A Chemical Acetylation-Based Mass Spectrometry Platform for Histone Methylation Profiling.

Histones are highly posttranslationally modified proteins that regulate gene expression by modulating chromatin structure and function. Acetylation and methylation are the most abundant histone modifications, with methylation occurring on lysine (mono-, di-, and trimethylation) and arginine (mono- and dimethylation) predominately on histones H3 and H4. In addition, arginine dimethylation can occur either symmetrically (SDMA) or asymmetrically (ADMA) conferring different biological functions. Despite the importance of histone methylation on gene regulation, characterization and quantitation of this modification have proven to be quite challenging. Great advances have been made in the analysis of histone modification using both bottom-up and top-down mass spectrometry (MS). However, MS-based analysis of histone posttranslational modifications (PTMs) is still problematic, due both to the basic nature of the histone N-terminal tails and to the combinatorial complexity of the histone PTMs. In this report, we describe a simplified MS-based platform for histone methylation analysis. The strategy uses chemical acetylation with d0-acetic anhydride to collapse all the differently acetylated histone forms into one form, greatly reducing the complexity of the peptide mixture and improving sensitivity for the detection of methylation via summation of all the differently acetylated forms. We have used this strategy for the robust identification and relative quantitation of H4R3 methylation, for which stoichiometry and symmetry status were determined, providing an antibody-independent evidence that H4R3 is a substrate for both Type I and Type II PRMTs. Additionally, this approach permitted the robust detection of H4K5 monomethylation, a very low stoichiometry methylation event (0.02% methylation). In an independent example, we developed an in vitro assay to profile H3K27 methylation and applied it to an EZH2 mutant xenograft model following small-molecule inhibition of the EZH2 methyltransferase. These specific examples highlight the utility of this simplified MS-based approach to quantify histone methylation profiles.

Ubiquitination of the scaffold protein IQGAP1 diminishes its interaction with and activation of the Rho GTPase CDC42.

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a scaffold protein that interacts with numerous binding partners and thereby regulates fundamental biological processes. The functions of IQGAP1 are modulated by several mechanisms, including protein binding, self-association, subcellular localization, and phosphorylation. Proteome-wide screens have indicated that IQGAP1 is ubiquitinated, but the possible effects of this post-translational modification on its function are unknown. Here we characterized and evaluated the function of IQGAP1 ubiquitination. Using MS-based analysis in HEK293 cells, we identified six lysine residues (Lys-556, -1155, -1230, -1465, -1475, and -1528) as ubiquitination sites in IQGAP1. To elucidate the biological consequences of IQGAP1 ubiquitination, we converted each of these lysines to arginine and found that replacing two of these residues, Lys-1155 and Lys-1230, in the GAP-related domain of IQGAP1 (termed IQGAP1 GRD-2K) reduces its ubiquitination. Moreover, IQGAP1 GRD-2K bound a significantly greater proportion of the two Rho GTPases cell division cycle 42 (CDC42) and Rac family small GTPase 1 (RAC1) than did WT IQGAP1. Consistent with this observation, reconstitution of IQGAP1-null cells with IQGAP1 GRD-2K significantly increased the amount of active CDC42 and enhanced cell migration significantly more than WT IQGAP1. Our results reveal that ubiquitination of the CDC42 regulator IQGAP1 alters its ability to bind to and activate this GTPase, leading to physiological effects. Collectively, these findings expand our view of the role of ubiquitination in cell signaling and provide additional insight into CDC42 regulation.

One-Step Synthesis of Photoaffinity Probes for Live-Cell MS-Based Proteomics.

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

A Photoaffinity-Based Fragment-Screening Platform for Efficient Identification of Protein Ligands.

Advances in genomic analyses enable the identification of new proteins that are associated with disease. To validate these targets, tool molecules are required to demonstrate that a ligand can have a disease-modifying effect. Currently, as tools are reported for only a fraction of the proteome, platforms for ligand discovery are essential to leverage insights from genomic analyses. Fragment screening offers an efficient approach to explore chemical space. Presented here is a fragment-screening platform, termed PhABits (PhotoAffinity Bits), which utilizes a library of photoreactive fragments to covalently capture fragment-protein interactions. Hits can be profiled to determine potency and the site of crosslinking, and subsequently developed as reporters in a competitive displacement assay to identify novel hit matter. The PhABit platform is envisioned to be widely applicable to novel protein targets, identifying starting points in the development of therapeutics.

A Photoaffinity Displacement Assay and Probes to Study the Cyclin-Dependent Kinase Family.

The CDK family plays a crucial role in the control of the cell cycle. Dysregulation and mutation of the CDKs has been implicated in cancer and the CDKs have been investigated extensively as potential therapeutic targets. Selective inhibition of specific isoforms of the CDKs is crucial to achieve therapeutic effect while minimising toxicity. We present a group of photoaffinity probes designed to bind to the family of CDKs. The site of crosslinking of the optimised probe, as well as its ability to enrich members of the CDK family from cell lysates, was investigated. In a proof of concept study, we subsequently developed a photoaffinity probe-based competition assay to profile CDK inhibitors. We anticipate that this approach will be widely applicable to the study of small molecule binding to protein families of interest.

An optimized platform for hydrophilic interaction chromatography-immobilized metal affinity chromatography enables deep coverage of the rat liver phosphoproteome.

While analysis of the phosphoproteome has become an important component of understanding how cells function, it remains a nontrivial task in terms of the number of sample preparation steps and instrument time needed to achieve sufficient depth of coverage to produce meaningful results. We previously described a multidimensional method that uses hydrophilic interaction chromatography (HILIC) followed by Fe(3+) immobilized metal affinity chromatography (IMAC) to reduce complexity, improve selectivity, and increase phosphopeptide identifications. Here we present refinements to our overall protocol that make it simpler and more efficient, while they provide greater coverage of the phosphoproteome. We introduce filter-aided sample prep (FASP) for cell lysis and trypsin digestion. Following HILIC separation, fractions are IMAC enriched using a 96-well filter plate. Finally, enriched samples are analyzed using an LC-MS strategy optimized for the fractionation scheme. The optimized protocol improves protein recovery, simplifies phosphopeptide enrichment, and optimizes instrument time, while it maintains deep coverage of the phosphoproteome. By using the refined protocol, we identified more than 16,000 unique phosphosites from rat liver in a single experiment, which used approximately 1 day of instrument time. All together, we present evidence for 24,485 rat liver phosphosites that represents the deepest coverage of a tissue phosphoproteome to date.

Novel WRN Helicase Inhibitors Selectively Target Microsatellite-Unstable Cancer Cells.

Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumors, and WRN inhibitors are in development. In this study, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth in vitro and in vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Significance: We report the discovery and characterization of potent, selective WRN helicase inhibitors for MSI cancer treatment, with biomarker analysis and evaluation of efficacy in vivo and in immunotherapy-refractory preclinical models. These findings pave the way to translate WRN inhibition into MSI cancer therapies and provide tools to investigate WRN biology. See related commentary by Wainberg, p. 1369.

Efficient Ligand Discovery Using Sulfur(VI) Fluoride Reactive Fragments.

Sulfur(VI) fluorides (SFs) have emerged as valuable electrophiles for the design of "beyond-cysteine" covalent inhibitors and offer potential for expansion of the liganded proteome. Since SFs target a broad range of nucleophilic amino acids, they deliver an approach for the covalent modification of proteins without requirement for a proximal cysteine residue. Further to this, libraries of reactive fragments present an innovative approach for the discovery of ligands and tools for proteins of interest by leveraging a breadth of mass spectrometry analytical approaches. Herein, we report a screening approach that exploits the unique properties of SFs for this purpose. Libraries of SF-containing reactive fragments were synthesized, and a direct-to-biology workflow was taken to efficiently identify hit compounds for CAII and BCL6. The most promising hits were further characterized to establish the site(s) of covalent modification, modification kinetics, and target engagement in cells. Crystallography was used to gain a detailed molecular understanding of how these reactive fragments bind to their target. It is anticipated that this screening protocol can be used for the accelerated discovery of "beyond-cysteine" covalent inhibitors.

Reactive fragments targeting carboxylate residues employing direct to biology, high-throughput chemistry.

The screening of covalent or 'reactive' fragment libraries against proteins is becoming an integral approach in hit identification, enabling the development of targeted covalent inhibitors and tools. To date, reactive fragment screening has been limited to targeting cysteine residues, thus restricting applicability across the proteome. Carboxylate residues present a unique opportunity to expand the accessible residues due to high proteome occurrence (∼12%). Herein, we present the development of a carboxylate-targeting reactive fragment screening platform utilising 2-aryl-5-carboxytetrazole (ACT) as the photoreactive functionality. The utility of ACT photoreactive fragments (ACT-PhABits) was evaluated by screening a 546-membered library with a small panel of purified proteins. Hits identified for BCL6 and KRASG12D were characterised by LC-MS/MS studies, revealing the selectivity of the ACT group. Finally, a photosensitised approach to ACT activation was developed, obviating the need for high energy UV-B light.

Orthogonal quantification of soluble inducible T-cell costimulator (ICOS) in healthy and diseased human serum.

Inducible T-cell costimulator (ICOS), a homodimeric protein expressed on the surface of activated T-cells, is being investigated as a potential therapeutic target to treat various cancers. Recent studies have reported aberrant increases in the soluble form of ICOS (sICOS) in human serum in disease-state patients, primarily using commercial ELISA kits. However, results from our in-house immunoassay did not show these aberrant increases, leading us to speculate that commercial sICOS ELISAs may be prone to interference. We directly tested that hypothesis and found that one widely used commercial kit yields false-positives and is prone to human anti-mouse antibody interference. We then analyzed a panel of healthy, cancer, chronic hepatitis C virus, systemic lupus erythematosus, and diffuse cutaneous systemic sclerosis human serum using our in-house immunoassay and reported the measured sICOS concentrations in these populations. Since even well characterized immunoassay methods are prone to non-specific interference, we also developed a novel sICOS LC-MS/MS method to confirm the results. Using these orthogonal approaches, we show that sICOS is a low abundance soluble protein that cannot be measured above approximately 20 pg/mL in human serum.

Complement membrane attack complex is an immunometabolic regulator of NLRP3 activation and IL-18 secretion in human macrophages.

The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type.

Acetylation of the Catalytic Lysine Inhibits Kinase Activity in PI3Kδ.

Covalent inhibition is a powerful strategy to develop potent and selective small molecule kinase inhibitors. Targeting the conserved catalytic lysine is an attractive method for selective kinase inactivation. We have developed novel, selective inhibitors of phosphoinositide 3-kinase δ (PI3Kδ) which acylate the catalytic lysine, Lys779, using activated esters as the reactive electrophiles. The acylating agents were prepared by adding the activated ester motif to a known selective dihydroisobenzofuran PI3Kδ inhibitor. Three esters were designed, including an acetate ester which was the smallest lysine modification evaluated in this work. Covalent binding to the enzyme was characterized by intact protein mass spectrometry of the PI3Kδ-ester adducts. An enzymatic digest coupled with tandem mass spectrometry identified Lys779 as the covalent binding site, and a biochemical activity assay confirmed that PI3Kδ inhibition was a direct result of covalent lysine acylation. These results indicate that a simple chemical modification such as lysine acetylation is sufficient to inhibit kinase activity. The selectivity of the compounds was evaluated against lipid kinases in cell lysates using a chemoproteomic binding assay. Due to the conserved nature of the catalytic lysine across the kinome, we believe the covalent inhibition strategy presented here could be applicable to a broad range of clinically relevant targets.

A direct-to-biology high-throughput chemistry approach to reactive fragment screening.

Methods for rapid identification of chemical tools are essential for the validation of emerging targets and to provide medicinal chemistry starting points for the development of new medicines. Here, we report a screening platform that combines 'direct-to-biology' high-throughput chemistry (D2B-HTC) with photoreactive fragments. The platform enabled the rapid synthesis of >1000 PhotoAffinity Bits (HTC-PhABits) in 384-well plates in 24 h and their subsequent screening as crude reaction products with a protein target without purification. Screening the HTC-PhABit library with carbonic anhydrase I (CAI) afforded 7 hits (0.7% hit rate), which were found to covalently crosslink in the Zn2+ binding pocket. A powerful advantage of the D2B-HTC screening platform is the ability to rapidly perform iterative design-make-test cycles, accelerating the development and optimisation of chemical tools and medicinal chemistry starting points with little investment of resource.

The catalytic role of INCENP in Aurora B activation and the kinetic mechanism of Aurora B/INCENP.

Aurora kinases are a family of serine/threonine protein kinases that play essential roles in mitosis and cytokinesis. AurB (Aurora B kinase) has shown a clear link to cancer and is being pursued as an attractive cancer target. Multiple small molecules targeting AurB have entered the clinic for the treatment of cancer. A protein cofactor, INCENP (inner centromere protein), regulates the cellular localization and activation of AurB. In the present study, we examined the effect of INCENP on the activation kinetics of AurB and also elucidated the kinetic mechanism of AurB-catalysed substrate phosphorylation. We have concluded that: (i) substoichoimetric concentrations of INCENP are sufficient for AurB autophosphorylation at the activation loop residue Thr(232), and hence INCENP plays a catalytic role in AurB autophosphorylation; (ii) AurB/INCENP-catalysed phosphorylation of a peptide substrate proceeds through a rapid equilibrium random Bi Bi kinetic mechanism; and (iii) INCENP has relatively minor effects on the specific activity of AurB using a peptide substrate when compared with its role in AurB autoactivation. These results indicate that the effects of INCENP, and probably accessory proteins in general, may differ when enzymes are acting on different downstream targets.

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

Anti-tumor Activity of the Type I PRMT Inhibitor, GSK3368715, Synergizes with PRMT5 Inhibition through MTAP Loss.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of arginines on proteins. Type I PRMTs and their substrates have been implicated in human cancers, suggesting inhibition of type I PRMTs may offer a therapeutic approach for oncology. The current report describes GSK3368715 (EPZ019997), a potent, reversible type I PRMT inhibitor with anti-tumor effects in human cancer models. Inhibition of PRMT5, the predominant type II PRMT, produces synergistic cancer cell growth inhibition when combined with GSK3368715. Interestingly, deletion of the methylthioadenosine phosphorylase gene (MTAP) results in accumulation of the metabolite 2-methylthioadenosine, an endogenous inhibitor of PRMT5, and correlates with sensitivity to GSK3368715 in cell lines. These data provide rationale to explore MTAP status as a biomarker strategy for patient selection.

Interaction between Brk kinase and insulin receptor substrate-4.

Breast tumor kinase (Brk) is a member of the Frk family of nonreceptor tyrosine kinases that is overexpressed in a high percentage of human breast tumors. The downstream substrates and effectors of Brk remain largely unidentified. In this study, we carried out immunoprecipitation and mass spectrometry experiments to identify new Brk binding partners. One interacting protein was insulin receptor substrate 4 (IRS-4), a member of the IRS family. We confirmed that Brk associates with IRS-4 in resting and insulin-like growth factor 1 (IGF-1)-stimulated HEK 293 cells. The SH3 and SH2 domains of Brk are both involved in the association. The tyrosine phosphorylation of Brk increases after stimulation with IGF-1, and in MCF-7 breast cancer cells we show that the presence of IRS-4 enhances this effect. Finally, we demonstrate that endogenous Brk and IRS-4 interact in A431 human epidermoid carcinoma cells.

Structure-Based Design of a Novel SMYD3 Inhibitor that Bridges the SAM-and MEKK2-Binding Pockets.

SMYD3 is a lysine methyltransferase overexpressed in colorectal, breast, prostate, and hepatocellular tumors, and has been implicated as an oncogene in human malignancies. Methylation of MEKK2 by SMYD3 is important for regulation of the MEK/ERK pathway, suggesting the possibility of selectively targeting SMYD3 in RAS-driven cancers. Structural and kinetic characterization of SMYD3 was undertaken leading to a co-crystal structure of SMYD3 with a MEKK2-peptide substrate bound, and the observation that SMYD3 follows a partially processive mechanism. These insights allowed for the design of GSK2807, a potent and selective, SAM-competitive inhibitor of SMYD3 (Ki = 14 nM). A high-resolution crystal structure reveals that GSK2807 bridges the gap between the SAM-binding pocket and the substrate lysine tunnel of SMYD3. Taken together, our data demonstrate that small-molecule inhibitors of SMYD3 can be designed to prevent methylation of MEKK2 and these could have potential use as anticancer therapeutics.

Comparative phosphorylation site mapping from gel-derived proteins using a multidimensional ES/MS-based approach.

Understanding how phosphorylation regulates the behavior of individual proteins is critical to understanding signaling pathways. These studies usually involve knowledge of which amino acid residues are phosphorylated on a given protein and the extent of such a modification. This is often a rather difficult task in that most phosphoproteins contain multiple substoichiometric sites of phosphorylation. Here we describe the multidimensional electrospray (ES) mass spectrometry (MS)-based phosphopeptide-mapping strategy developed in our laboratory. In the first dimension of the process, phosphopeptides present in a protein digest are selectively detected and collected into fractions during on-line liquid chromatography (LC)/ES/MS, which monitors for phosphopeptide-specific marker ions. This analysis generates a phosphorylation profile that can be used to assess changes in the phosphorylation state of a protein pointing to those phosphopeptides that require further investigation. The phosphopeptide-containing fractions are then analyzed in the second dimension by nano-ES with precursor-ion scan for the marker ion m/z 79. As the final step, direct sequencing of the phosphopeptides is performed by LC/ES/MS/MS. Merits and limitations of the strategy, as well as experimental details and suggestions, are described here.

Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

Sickle cell anemia (SCA) is a genetic disorder of the ß-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.