Deep thermal profiling for detection of functional proteoform groups.

The complexity of the functional proteome extends considerably beyond the coding genome, resulting in millions of proteoforms. Investigation of proteoforms and their functional roles is important to understand cellular physiology and its deregulation in diseases but challenging to perform systematically. Here we applied thermal proteome profiling with deep peptide coverage to detect functional proteoform groups in acute lymphoblastic leukemia cell lines with different cytogenetic aberrations. We detected 15,846 proteoforms, capturing differently spliced, cleaved and post-translationally modified proteins expressed from 9,290 genes. We identified differential co-aggregation of proteoform pairs and established links to disease biology. Moreover, we systematically made use of measured biophysical proteoform states to find specific biomarkers of drug sensitivity. Our approach, thus, provides a powerful and unique tool for systematic detection and functional annotation of proteoform groups.

Preclinical efficacy of azacitidine and venetoclax for infant KMT2A-rearranged acute lymphoblastic leukemia reveals a new therapeutic strategy.

Infants with KMT2A-rearranged B-cell acute lymphoblastic leukemia (ALL) have a dismal prognosis. Survival outcomes have remained static in recent decades despite treatment intensification and novel therapies are urgently required. KMT2A-rearranged infant ALL cells are characterized by an abundance of promoter hypermethylation and exhibit high BCL-2 expression, highlighting potential for therapeutic targeting. Here, we show that hypomethylating agents exhibit in vitro additivity when combined with most conventional chemotherapeutic agents. However, in a subset of samples an antagonistic effect was seen between several agents. This was most evident when hypomethylating agents were combined with methotrexate, with upregulation of ATP-binding cassette transporters identified as a potential mechanism. Single agent treatment with azacitidine and decitabine significantly prolonged in vivo survival in KMT2A-rearranged infant ALL xenografts. Treatment of KMT2A-rearranged infant ALL cell lines with azacitidine and decitabine led to differential genome-wide DNA methylation, changes in gene expression and thermal proteome profiling revealed the target protein-binding landscape of these agents. The selective BCL-2 inhibitor, venetoclax, exhibited in vitro additivity in combination with hypomethylating or conventional chemotherapeutic agents. The addition of venetoclax to azacitidine resulted in a significant in vivo survival advantage indicating the therapeutic potential of this combination to improve outcome for infants with KMT2A-rearranged ALL.

Integrative multi-omics and drug response profiling of childhood acute lymphoblastic leukemia cell lines.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Although standard-of-care chemotherapeutics are sufficient for most ALL cases, there are subsets of patients with poor response who relapse in disease. The biology underlying differences between subtypes and their response to therapy has only partially been explained by genetic and transcriptomic profiling. Here, we perform comprehensive multi-omic analyses of 49 readily available childhood ALL cell lines, using proteomics, transcriptomics, and pharmacoproteomic characterization. We connect the molecular phenotypes with drug responses to 528 oncology drugs, identifying drug correlations as well as lineage-dependent correlations. We also identify the diacylglycerol-analog bryostatin-1 as a therapeutic candidate in the MEF2D-HNRNPUL1 fusion high-risk subtype, for which this drug activates pro-apoptotic ERK signaling associated with molecular mediators of pre-B cell negative selection. Our data is the foundation for the interactive online Functional Omics Resource of ALL (FORALL) with navigable proteomics, transcriptomics, and drug sensitivity profiles at https://proteomics.se/forall .

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.

Chemical Phosphoproteomics Sheds New Light on the Targets and Modes of Action of AKT Inhibitors.

Due to its important roles in oncogenic signaling, AKT has been subjected to extensive drug discovery efforts leading to small molecule inhibitors investigated in advanced clinical trials. To better understand how these drugs exert their therapeutic effects at the molecular level, we combined chemoproteomic target affinity profiling using kinobeads and phosphoproteomics to analyze the five clinical AKT inhibitors AZD5363 (Capivasertib), GSK2110183 (Afuresertib), GSK690693, Ipatasertib, and MK-2206 in BT-474 breast cancer cells. Kinobead profiling identified between four and 29 nM targets for these compounds and showed that AKT1 and AKT2 were the only common targets. Similarly, measuring the response of the phosphoproteome to the same inhibitors identified ∼1700 regulated phosphorylation sites, 276 of which were perturbed by all five compounds. This analysis expanded the known AKT signaling network by 119 phosphoproteins that may represent direct or indirect targets of AKT. Within this new network, 41 regulated phosphorylation sites harbor the AKT substrate motif, and recombinant kinase assays validated 16 as novel AKT substrates. These included CEP170 and FAM83H, suggesting a regulatory function of AKT in mitosis and cytoskeleton organization. In addition, a specific phosphorylation pattern on the ULK1-FIP200-ATG13-VAPB complex was found to determine the active state of ULK1, leading to elevated autophagy in response to AKT inhibition.

Novel Broad-Spectrum Antiviral Inhibitors Targeting Host Factors Essential for Replication of Pathogenic RNA Viruses.

Recent RNA virus outbreaks such as Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV) have caused worldwide health emergencies highlighting the urgent need for new antiviral strategies. Targeting host cell pathways supporting viral replication is an attractive approach for development of antiviral compounds, especially with new, unexplored viruses where knowledge of virus biology is limited. Here, we present a strategy to identify host-targeted small molecule inhibitors using an image-based phenotypic antiviral screening assay followed by extensive target identification efforts revealing altered cellular pathways upon antiviral compound treatment. The newly discovered antiviral compounds showed broad-range antiviral activity against pathogenic RNA viruses such as SARS-CoV-2, EBOV and Crimean-Congo hemorrhagic fever virus (CCHFV). Target identification of the antiviral compounds by thermal protein profiling revealed major effects on proteostasis pathways and disturbance in interactions between cellular HSP70 complex and viral proteins, illustrating the supportive role of HSP70 on many RNA viruses across virus families. Collectively, this strategy identifies new small molecule inhibitors with broad antiviral activity against pathogenic RNA viruses, but also uncovers novel virus biology urgently needed for design of new antiviral therapies.

Repurposing human kinase inhibitors to create an antibiotic active against drug-resistant Staphylococcus aureus, persisters and biofilms.

New drugs are desperately needed to combat methicillin-resistant Staphylococcus aureus (MRSA) infections. Here, we report screening commercial kinase inhibitors for antibacterial activity and found the anticancer drug sorafenib as major hit that effectively kills MRSA strains. Varying the key structural features led to the identification of a potent analogue, PK150, that showed antibacterial activity against several pathogenic strains at submicromolar concentrations. Furthermore, this antibiotic eliminated challenging persisters as well as established biofilms. PK150 holds promising therapeutic potential as it did not induce in vitro resistance, and shows oral bioavailability and in vivo efficacy. Analysis of the mode of action using chemical proteomics revealed several targets, which included interference with menaquinone biosynthesis by inhibiting demethylmenaquinone methyltransferase and the stimulation of protein secretion by altering the activity of signal peptidase IB. Reduced endogenous menaquinone levels along with enhanced levels of extracellular proteins of PK150-treated bacteria support this target hypothesis. The associated antibiotic effects, especially the lack of resistance development, probably stem from the compound's polypharmacology.

The Heat Shock Response in Yeast Maintains Protein Homeostasis by Chaperoning and Replenishing Proteins.

Life is resilient because living systems are able to respond to elevated temperatures with an ancient gene expression program called the heat shock response (HSR). In yeast, the transcription of hundreds of genes is upregulated at stress temperatures. Besides stress protection conferred by chaperones, the function of the majority of the upregulated genes under stress has remained enigmatic. We show that those genes are required to directly counterbalance increased protein turnover at stress temperatures and to maintain the metabolism. This anaplerotic reaction together with molecular chaperones allows yeast to efficiently buffer proteotoxic stress. When the capacity of this system is exhausted at extreme temperatures, aggregation processes stop translation and growth pauses. The emerging concept is that the HSR is modular with distinct programs dependent on the severity of the stress.

Biomarkers for tissue engineering of the tendon-bone interface.

The tendon-bone interface (enthesis) is a highly sophisticated biomaterial junction that allows stress transfer between mechanically dissimilar materials. The enthesis encounters very high mechanical demands and the regenerative capacity is very low resulting in high rupture recurrence rates after surgery. Tissue engineering offers the potential to recover the functional integrity of entheses. However, recent enthesis tissue engineering approaches have been limited by the lack of knowledge about the cells present at this interface. Here we investigated the cellular differentiation of enthesis cells and compared the cellular pattern of enthesis cells to tendon and cartilage cells in a next generation sequencing transcriptome study. We integrated the transcriptome data with proteome data of a previous study to identify biomarkers of enthesis cell differentiation. Transcriptomics detected 34468 transcripts in total in enthesis, tendon, and cartilage. Transcriptome comparisons revealed 3980 differentially regulated candidates for enthesis and tendon, 395 for enthesis and cartilage, and 946 for cartilage and tendon. An asymmetric distribution of enriched genes was observed in enthesis and cartilage transcriptome comparison suggesting that enthesis cells are more chondrocyte-like than tenocyte-like. Integrative analysis of transcriptome and proteome data identified ten enthesis biomarkers and six tendon biomarkers. The observed gene expression characteristics and differentiation markers shed light into the nature of the cells present at the enthesis. The presented markers will foster enthesis tissue engineering approaches by setting a bench-mark for differentiation of seeded cells towards a physiologically relevant phenotype.

Natural-Product-Inspired Aminoepoxybenzoquinones Kill Members of the Gram-Negative Pathogen Salmonella by Attenuating Cellular Stress Response.

Gram-negative bacteria represent a challenging task for antibacterial drug discovery owing to their impermeable cell membrane and restricted uptake of small molecules. We herein describe the synthesis of natural-product-derived epoxycyclohexenones and explore their antibiotic activity against several pathogenic bacteria. A compound with activity against Salmonella Typhimurium was identified, and the target enzymes were unraveled by quantitative chemical proteomics. Importantly, two protein hits were linked to bacterial stress response, and corresponding assays revealed an elevated susceptibility to reactive oxygen species upon compound treatment. The consolidated inhibition of these targets provides a rationale for antibacterial activity and highlights epoxycyclohexenones as natural product scaffolds with suitable properties for killing Gram-negative Salmonella.

Alkynol natural products target ALDH2 in cancer cells by irreversible binding to the active site.

Falcarinol and stipudiol are natural products with potent anti-cancer activity found in several vegetables. Here, we use a chemical proteomic strategy to identify ALDH2 as a molecular target of falcarinol in cancer cells and confirm enzyme inhibition via covalent alkylation of the active site. Furthermore, the synthesis of stipudiol led to the observation that ALDH2 exhibits preference for alkynol-based binders. Inhibition of ALDH2 impairs detoxification of reactive aldehydes and limits oxidative stress response, two crucial pathways for cellular viability.

Phosphorylation site localization in peptides by MALDI MS/MS and the Mascot Delta Score.

Owing to its broad biological significance, the large-scale analysis of protein phosphorylation is more and more getting into the focus of proteomic research. Thousands of phosphopeptides can nowadays be identified using state-of-the-art tandem mass spectrometers in conjunction with sequence database searching, but localizing the phosphate group to a particular amino acid in the peptide sequence is often still difficult. Using 180 individually synthesized phosphopeptides with precisely known phosphorylation sites (p-sites), we have assessed the merits of the Mascot Delta Score (MD score) for the assignment of phosphorylation sites from tandem mass spectra (MS/MS) generated on four different matrix-assisted laser desorption ionization (MALDI) mass spectrometers including tandem time-of-flight (TOF/TOF), quadrupole time-of-flight, and ion trap mass analyzers. The results show that phosphorylation site identification is generally possible with false localization rates of about 10%. However, a comparison to previous work also revealed that phosphorylation site determination by MALDI MS/MS is less accurate than by ESI-MS/MS particularly if several and/or adjacent possible phosphorylation acceptor sites exist in a peptide sequence. We are making the tandem MS spectra and phosphopeptide collection available to the community so that scientists may adapt the MD scores reported here to their analytical environment and so that informatics developers may integrate the MD score into proteomic data analysis pipelines.