Marion Rusch

Small-molecule inhibition of APT1 affects Ras localization and signaling

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.

Identification of Acyl Protein Thioesterases 1 and 2 as the Cellular Targets of the Ras-Signaling Modulators Palmostatin B and M

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.

Development of Highly Potent Inhibitors of the Ras‐Targeting Human Acyl Protein Thioesterases Based on Substrate Similarity Design

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.

The mechanism of caseinolytic protease (ClpP) inhibition.

Maintaining homeostasis at the protein level is an important prerequisite for cellular viability for which prokaryotes exhibit several proteolytic machineries, including ClpXP. In 2008, we reported the first small-molecule inhibitor for the proteolytic subunit ClpP and demonstrated that the inhibition of the enzyme in living bacteria significantly attenuates their capability to produce virulence factors, such as life-threatening toxins. Although ClpP has been extensively studied by biochemical and structural methods, the mechanism of small-molecule inhibition of this enzyme is currently poorly understood. Because chemical inhibition may lead to a novel antibacterial therapy, it is important to systematically analyze the binding site, the mechanism of inhibition, the stereogenic preference of the enzyme for inhibitors, the chemical space of putative inhibitors, and how other members of the ClpP family can be inhibited. One major step towards these aims was accomplished by the recently solved crystal structure of homotetradecameric ClpP from Staphylococcus aureus (SaClpP) in its active conformation. With the structural data at hand, we herein report an in-depth mechanistic analysis of S. aureus ClpP inhibition by b-lactones. A screen of a focused library of enantiopure b-lactones revealed the S,Sstereopreference of the protease, which was rationalized by molecular docking. Docking experiments also gave insight into a hitherto unnoted deep hydrophobic pocket next to the active site that accommodates b-lactone substituents in the aposition to the carbonyl group. The binding hypothesis was verified by binding studies with model compounds, detailed kinetic analysis, and protein mutagenesis studies. Furthermore, the replacement of the b-lactone core by other scaffolds resulted in the loss of inhibitory potency, thereby highlighting the importance of a b-lactone moiety for mechanism-based ClpP inhibition. Taken together, these results open intriguing perspectives in the mechanistic understanding of ClpP inhibition and provide direction for the design of potent and pharmacologically optimized inhibitors. We started by testing 22 enantiopure trans-substituted blactones 1–22 for ClpP inhibition (Supporting Information, Figure S1 A). These molecules share a high structural similarity with our previous b-lactone candidates. They feature a decyl chain as R substituent and structural variations in chain lengths as well as in functional groups at the R position (Figure 1A). For all of the compounds, both trans-configured enantiomers (that is, R,R and S,S) were tested for inhibition of recombinantly expressed SaClpP in an assay monitoring the cleavage of a fluorogenic substrate. Almost all of the compounds inhibited SaClpP at 100 mm concentration (100-fold excess over enzyme) after 15 min incubation at 32 8C (Supporting Information, Figure S1 A). By lowering the inhibitor concentration to 10 mm, we were able to differentiate the compounds tested. While most S,Sconfigured lactones lead to inhibition below 10% residual activity, R,R-configured lactones showed essentially no inhibition (Figure 1 B). Incubation of SaClpP with 1.3-fold molar excess of the most potent compound, 2, led to modification of all 14 subunits as revealed by intact-protein mass spectrometry (Figure 1 C). To investigate if the potent in vitro inhibition correlates with ClpP binding in living cells we applied the structurally related alkynylated probe 23 with S,S-configuration for an

Mirs-138 and -424 Control Palmitoylation-Dependent CD95-Mediated Cell Death By Targeting Acyl Protein Thioesterases 1 and 2 in Chronic Lymphocytic Leukemia

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.

Omuralide and Vibralactone: Differences in the Proteasome‐ β‐Lactone‐γ‐Lactam Binding Scaffold Alter Target Preferences

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.

Characterization of a Serine Hydrolase Targeted by Acyl-protein Thioesterase Inhibitors in Toxoplasma gondii

Background: S-Palmitoylation is an important reversible modification that involves the action of an acyl-protein thioesterase (APT). Results: We identified an active serine hydrolase (TgASH1) specifically targeted by human APT1 inhibitors in Toxoplasma gondii. Conclusion: TgASH1 is dispensable and cannot be solely responsible for S-depalmitoylation in Apicomplexa. Significance: β-Lactone-based APT1 inhibitors hit multiple targets in T. gondii and severely compromise parasite survival. 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 β-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 β-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.

Design, synthesis and evaluation of polar head group containing 2-keto-oxazole inhibitors of FAAH.

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.