Affinity Selection Mass Spectrometry Research Articles

Improved Rapid Equilibrium Dialysis-Mass Spectrometry (RED-MS) Method for Measuring Small Molecule-Protein Complex Binding Affinities in Solution.

Rapid equilibrium dialysis (RED) is predominantly used for the characterization of drug absorption, distribution, metabolism, and excretion (ADME) properties in plasma and biological fluids. We describe herein improvements in the use of RED in conjunction with mass spectrometry (RED-MS) to enable robust binding affinity measurements of small molecules for recombinant proteins and complexes from a single dialysis data set. The affinities calculated from RED-MS correlated well with measurements by both surface plasmon resonance (SPR) and affinity selection mass spectrometry (AS-MS). The method was particularly useful for quantifying the binding of small molecules to large protein complexes that were not amendable by common biophysical characterization techniques. Compound pooling and integration with automated liquid handling increased assay throughput and enabled the analysis of hundreds of measurements per week. RED-MS offers a viable option for measuring compound binding in solution and may facilitate small molecule affinity optimization toward difficult-to-drug protein complexes.

The quick screening of binding compounds and proteins for drug discovery and pharmacological research

In drug discovery and pharmacological research, early identification of target molecules for compounds with pharmacological effects is crucial. However, this process often requires significant effort and can be rate-limiting, thereby slowing down research progress. This paper introduces a simplified and rapid method for quick screening of binding compounds or proteins. Utilizing Affinity Selection Mass Spectrometry (ASMS), this technique efficiently detects compound-target binding through size-exclusion chromatography and mass spectrometry. ASMS offers high sensitivity and specificity, making it ideal for accurate identification of binding interactions. We have further enhanced ASMS to handle membrane proteins without solubilization, creating Binder Selection Technology (BST). BST allows screening against both soluble and challenging membrane proteins such as GPCRs and SLC transporters. By using cell membrane fractions or organelle fractions with high target molecule expression, BST efficiently identifies potential binding compounds. This innovative method constructs a comprehensive database of binding compounds for various targets, facilitating rapid hypothesis testing and pharmacological evaluation. Additionally, BST screens 17,000 proteins, including membrane proteins, using wheat germ cell-free and animal cell expression systems. This approach allows exploration of binding interactions without labeling compounds or immobilizing proteins, preserving their native state. BST is powerful for identifying targets of compounds with known pharmacological effects but unknown targets in animal or cell-based assays. By utilizing BST, researchers can overcome bottlenecks in early drug discovery, significantly enhancing research speed and success rates. This method represents a major advancement, providing an efficient and effective way to identify and validate target molecules in drug discovery.

Discovery of ICOS-Targeted Small Molecules Using Affinity Selection Mass Spectrometry Screening.

Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules. To the best of our knowledge, this is the first study that utilizes ASMS screening to discover small molecules targeting immune checkpoints. Compound 9 with a promising ICOS/ICOS-L inhibitory profile (IC50=29.38±3.41 μM) was selected as the template for the modification. Following preliminary structure-activity relationship (SAR) study and molecular dynamic (MD) simulation revealed the critical role of the ortho-hydroxy group on compound 9 in the ICOS binding, as it could stabilize the interaction via the hydrogen bond formation with residuals on the glycan, and the depletion could lead to an activity lost. This work validates a promising inhibitor for the ICOS/ICOS-L interaction, and we anticipate future modifications could provide more potent modulators for this interaction.

Discovery of ICOS-targeted small molecules using affinity selection mass spectrometry screening.

Inducible T cell co-stimulator (ICOS) is a positive immune checkpoint receptor expressed on the surface of activated T cells, which could promote cell function after being stimulated with ICOS ligand (ICOS-L). Although clinical benefits have been reported in the ICOS modulation-based treatment for cancer and autoimmune disease, current modulators are restricted in biologics, whereas ICOS-targeted small molecules are lacking. To fill this gap, we performed an affinity selection mass spectrometry (ASMS) screening for ICOS binding using a library of 15,600 molecules. To the best of our knowledge, this is the first study that utilizes ASMS screening to discover small molecules targeting immune checkpoints. Compound 9 with a promising ICOS/ICOS-L inhibitory profile (IC50 = 29.38 ± 3.41 μM) was selected as the template for the modification. Following preliminary structure-activity relationship (SAR) study and molecular dynamic (MD) simulation revealed the critical role of the ortho-hydroxy group on compound 9 in the ICOS binding, as it could stabilize the interaction via the hydrogen bond formation with residuals on the glycan, and the depletion could lead to an activity lost. This work validates a promising inhibitor for the ICOS/ICOS-L interaction, and we anticipate future modifications could provide more potent modulators for this interaction.

Optimizing the affinity selection mass spectrometry workflow for efficient identification and ranking of potent USP1 inhibitors

An optimized Affinity Selection-Mass Spectrometry (AS-MS) workflow has been developed for the efficient identification of potent USP1 inhibitors. USP1 was immobilized on agarose beads, ensuring low small molecule retention, efficient protein capture, and protein stability. The binding affinity of 49 compounds to USP1 was evaluated using the optimized AS-MS method, calculating binding index (BI) values for each compound. Biochemical inhibition assays validated the AS-MS results, revealing a potential correlation between higher BI values and lower IC50 values. This optimized workflow enables rapid identification of high-quality USP1 inhibitor hits, facilitating structure-activity relationship studies and accelerating the discovery of potential cancer therapeutics.

A duplexed high-throughput mass spectrometry assay for bifunctional POLB polymerase and lyase activity

Polymerase β (POLB), with dual functionality as a lyase and polymerase, plays a critical role in the base excision repair (BER) pathway to maintain genomic stability. POLB knockout and rescue studies in BRCA1/2-mutant cancer cell lines revealed that inhibition of lyase and polymerase activity is required for the synthetic lethal interaction observed with PARP inhibitors, highlighting POLB as a valuable therapeutic target. Traditional biochemical assays to screen for enzyme inhibitors focus on a single substrate to product relationship and limit the comprehensive analysis of enzymes such as POLB that utilize multiple substrates or catalyze a multi-step reaction. This report describes the first high-throughput mass spectrometry-based screen to measure the two distinct biochemical activities of POLB in a single assay using a duplexed self-assembled monolayer desorption ionization (SAMDI) mass spectrometry methodology. A multiplexed assay for POLB dual enzymatic activities was developed optimizing for kinetically balanced conditions and a collection of 200,000 diverse small molecules was screened in the duplexed format. Small molecule modulators identified in the screen were confirmed in a traditional fluorescence-based polymerase strand-displacement assay and an orthogonal label-free binding assay using SAMDI affinity selection mass spectrometry (ASMS). This work demonstrates the flexibility of high-throughput mass spectrometry approaches in drug discovery and highlights a novel application of SAMDI technology that opens new avenues for multiplexed high-throughput screening.

High-throughput screening, “protein–metabolite” interaction, and hypoglycemic effect investigations of α-amylase inhibitors in teas using an affinity selection-mass spectrometry method

Tea is consumed worldwide and can reduce the risk of diabetes whereas the hypoglycemic compounds in tea remain unclear. Herein, the chemical compositions comparison and inhibition assays indicated that the inhibitory effect of tea against α-amylase was positively related with the enzyme-mediated oxidation degree of tea and black teas containing high contents of theaflavins exhibited the strongest in vitro inhibitory effect. Affinity selection-mass spectrometry-based high-throughput screening revealed that four theaflavins specifically bind to α-amylase. Compared to other compounds, theaflavins showed one order of magnitude higher in vitro inhibitory effects; thus, theaflavins are possibly the dominant α-amylase inhibitors in tea. A series of “α-amylase–theaflavins” interaction studies jointly demonstrated that theaflavins noncompetitively inhibit α-amylase activity by interacting with the amino acid residues of α-amylase and then inducing changes in the secondary structure. Furthermore, theaflavins (10 mg/kg/d) and black tea extracts (100 mg/kg/d) exhibited comparable hypoglycemic effects as acarbose in diabetic mice.

Reversibly Reactive Affinity Selection-Mass Spectrometry Enables Identification of Covalent Peptide Binders.

Covalent peptide binders have found applications as activity-based probes and as irreversible therapeutic inhibitors. Currently, there is no rapid, label-free, and tunable affinity selection platform to enrich covalent reactive peptide binders from synthetic libraries. We address this challenge by developing a reversibly reactive affinity selection platform termed ReAct-ASMS enabled by tandem high-resolution mass spectrometry (MS/MS) to identify covalent peptide binders to native protein targets. It uses mixed disulfide-containing peptides to build reversible peptide-protein conjugates that can enrich for covalent variants, which can be sequenced by MS/MS after reduction. Using this platform, we identified covalent peptide binders against two oncoproteins, human papillomavirus 16 early protein 6 (HPV16 E6) and peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 protein (Pin1). The resulting peptide binders efficiently and selectively cross-link Cys58 of E6 at 37 °C and Cys113 of Pin1 at room temperature, respectively. ReAct-ASMS enables the identification of highly selective covalent peptide binders for diverse molecular targets, introducing an applicable platform to assist preclinical therapeutic development pipelines.

Development of an α-Klotho Recognizing High-Affinity Peptide Probe from In-Solution Enrichment.

The kidney, parathyroid gland, and choroid plexus express the aging-related transmembrane protein α-Klotho, a coreceptor of the fibroblast growth factor 23 (FGF23) receptor complex. Reduced α-Klotho levels are correlated with chronic kidney disease and other age-related diseases, wherein they are released from membranes into circulation. Klotho's potential physiological action as a hormone is of current scientific interest. Part of the challenges associated with advancing these studies, however, has been the long-standing difficulty in detecting soluble α-Klotho in biofluids. Here, we describe the discovery of peptides that recognize α-Klotho with high affinity and selectivity by applying in-solution size-exclusion-based affinity selection-mass spectrometry (AS-MS). After two rounds of AS-MS and subsequent N-terminal modifications, the peptides improved their binding affinity to α-Klotho by approximately 2300-fold compared to the reported starting peptide Pep-10, previously designed based on the C-terminal region of FGF23. The lead peptide binders were shown to enrich α-Klotho from cell lysates and to label α-Klotho in kidney cells. Our results further support the utility of in-solution, label-free AS-MS protocols to discover peptide-based binders to target proteins of interest with high affinity and selectivity, resulting in functional probes for biological studies.

Advancing Kir4.2 Channel Ligand Identification through Collision-Induced Affinity Selection Mass Spectrometry.

The inwardly rectifying potassium Kir4.2 channel plays a crucial role in regulating membrane potentials and maintaining potassium homeostasis. Kir4.2 has been implicated in various physiological processes, including insulin secretion, gastric acid regulation, and the pathogenesis of central nervous system diseases. Despite its significance, the number of identified ligands for Kir4.2 remains limited. In this study, we established a method to directly observe ligands avoiding a requirement to observe the high-mass ligand-membrane protein-detergent complexes. This method used collision-induced affinity selection mass spectrometry (CIAS-MS) to identify ligands dissociated from the Kir4.2 channel-detergent complex. The CIAS-MS approach integrated all stages of affinity selection within the mass spectrometer, offering advantages in terms of time efficiency and cost-effectiveness. Additionally, we explored the effect of collisional voltage ramps on the dissociation behavior of the ligand and the ligand at different concentrations, demonstrating dose dependency.

Identification of Ligands for Ion Channels: TRPM2.

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable, nonselective cation channel with a widespread distribution throughout the body. It is involved in many pathological and physiological processes, making it a potential therapeutic target for various diseases, including Alzheimer's disease, Parkinson's disease, and cancers. New analytical techniques are beneficial for gaining a deeper understanding of its involvement in disease pathogenesis and for advancing the drug discovery for TRPM2-related diseases. In this work, we present the application of collision-induced affinity selection mass spectrometry (CIAS-MS) for the direct identification of ligands binding to TRPM2. CIAS-MS circumvents the need for high mass detection typically associated with mass spectrometry of large membrane proteins. Instead, it focuses on the detection of small molecules dissociated from the ligand-protein-detergent complexes. This affinity selection approach consolidates all affinity selection steps within the mass spectrometer, resulting in a streamlined process. We showed the direct identification of a known TRPM2 ligand dissociated from the protein-ligand complex. We demonstrated that CIAS-MS can identify binding ligands from complex mixtures of compounds and screened a compound library against TRPM2. We investigated the impact of voltage increments and ligand concentrations on the dissociation behavior of the binding ligand, revealing a dose-dependent relationship.

High-throughput screening and investigation of the inhibitory mechanism of α-glucosidase inhibitors in teas using an affinity selection-mass spectrometry method.

An affinity selection-mass spectrometry method was applied for high-throughput screening of α-glucosidase (AGH) inhibitors from teas. Fourteen out of nineteen screened AGH inhibitor candidates were clustered as galloylated polyphenols (GPs). "AGH-GPs" interaction studies, including enzyme kinetics, fluorescence spectroscopy, circular dichroism, and molecular docking, jointly suggested that GPs noncompetitively inhibit AGH activity by interacting with amino acid residues near the active site of AGH and inducing changes in AGH secondary structure. Representative GPs and white tea extract (WTE) showed comparable AGH inhibition effects in Caco2 cells and postprandial hypoglycemic efficacy in diabetic mice as acarbose. The area under the curve of oral sucrose tolerance test was lower by 8.16%, 6.17%, and 7.37% than control group in 15mg/kg EGCG, 15mg/kg strictinin, and 150mg/kg WTE group, respectively. Our study presents a high-efficiency approach to discover novel AGH inhibitors and elucidates a potential mechanism by which tea decreases diabetes risks.

Women in (Bio)Analytical Chemistry: Moving Towards Equity and Inclusion

Women's participation in science has been an increasingly relevant and discussed topic in recent decades. Throughout history, women have faced numerous barriers and challenges to enter and stand out in this field of knowledge. However, despite the difficulties, women have contributed significantly to scientific advancement in all areas. In Brazil, the discussion about the participation of women in science has been highlighted in national events around analytical chemistry and academia. Thus, some changes have been implemented to minimize the differences discussed herein. Today we see more and more women engaged in science, holding prominent positions as researchers, professors, and leaders in their fields. They exist in many scientific disciplines, including physics, chemistry, biology, mathematics, engineering, and technology. In addition, women have also been involved in emerging and interdisciplinary areas such as computer science, artificial intelligence, and biotechnology. It is crucial to promote gender equality in chemistry by providing an inclusive environment and encouraging the active participation of women. This can be achieved by implementing policies that promote equal opportunities, encouraging female role models and mentors, supporting women chemists' training and professional development, and valuing and recognizing their contributions. Women's participation in chemistry is essential to scientific advancement in this field. We bring unique perspectives, creativity, and skills to address chemical challenges and contribute to a more comprehensive understanding of chemical processes and their practical applications. Gender diversity in chemistry drives innovation and promotes a more prosperous and inclusive scientific environment. It is essential to provide equal opportunities, support, and encouragement for women to enter and thrive in academic careers in the exact sciences, thus strengthening female representation in this field and boosting scientific excellence. In this context, the BrJAC journal worked on organizing an issue dedicated to women researchers in the field of (bio)analytical chemistry. The central idea is to celebrate those female researchers who have worked hard and dedicatedly in analytical chemistry. The interview was with Dr. Joanna Szpunar from the National Research Council of France (CNRS). She talked about her career and experience as a prominent researcher in the spectroanalytical chemistry field. The point of view was with Dr. Maria Valnice Boldrin Zanoni from UNESP, who excels in electroanalytical chemistry and shares a new perspective on artificial photosynthesis technology. The Letter written by Dr. Quezia Cass from UFSCar presented us with text on separation techniques highlighting affinity selection mass spectrometry. The issue will also feature three reviews, eight articles, and three technical notes, all led by women in (bio)analytical chemistry. It is imperative to thank all the reviewers who participated in this process by giving their time and expertise in evaluating the manuscripts. Many thanks! We hope you enjoy the reading and learn some of the work these researchers have been developing in (bio)analytical chemistry.

Affinity Selection Mass Spectrometry (AS-MS) as a Tool for Prospecting Target Ligands

Affinity selection mass spectrometry (AS-MS) has been shown to be a powerful tool for identifying bioactive molecules in synthetic and/or natural libraries. The selection provided by the formation of the target-ligand complex allows the identification of hits irrespective of their functional effect. Moreover, it precludes the use of label, since the binders are identified by their exact mass.1 The binders are determined by an affinity or index ratio calculated through control assays. The target protein can be used in solution or immobilized in a solid support (Figure 1). Both approaches have pros and cons. Unlike most conventional high-throughput screening assays, AS-MS has fewer or no limitations when it comes to target selection. It is important, however, to understand the implications of choosing membrane proteins as targets. Membrane proteins correspond to 42% of all drug targets listed in DrugBank. Moreover, they are likely to be selected as protein targets due to their participation in many disease pathways, acting as ion channels, molecular transporters, solute carriers, receptors, and anchors. One of the bottlenecks in working with membrane proteins comes from the need to use a detergent for solubilization, folding, and structure maintenance. Detergents are usually used above the critical micelle concentration, which can lead to empty micelles and thus to false positive results, caused by nonspecific interactions with the detergent micelles.8 Interference in the ionization of the binders also needs to be examined.

Affinity selection of double-click triazole libraries for rapid discovery of allosteric modulators for GLP-1 receptor

The recently developed double-click reaction sequence [G. Meng et al.,Nature 574, 86-89 (2019)] is expected to vastly expand the number and diversity of synthetically accessible 1,2,3-triazole derivatives. However, it remains elusive how to rapidly navigate the extensive chemical space created by double-click chemistry for bioactive compound discovery. In this study, we selected a particularly challenging drug target, the glucagon-like-peptide-1 receptor (GLP-1R), to benchmark our new platform for the design, synthesis, and screening of double-click triazole libraries. First, we achieved a streamlined synthesis of customized triazole libraries on an unprecedented scale (composed of 38,400 new compounds). By interfacing affinity-selection mass spectrometry and functional assays, we identified a series of positive allosteric modulators (PAMs) with unreported scaffolds that can selectively and robustly enhance the signaling activity of the endogenous GLP-1(9-36) peptide. Intriguingly, we further revealed an unexpected binding mode of new PAMs which likely act as a molecular glue between the receptor and the peptide agonist. We anticipate the merger of double-click library synthesis with the hybrid screening platform allows for efficient and economic discovery of drug candidates or chemical probes for various therapeutic targets.

Targeting RNA with small molecules: lessons learned from Xist RNA.

Although more than 98% of the human genome is noncoding, nearly all drugs on the market target one of about 700 disease-related proteins. However, an increasing number of diseases are now being attributed to noncoding RNA and the ability to target them would vastly expand the chemical space for drug development. We recently devised a screening strategy based upon affinity-selection mass spectrometry and succeeded in identifying bioactive compounds for the noncoding RNA prototype, Xist. One such compound, termed X1, has drug-like properties and binds specifically to the RepA motif of Xist in vitro and in vivo. Small-angle X-ray scattering analysis reveals that X1 changes the conformation of RepA in solution, thereby explaining the displacement of cognate interacting protein factors (PRC2 and SPEN) and inhibition of X-chromosome inactivation. In this Perspective, we discuss lessons learned from these proof-of-concept experiments and suggest that RNA can be systematically targeted by drug-like compounds to disrupt RNA structure and function.

A high-throughput MALDI-TOF MS biochemical screen for small molecule inhibitors of the antigen aminopeptidase ERAP1.

MALDI-TOF MS is a powerful analytical technique that provides a fast and label-free readout for in vitro assays in the high-throughput screening (HTS) environment. Here, we describe the development of a novel, HTS compatible, MALDI-TOF MS-based drug discovery assay for the endoplasmic reticulum aminopeptidase 1 (ERAP1), an important target in immuno-oncology and auto-immune diseases. A MALDI-TOF MS assay was developed beginning with an already established ERAP1 RapidFire MS (RF MS) assay, where the peptide YTAFTIPSI is trimmed into the product TAFTIPSI. We noted low ionisation efficiency of these peptides in MALDI-TOF MS and hence incorporated arginine residues into the peptide sequences to improve ionisation. The optimal assay conditions were established with these new basic assay peptides on the MALDI-TOF MS platform and validated with known ERAP1 inhibitors. Assay stability, reproducibility and robustness was demonstrated on the MALDI-TOF MS platform. From a set of 699 confirmed ERAP1 binders, identified in a prior affinity selection mass spectrometry (ASMS) screen, active compounds were determined at single concentration and in a dose-response format with the new MALDI-TOF MS setup. Furthermore, to allow for platform performance comparison, the same compound set was tested on the established RF MS setup, as the new basic peptides showed fragmentation in ESI-MS. The two platforms showed a comparable performance, but the MALDI-TOF MS platform had several advantages, such as shorter sample cycle times, reduced reagent consumption, and a lower tight-binding limit.

Dipyridamole interacts with the N-terminal domain of HSP90 and antagonizes the function of the chaperone in multiple cancer cell lines

Molecular chaperone HSP90 has been considered as a promising target for anti-cancer drug development for years. However, due to the heat shock response induced by the ATP competitive inhibitors against HSP90, the therapeutic efficacies of the compounds are compromised, which consequently restricts the clinical use of HSP90-targeted inhibitors. Therefore, there is a need to discover novel HSP90-targeted modulators which exhibit acceptable inhibition activity against the chaperone and do not induce significant heat shock response in the meantime. Here in this study, we firstly developed a tip-based affinity selection-mass spectrometry platform with optimized experimental conditions/parameters for HSP90-targeted active compound screening, and then applied it to fish out inhibitors against HSP90 from a collection of 2,395 compounds composed of FDA-approved drugs and drug candidates. Dipyridamole, which acts as an anti-thrombotic agent by modulating multiple targets and has a long history of safe use, was identified to interact with HSP90′s N-terminal domain. The following conducted biophysical and biochemical experiments demonstrated that Dipyridamole could bind to HSP90′s ATP binding pocket and function as an ATP competitive inhibitor of the chaperone. Finally, cellular-based assays including CESTA, cell viability assessment and proteomic analysis etc. were performed to evaluate whether the interaction between HSP90 and Dipyridamole contributes to the anti-tumor effects of the compound. We then found that Dipyridamole inhibits the growth and proliferation of human cancer cells by downregulating cell cycle regulators and upregulating apoptotic cell signaling, which are potentially mediated by the binding of Dipyridamole to HSP90 and to PDEs (phosphodiesterases), respectively.

Advancing ASMS with LC-MS/MS for the discovery of novel PDCL2 ligands from DNA-encoded chemical library selections.

A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure. To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy. We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS). After a screening process of PDCL2 with DECLs containing>440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory andwhich were further confirmed by a thermal shift assay. With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive.

The long non-coding RNA Heat4 is elevated in heart failure patients and mediates anti-inflammatory functions thereby promoting vascular regeneration

Abstract Background and purpose Activation of the immune system correlates with the severity and the prognosis of patients with heart failure (HF). Here, we aim to identify and characterize long non-coding RNAs (lncRNAs) as a potential mechanistic link between the activation of the immune system and the pathophysiology of HF. Methods and results Using next-generation sequencing we found a yet uncharacterized lncRNA to be significantly upregulated in peripheral blood mononuclear cells of ischemic cardiomyopathy patients compared to controls, which we named Heat4 – Heart-disease associated transcript 4 (N=4; 2.05-fold increase; p<0.05). In the blood, monocytes show the highest expression of Heat4 and here in particular the non-classical monocytes compared to classical monocytes (N=4; 3.37-fold; p<0.05). Matching the known anti-inflammatory properties of this monocyte subpopulation we found that overexpression of Heat4 in monocytes resulted in decreased levels of inflammation (TNFα: −38.6%; p<0.05). Accordingly, a knockdown of Heat4 increased levels of inflammatory cytokine expression (TNFα: +4.14-fold; p<0.05). Non-classical monocytes are known to maintain vascular homeostasis by patrolling the endothelium in search of injury. Indeed, overexpression of Heat4 in human monocytes increased vascular regeneration after injury of the carotid artery in NOD-SCID mice (N=6; +1.85-fold compared to injection of control monocytes; p<0.05). We found Heat4 enriched in the cytoplasm of monocytes compared to the nuclear fraction. Using biotin-labelled RNA probes containing 2$'$O-Me-RNA oligonucleotides we performed RNA antisense affinity selection and subsequent mass spectrometry to identify proteins interacting with Heat4. We found two proteins, namely IP1 and IP2, enriched in the Heat4 fraction (+1.20 and +1.45-fold, respectively compared to the control probe). Knockdown of IP1 resulted in reduced induction of inflammatory gene expression (IL-6: −49.2%; p<0.05) after stimulation of monocytes with TNFα. Mechanistically, overexpression of Heat4 resulted in reduced extracellular levels of the IP1/IP2 heterodimer (IP1/IP2: −23.6%; p<0.05) as determined by ELISA. Conclusion The lncRNA Heat4 is elevated in the blood of patients with HF. Heat4 limits the extent of the inflammatory response of non-classical monocytes and leads to a faster regeneration after vascular injury. Heat4 is located in the cytoplasm of monocytes interacting with the pro-inflammatory proteins IP1/IP2 and repealing their extracellular release. Modulating Heat4 levels may represent a novel strategy for treatment of cardiovascular diseases with impaired vascular functions. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Projektfoerderung im Bereich der Herzmedizin, Leipzig