Modulation of Protein-Interaction States through the Cell Cycle.

Global profiling of protein expression through the cell cycle has revealed subsets of periodically expressed proteins. However, expression levels alone only give a partial view of the biochemical processes determining cellular events. Using a proteome-wide implementation of the cellular thermal shift assay (CETSA) to study specific cell-cycle phases, we uncover changes of interaction states for more than 750 proteins during the cell cycle. Notably, many protein complexes are modulated in specific cell-cycle phases, reflecting their roles in processes such as DNA replication, chromatin remodeling, transcription, translation, and disintegration of the nuclear envelope. Surprisingly, only small differences in the interaction states were seen between the G1 and the G2 phase, suggesting similar hardwiring of biochemical processes in these two phases. The present work reveals novel molecular details of the cell cycle and establishes proteome-wide CETSA as a new strategy to study modulation of protein-interaction states in intact cells.

NDUFA12 as a Functional Target of the Anticancer Compound Ertredin in Human Hepatoma Cells As Revealed by Label-Free Chemical Proteomics.

Many attempts have been made to develop new agents that target EGFR mutants or regulate downstream factors in various cancers. Cell-based screening showed that a natural small molecule, Ertredin, inhibited the growth of EGFRvIII mutant cancer cells. Previous studies have shown that Ertredin effectively inhibits anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII mutant cDNA. However, the underlying mechanism remains unclear. In this study, we investigated the target protein of Ertredin by combining drug affinity-responsive target stability (DARTS) assays with liquid chromatography-mass spectrometry using label-free Ertredin as a bait and HepG2 cell lysates as a proteome pool. NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 12 (NDUFA12) was identified as an Ertredin-binding protein that was responsible for its biological activity. The interaction between NDUFA12 and Ertredin was validated by DARTS and cellular thermal shift assays. In addition, the genetic knockdown of the identified target, NDUFA12, was shown to suppress cell proliferation. NDUFA12 was identified as a biologically relevant target protein of Ertredin that is responsible for its antitumor activity, and these results provide insights into the role of NDUFA12 as a downstream factor in EGFRvIII mutants.

Discovery of novel thiophene-3-carboxamide derivatives as potential VEGFR-2 inhibitors with anti-angiogenic properties.

VEGFR-2 is an attractive target for the development of anti-tumor drugs and plays a crucial role in tumor angiogenesis. This study reports a series of novel thiophene-3-carboxamide derivatives based on PAN-90806 as VEGFR-2 inhibitors, among which compound 14d exhibits excellent anti-proliferative activity against HCT116, MCF7, PC3, and A549 cell lines, and has effective VEGFR-2 inhibitory activity with an IC50 value of 191.1 nM. Additionally, CETSA results indicated that VEGFR-2 was a relevant target of compound 14d in the cell lines, and compound 14d could also inhibit VEGFR-2 protein phosphorylation in A549 cell line. Furthermore, compound 14d inhibited colony formation, cell migration, and HUVECs tube formation in a dose-dependent manner. The mechanism by which 14d induced cancer cell death involves blocking the cell cycle, increasing ROS production, inducing apoptosis, and dose-dependently reducing the levels of phosphorylated ERK and MEK. Molecular docking and molecular dynamics simulations had shown that compound 14d could stably bind to the active site of VEGFR-2. These results confirmed that compound 14d might be a promising lead compound for anti-angiogenesis.

Application of the Cellular Thermal Shift Assay (CETSA) to validate drug target engagement in platelets.

Small molecule drugs play a major role in the study of human platelets. Effective action of a drug requires it to bind to one or more targets within the platelet (target engagement). However, although in vitro assays with isolated proteins can be used to determine drug affinity to these targets, additional factors affect target engagement and its consequences in an intact platelet, including plasma membrane permeability, intracellular metabolism or compartmentalization, and level of target expression. Mechanistic interpretation of the effect of drugs on platelet activity requires comprehensive investigation of drug binding in the proper cellular context, i.e. in intact platelets. The Cellular Thermal Shift Assay (CETSA) is a valuable method to investigate target engagement within complex cellular environments. The assay is based on the principle that drug binding to a target protein increases that protein's thermal stability. In this technical report, we describe the application of CETSA to platelets. We highlight CETSA as a quick and informative technique for confirming the direct binding of drugs to platelet protein targets, providing a platform for understanding the mechanism of action of drugs in platelets, and which will be a valuable tool for investigating platelet signaling and function.

Platelets control blood clotting in health and disease. Small molecule drugs are often used to study human platelets. Here, describe how Cellular Thermal Shift Assay (CETSA) can be used in platelets to investigate the binding between these drugs and their targets inside platelets. This technique can be used to increase our understanding of how existing and future drugs work in platelets.

The pharmacologic and toxicologic characterization of the potent and selective KRAS G12D inhibitors ERAS-4693 and ERAS-5024.

Two potent and selective KRASG12D inhibitors, ERAS-4693 and ERAS-5024, were generated as possible clinical candidates to treat patients harboring G12D mutations in solid tumors. Both molecules exhibited strong anti-tumor activity in the KRASG12D mutant PDAC xenograft mouse models while ERAS-5024 also showed tumor growth inhibition when administered on an intermittent dosing regimen. Acute dose-limiting toxicity consistent with an allergic reaction was observed for both molecules shortly after administration at doses just above those which demonstrated anti-tumor activity, indicative of a narrow therapeutic index. A series of studies were subsequently conducted to identify a common underlying mechanism for the observed toxicity, including CETSA® (Cellular Thermal Shift Assay) as well as several functional off-target screens. Both ERAS-4693 and ERAS-5024 were identified to agonize MRGPRX2 which has been linked to pseudo-allergic reactions. In vivo toxicologic characterization of both molecules included repeat-dose studies in the rat and dog. Dose-limiting toxicities were observed in both species with ERAS-4693 and ERAS-5024 and plasma exposure levels at the maximum tolerated doses were generally below that which caused strong anti-tumor activity, supporting the initial observation of a narrow therapeutic index. Additional overlapping toxicities included a reduction in reticulocytes and clinical pathological changes suggestive of an inflammatory response. Furthermore, increases in plasma histamine were observed in dogs administered ERAS-5024, supporting the hypothesis that MRGPRX2 agonism may be the cause of the pseudo-allergic reaction. This work highlights the importance of balancing both the safety and efficacy of KRASG12D inhibitors as this class of molecules begins to enter clinical development.

Assessing the role of intrinsic disorder in RNA-binding protein function: hnRNP K as a case study.

RNA-binding proteins (RBPs) typically bind to RNA in a sequence-specific manner, resulting in post-transcriptional gene regulation. While the various classes of RNA-binding domains are largely structured, flexible linkers are frequently observed between them. Emerging evidence suggests that these unstructured regions may help spatially position the RNA-binding domains allowing for RNA binding and/or may contribute directly to RNA association via certain sequence motifs contained within them. The importance of these unstructured regions is widely appreciated; however, understanding their contribution to RNA binding, protein stability, and function has been difficult to ascertain. Thus, it is crucial to have a set of rapid and economical assays that do not require specialized instrumentation to study their impact on RBP function. Herein, we discuss the use of plate-based and cell-based thermal shift assays to study the impact of the intrinsically disordered region on the function of a highly conserved RBP, hnRNP K.

New insights into the molecular mechanisms of ROR1, ROR2, and PTK7 signaling from the proteomics and pharmacological modulation of ROR1 interactome.

ROR1, ROR2, and PTK7 are Wnt ligand-binding members of the receptor tyrosine kinase family. Despite their lack of catalytic activity, these receptors regulate skeletal, cardiorespiratory, and neurological development during embryonic and fetal stages. However, their overexpression in adult tissue is strongly connected to tumor development and metastasis, suggesting a strong pharmacological potential for these molecules. Wnt5a ligand can activate these receptors, but lead to divergent signaling and functional outcomes through mechanisms that remain largely unknown. Here, we developed a cellular model by stably expressing ROR1, ROR2, and PTK7 in BaF3 cells that allowed us to readily investigate side-by-side their signaling capability and functional outcome. We applied proteomic profiling to BaF3 clones and identified distinctive roles for ROR1, ROR2, and PTK7 pseudokinases in modulating the expression of proteins involved in cytoskeleton dynamics, apoptotic, and metabolic signaling. Functionally, we show that ROR1 expression enhances cell survival and Wnt-mediated cell proliferation, while ROR2 and PTK7 expression is linked to cell migration. We also demonstrate that the distal C-terminal regions of ROR1 and ROR2 are required for receptors stability and downstream signaling. To probe the pharmacological modulation of ROR1 oncogenic signaling, we used affinity purification coupled to mass spectrometry (AP-MS) and proximity-dependent biotin identification (BioID) to map its interactome before and after binding of GZD824, a small molecule inhibitor previously shown to bind to the ROR1 pseudokinase domain. Our findings bring new insight into the molecular mechanisms of ROR1, ROR2, and PTK7, and highlight the therapeutic potential of targeting ROR1 with small molecule inhibitors binding to its vestigial ATP-binding site.

Thermal proteome profiling identifies PIP4K2A and ZADH2 as off-targets of Polo-like kinase 1 inhibitor volasertib.

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.

Small Molecule Arranged Thermal Proximity Coaggregation (smarTPCA)-A Novel Approach to Characterize Protein-Protein Interactions in Living Cells by Similar Isothermal Dose-Responses.

Chemical biology and the application of small molecules has proven to be a potent perturbation strategy, especially for the functional elucidation of proteins, their networks, and regulators. In recent years, the cellular thermal shift assay (CETSA) and its proteome-wide extension, thermal proteome profiling (TPP), have proven to be effective tools for identifying interactions of small molecules with their target proteins, as well as off-targets in living cells. Here, we asked the question whether isothermal dose-response (ITDR) CETSA can be exploited to characterize secondary effects downstream of the primary binding event, such as changes in post-translational modifications or protein-protein interactions (PPI). By applying ITDR-CETSA to MAPK14 kinase inhibitor treatment of living HL-60 cells, we found similar dose-responses for the direct inhibitor target and its known interaction partners MAPKAPK2 and MAPKAPK3. Extension of the dose-response similarity comparison to the proteome wide level using TPP with compound concentration range (TPP-CCR) revealed not only the known MAPK14 interaction partners MAPKAPK2 and MAPKAPK3, but also the potentially new intracellular interaction partner MYLK. We are confident that dose-dependent small molecule treatment in combination with ITDR-CETSA or TPP-CCR similarity assessment will not only allow discrimination between primary and secondary effects, but will also provide a novel method to study PPI in living cells without perturbation by protein modification, which we named "small molecule arranged thermal proximity coaggregation" (smarTPCA).

Inflect: Optimizing Computational Workflows for Thermal Proteome Profiling Data Analysis.

The CETSA and Thermal Proteome Profiling (TPP) analytical methods are invaluable for the study of protein-ligand interactions and protein stability in a cellular context. These tools have increasingly been leveraged in work ranging from understanding signaling paradigms to drug discovery. Consequently, there is an important need to optimize the data analysis pipeline that is used to calculate protein melt temperatures (Tm) and relative melt shifts from proteomics abundance data. Here, we report a user-friendly analysis of the melt shift calculation workflow where we describe the impact of each individual calculation step on the final output list of stabilized and destabilized proteins. This report also includes a description of how key steps in the analysis workflow quantitatively impact the list of stabilized/destabilized proteins from an experiment. We applied our findings to develop a more optimized analysis workflow that illustrates the dramatic sensitivity of chosen calculation steps on the final list of reported proteins of interest in a study and have made the R based program Inflect available for research community use through the CRAN repository [McCracken, N. Inflect: Melt Curve Fitting and Melt Shift Analysis. R package version 1.0.3, 2021]. The Inflect outputs include melt curves for each protein which passes filtering criteria in addition to a data matrix which is directly compatible with downstream packages such as UpsetR for replicate comparisons and identification of biologically relevant changes. Overall, this work provides an essential resource for scientists as they analyze data from TPP and CETSA experiments and implement their own analysis pipelines geared toward specific applications.

ABCG2 requires a single aromatic amino acid to "clamp" substrates and inhibitors into the binding pocket.

ATP-binding cassette sub-family G member 2 (ABCG2) is a homodimeric ATP-binding cassette (ABC) transporter that not only has a key role in helping cancer cells to evade the cytotoxic effects of chemotherapy, but also in protecting organisms from multiple xeno- and endobiotics. Structural studies indicate that substrate and inhibitor (ligands) binding to ABCG2 can be differentiated quantitatively by the number of amino acid contacts, with inhibitors displaying more contacts. Although binding is the obligate initial step in the transport cycle, there is no empirical evidence for one amino acid being primarily responsible for ligand binding. By mutagenesis and biochemical studies, we demonstrated that the phylogenetically conserved amino acid residue, F439, was critical for both transport and the binding of multiple substrates and inhibitors. Structural modeling implied that the π-π interactions from each F439 monomer mediated the binding of a surprisingly diverse array of structurally unrelated substrates and inhibitors and that this symmetrical π-π interaction "clamps" the ligand into the binding pocket. Key molecular features of diverse ABCG2 ligands using the π-π clamp along with structural studies created a pharmacophore model. These novel findings have important therapeutic implications because key properties of ligands interacting with ABCG2 have been disovered. Furthermore, mechanistic insights have been revealed by demonstrating that for ABCG2 a single amino acid is essential for engaging and initiating transport of multiple drugs and xenobiotics.

The use of the cellular thermal shift assay for the detection of intracellular beta-site amyloid precursor protein cleaving enzyme-1 ligand binding.

Inhibition of the Alzheimer's disease associated protein ß-site amyloid precursor protein cleaving enzyme-1 (BACE1) remains a potential avenue for treatment of this disease. The cellular thermal shift assay (CETSA) is an attractive method of screening for protein binding molecules due to its ability to detect intracellular binding while avoiding the need to purify the protein in question. Here, the CETSA was carried out using the known BACE1 inhibitor verubecestat, where an increase in Tagg to 53.27 ± 0.89 °C from 49.53 ± 0.69 °C was observed. Three test compounds from the ChemBridge DiverSet compound library, identified to bind BACE1 using differential scanning fluorimetry, were then screened using the CETSA. Only compound C34 yielded a significant increase in Tagg (p value ≤ 0.05), indicative of intracellular binding. This is the first description of the cellular thermal shift assay being used to detect BACE1 binding molecules, with one novel BACE1 binding molecule being validated.

Mass spectrometry-based Cellular Thermal Shift Assay (CETSA®) for target deconvolution in phenotypic drug discovery.

The recent renewed interest in phenotypic drug discovery has concomitantly put a focus on target deconvolution in order to achieve drug-target identification. Even though there are prescribed therapies whose mode of action is not fully understood, knowledge of the primary target will inevitably facilitate the discovery and translation of efficacy from bench to bedside. Elucidating targets and subsequent pathways engaged will also facilitate safety studies and overall development of novel drug candidates. Today, there are several techniques available for identifying the primary target, many of which rely on mass spectrometry (MS) to identify compound - target protein interactions. The Cellular Thermal Shift Assay (CETSA®) is well suited for identifying target engagement between ligands and their protein targets. Several studies have shown that CETSA combined with MS is a powerful technique that allows unlabeled target deconvolution in complex samples such as intact cells and tissues in addition to cell lysates and other protein suspensions. The applicability of CETSA MS for target deconvolution purposes will be discussed and exemplified in this mini review.

The amyloid precursor protein affects glyceraldehyde 3-phosphate dehydrogenase levels, organelle localisation and thermal stability.

Glyceraldehyde 3-phosphate dehydrogenase's (GAPDH) proapoptotic response to cellular oxidative stress has suspected implication for Alzheimer's disease (AD). Interestingly, the overexpression of the amyloid precursor protein (APP) can initiate oxidative stress responses within mammalian cell lines. Here, APP695 and APP770 overexpression significantly increased the level of GAPDH, while no effect was observed when the APP homologues APLP1 or APLP2 were used. Heterologous expression of APP695 was shown to increase the level of GAPDH within the cytoplasm by over 100% and within the mitochondria by approximately 50%. Moreover, a shift in organelle distribution from cytoplasm > nucleus > mitochondria in control cell lines to cytoplasm > mitochondria > nucleus in the APP695 overexpressing cell line was also observed. Further, the overexpression of APP695 increased GAPDH aggregation temperature by 3.09 ± 0.46 °C, indicative of greater thermal stability. These results demonstrate a clear correlation between APP overexpression and GAPDH levels, organelle distribution and thermal stability.

New use for CETSA: monitoring innate immune receptor stability via post-translational modification by OGT.

O-GlcNAcylation is a dynamic and functionally diverse post-translational modification shown to affect thousands of proteins, including the innate immune receptor nucleotide-binding oligomerization domain-containing protein 2 (Nod2). Mutations of Nod2 (R702W, G908R and 1007 fs) are associated with Crohn's disease and have lower stabilities compared to wild type. Cycloheximide (CHX)-chase half-life assays have been used to show that O-GlcNAcylation increases the stability and response of both wild type and Crohn's variant Nod2, R702W. A more rapid method to assess stability afforded by post-translational modifications is necessary to fully comprehend the correlation between NLR stability and O-GlcNAcylation. Here, a recently developed cellular thermal shift assay (CETSA) that is typically used to demonstrate protein-ligand binding was adapted to detect shifts in protein stabilization upon increasing O-GlcNAcylation levels in Nod2. This assay was used as a method to predict if other Crohn's associated Nod2 variants were O-GlcNAcylated, and also identified the modification on another NLR, Nod1. Classical immunoprecipitations and NF-κB transcriptional assays were used to confirm the presence and effect of this modification on these proteins. The results presented here demonstrate that CETSA is a convenient method that can be used to detect the stability effect of O-GlcNAcylation on O-GlcNAc-transferase (OGT) client proteins and will be a powerful tool in studying post-translational modification.

Novel approaches to map small molecule-target interactions.

The quest for small molecule perturbators of protein function or a given cellular process lies at the heart of chemical biology and pharmaceutical research. Bioactive compounds need to be extensively characterized in the context of the modulated protein(s) or process(es) in living systems to unravel and confirm their mode of action. A crucial step in this workflow is the identification of the molecular targets for these small molecules, for which a generic methodology is lacking. Herein we summarize recently developed approaches for target identification spurred by advances in omics techniques and chemo- and bioinformatics analysis.

Target engagement in lead generation.

The pharmaceutical industry is currently facing multiple challenges, in particular the low number of new drug approvals in spite of the high level of R&D investment. In order to improve target selection and assess properly the clinical hypothesis, it is important to start building an integrated drug discovery approach during Lead Generation. This should include special emphasis on evaluating target engagement in the target tissue and linking preclinical to clinical readouts. In this review, we would like to illustrate several strategies and technologies for assessing target engagement and the value of its application to medicinal chemistry efforts.

NQO2 is a reactive oxygen species generating off-target for acetaminophen.

The analgesic and antipyretic compound acetaminophen (paracetamol) is one of the most used drugs worldwide. Acetaminophen overdose is also the most common cause for acute liver toxicity. Here we show that acetaminophen and many structurally related compounds bind quinone reductase 2 (NQO2) in vitro and in live cells, establishing NQO2 as a novel off-target. NQO2 modulates the levels of acetaminophen derived reactive oxygen species, more specifically superoxide anions, in cultured cells. In humans, NQO2 is highly expressed in liver and kidney, the main sites of acetaminophen toxicity. We suggest that NQO2 mediated superoxide production may function as a novel mechanism augmenting acetaminophen toxicity.

Quantitative target engagement of RIPK1 in human whole blood via the cellular thermal shift assay for potential pre-clinical and clinical applications.

The cellular thermal shift assay (CETSA®) is a target engagement method widely used for preclinical characterization of small molecule compounds. CETSA® has been used for semi-quantitative readouts in whole blood with PBMC isolation, and quantitative, plate-based readouts using cell lines. However, there has been no quantitative evaluation of CETSA® in unprocessed human whole blood, which is preferred for clinical applications. Here we report two separate assay formats - Alpha CETSA® and MSD CETSA® - that require less than 100 µL of whole blood per sample without PBMC isolation. We chose RIPK1 as a proof-of-concept target and, by measuring engagement of seven different inhibitors, demonstrate high assay sensitivity and robustness. These quantitative CETSA® platforms enable possible applications in preclinical pharmacokinetic-pharmacodynamic studies, and direct target engagement with small molecules in clinical trials.

Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay.

Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.