Selective Activity-based Probes Research Articles

Discovery of potent and selective activity-based probes (ABPs) for the deubiquitinating enzyme USP30.

Ubiquitin-specific protease 30 (USP30) is a deubiquitinating enzyme (DUB) localized at the mitochondrial outer membrane and involved in PINK1/Parkin-mediated mitophagy, pexophagy, BAX/BAK-dependent apoptosis, and IKKβ-USP30-ACLY-regulated lipogenesis/tumorigenesis. A USP30 inhibitor, MTX652, has recently entered clinical trials as a potential treatment for mitochondrial dysfunction. Small molecule activity-based probes (ABPs) for DUBs have recently emerged as powerful tools for in-cell inhibitor screening and DUB activity analysis, and here, we report the first small molecule ABPs (IMP-2587 and IMP-2586) which can profile USP30 activity in cells. Target engagement studies demonstrate that IMP-2587 and IMP-2586 engage active USP30 at nanomolar concentration after only 10 min incubation time in intact cells, dependent on the presence of the USP30 catalytic cysteine. Interestingly, proteomics analyses revealed that DESI1 and DESI2, small ubiquitin-related modifier (SUMO) proteases, can also be engaged by these probes, further suggesting a novel approach to develop DESI ABPs.

An activity-based probe targeting the streptococcal virulence factor C5a peptidase.

Development of profiling strategies to provide high resolution understanding of enzymes involved in bacterial infections remains an important need. These strategies help resolve enzyme mechanisms of actions and can guide therapeutic development. We have developed a selective new activity-based probe (ABP) targeting a highly conserved surface bound enzyme, C5a peptidase, present in several pathogenic Streptococci. We demonstrate our probe inhibits C5a peptidase activity and enables detection of C5a peptidase expressing pathogens in microbial mixtures. Our profiling strategy selectively labels the pathogen by phenotype and enables specific isolation of the live bacteria providing a route for further in-depth investigation. This study paves the way towards a rapid detection, isolation, and characterization pipeline for existing and emerging strains of most common pathogenic Streptococci.

Rapid Solid-Phase Construction of Serine Hydrolase Probes Results in Selective Activity-Based Probes for Acyl Protein Thioesterases-1/2.

Serine hydrolases (SHs) are a large, diverse family of enzymes that play various biomedically important roles. Their study has been substantially advanced by activity-based protein profiling, which makes use of covalent chemical probes for labeling the active site and detection by various methodologies. However, highly selective probes for individual SHs are scarce because probe synthesis usually takes place by time-consuming solution phase chemistry. We here report a general solid-phase synthesis toward SH chemical probes, which will speed up probe library synthesis. It involves the construction of a recognition element ending in a secondary amine followed by capping with different electrophiles. We illustrate the power of this approach by the discovery of selective chemical probes for the depalmitoylating enzymes APT-1/2. Overall, this study reports new methodologies to synthesize SH probes, while providing new reagents to study protein depalmitoylation.

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2*.

The global pandemic caused by SARS-CoV-2 calls for the fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high-throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including unnatural amino acids that targets the nonprimed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within a human proteome without background. We expect that these reagents will be useful in the drug-development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.

Development of Chemical Tools to Monitor Human Kallikrein 13 (KLK13) Activity.

Kallikrein 13 (KLK13) was first identified as an enzyme that is downregulated in a subset of breast tumors. This serine protease has since been implicated in a number of pathological processes including ovarian, lung and gastric cancers. Here we report the design, synthesis and deconvolution of libraries of internally quenched fluorogenic peptide substrates to determine the specificity of substrate binding subsites of KLK13 in prime and non-prime regions (according to the Schechter and Berger convention). The substrate with the consensus sequential motive ABZ-Val-Arg-Phe-Arg-ANB-NH2 demonstrated selectivity towards KLK13 and was successfully converted into an activity-based probe by the incorporation of a chloromethylketone warhead and biotin bait. The compounds described may serve as suitable tools to detect KLK13 activity in diverse biological samples, as exemplified by overexpression experiments and targeted labeling of KLK13 in cell lysates and saliva. In addition, we describe the development of selective activity-based probes targeting KLK13, to our knowledge the first tool to analyze the presence of the active enzyme in biological samples.

Development of an advanced nanoformulation for the intracellular delivery of a caspase-3 selective activity-based probe.

The ability to label active caspase-3 represents a useful pharmacodynamic strategy to determine the efficacy of anti-tumour drugs. Activity-based probes (ABPs) provide a method for the labelling of activated caspases and the recent development of hybrid combinatorial substrate libraries (HyCoSuL) has allowed for the generation of highly selective ABPs to discriminately label these proteases. Here using this approach, a novel caspase-3 selective ABP (CS1) has been developed and validated in apoptotic cells to selectively bind caspase-3 over the closely related caspase-7. However, a critical bottleneck for ABPs is their cell penetrance and therefore this cell-impermeable CS1 probe was subsequently formulated into PLGA-based nanoparticles (CS1-NPs). We demonstrate the ability of these particles to be taken up by the cells and facilitate intracellular delivery of the ABP to effectively label caspase 3 in response to apoptotic stimuli. This work forms the foundation of a novel approach for the labelling of caspase 3 and may have downstream utility to measure real time apoptosis in tumours and other organs.

A Probe-Enabled Approach for the Selective Isolation and Characterization of Functionally Active Subpopulations in the Gut Microbiome

Commensal microorganisms in the mammalian gut play important roles in host health and physiology, but a central challenge remains in achieving a detailed mechanistic understanding of specific microbial contributions to host biochemistry. New function-based approaches are needed that analyze gut microbial function at the molecular level by coupling detection and measurements of in situ biochemical activity with identification of the responsible microbes and enzymes. We developed a platform employing β-glucuronidase selective activity-based probes to detect, isolate, and identify microbial subpopulations in the gut responsible for this xenobiotic metabolism. We find that metabolic activity of gut microbiota can be plastic and that between individuals and during perturbation, phylogenetically disparate populations can provide β-glucuronidase activity. Our work links biochemical activity with molecular-scale resolution without relying on genomic inference.

Glycosylation is important for legumain localization and processing to active forms but not for cystatin E/M inhibitory functions

The asparaginyl endopeptidase legumain and its inhibitor cystatin E/M are endogenously glycosylated. However, little is known about the nature of the carbohydrate groups and whether they affect the functions of these proteins. In this study both glycosylated and unglycosylated forms of legumain and cystatin E/M were studied. HEK293 cell lines stably over-expressing legumain or cystatin E/M, and HCT116 cells were used as cell models, and mature legumain was purified from bovine kidneys. To obtain unglycosylated proteins, cells were treated with tunicamycin, an inhibitor of N-linked glycosylation, whereas PNGase F and Endo H were used to characterize the glycosylation types. Cells were incubated with glycosylated, unglycosylated proteins and/or legumain selective activity-based probe, and legumain and/or cystatin E/M was studied by activity measurement, ELISA or immunoblotting in cell lysates or conditioned media. Legumain and probe in whole cells were studied by immunofluorescence. The carbohydrates on legumain were shown to be of the hybrid or high mannose type, whereas cystatin E/M was characterized as complex mannose-linked. While glycosylated prolegumain was able to autoactivate, the unglycosylated form was not, and addition of glycosaminoglycans did not facilitate autoactivation of unglycosylated prolegumain. Glycosylated prolegumain was internalized and processed to the mature active form, but no internalization of unglycosylated prolegumain was observed. A Cy5-labelled legumain specific activity-based probe (MP-L09) was synthesized and shown to be a novel tool to study intracellular legumain. Also, internalization of mature legumain (36 kDa) was visualized both alone and complexed with probe. Contrary to the importance of legumain glycosylation, both glycosylated and unglycosylated cystatin E/M showed similar capacity to inhibit legumain. In conclusion, glycosylation of prolegumain is necessary for correct processing to active forms and internalization, whereas the inhibitory property of cystatin E/M is independent of the glycosylation status.

Development of a selective activity-based probe for glycosylated LIPA

Loss of LIPA activity leads to diseases such as Wolman’s Disease and Cholesterol Ester Storage Disease. While it is possible to measure defects in LIPA protein levels, it is difficult to directly measure LIPA activity in cells. In order to measure LIPA activity directly we developed a LIPA specific activity based probe. LIPA is heavily glycosylated although it is unclear how glycosylation affects LIPA activity or function. Our probe is specific for a glycosylated form of LIPA in cells, although it labels purified LIPA regardless of glycosylation.

Potent and selective activity-based probes for GH27 human retaining α-galactosidases.

Lysosomal degradation of glycosphingolipids is mediated by the consecutive action of several glycosidases. Malfunctioning of one of these hydrolases can lead to a lysosomal storage disorder such as Fabry disease, which is caused by a deficiency in α-galactosidase A. Herein we describe the development of potent and selective activity-based probes that target retaining α-galactosidases. The fluorescently labeled aziridine-based probes 3 and 4 inhibit the two human retaining α-galactosidases αGal A and αGal B covalently and with high affinity. Moreover, they enable the visualization of the endogenous activity of both α-galactosidases in cell extracts, thereby providing a means to study the presence and location of active enzyme levels in different cell types, such as healthy cells versus those derived from Fabry patients.

Development of a Selective Activity-Based Probe for Adenylating Enzymes: Profiling MbtA Involved in Siderophore Biosynthesis from Mycobacterium tuberculosis

MbtA is an adenylating enzyme from Mycobacterium tuberculosis that catalyzes the first step in the biosynthesis of the mycobactins. A bisubstrate inhibitor of MbtA (Sal-AMS) was previously described that displays potent antitubercular activity under iron-replete as well as iron-deficient growth conditions. This finding is surprising since mycobactin biosynthesis is not required under iron-replete conditions and suggests off-target inhibition of additional biochemical pathways. As a first step toward a complete understanding of the mechanism of action of Sal-AMS, we have designed and validated an activity-based probe (ABP) for studying Sal-AMS inhibition in M. tuberculosis. This probe labels pure MbtA as well as MbtA in mycobacterial lysate, and labeling can be completely inhibited by preincubation with Sal-AMS. Furthermore, this probe provides a prototypical core scaffold for the creation of ABPs to profile any of the other 66 adenylating enzymes in Mtb or the multitude of adenylating enzymes in other pathogenic bacteria.

Selective Fluorescence Probes for Dipeptidyl Peptidase ActivityFibroblast Activation Protein and Dipeptidyl Peptidase IV

Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion, and metastasis. Here, we validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.

A Selective Activity-Based Probe for the Papain Family Cysteine Protease Dipeptidyl Peptidase I/Cathepsin C

Dipeptidyl peptidase I is involved in the activation of a number of disease-related proteases by removal of N-terminal prodipeptides. We here report a selective activity-based probe for monitoring dipeptidyl peptidase I activity in whole proteomes as well as in intact cells, without labeling of closely related enzyme family members.