysfunction, nephropathy, retinopathy, and neuropathy. Strategies for discovering molecular targets for CBD in the treatment of diabetes and its complications are also proposed. Moreover, ways to optimize the structure of CBD based on known targets to generate new CBD analogues are explored.

Metals are indispensable for the life of all organisms, and their dysregulation leads to various disorders due to the disruption of their homeostasis. Nowadays, various transition metals are used in pharmaceutical products as diagnostic and therapeutic agents because their electronic structure allows them to adjust the properties of molecules differently from organic molecules. Therefore, interest in the study of metal–drug complexes from different aspects has been aroused, and numerous approaches have been developed to characterize, activate, deliver, and clarify molecular mechanisms. The integration of these different approaches, ranging from chemoproteomics to nanoparticle systems and various activation strategies, enables the understanding of the cellular responses to metal drugs, which may form the basis for the development of new drugs and/or the modification of currently used drugs. The purpose of this review is to briefly summarize the recent advances in this field by describing the technological platforms and their potential applications for identifying protein targets for discovering the mechanisms of action of metallodrugs and improving their efficiency during delivery. [ABSTRACT FROM AUTHOR]

Although the use of detergents in thermal proteome profiling (TPP) has become a common practice to identify membrane protein targets in complex biological samples, surprisingly, there is no proteome-wide investigation into the impacts of detergent introduction on the target identification performance of TPP. In this study, we assessed the target identification performance of TPP in the presence of a commonly used non-ionic detergent or a zwitterionic detergent using a pan-kinase inhibitor staurosporine, our results showed that the addition of either of these detergents significantly impaired the identification performance of TPP at the optimal temperature for soluble target protein identification. Further investigation showed that detergents destabilized the proteome and increased protein precipitation. By lowering the applied temperature point, the target identification performance of TPP with detergents is significantly improved and is comparable to that in the absence of detergents. Our findings provide valuable insight into how to select the appropriate temperature range when detergents are used in TPP. In addition, our results also suggest that the combination of detergent and heat may serve as a novel precipitation-inducing force that can be applied for target protein identification. [ABSTRACT FROM AUTHOR]

Kinases are key regulatory signalling proteins governing numerous essential biological processes and cellular functions. Dysregulation of many protein kinases is associated with cancer initiation and progression. Given their crucial roles, there has been increasing interest in harnessing kinases as prospective drug targets for cancer. In recent decades, numerous small‐molecule kinase inhibitors have been developed and revolutionized the cancer treatment landscape. Despite their great potential, challenges remain in developing highly selective and effective kinase inhibitors, with toxicity and resistance issues frequently arising. In this review, we first provide an overview of the role of kinases in carcinogenesis and describe the current progress with small‐molecule kinase inhibitors that have been approved for clinical use. We then discuss the application of mass spectrometry (MS)‐based proteomics strategies to help in the design of kinase inhibitors. Finally, we discuss the challenges and outlook concerning MS‐based proteomics techniques for kinase drug research. [ABSTRACT FROM AUTHOR]

Proteins are essential macromolecules that carry out a plethora of biological functions. The thermal stability of proteins is an important property that affects their function and determines their suitability for various applications. However, current experimental approaches, primarily thermal proteome profiling, are expensive, labor-intensive, and have limited proteome and species coverage. To close the gap between available experimental data and sequence information, a novel protein thermal stability predictor called DeepSTABp has been developed. DeepSTABp uses a transformer-based protein language model for sequence embedding and state-of-the-art feature extraction in combination with other deep learning techniques for end-to-end protein melting temperature prediction. DeepSTABp can predict the thermal stability of a wide range of proteins, making it a powerful and efficient tool for large-scale prediction. The model captures the structural and biological properties that impact protein stability, and it allows for the identification of the structural features that contribute to protein stability. DeepSTABp is available to the public via a user-friendly web interface, making it accessible to researchers in various fields. [ABSTRACT FROM AUTHOR]

Purpose: Aberrant activation of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases drives oncogenic signaling through its proximal adaptor protein FRS2. Precise disruption of this disease-causing signal transmission in metastatic cancers could stall tumor growth and progression. The purpose of this study was to identify a small molecule ligand of FRS2 to interrupt oncogenic signal transmission from activated FGFRs. Methods: We used pharmacophore-based computational screening to identify potential small molecule ligands of the PTB domain of FRS2, which couples FRS2 to FGFRs. We confirmed PTB domain binding of molecules identified with biophysical binding assays and validated compound activity in cell-based functional assays in vitro and in an ovarian cancer model in vivo. We used thermal proteome profiling to identify potential off-targets of the lead compound. Results: We describe a small molecule ligand of the PTB domain of FRS2 that prevents FRS2 activation and interrupts FGFR signaling. This PTB-domain ligand displays on-target activity in cells and stalls FGFR-dependent matrix invasion in various cancer models. The small molecule ligand is detectable in the serum of mice at the effective concentration for prolonged time and reduces growth of the ovarian cancer model in vivo. Using thermal proteome profiling, we furthermore identified potential off-targets of the lead compound that will guide further compound refinement and drug development. Conclusions: Our results illustrate a phenotype-guided drug discovery strategy that identified a novel mechanism to repress FGFR-driven invasiveness and growth in human cancers. The here identified bioactive leads targeting FGF signaling and cell dissemination provide a novel structural basis for further development as a tumor agnostic strategy to repress FGFR- and FRS2-driven tumors. [ABSTRACT FROM AUTHOR]

The impact of exposure to multiple chemicals raises concerns for human and environmental health. The adverse outcome pathway method offers a framework to support mechanism-based assessment in environmental health starting by describing which mechanisms are triggered upon interaction with different stressors. The identification of the molecular initiating event and the molecular interaction between a chemical and a protein target is still a challenge for the development of adverse outcome pathways. The cellular response to chemical exposure studied with omics could not directly identify the protein targets. However, recent mass spectrometry-based methods are offering a proteome-wide identification of protein targets interacting with s but unrevealing a molecular initiating event from a set of targets is still dependent on available knowledge. Here, we directly coupled the target identification findings from the proteome integral solubility alteration assay with an analytical hierarchy process for the prediction of a prioritized molecular initiating event. We demonstrate the applicability of this combination of methodologies with a test compound (TCDD), and it could be further studied and integrated into AOPs. From the eight protein targets identified by the proteome integral solubility alteration assay after analyzing 2824 human hepatic proteins, the analytical hierarchy process can select the most suitable protein for an AOP. Our combined method solves the missing links between high-throughput target identification and prediction of the molecular initiating event. We anticipate its utility to decipher new molecular initiating events and support more sustainable methodologies to gain time and resources in chemical assessment. [ABSTRACT FROM AUTHOR]

Nonalcoholic steatohepatitis (NASH) is the severe subtype of nonalcoholic fatty diseases (NAFLD) with few options for treatment. Patients with NASH exhibit partial responses to the current therapeutics and adverse effects. Identification of the binding proteins for the drugs is essential to understanding the mechanism and adverse effects of the drugs and fuels the discovery of potent and safe drugs. This paper aims to critically discuss recent advances in covalent and noncovalent approaches for identifying binding proteins that mediate NASH progression, along with an in-depth analysis of the mechanisms by which these targets regulate NASH. A literature search was conducted to identify the relevant studies in the database of PubMed and the American Chemical Society. The search covered articles published from January 1990 to July 2024, using the search terms with keywords such as NASH, benzophenone, diazirine, photo-affinity labeling, thermal protein profiling, CETSA, target identification. The covalent approaches utilize drugs modified with diazirine and benzophenone to covalently crosslink with the target proteins, which facilitates the purification and identification of target proteins. In addition, they map the binding sites in the target proteins. By contrast, noncovalent approaches identify the binding targets of unmodified drugs in the intact cell proteome. The advantages and limitations of both approaches have been compared, along with a comprehensive analysis of recent innovations that further enhance the efficiency and specificity. The analyses of the applicability of these approaches provide novel tools to delineate NASH pathogenesis and promote drug discovery. [Display omitted] • Photoaffinity labeling approaches identify the drug targets that regulate NASH. • Target identification delineates the adverse effects and toxicity of anti-NASH drugs. • Noncovalent approaches identify the in situ binding targets for small molecules. • Multidisciplinary advances greatly facilitate target identification to treat NASH. [ABSTRACT FROM AUTHOR]

Our capability to predict the impact of exposure to chemical mixtures on environmental and human health is limited in comparison to the advances on the chemical characterization of the exposome. Current approaches, such as new approach methodologies, rely on the characterization of the chemicals and the available toxicological knowledge of individual compounds. In this study, we show a new methodological approach for the assessment of chemical mixtures based on a proteome-wide identification of the protein targets and revealing the relevance of new targets based on their role in the cellular function. We applied a proteome integral solubility alteration assay to identify 24 protein targets from a chemical mixture of 2,3,7,8-tetrachlorodibenzo- p- dioxin, alpha-endosulfan, and bisphenol A among the HepG2 soluble proteome, and validated the chemical mixture-target interaction orthogonally. To define the range of interactive capability of the new targets, the data from intrinsic properties of the targets were retrieved. Introducing the target properties as criteria for a multi-criteria decision-making analysis called the analytical hierarchy process, the prioritization of targets was based on their involvement in multiple pathways. This methodological approach that we present here opens a more realistic and achievable scenario to address the impact of complex and uncharacterized chemical mixtures in biological systems. [Display omitted] • New protein targets for a chemical mixture were identified. • Targets were ranked according to disturbance of cellular function. • Incorporating data from intrinsic properties of proteins prevents the exclusion of unstudied targets. • This approach improves the prediction of the impact of complex or uncharacterized mixtures. [ABSTRACT FROM AUTHOR]

Parasitic roundworms (nematodes) cause destructive diseases, and immense suffering in humans and other animals around the world. The control of these parasites relies heavily on anthelmintic therapy, but treatment failures and resistance to these drugs are widespread. As efforts to develop vaccines against parasitic nematodes have been largely unsuccessful, there is an increased focus on discovering new anthelmintic entities to combat drug resistant worms. Here, we employed thermal proteome profiling (TPP) to explore hit pharmacology and to support optimisation of a hit compound (UMW-868), identified in a high-throughput whole-worm, phenotypic screen. Using advanced structural prediction and docking tools, we inferred an entirely novel, parasite-specific target (HCO_011565) of this anthelmintic small molecule in the highly pathogenic, blood-feeding barber’s pole worm, and in other socioeconomically important parasitic nematodes. The “hit-to-target” workflow constructed here provides a unique prospect of accelerating the simultaneous discovery of novel anthelmintics and associated parasite-specific targets.

Metals are indispensable for the life of all organisms, and their dysregulation leads to various disorders due to the disruption of their homeostasis. Nowadays, various transition metals are used in pharmaceutical products as diagnostic and therapeutic agents because their electronic structure allows them to adjust the properties of molecules differently from organic molecules. Therefore, interest in the study of metal–drug complexes from different aspects has been aroused, and numerous approaches have been developed to characterize, activate, deliver, and clarify molecular mechanisms. The integration of these different approaches, ranging from chemoproteomics to nanoparticle systems and various activation strategies, enables the understanding of the cellular responses to metal drugs, which may form the basis for the development of new drugs and/or the modification of currently used drugs. The purpose of this review is to briefly summarize the recent advances in this field by describing the technological platforms and their potential applications for identifying protein targets for discovering the mechanisms of action of metallodrugs and improving their efficiency during delivery.

Although the use of detergents in thermal proteome profiling (TPP) has become a common practice to identify membrane protein targets in complex biological samples, surprisingly, there is no proteome-wide investigation into the impacts of detergent introduction on the target identification performance of TPP. In this study, we assessed the target identification performance of TPP in the presence of a commonly used non-ionic detergent or a zwitterionic detergent using a pan-kinase inhibitor staurosporine, our results showed that the addition of either of these detergents significantly impaired the identification performance of TPP at the optimal temperature for soluble target protein identification. Further investigation showed that detergents destabilized the proteome and increased protein precipitation. By lowering the applied temperature point, the target identification performance of TPP with detergents is significantly improved and is comparable to that in the absence of detergents. Our findings provide valuable insight into how to select the appropriate temperature range when detergents are used in TPP. In addition, our results also suggest that the combination of detergent and heat may serve as a novel precipitation-inducing force that can be applied for target protein identification.

Hydroxychloroquine (HCQ) is an autophagy inhibitor that has been used for the treatment of many diseases, such as malaria, rheumatoid arthritis, systemic lupus erythematosus, and cancer. Despite the therapeutic advances in these diseases, the underlying mechanisms have not been well determined and hinder the rational use of this drug in the future. Here, we explored the possible mechanisms and identified the potential binding targets of HCQ by performing quantitative proteomics and thermal proteome profiling on MIA PaCa-2 cells. This study revealed that HCQ may exert its functions by targeting some autophagy-related proteins such as ribosyldihydronicotinamide dehydrogenase (NQO2) and transport protein Sec23A (SEC23A), or regulating the expression of galectin-8 (LGALS8), mitogen-activated protein kinase 8 (MAPK8), and so on. Furthermore, HCQ may prevent the progression of pancreatic cancer by regulating the expression of nesprin-2 (SYNE2), protein-S-isoprenylcysteine O-methyltransferase (ICMT), and cotranscriptional regulator FAM172A (FAM172A). Together, these findings not only identified potential binding targets for HCQ but also revealed the non-canonical mechanisms of HCQ that may contribute to pancreatic cancer treatment. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an oft-fatal neglected tropical disease. In the absence of an effective vaccine, the control of leishmaniasis relies exclusively on chemotherapy. Due to the lack of established molecular/genetic markers denoting parasite resistance, clinical treatment failure is often used as an indicator. Antimony-based drugs have been the standard antileishmanial treatment for more than seven decades, leading to major drug resistance in certain regions. Likewise, drug resistance to miltefosine and amphotericin B continues to spread at alarming rates. In consequence, innovative approaches are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. To this end, we have implemented a novel approach based on thermal proteome profiling (TPP) to further characterize the mode of action of antileishmanials antimony, miltefosine and amphotericin B, as well as to better understand the mechanisms of drug resistance deployed by Leishmania. Proteins become more resistant to heat-induced denaturation when complexed with a ligand. In this way, we used multiplexed quantitative mass spectrometry-based proteomics to monitor the melting profile of thousands of expressed soluble proteins in WT, antimony-resistant, miltefosine-resistant, and amphotericin Bresistant L. infantum parasites, in the presence (or absence) of the abovementioned drugs. Bioinformatics analyses were performed, including data normalization, melting profile fitting, and identification of proteins that underwent changes (fold change > 4) caused by complexation with a drug. With this unique approach, we were able to narrow down the regions of the L. infantum proteome that interact with antimony, miltefosine, and amphotericin B; validating previously-identified and unveiling novel drug targets. Moreover, analyses revealed candidate proteins potentially involved in drug resistance. Interestingly, we detected thermal proximity coaggregation for several proteins belonging to the same metabolic pathway (i.e., tryparedoxin peroxidase and aspartate aminotransferase in proteins exposed to antimony), highlighting the importance of these pathways. Collectively, our results could serve as a jumping-off point for the future development of innovative diagnostic tools for the detection and evaluation of antimicrobial-resistant Leishmania populations, as well as open the door for new on-target therapies. [ABSTRACT FROM AUTHOR]

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an oft-fatal neglected tropical disease. In the absence of an effective vaccine, the control of leishmaniasis relies exclusively on chemotherapy. Due to the lack of established molecular/genetic markers denoting parasite resistance, clinical treatment failure is often used as an indicator. Antimony-based drugs have been the standard antileishmanial treatment for more than seven decades, leading to major drug resistance in certain regions. Likewise, drug resistance to miltefosine and amphotericin B continues to spread at alarming rates. In consequence, innovative approaches are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. To this end, we have implemented a novel approach based on thermal proteome profiling (TPP) to further characterize the mode of action of antileishmanials antimony, miltefosine and amphotericin B, as well as to better understand the mechanisms of drug resistance deployed by Leishmania. Proteins become more resistant to heat-induced denaturation when complexed with a ligand. In this way, we used multiplexed quantitative mass spectrometry-based proteomics to monitor the melting profile of thousands of expressed soluble proteins in WT, antimony-resistant, miltefosine-resistant, and amphotericin B-resistant L. infantum parasites, in the presence (or absence) of the above-mentioned drugs. Bioinformatics analyses were performed, including data normalization, melting profile fitting, and identification of proteins that underwent changes (fold change > 4) caused by complexation with a drug. With this unique approach, we were able to narrow down the regions of the L. infantum proteome that interact with antimony, miltefosine, and amphotericin B; validating previously-identified and unveiling novel drug targets. Moreover, analyses revealed candidate proteins potentially involved in drug resistance. Interestingly, we detected thermal proximity coaggregation for several proteins belonging to the same metabolic pathway (i.e., tryparedoxin peroxidase and aspartate aminotransferase in proteins exposed to antimony), highlighting the importance of these pathways. Collectively, our results could serve as a jumping-off point for the future development of innovative diagnostic tools for the detection and evaluation of antimicrobial-resistant Leishmania populations, as well as open the door for new on-target therapies.

Apicomplexan parasites cause persistent mortality and morbidity worldwide through diseases including malaria, toxoplasmosis, and cryptosporidiosis. Ca2+ signaling pathways have been repurposed in these eukaryotic pathogens to regulate parasite-specific cellular processes governing the replicative and lytic phases of the infectious cycle, as well as the transition between them. Despite the presence of conserved Ca2+-responsive proteins, little is known about how specific signaling elements interact to impact pathogenesis. We mapped the Ca2+-responsive proteome of the model apicomplexan Taxoplasma gondii via time-resolved phosphoproteomics and thermal proteome profiling. The waves of phosphoregulation following PKG activation and stimulated Ca2+ release corroborate known physiological changes but identify specific proteins operating in these pathways. Thermal profiling of parasite extracts identified many expected Ca2+-responsive proteins, such as parasite Ca2+-dependent protein kinases. Our approach also identified numerous Ca2+-responsive proteins that are not predicted to bind Ca2+, yet are critical components of the parasite signaling network. We characterized protein phosphatase 1 (PP1) as a Ca2+-responsive enzyme that relocalized to the parasite apex upon Ca2+ store release. Conditional depletion of PP1 revealed that the phosphatase regulates Ca2+ uptake to promote parasite motility. PP1 may thus be partly responsible for Ca2+-regulated serine/threonine phosphatase activity in apicomplexan parasites.

Infections and diseases caused by parasitic nematodes have a major adverse impact on the health and productivity of animals and humans worldwide. The control of these parasites often relies heavily on the treatment with commercially available chemical compounds (anthelmintics). However, the excessive or uncontrolled use of these compounds in livestock animals has led to major challenges linked to drug resistance in nematodes. Therefore, there is a need to develop new anthelmintics with novel mechanism(s) of action. Recently, we identified a small molecule, designated UMW-9729, with nematocidal activity against the free-living model organism Caenorhabditis elegans. Here, we evaluated UMW-9729's potential as an anthelmintic in a structure-activity relationship (SAR) study in C. elegans and the highly pathogenic, blood-feeding Haemonchus contortus (barber's pole worm), and explored the compound-target relationship using thermal proteome profiling (TPP). First, we synthesised and tested 25 analogues of UMW-9729 for their nematocidal activity in both H. contortus (larvae and adults) and C. elegans (young adults), establishing a preliminary nematocidal pharmacophore for both species. We identified several compounds with marked activity against either H. contortus or C. elegans which had greater efficacy than UMW-9729, and found a significant divergence in compound bioactivity between these two nematode species. We also identified a UMW-9729 analogue, designated 25 , that moderately inhibited the motility of adult female H. contortus in vitro. Subsequently, we inferred three H. contortus proteins (HCON_00134350, HCON_00021470 and HCON_00099760) and five C. elegans proteins (F30A10.9, F15B9.8, B0361.6, DNC-4 and UNC-11) that interacted directly with UMW-9729; however, no conserved protein target was shared between the two nematode species. Future work aims to extend the SAR investigation in these and other parasitic nematode species, and validate individual proteins identified here as possible targets of UMW-9729. Overall, the present study evaluates this anthelmintic candidate and highlights some challenges associated with early anthelmintic investigation. [Display omitted] • A new chemical entity, UMW-9729, has anthelmintic efficacy on H. contortus and C. elegans. • UMW-9729 and analogues display a significant bioactivity divergence across nematode species. • One non-toxic analogue, 25 , shows moderated inhibition of H. contortus adult motility. • Several H. contortus and C. elegans protein targets investigated via thermal proteome profiling. • No conserved protein target suggests UMW-9729 has a unique antiparasitic mechanism of action. [ABSTRACT FROM AUTHOR]

Proteins are essential macromolecules that carry out a plethora of biological functions. The thermal stability of proteins is an important property that affects their function and determines their suitability for various applications. However, current experimental approaches, primarily thermal proteome profiling, are expensive, labor-intensive, and have limited proteome and species coverage. To close the gap between available experimental data and sequence information, a novel protein thermal stability predictor called DeepSTABp has been developed. DeepSTABp uses a transformer-based protein language model for sequence embedding and state-of-the-art feature extraction in combination with other deep learning techniques for end-to-end protein melting temperature prediction. DeepSTABp can predict the thermal stability of a wide range of proteins, making it a powerful and efficient tool for large-scale prediction. The model captures the structural and biological properties that impact protein stability, and it allows for the identification of the structural features that contribute to protein stability. DeepSTABp is available to the public via a user-friendly web interface, making it accessible to researchers in various fields.

The Cellular Thermal Shift Assay (CETSA) plays an important role in drug-target identification, and statistical analysis is a crucial step significantly affecting conclusion. We put forward ProSAP (Protein Stability Analysis Pod), an open-source, cross-platform and user-friendly software tool, which provides multiple methods for thermal proteome profiling (TPP) analysis, nonparametric analysis (NPA), proteome integral solubility alteration and isothermal shift assay (iTSA). For testing the performance of ProSAP, we processed several datasets and compare the performance of different algorithms. Overall, TPP analysis is more accurate with fewer false positive targets, but NPA methods are flexible and free from parameters. For iTSA, edgeR and DESeq2 identify more true targets than t -test and Limma, but when it comes to ranking, the four methods show not much difference. ProSAP software is available at https://github.com/hcji/ProSAP and https://zenodo.org/record/5763315. [ABSTRACT FROM AUTHOR]

Drug target deconvolution can accelerate the drug discovery process by identifying a drug's targets (facilitating medicinal chemistry efforts) and off-targets (anticipating toxicity effects or adverse drug reactions). Multiple mass spectrometry–based approaches have been developed for this purpose, but thermal proteome profiling (TPP) remains to date the only one that does not require compound modification and can be used to identify intracellular targets in living cells. TPP is based on the principle that the thermal stability of a protein can be affected by its interactions. Recent developments of this approach have expanded its applications beyond drugs and cell cultures to studying protein-drug interactions and biological phenomena in tissues. These developments open up the possibility of studying drug treatment or mechanisms of disease in a holistic fashion, which can result in the design of better drugs and lead to a better understanding of fundamental biology. [ABSTRACT FROM AUTHOR]

Abstract Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry‐based proteomics to characterize aggregation and disaggregation in human cells after non‐lethal heat shock. Aggregation‐prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point, and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions were associated with faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared with control condition. Therefore, our results provide a rich resource of heat stress‐related protein solubility data and can foster further studies related to protein aggregation diseases.

Abstract Thermal proteome profiling (TPP) is based on the principle that, when subjected to heat, proteins denature and become insoluble. Proteins can change their thermal stability upon interactions with small molecules (such as drugs or metabolites), nucleic acids or other proteins, or upon post‐translational modifications. TPP uses multiplexed quantitative mass spectrometry‐based proteomics to monitor the melting profile of thousands of expressed proteins. Importantly, this approach can be performed in vitro, in situ, or in vivo. It has been successfully applied to identify targets and off‐targets of drugs, or to study protein–metabolite and protein–protein interactions. Therefore, TPP provides a unique insight into protein state and interactions in their native context and at a proteome‐wide level, allowing to study basic biological processes and their underlying mechanisms.

The impact of exposure to multiple chemicals raises concerns for human and environmental health. The adverse outcome pathway method offers a framework to support mechanism-based assessment in environmental health starting by describing which mechanisms are triggered upon interaction with different stressors. The identification of the molecular initiating event and the molecular interaction between a chemical and a protein target is still a challenge for the development of adverse outcome pathways. The cellular response to chemical exposure studied with omics could not directly identify the protein targets. However, recent mass spectrometry-based methods are offering a proteome-wide identification of protein targets interacting with s but unrevealing a molecular initiating event from a set of targets is still dependent on available knowledge. Here, we directly coupled the target identification findings from the proteome integral solubility alteration assay with an analytical hierarchy process for the prediction of a prioritized molecular initiating event. We demonstrate the applicability of this combination of methodologies with a test compound (TCDD), and it could be further studied and integrated into AOPs. From the eight protein targets identified by the proteome integral solubility alteration assay after analyzing 2824 human hepatic proteins, the analytical hierarchy process can select the most suitable protein for an AOP. Our combined method solves the missing links between high-throughput target identification and prediction of the molecular initiating event. We anticipate its utility to decipher new molecular initiating events and support more sustainable methodologies to gain time and resources in chemical assessment.

Thermal proteome profiling (TPP) is a powerful tool for drug target deconvolution. Recently, data-independent acquisition mass spectrometry (DIA-MS) approaches have demonstrated significant improvements to depth and missingness in proteome data, but traditional TPP (a.k.a. CEllular Thermal Shift Assay "CETSA") workflows typically employ multiplexing reagents reliant on data-dependent acquisition (DDA). Herein, we introduce a new experimental design for the Proteome Integral Solubility Alteration via label-free DIA approach (PISA-DIA). We highlight the proteome coverage and sensitivity achieved by using multiple overlapping thermal gradients alongside DIA-MS, which maximizes efficiencies in PISA sample concatenation and safeguards against missing protein targets that exist at high melting temperatures. We demonstrate our extended PISA-DIA design has superior proteome coverage as compared to using tandem-mass tags (TMT) necessitating DDA-MS analysis. Importantly, we demonstrate our PISA-DIA approach has the quantitative and statistical rigor using A-1331852, a specific inhibitor of BCL-xL. Due to the high melt temperature of this protein target, we utilized our extended multiple gradient PISA-DIA workflow to identify BCL-xL. We assert our novel overlapping gradient PISA-DIA-MS approach is ideal for unbiased drug target deconvolution, spanning a large temperature range whilst minimizing target dropout between gradients, increasing the likelihood of resolving the protein targets of novel compounds., (© 2024 The Authors. Proteomics published by Wiley‐VCH GmbH.)

Gemcitabine (GEM) is widely employed in the treatment of various cancers, including pancreatic cancer. Despite their clinical success, challenges related to GEM resistance and toxicity persist. Therefore, a deeper understanding of its intracellular mechanisms and potential targets is urgently needed. In this study, through mass spectrometry analysis in data-dependent acquisition mode, we carried out quantitative proteomics (three independent replications) and thermal proteome profiling (TPP, two independent replications) on MIA PaCa-2 cells to explore the effects of GEM. Our proteomic analysis revealed that GEM led to the upregulation of the cell cycle and DNA replication proteins. Notably, we observed the upregulation of S-phase kinase-associated protein 2 (SKP2), a cell cycle and chemoresistance regulator. Combining SKP2 inhibition with GEM showed synergistic effects, suggesting SKP2 as a potential target for enhancing the GEM sensitivity. Through TPP, we pinpointed four potential GEM binding targets implicated in tumor development, including in breast and liver cancers, underscoring GEM's broad-spectrum antitumor capabilities. These findings provide valuable insights into GEM's molecular mechanisms and offer potential targets for improving treatment efficacy.

Flavonoids are natural polyphenolic compounds with a diverse array of biological activities and health-promoting effects. Recent studies have found that 4,4′-dimethoxychalcone (DMC) promoted longevity via autophagy; however, its targets are currently unknown. Herein, we employed an unbiased thermal proteome profiling (TPP) method and identified multiple targets of DMC, including ALDH1A3, ALDH2, and PTGES2. We further determined the dissociation constant (Kd) of DMC and ALDH1A3 to be 2.8 μM using microscale thermophoresis (MST) analysis, which indicated that DMC inhibited ALDH1A3 activity and aggravated cellular oxidative stress. DMC treatment significantly increased cellular reactive oxygen species (ROS) production and inhibited cancer cell growth. Quantitative proteomic analysis showed that DMC upregulated proteins associated with stress-responses and downregulated proteins associated with cell cycle progression, and this was confirmed using cell cycle analysis. Taken together, we showed that TPP is an effective tool with which to identify flavonoid targets and set a precedent for deciphering flavonoid function in the future. We have demonstrated that DMC inhibited cell proliferation via ROS-induced cell cycle arrest and is an anti-proliferative agent in cancer treatment. [Display omitted] - Identification of the targets of flavonoid 4,4′-dimethoxychalcone (DMC) by thermal proteome profiling (TPP). - ALDH1A3 was validated as a target of DMC with a dissociation constant of 2.8 μM. - DMC enhanced ROS production and aggravated oxidative stress in multiple cancer cells. - DMC induced G2/M phase arrest and inhibited cancer cell proliferation and colony formation. [ABSTRACT FROM AUTHOR]

Target identification of bioactive molecules is important for deciphering their functions and action mechanism in cells, as well as for drug development. Thermal proteome profiling (TPP), based on the principle that protein-ligand binding can increase the thermal stability of proteins, is a new technology for identifying target proteins of bioactive molecule. One of the advantages of TPP is that it doesn’t require functionalization of a bioactive compound, which significantly broadens its application in drug discovery and development. TH588 is an inhibitor of 7,8-dihydro-8-oxoguanine triphosphatase (MTH1), and it inhibits the proliferation of tumor cells by increasing the oxidative damage of DNA. However, previous studies showed that TH588 also executed MTH1-independent anti-tumor effects on multiple cancers by depolymerizing tubulin, increasing oxidative damage, and inactivating PI3K-AKT-mTOR signaling pathway. Therefore, identification of off-targets for TH588 is essential for understanding TH588 functions and its further chemical modification in drug development. Herein, a detailed experiment procedure of TPP workflow was presented, and 28 directly binding proteins and 53 indirectly binding proteins of TH588 was identified for understanding its anti-proliferative functions. [ABSTRACT FROM AUTHOR]

Polo‐like kinase 1 (PLK1) is an important cell cycle kinase and an attractive target for anticancer treatments. An ATP‐competitive small molecular PLK1 inhibitor, volasertib, has reached phase III in clinical trials in patients with refractory acute myeloid leukemia as a combination treatment with cytarabine. However, severe side effects limited its use. The origin of the side effects is unclear and might be due to insufficient specificity of the drug. Thus, identifying potential off‐targets to volasertib is important for future clinical trials and for the development of more specific drugs. In this study, we used thermal proteome profiling (TPP) to identify proteome‐wide targets of volasertib. Apart from PLK1 and proteins regulated by PLK1, we identified about 200 potential volasertib off‐targets. Comparison of this result with the mass‐spectrometry analysis of volasertib‐treated cells showed that phosphatidylinositol phosphate and prostaglandin metabolism pathways are affected by volasertib. We confirmed that PIP4K2A and ZADH2—marker proteins for these pathways—are, indeed, stabilized by volasertib. PIP4K2A, however, was not affected by another PLK1 inhibitor onvansertib, suggesting that PIP4K2A is a true off‐target of volasertib. Inhibition of these proteins is known to impact both the immune response and fatty acid metabolism and could explain some of the side effects seen in volasertib‐treated patients. [ABSTRACT FROM AUTHOR]

Within the context of our anthelmintic discovery program, we recently identified and evaluated a quinoline derivative, called ABX464 or obefazimod, as a nematocidal candidate; synthesised a series of analogues which were assessed for activity against the free-living nematode Caenorhabditis elegans ; and predicted compound-target relationships by thermal proteome profiling (TPP) and in silico docking. Here, we logically extended this work and critically evaluated the anthelmintic activity of ABX464 analogues on Haemonchus contortus (barber's pole worm) – a highly pathogenic nematode of ruminant livestock. First, we tested a series of 44 analogues on H. contortus (larvae and adults) to investigate the nematocidal pharmacophore of ABX464, and identified one compound with greater potency than the parent compound and showed moderate activity against a select number of other parasitic nematodes (including Ancylostoma, Heligmosomoides and Strongyloides species). Using TPP and in silico modelling studies, we predicted protein HCON_00074590 (a predicted aldo-keto reductase) as a target candidate for ABX464 in H. contortus. Future work aims to optimise this compound as a nematocidal candidate and investigate its pharmacokinetic properties. Overall, this study presents a first step toward the development of a new nematocide. [Display omitted] • ABX464, a quinoline molecule, displays anthelmintic activity against H. contortus. • ABX464 and one particular analogue, 36, are also active against several parasitic nematodes. • HCON_00074590, an aldo-keto reductase, is identified as a nematode drug target. • In silico docking predicts ligand interactions with H. contortus protein HCON_00074590. [ABSTRACT FROM AUTHOR]

Quercetin is widely distributed in nature and abundant in the human diet, which exhibits diverse biological activities and potential medical benefits. However, there remains a lack of comprehensive understanding about its cellular targets, impeding its in-depth mechanistic studies and clinical applications. This study aimed to profile protein targets of quercetin at the proteome level. A label-free CETSA-MS proteomics technique was employed for target enrichment and identification. The R package Inflect was used for melting curve fitting and target selection. D3Pocket and LiBiSco tools were used for binding pocket prediction and binding pocket analysis. Western blotting, molecular docking, site-directed mutagenesis and pull-down assays were used for target verification and validation. We curated a library of direct binding targets of quercetin in cells. This library comprises 37 proteins that show increased thermal stability upon quercetin binding and 33 proteins that display decreased thermal stability. Through Western blotting, molecular docking, site-directed mutagenesis and pull-down assays, we validated CBR1 and GSK3A from the stabilized protein group and MAPK1 from the destabilized group as direct binding targets of quercetin. Moreover, we characterized the shared chemical properties of the binding pockets of quercetin with targets. Our findings deepen our understanding of the proteins pivotal to the bioactivity of quercetin and lay the groundwork for further exploration into its mechanisms of action and potential clinical applications. [Display omitted] • Identification of quercetin's cellular protein targets using CETSA-MS. • Creation of a curated library with 37 stabilized and 33 destabilized proteins. • Identification of CBR1, GSK3A, and MAPK1 as direct quercetin binding targets. • Revelation of shared chemical properties in quercetin-target binding pockets. [ABSTRACT FROM AUTHOR]

Hydroxychloroquine (HCQ) is an autophagy inhibitor that has been used for the treatment of many diseases, such as malaria, rheumatoid arthritis, systemic lupus erythematosus, and cancer. Despite the therapeutic advances in these diseases, the underlying mechanisms have not been well determined and hinder the rational use of this drug in the future. Here, we explored the possible mechanisms and identified the potential binding targets of HCQ by performing quantitative proteomics and thermal proteome profiling on MIA PaCa-2 cells. This study revealed that HCQ may exert its functions by targeting some autophagy-related proteins such as ribosyldihydronicotinamide dehydrogenase (NQO2) and transport protein Sec23A (SEC23A), or regulating the expression of galectin-8 (LGALS8), mitogen-activated protein kinase 8 (MAPK8), and so on. Furthermore, HCQ may prevent the progression of pancreatic cancer by regulating the expression of nesprin-2 (SYNE2), protein-S-isoprenylcysteine O-methyltransferase (ICMT), and cotranscriptional regulator FAM172A (FAM172A). Together, these findings not only identified potential binding targets for HCQ but also revealed the non-canonical mechanisms of HCQ that may contribute to pancreatic cancer treatment.

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The assay is based on the concept that ligand binding affects protein stability. It can be used to detect the binding of label free drugs to target proteins in various media such as cell lysates, whole cells and tissues • In recent years combined with Western blot, two-dimensional difference gel electrophoresis, and quantitative protein mass spectrometry, CETSA method has been successfully used in drug target verification, clinical drug target discovery, off-tar get identification and so on. With development of this technique, it is now further used in the analysis of metabolites, nucleic acids and protein-protein interactions. This paper chiefly summarizes the principle and method evolution of CETSA and its application in drug target discovery. [ABSTRACT FROM AUTHOR]

The ID of disease-modifying, chemically accessible targets remains a central priority of modern therapeutic discovery. The phenotypic screening of small-molecule libraries not only represents an attractive approach to identify compounds that may serve as drug leads but also serves as an opportunity to uncover compounds with novel mechanisms of action (MoAs). However, a major bottleneck of phenotypic screens continues to be the ID of pharmacologically relevant target(s) for compounds of interest. The field of chemoproteomics aims to map proteome-wide small-molecule interactions in complex, native systems, and has proved a key technology to unravel the protein targets of pharmacological modulators. In this review, we discuss the application of modern chemoproteomic methods to identify protein targets of phenotypic screening hits and investigate MoAs, with a specific focus on the development of chemoproteomic-enabled compound libraries to streamline target discovery. [Display omitted] Phenotypic screening is a powerful approach in drug discovery; however, target identification (ID) remains a significant hurdle. In this review, Conway, Li, and Parker survey examples of coupled phenotypic screening and chemoproteomic target ID as well as provide their perspective on the development of chemoproteomic-enabled libraries for expedited target ID. [ABSTRACT FROM AUTHOR]

Target deconvolution can help understand how compounds exert therapeutic effects and can accelerate drug discovery by helping optimise safety and efficacy, revealing mechanisms of action, anticipate off-target effects and identifying opportunities for therapeutic expansion. Chemoproteomics, a combination of chemical biology with mass spectrometry has transformed target deconvolution. This review discusses modification-free chemoproteomic approaches that leverage the change in protein thermodynamics induced by small molecule ligand binding. Unlike modification-based methods relying on enriching specific protein targets, these approaches offer proteome-wide evaluations, driven by advancements in mass spectrometry sensitivity, increasing proteome coverage and quantitation methods. Advances in methods based on denaturation/precipitation by thermal or chemical denaturation, or by protease degradation are evaluated, emphasising the evolving landscape of chemoproteomics and its potential impact on future drug-development strategies.

Thermophilic microorganisms that thrive in extreme environments are of great importance because they express heat-resistant enzymes with the potential to serve as biocatalysts in industrial applications. Thermal proteome profiling (TPP) is a multiplexed quantitative mass spectrometry method for analyses of structural information and melting behavior of thousands of proteins, simultaneously determining the thermal denaturation profiles of each protein. We report, in this study, TPP applied to a thermophilic bacterial proteome, a recently isolated strain of Geobacillus thermoleovorans named as ARTRW1. The proteome was investigated in terms of thermostable enzymes that are relevant to industrial applications. In this study, we present the thermostability profiles of its 868 proteins. The majority of G. thermoleovorans proteome was observed to melt between 62.5°C and 72°C, with melting point (Tm) mean value of 68.1°C ± 6.6°C. Unfolding characteristics of several enzymes, including amylase, protease, and lipase, were demonstrated which are highly informative in terms of their applicability to specific industrial processes. A significant correlation was observed between protein melting temperature and the structural features such as molecular weight and abundance, whereas correlations were modest or weak in relation to the α-helix structure percentages. Taken together, we demonstrated a system-wide melting profile analysis of a thermal proteome and listed proteins with elevated Tm values that are highly promising for applications in medicine, food engineering, and cosmetics in particular. The extracted Tm values were found similar to those obtained by biophysical methods applied to purified proteins. TPP analysis has significant industrial and biomedical potentials to accelerate thermophilic enzyme research and innovation. [ABSTRACT FROM AUTHOR]

Abstract Increasing antibiotic resistance urges for new technologies for studying microbes and antimicrobial mechanism of action. We adapted thermal proteome profiling (TPP) to probe the thermostability of Escherichia coli proteins in vivo. E. coli had a more thermostable proteome than human cells, with protein thermostability depending on subcellular location—forming a high‐to‐low gradient from the cell surface to the cytoplasm. While subunits of protein complexes residing in one compartment melted similarly, protein complexes spanning compartments often had their subunits melting in a location‐wise manner. Monitoring the E. coli meltome and proteome at different growth phases captured changes in metabolism. Cells lacking TolC, a component of multiple efflux pumps, exhibited major physiological changes, including differential thermostability and levels of its interaction partners, signaling cascades, and periplasmic quality control. Finally, we combined in vitro and in vivo TPP to identify targets of known antimicrobial drugs and to map their downstream effects. In conclusion, we demonstrate that TPP can be used in bacteria to probe protein complex architecture, metabolic pathways, and intracellular drug target engagement.

Protein aggregates have negative implications in disease. While reductionist experiments have increased our understanding of aggregation processes, the systemic view in biological context is still limited. To extend this understanding, we used mass spectrometry‐based proteomics to characterize aggregation and disaggregation in human cells after non‐lethal heat shock. Aggregation‐prone proteins were enriched in nuclear proteins, high proportion of intrinsically disordered regions, high molecular mass, high isoelectric point, and hydrophilic amino acids. During recovery, most aggregating proteins disaggregated with a rate proportional to the aggregation propensity: larger loss in solubility was counteracted by faster disaggregation. High amount of intrinsically disordered regions were associated with faster disaggregation. However, other characteristics enriched in aggregating proteins did not correlate with the disaggregation rates. In addition, we analyzed changes in protein thermal stability after heat shock. Soluble remnants of aggregated proteins were more thermally stable compared with control condition. Therefore, our results provide a rich resource of heat stress‐related protein solubility data and can foster further studies related to protein aggregation diseases. Synopsis: An in situ proteome‐wide analysis of protein solubility upon heat shock and during recovery reveals protein aggregation and disaggregation features in human cells. Large, hydrophilic and disordered proteins are prone to aggregation.Slow disaggregation is the main strategy for human cells to deal with aggregates.An aggregation‐resistant protein pool exists within aggregating proteins. [ABSTRACT FROM AUTHOR]

Thermal proteome profiling (TPP) is based on the principle that, when subjected to heat, proteins denature and become insoluble. Proteins can change their thermal stability upon interactions with small molecules (such as drugs or metabolites), nucleic acids or other proteins, or upon post‐translational modifications. TPP uses multiplexed quantitative mass spectrometry‐based proteomics to monitor the melting profile of thousands of expressed proteins. Importantly, this approach can be performed in vitro, in situ, or in vivo. It has been successfully applied to identify targets and off‐targets of drugs, or to study protein–metabolite and protein–protein interactions. Therefore, TPP provides a unique insight into protein state and interactions in their native context and at a proteome‐wide level, allowing to study basic biological processes and their underlying mechanisms. [ABSTRACT FROM AUTHOR]

[Display omitted] Global challenges with treatment failures and/or widespread resistance in parasitic worms against commercially available anthelmintics lend impetus to the development of new anthelmintics with novel mechanism(s) of action. The free-living nematode Caenorhabditis elegans is an important model organism used for drug discovery, including the screening and structure-activity investigation of new compounds, and target deconvolution. Previously, we conducted a whole-organism phenotypic screen of the ' Pandemic Response Box ' (from Medicines for Malaria Venture, MMV) and identified a hit compound, called ABX464, with activity against C. elegans and a related, parasitic nematode, Haemonchus contortus. Here, we tested a series of 44 synthesized analogues to explore the pharmacophore of activity on C. elegans and revealed five compounds whose potency was similar or greater than that of ABX464, but which were not toxic to human hepatoma (HepG2) cells. Subsequently, we employed thermal proteome profiling (TPP), protein structure prediction and an in silico- docking algorithm to predict ABX464-target candidates. Taken together, the findings from this study contribute significantly to the early-stage drug discovery of a new nematocide based on ABX464. Future work is aimed at validating the ABX464-protein interactions identified here, and at assessing ABX464 and associated analogues against a panel of parasitic nematodes, towards developing a new anthelmintic with a mechanism of action that is distinct from any of the compounds currently-available commercially. [ABSTRACT FROM AUTHOR]

Obesity is a worldwide epidemic and natural products may hold promise in its treatment. The chlorophyll derivative 13-2-hydroxypheophytine (hpa) was isolated in a screen with zebrafish larvae to identify lipid reducing molecules from cyanobacteria. However, the mechanisms underlying the lipid-reducing effects of hpa in zebrafish larvae remain poorly understood. Thus, investigating the mechanism of action of hpa and validation in other model organisms such as mice represents important initial steps. In this study, we identified 14 protein targets of hpa in zebrafish larvae by thermal proteome profiling, and selected two targets (malate dehydrogenase and pyruvate kinase) involved in cellular metabolism for further validation by enzymatic measurements. Our findings revealed a dose-dependent inhibition of pyruvate kinase by hpa exposure using protein extracts of zebrafish larvae in vitro, and in exposure experiments from 3 to 5 days post fertilization in vivo. Analysis of untargeted metabolomics of zebrafish larvae detected 940 mass peaks (66 increased, 129 decreased) and revealed that hpa induced the formation of various phospholipid species (phosphoinositol, phosphoethanolamine, phosphatidic acid). Inter-species validation showed that brown adipocytes exposed to hpa significantly reduced the size of lipid droplets, increased maximal mitochondrial respiratory capacity, and the expression of PPARy during adipocyte differentiation. In line with our data, previous work described that reduced pyruvate kinase activity lowered hepatic lipid content via reduced pyruvate and citrate, and improved mitochondrial function via phospholipids. Thus, our data provide new insights into the molecular mechanism underlying the lipid reducing activities of hpa in zebrafish larvae, and species overlapping functions in reduction of lipids. [Display omitted] • Insights into the molecular mechanism underlying the lipid reducing activity of hpa. • 14 potential protein ligands of hpa were identified by thermal protein profiling. • Further validation revealed dose-dependent inhibition of pyruvate kinase. • Untargeted metabolomics revealed the formation of various phospholipid species. • Hpa increased mitochondrial respiration and ppary mRNA in brown adipocytes. [ABSTRACT FROM AUTHOR]