Marcin Drag

Emerging principles in protease-based drug discovery

Proteases have an important role in many signalling pathways, and represent potential drug targets for diseases ranging from cardiovascular disorders to cancer, as well as for combating many parasites and viruses. Although inhibitors of well-established protease targets such as angiotensin-converting enzyme and HIV protease have shown substantial therapeutic success, developing drugs for new protease targets has proved challenging in recent years. This in part could be due to issues such as the difficulty of achieving selectivity when targeting protease active sites. This Perspective discusses the general principles in protease-based drug discovery, highlighting the lessons learned and the emerging strategies, such as targeting allosteric sites, which could help harness the therapeutic potential of new protease targets.

FLIPL induces caspase-8 activity in the absence of interdomain caspase-8 cleavage and alters substrate specificity

Caspase 8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase 8 activation involves dimerization and subsequent interdomain autoprocessing of caspase 8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase 8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase 8-deficient mice are shared by mice lacking the dimerization adapter FADD (Fas-associated death domain) or the caspase paralogue FLIP(L) [FLICE (FADD-like interleukin 1β-converting enzyme)-inhibitory protein, long form] has led to the hypothesis that FADD-dependent formation of heterodimers between caspase 8 and FLIP(L) could mediate the developmental role of caspase 8. In the present study, using an inducible dimerization system we demonstrate that cleavage of the catalytic domain of caspase 8 is crucial for its activity in the context of activation by homodimerization. However, we find that use of FLIP(L) as a partner for caspase 8 in dimerization-induced activation rescues the requirement for intersubunit linker proteolysis in both protomers. Moreover, before processing, caspase 8 in complex with FLIP(L) does not generate a fully active enzyme, but an attenuated species able to process only selected natural substrates. Based on these results we propose a mechanism of caspase 8 activation by dimerization in the presence of FLIP(L), as well as a mechanism of caspase 8 functional divergence in apoptotic and non-apoptotic pathways.

A constitutively active and uninhibitable caspase-3 zymogen efficiently induces apoptosis

The caspase-3 zymogen has essentially zero activity until it is cleaved by initiator caspases during apoptosis. However, a mutation of V266E in the dimer interface activates the protease in the absence of chain cleavage. We show that low concentrations of the pseudo-activated procaspase-3 kill mammalian cells rapidly and, importantly, this protein is not cleaved nor is it inhibited efficiently by the endogenous regulator XIAP (X-linked inhibitor of apoptosis). The 1.63 Å (1 Å = 0.1 nm) structure of the variant demonstrates that the mutation is accommodated at the dimer interface to generate an enzyme with substantially the same activity and specificity as wild-type caspase-3. Structural modelling predicts that the interface mutation prevents the intersubunit linker from binding in the dimer interface, allowing the active sites to form in the procaspase in the absence of cleavage. The direct activation of procaspase-3 through a conformational switch rather than by chain cleavage may lead to novel therapeutic strategies for inducing cell death.

Small Ubiquitin-related Modifier (SUMO)-specific Proteases PROFILING THE SPECIFICITIES AND ACTIVITIES OF HUMAN SENPs

SENPs are proteases that participate in the regulation of SUMOylation by generating mature small ubiquitin-related modifiers (SUMO) for protein conjugation (endopeptidase activity) and removing conjugated SUMO from targets (isopeptidase activity). Using purified recombinant catalytic domains of 6 of the 7 human SENPs, we demonstrate the specificity of their respective activities on SUMO-1, -2, and -3. The primary mode of recognition of substrates is via the SUMO domain, and the C-terminal tails direct endopeptidase specificity. Broadly speaking, SENP1 is the most efficient endopeptidase, whereas SENP2 and -5–7 have substantially higher isopeptidase than endopeptidase activities. We developed fluorogenic tetrapeptide substrates that are cleaved by SENPs, enabling us to characterize the environmental profiles of each enzyme. Using these synthetic substrates we reveal that the SUMO domain enhances catalysis of SENP1, -2, -5, -6, and -7, demonstrating substrate-induced activation of SENPs by SUMOs.

DeSUMOylating enzymes—SENPs

Modification of proteins by ubiquitin and SUMO (small ubiquitin‐like modifiers) is a dynamic and reversible process. Similar to the ubiquitin pathway, where the action of deubiquitinating enzymes removes ubiquitin from ubiquitin‐adducts, SUMO is also removed intact from its substrates by proteases belonging to the sentrin‐specific proteases (SENPs) family. In addition to their isopeptidase activity, SENPs also execute another essential function as endopeptidases by removing the short C‐terminal extension from immature SUMOs. The defining characteristics of SENPs are their predicted conserved molecular scaffold—defined as members of peptidase Clan CE, conserved catalytic mechanism, and their reported activity on SUMO or Nedd8 conjugated proteins (or the respective precursors). We discuss recent progress on the human SENPs and their substrates.

Distribution and paralogue specificity of mammalian deSUMOylating enzymes

The covalent attachment of SUMO (small ubiquitin-like protein modifier) to target proteins results in modifications in their activity, binding interactions, localization or half-life. The reversal of this modification is catalysed by SENPs (SUMO-specific processing proteases). Mammals contain four SUMO paralogues and six SENP enzymes. In the present paper, we describe a systematic analysis of human SENPs, integrating estimates of relative selectivity for SUMO1 and SUMO2, and kinetic measurements of recombinant C-terminal cSENPs (SENP catalytic domains). We first characterized the reaction of each endogenous SENP and cSENPs with HA-SUMO-VS [HA (haemagglutinin)-tagged SUMO-vinyl sulfones], active-site-directed irreversible inhibitors of SENPs. We found that all cSENPs and endogenous SENP1 react with both SUMO paralogues, whereas all other endogenous SENPs in mammalian cells and tissues display high selectivity for SUMO2-VS. To obtain more quantitative data, the kinetic properties of purified cSENPs were determined using SUMO1- or SUMO2-AMC (7-amino-4-methylcoumarin) as substrate. All enzymes bind their respective substrates with high affinity. cSENP1 and cSENP2 process either SUMO substrate with similar affinity and catalytic efficiency; cSENP5 and cSENP6 show marked catalytic specificity for SUMO2 as measured by Km and kcat, whereas cSENP7 works only on SUMO2. Compared with cSENPs, recombinant full-length SENP1 and SENP2 show differences in SUMO selectivity, indicating that paralogue specificity is influenced by the presence of the variable N-terminal domain of each SENP. Our data suggest that SUMO2 metabolism is more dynamic than that of SUMO1 since most SENPs display a marked preference for SUMO2.

Aminopeptidase Fingerprints, an Integrated Approach for Identification of Good Substrates and Optimal Inhibitors

Aminopeptidases process the N-terminal amino acids of target substrates by sequential cleavage of one residue at a time. They are found in all cell compartments of prokaryotes and eukaryotes, being implicated in the major proteolytic events of cell survival, defense, growth, and development. We present a new approach for the fast and reliable evaluation of the substrate specificity of individual aminopeptidases. Using solid phase chemistry with the 7-amino-4-carbamoylmethylcoumarin fluorophore, we have synthesized a library of 61 individual natural and unnatural amino acids substrates, chosen to cover a broad spectrum of the possible interactions in the S1 pocket of this type of protease. As proof of concept, we determined the substrate specificity of human, pig, and rat orthologs of aminopeptidase N (CD13), a highly conserved cell surface protease that inactivates enkephalins and other bioactive peptides. Our data reveal a large and hydrophobic character for the S1 pocket of aminopeptidase N that is conserved with aminopeptidase Ns. Our approach, which can be applied in principle to all aminopeptidases, yields useful information for the design of specific inhibitors, and more importantly, reveals a relationship between the kinetics of substrate hydrolysis and the kinetics of enzyme inhibition.

Metallo-aminopeptidase inhibitors.

Abstract Aminopeptidases are enzymes that selectively hydrolyze an amino acid residue from the N-terminus of proteins and peptides. They are important for the proper functioning of prokaryotic and eukaryotic cells, but very often are central players in the devastating human diseases like cancer, malaria and diabetes. The largest aminopeptidase group include enzymes containing metal ion(s) in their active centers, which often determines the type of inhibitors that are the most suitable for them. Effective ligands mostly bind in a non-covalent mode by forming complexes with the metal ion(s). Here, we present several approaches for the design of inhibitors for metallo-aminopeptidases. The optimized structures should be considered as potential leads in the drug discovery process against endogenous and infectious diseases.

Mechanism and specificity of the human paracaspase MALT1

The paracaspase domain of MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1) is a component of a gene translocation fused to the N-terminal domains of the cellular inhibitor of apoptosis protein 2. The paracaspase itself, commonly known as MALT1, participates in the NF-κB (nuclear factor κB) pathway, probably by driving survival signals downstream of the B-cell antigen receptor through MALT1 proteolytic activity. We have developed methods for the expression and purification of recombinant full-length MALT1 and its constituent catalytic domain alone. Both are activated by dimerization without cleavage, with a similar dimerization barrier to the distantly related cousins, the apical caspases. By using positional-scanning peptidyl substrate libraries we demonstrate that the activity and specificity of full-length MALT1 is recapitulated by the catalytic domain alone, showing a stringent requirement for cleaving after arginine, and with striking peptide length constraints for efficient hydrolysis. Rates of cleavage (kcat/Km values) of optimal peptidyl substrates are in the same order (103–104 M−1·s−1) as for a putative target protein CYLD. Thus MALT1 has many similarities to caspase 8, even cleaving the putative target protein CYLD with comparable efficiencies, but with diametrically opposite primary substrate specificity.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

Significance The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Using this approach, we have designed an extremely active substrate of NE and subsequently converted it into an ultrasensitive activity-based probe for imaging active elastase during the process of neutrophil extracellular trap formation. Our study could have a substantial effect on the design of substrates, inhibitors, and probes for any endopeptidase. The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes.