Fast prediction of the mode of action (MoA) for bioactive compounds would immensely foster bioactivity annotation in compound collections and may early on reveal off-targets in chemical biology research and drug discovery. Morphological profiling, e.g., using the Cell Painting assay, offers a fast, unbiased assessment of compound activity on various targets in one experiment. However, due to incomplete bioactivity annotation and unknown activities of reference compounds, prediction of bioactivity is not straightforward. Here we introduce the concept of subprofile analysis to map the MoA for both, reference and unexplored compounds. We defined MoA clusters and extracted cluster subprofiles that contain only a subset of morphological features. Subprofile analysis allows for the assignment of compounds to, currently, twelve targets or MoA. This approach enables rapid bioactivity annotation of compounds and will be extended to further clusters in the future.

Resistance toward CD95-mediated apoptosis is a hallmark of many different malignancies, as it is known from primary chronic lymphocytic leukemia (CLL) cells. Previously, we could show that miR-138 and -424 are downregulated in CLL cells. Here, we identified 2 new target genes, namely acyl protein thioesterase (APT) 1 and 2, which are under control of both miRs and thereby significantly overexpressed in CLL cells. APTs are the only enzymes known to promote depalmitoylation. Indeed, membrane proteins are significantly less palmitoylated in CLL cells compared with normal B cells. We identified APTs to directly interact with CD95 to promote depalmitoylation, thus impairing apoptosis mediated through CD95. Specific inhibition of APTs by siRNAs, treatment with miRs-138/-424, and pharmacologic approaches restore CD95-mediated apoptosis in CLL cells and other cancer cells, pointing to an important regulatory role of APTs in CD95 apoptosis. The identification of the depalmitoylation reaction of CD95 by APTs as a microRNA (miRNA) target provides a novel molecular mechanism for how malignant cells escape from CD95-mediated apoptosis. Here, we introduce palmitoylation as a novel posttranslational modification in CLL, which might impact on localization, mobility, and function of molecules, survival signaling, and migration.

Catch me if you can: The ClpP protease mediates protein homeostasis and can be efficiently inhibited by β-lactones. A combination of molecular docking, mutagenesis, activity-based protein profiling, and kinetics studies now reveals the mechanism of ClpP inhibition. A hydrophobic pocket next to the active site allows binding of long aliphatic and aromatic residues. The preferred stereoisomer binds into the oxyanion hole. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

In eukaryotic organisms, cysteine palmitoylation is an important reversible modification that impacts protein targeting, folding, stability, and interactions with partners. Evidence suggests that protein palmitoylation contributes to key biological processes in Apicomplexa with the recent palmitome of the malaria parasite Plasmodium falciparum reporting over 400 substrates that are modified with palmitate by a broad range of protein S-acyl transferases. Dynamic palmitoylation cycles require the action of an acyl-protein thioesterase (APT) that cleaves palmitate from substrates and conveys reversibility to this posttranslational modification. In this work, we identified candidates for APT activity in Toxoplasma gondii. Treatment of parasites with low micromolar concentrations of beta-lactone- or triazole urea-based inhibitors that target human APT1 showed varied detrimental effects at multiple steps of the parasite lytic cycle. The use of an activity-based probe in combination with these inhibitors revealed the existence of several serine hydrolases that are targeted by APT1 inhibitors. The active serine hydrolase, TgASH1, identified as the homologue closest to human APT1 and APT2, was characterized further. Biochemical analysis of TgASH1 indicated that this enzyme cleaves substrates with a specificity similar to APTs, and homology modeling points toward an APT-like enzyme. TgASH1 is dispensable for parasite survival, which indicates that the severe effects observed with the beta-lactone inhibitors are caused by the inhibition of non-TgASH1 targets. Other ASH candidates for APT activity were functionally characterized, and one of them was found to be resistant to gene disruption due to the potential essential nature of the protein.

Despite their structural similarity, the natural products omuralide and vibralactone have different biological targets. While omuralide blocks the chymotryptic activity of the proteasome with an IC50 value of 47 nM, vibralactone does not have any effect at this protease up to a concentration of 1 mM. Activity-based protein profiling in HeLa cells revealed that the major targets of vibralactone are APT1 and APT2.

Abstract Introduction CD95-mediated apoptosis is a central physiologic mechanism to eliminate e.g. auto-reactive and malignant cells. However, its mode of action remains still not fully understood. Recently, it could be shown that palmitoylation of CD95 alters its apoptotic function. However, the role, regulation and precise molecular function of palmitoylated-CD95 need to be determined. Methods and Results Applying acyl-biotin exchange (ABE) assays and click chemistry we uncovered, that CD95 is palmitoylated in weakly palmitoylated in primary CLL cells and other malignant cell types. Via mutational analysis and ABE assays we identified the palmitoylation site of CD95 and applied a mutant as control in further experiments. Interestingly, we could show that the de-palmitoylating enzymes LYPLA1 and LYPLA2 are significantly over-expressed on gene and protein level in primary CLL cells. Importantly, FLIM-FRET experiments (Fluorescence Lifetime Imaging Microscopy - Fluorescence Resonance Energy Transfer) reveal direct interactions between LYPLAs and CD95 for the first time. To uncover how LYPLA1 and LYPLA2 are regulated, we determined differentially expressed miRNAs between CLL cells and normal B cells via bead chip arrays, confirmed their expression via qPCR and checked their binding to both enzymes via luciferase reporter-assays. Over-expression of those finally four miRNAs lead to down-regulation of both enzymes in malignant cells on protein level. Moreover, our data reveal, that these miRNAs are down-regulated due to epigenetics, as these miRNAs were up-regulated after 5-AZA treatment and in DNMT knockout cells. Most remarkable, pharmacological inhibition and siRNA-mediated knockdown of LYPLA1 and LYPLA2 resulted in increased CD95 palmitoylation and subsequently in increased CD95-mediated apoptosis. Interestingly, also over-expression of miRNAs increased susceptibility towards CD95-mediated apoptosis significantly. These results show that the interaction between LYPLA1/LYPLA2 and CD95 is essential for a proper apoptotic signaling. To understand the functional relevance of the palmitoylation site during the apoptotic process, we analyzed the receptor by FACS and microscopy (FRAP, Fluorescence Recovery After Photobleaching) and revealed that the precise localization of CD95 on the plasma membrane might be responsible for the effects observed on CLL cells and other tumor cells. Conclusion Here we uncovered the complexity of CD95 signaling in CLL and malignant cells in general. We identified novel interaction partners of CD95, which account for the molecular switch between survival and apoptosis mediated by CD95. Moreover, our data reveal that susceptibility towards CD95 is dramatically altered by a molecular network of epigenetics, miRNAs and de-palmitoylating enzymes. Importantly, we can show that de-palmitoylating enzymes are drugable and their inhibition restores CD95 apoptotic signaling and improves thereby immunogenicity of CLL cells. L.P.F. and C-M.W. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

Catch me if you can! Die ClpP-Protease ist ein integraler Bestandteil der Protein-Homöostase und kann effizient durch β-Lactone gehemmt werden. Eine Kombination aus molekularem Modelling, Mutagenese und Kinetikstudien enthüllt jetzt den Mechanismus der ClpP-Inhibition: Eine hydrophobe Tasche neben dem aktiven Zentrum ermöglicht die Bindung von aliphatischen und aromatischen Resten. Das bevorzugte Stereoisomer adressiert das Oxyanion-Loch.

AbstractTrotz der strukturellen Ähnlichkeit der beiden Naturstoffe Omuralid und Vibralacton zeigen sich unerwartete Unterschiede in den Präferenzen ihrer jeweiligen Zielenzyme. Während Omuralid das Chymotrypsin‐ähnliche aktive Zentrum des Proteasoms mit IC50 = 47 nM inhibiert, hat Vibralacton auch bei Konzentrationen von bis zu 1 mM keinen Effekt auf diese Protease. Aktivitätsbasiertes Protein‐Profiling in HeLa‐Zellen identifizierte APT1 und APT2 als zelluläre Angriffsziele von Vibralacton.

2-α-Keto oxazoles containing polar head groups in their C5-side chains were designed as fatty acid amide hydrolase (FAAH) inhibitors. Variation in the spacer length resulted in submicromolar α-keto-oxazole FAAH inhibitor (IC(50)=436 nM) presenting electrostatic stabilizing interactions between its polar head group contained in the C5-side chain and the hydrophilic pocket of the enzyme.

Finding the target: activity-based proteomic profiling probes based on the depalmitoylation inhibitors palmostatin B and M have been synthesized and were found to target acyl protein thioesterase 1 (APT1) and 2 (APT2) in cells.

Abstract Abstract 2829 Introduction: Post-translational modifications are important fine-tuning elements for controlling protein activity and signaling. Palmitoylation is a common post-translational modification and defined as the addition of palmitic acid to internal cysteins. Interestingly, in contrast to other lipid modifications, it is reversible. Control over the palmitoylation cycle therefore provides indirect control over protein localization and function. While a number of proteins with palmitoyl transferase activity are known, LYPLA1 (lysophospholipase 1) is the only enzyme known to be responsible for the process of depalmitoylation. CLL cells are known to be resistant to TRAIL-mediated apoptosis. While TRAIL-R1 is reported to be palmitoylated, TRAIL-R2 seems to contain a region with basic amino acids in its membrane-proximal cytoplasmatic domain. Some studies showed that palmitoylation is crucial for several steps of death receptor signaling. Therefore, regulation of depalmitoylation by LYPLA1 seems to be an important tool for the regulation of death receptor function. Methods and Results: Global palmitoylation in CLL cells was investigated by screening for all palmitoylated proteins via a click chemistry assay. There, cells were metabolically labeled, coupled to a specific reporter group and then analyzed by in-gel fluorescence. Comparison of healthy B cells, healthy PBMCs and CLL cells revealed a significant difference in global palmitoylation (+38.5 % in B cells, n=6, p<0.001; +57.8 % in healthy PBMCs, n=6, p<0.001 compared to CLL cells, n=10). We identified LYPLA1 as overexpressed in CLL compared to healthy controls on both protein and mRNA level. We generated a potent LYPLA1 inhibitor. We could show, that inhibition of LYPLA1 led to a significant increase of the overall protein palmitoylation level in CLL cells (+24.7 % n=6, p=0.0118). Ours and other groups have shown, that treatment of cancer cells with TRAIL and X-linked inhibitor of apoptosis protein (XIAP)-inhibition lead to apoptosis in otherwise TRAIL resistant CLL cells. Since death receptors might be palmitoylated, we extended these studies. Treatment of CLL cells with TRAIL, XIAP- and LYPLA1-inhibition led to significantly increased apoptosis compared to TRAIL treatment and XIAP-inhibition alone (+43.2 %, n=12, p=0.0089). Palmitoylation of death receptors was investigated with the help of acyl-biotin exchange chemistry. We could show that palmitoylation of TRAIL-R1 was significantly increased after LYPLA1-inhibiton (+58.7 %, n=3, p=0.0169). It could be demonstrated, that inhibition of LYPLA1 in combination with death receptor stimulation increased the amount of activated caspase-8 in comparison to solely TRAIL and DMSO treated cells (+41.8 %, n=3, p=0.0199), indicating that palmitoylation plays a crucial role in apoptotic signaling far from XIAP. In addition to that, we could show that inhibition of depalmitoylation of TRAIL-R1 led to more death receptor located to lipid rafts. To understand how LYPLA1 is regulated, we investigated two highly conserved miRNAs which were predicted as key regulators of LYPLA1 and which are significantly downregulated in CLL. Indeed, luciferase assays revealed that both miRNAs were able to downregulate LYPLA1 expression. Conclusion: We show for the first time, that LYPLA1 is a central enzyme which regulates the apoptotic signaling of TRAIL. Furthermore, we identified LYPLA1 to be regulated by miRNAs, which are deregulated in CLL. These novel findings allow speculation, that LYPLA1 inhibitors could be used for the treatment of CLL. Future experiments should therefore aim at investigating the LYPLA1 signaling pathway as a potential target for CLL/ cancer therapy. L.P.F. and V.F. contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

A matter of common sense: a common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two native acyl protein thioesterase 1 (APT1) substrates. This similarity led to the design of potent inhibitors of the Ras-depalmitoylating enzyme APT1.

Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.