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Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.

Additional file 1 : Figure S1. Activity-based protein profiling (ABPP) and the power of this approach to unveil SH activity in glioma. Figure S2. Characteristics of the rat BT4C gliosarcoma model. Figure S3. Coronal plane MRI images of glioma and control brains. Figure S4. Testing various fixation method for rodent brain sections. Figure S5. Effect of TAMRA-FP concentration on fluorescence signal in mouse brain sections. Figure S6. Effects of pH and buffer composition on TAMRA-FP signal and its inhibitor sensitivity. Figure S7. Comparative gel-based ABPP of seven animals confirming distinct SH activity profiles between glioma and control brain. Figure S8. Confocal imaging of SH activity in relation to proliferating tumor cells. Figure S9. Confocal imaging of SH activity in relation to astrocytes. Figure S10. Confocal imaging of SH activity in relation to blood vessels. Figure S11. Confocal imaging of SH activity in relation to heme oxygenase 1 (HO-1). Figure S12. Confocal imaging of SH activity in relation to microglial marker Iba1. Figure S13. Confocal imaging of SH activity in relation to hyaluronan (HA). Figure S14. Confocal imaging of SH activity in relation to HA receptor CD44. Figure S15. Confocal imaging of SH activity in relation to the stiffness marker pMLC2. Figure S16. Confocal imaging of SH activity in relation to the stiffness marker tenascin C. Figure S17. Confocal imaging of SH activity in relation to CD45, a marker for nucleated hematopoietic cells. Figure S18. Confocal imaging of SH activity in relation to CD11b/c, a marker for phagocytes. Figure S19. Confocal imaging of SH activity in relation to CD68, a marker for monocytes and macrophages. Figure S20. Confocal imaging of SH activity in relation to CD163, a marker for monocytes and macrophages. Figure S21. Confocal imaging of SH activity in relation to CD169, a marker for macrophages. Figure S22. Confocal imaging of SH activity in relation to T cell marker CD4. Figure S23. Confocal imaging of SH activity in relation to T cell marker CD8. Figure S24. Confocal imaging of SH activity in relation to FcεRIγ, a marker for mast cells, eosinophils, basophils and monocytes. Figure S25. Confocal imaging of SH activity in relation to chymase (CMA1), a marker for mast cells. Figure S26. TAMRA-FP signal at the site of injection in sham-operated animals. Figure S27. Gel-ABPP of rat glioma proteomes using Cy5-labeled serine protease activity probes PK-DPP and V-DPP. Figure S28. Tissue-ABPP of glioma sections using Cy5-labeled activity probes PK-DPP and V-DPP. Figure S29. ABPP of rat neutrophil and glioma samples using Cy5-labeled neutrophil serine protease (NSP) probes in combination with TAMRA-FP. Figure S30. Inhibitor profiles of human cathepsin G (hCTSG) and the prominent 25–30 kDa SH bands in rat bone-marrow-derived mononuclear cells and neutrophils. Figure S31. High-resolution imaging of TAMRA-FP hotspots and their inhibitor sensitivity in rat spleen. Figure S32. Confocal imaging of SH activity in rat spleen sections in relation to selected immunomarkers. Figure S33. Tissue-ABPP offers sufficient sensitivity to enable imaging of TAMRA-FP fluorescence in regions of the healthy brain.

Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. The ABPP approach utilizes cell/tissue proteomes and features the FP warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Here, we advance the ABPP methodology to glioma brain cryosections, enabling high-resolution confocal fluorescence imaging of SH activity in different cell types of the tumor microenvironment, identified by using extensive immunohistochemistry on activity probe labeled sections. We name this technique tissue-ABPP to distinguish it from conventional gel-based ABPP. We show heightened SH activity in glioma vs. normal brain and unveil activity hotspots originating from tumor-associated neutrophils. Thorough optimization and validation is provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Tissue-ABPP enables a wide range of applications for confocal imaging of SH activity in any type of tissue or animal species.

De novo synthesis of fatty acids is essential to maintain intensive proliferation of cancer cells. Unlike normal cells that utilize food-derived circulating lipids for their fuel, cancer cells rely on heightened lipogenesis irrespective of exogenous lipid availability. Overexpression and activity of the multidomain enzyme fatty acid synthase (FASN) is crucial in supplying palmitate for protumorigenic activity. Therefore, FASN has been proposed as an attractive target for drug development. As an effort to set up an effective toolkit to study FASN inhibitors in human and rodent tissues, we validated activity-based protein profiling (ABPP) as a viable approach to unveil inhibitors targeting FASN thioesterase domain (FASN-TE). ABPP was combined with multi-well plate-assays designed for classical substrate-based FASN activity analysis together with powerful monitoring of cancer cell proliferation using IncuCyte® Live Cell Analyzing System. FASN-TE inhibitors were identified by competitive ABPP using HEK293 cell lysates in a screen of in-house compounds (200+) designed to target serine hydrolase (SH) family. The identified compounds were tested for their inhibitor potencies in vitro using a substrate-based activity assay monitoring FASN-dependent NADPH consumption in LNCaP prostate cancer cell preparation, in parallel with selected reference inhibitors, including orlistat (THL), GSK2194069, GSK837149A, platensimycin and BI-99179. LNCaP lysate supernatant was validated as a reliable native preparation to monitor FASN-dependent NADPH consumption as opposed to human glioma GAMG cells, whereas FASN enrichment was a prerequisite for accurate assays. While inhibitor pharmacology was identical between human prostate and glioma cancer cell FASN preparations, notable differences were revealed between human and rodent FASN preparations, especially for inhibitors targeting FASN-TE. ABPP combined with substrate-based assays facilitated identification of pan thiol-reactive inhibitor scaffolds, exemplified by the 1,2,4-thiadiazole moiety. Finally, selected compounds were evaluated for their antiproliferative efficacy in situ using GAMG cells. These studies revealed that while the tested compounds acted as potent FASN inhibitors in vitro, only a few showed antiproliferative efficacy in situ. To conclude, we describe a versatile toolkit to study FASN inhibitors in vitro and in situ using human cancer cells and reveal dramatic pharmacological differences between human and rodent FASN preparations.

Inhibition of Autotaxin (ATX) is a potential treatment strategy for several diseases, including tumors with elevated ATX-lysophosphatidic acid (LPA) signaling. Combining structure-based virtual screening together with hen egg-white Autotaxin (ewATX) activity assays enabled the discovery of novel small-molecule ATX inhibitors with a 2,4-dihydropyrano[2,3-c]pyrazole scaffold. These compounds are suggested to bind to the lipophilic pocket, leaving the active site unrestrained. Our most potent compound, (S)-6-amino-4-(3,4-dichlorophenyl)-3-(4-[(4-fluorobenzyl)oxy]phenyl)-2,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile [(S)-25], inhibited human ATX (hATX) with an IC50-value of 134 nM. It also blocked ATX-evoked but not LPA-mediated A2058 melanoma cell migration. Noteworthy, molecular modeling correctly predicted the biologically active enantiomer of 2,4-dihydropyrano[2,3-c]pyrazoles, as verified by compound crystallization and activity assays. Our study established the ewATX activity assay as a valid and affordable tool in ATX inhibitor discovery. Overall, our study offers novel insights and approaches into design of novel ATX inhibitors targeting the hydrophobic pocket instead of the active site., final draft, peerReviewed

Many splicing regulators bind to their own pre-mRNAs to induce alternative splicing that leads to formation of unstable mRNA isoforms. This provides an autoregulatory feedback mechanism that regulates the cellular homeostasis of these factors. We have described such an autoregulatory mechanism for two core protein components, U11-48K and U11/U12-65K, of the U12-dependent spliceosome. This regulatory system uses an atypical splicing enhancer element termed USSE (U11 snRNP-binding splicing enhancer), which contains two U12-type consensus 5' splice sites (5'ss). Evolutionary analysis of the USSE element from a large number of animal and plant species indicate that USSE sequence must be located 25-50 nt downstream from the target 3' splice site (3'ss). Together with functional evidence showing a loss of USSE activity when this distance is reduced and a requirement for RS-domain of U11-35K protein for 3'ss activation, our data suggests that U11 snRNP bound to USSE uses exon definition interactions for regulating alternative splicing. However, unlike standard exon definition where the 5'ss bound by U1 or U11 will be subsequently activated for splicing, the USSE element functions similarly as an exonic splicing enhancer and is involved only in upstream splice site activation but does not function as a splicing donor. Additionally, our evolutionary and functional data suggests that the function of the 5'ss duplication within the USSE elements is to allow binding of two U11/U12 di-snRNPs that stabilize each others' binding through putative mutual interactions.