Small Molecule Active Site Directed Tools for Studying Human Caspases

Poręba, Marcin; Szalek, Aleksandra; Kasperkiewicz, Paulina; Rut, Wioletta; Salvesen, Guy S.; Drąg, Marcin

doi:10.1021/acs.chemrev.5b00434

Cited by 75 publications

(82 citation statements)

References 432 publications

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“…In our opinion, the S2 pocket is crucial for distinguishing between granzyme B and caspases since it is the only position in which these enzymes possess distinct specificity. With our peptide library screening of GrB, we confirmed that it recognizes glutamic acid at P3 and shares this feature with caspases 10,17,22,29 . The carboxyl group of glutamic acid interacts with Lys192 and Asn218 in the GrB S3 pocket, consistent with GrB specificity.…”

Section: Discussionsupporting

confidence: 55%

Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

Janiszewski

Kołt

Kaiserman

et al. 2019

Preprint

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Despite many studies on the cytotoxic protease granzyme B, key aspects of its function remain unexplored due to the lack of selective probes for its activity. In this study, we fully mapped the substrate preferences of GrB using a set of unnatural amino acids, demonstrating previously unknown GrB substrate preferences that we then used to design novel substrate-based inhibitors and a GrB-activatable activitybased probe. We showed that our GrB probes react poorly with caspases, making them ideal for the in-depth analysis of GrB localization and function in cells. With our quenched fluorescence substrate, we determined GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with the GrB attack MDA-MB-231 target cells, and active GrB influences its target cell killing efficiency.NK cell inhibitory receptors recognize MHC class I proteins and protect healthy host cells 2 . Among the various weapons of NK cells and cytotoxic T lymphocytes (CTLs), the most important, located in the cytotoxic granules, are perforin and granzymes (Granule associated enzymes, Grs). Grs are the family of homologous serine proteases and the five different human granzymes (A/B/H/K/M); the most studied and abundant are granzymes A and B 3, 4 . These enzymes are not only stored within the cytotoxic granules of immune killer cells but are also detected in primary breast carcinoma and in chondrocytes of articular cartilage 5 . Upon the activation of NK cells and binding to the target cell, the granule membrane fuses with the plasma membrane of the NK cell, and perforins (proteins that form pores in cell membranes) and granzymes are released from the cytotoxic effector cell into the intermembrane space 6, 7 . The mechanism of GrB entering the target cell through perforin-formed pores in the plasma membrane is still unclear 8 . GrB is transported into the target cell to carry out its effect. Within the cell, GrB initiates at least three distinct pathways of programmed cell death, namely, (1) the activation of caspase 3, which triggers apoptosis, (2) GrB caspase-3 substrate hydrolysis: the inhibitor of Caspase Activated DNAse (ICAD), Bid, (3) or the direct hydrolysis of lamin B. The detection of individual granzymes with antibody-related techniques is challenging due to their structural similarities; for example, GrB antibody cross-reacts with GrA and GrH 3 . For this reason, the detailed analysis of individual Grs is challenging. To overcome this obstacle, chemical approaches for developing enzymatic inhibitors and activity-based probes (ABP) as efficient tools for granzyme exploration have been attempted. However, one problem is that the substrate specificity of GrB is similar to that of some caspases (casp-8) 9 , cleaving the same substrates at the same cleavage site (synthetic: IEPD tetrapeptide 10 or natural ones: caspase-3 or BID 11 ); therefore, the reliable detection of individual Grs with either chemical or antibodyrelated techniques remains challenging 10, 12 . To date, only a few groups have...

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Section: Discussionsupporting

confidence: 55%

Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

Janiszewski

Kołt

Kaiserman

et al. 2019

Preprint

Self Cite

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“…These factors are consistent with known caspase active site inhibitors. The NSAID size restriction mirrors existing caspase inhibitors that often contain small core scaffolds (Poreba et al, 2015). It may be that larger NSAIDs are incapable of fitting into the S1 pocket, and are unable to compensate by forming sufficient contacts with the rest of the active site (Figure S2E).…”

Section: Resultsmentioning

confidence: 99%

“…Despite these functional differences, caspases are structurally analogous, posing a challenge for chemical targeting of individual caspases (Poreba et al, 2015; Wei et al, 2000). To explore NSAID specificity, we assayed the catalytic activity of caspases-1, -3, -4, -5, and -9 (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

Non-steroidal Anti-inflammatory Drugs Are Caspase Inhibitors

Smith

Soti

Jones

et al. 2017

Cell Chemical Biology

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Summary Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world. While the role of NSAIDs as cyclooxygenase (COX) inhibitors is well established, other targets may contribute to anti-inflammation. Here we report caspases as a new pharmacological target for NSAID-family drugs such as ibuprofen, naproxen, and ketorolac at physiologic concentrations both in vitro and in vivo. We characterize caspase activity both in vitro and in cell culture, and combine computational modeling and biophysical analysis to determine the mechanism of action. We observe that inhibition of caspase catalysis reduces cell death and the generation of pro-inflammatory cytokines. Further, NSAID inhibition of caspases is COX-independent, representing a new anti-inflammatory mechanism. This finding expands upon existing NSAID anti-inflammatory behaviors, with implications for patient safety and next-generation drug design.

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“…During the last step of apoptosis, caspase 3 cleaves one of its substrates, poly(ADP ribose) polymerase (PARP), after the DEVD (Asp–Glu–Val–Asp) sequence . This upstream sequence of the PARP cleavage site has been utilized as the basic scaffold for countless caspase 3 substrates, inhibitors, and activity‐based probes and in prodrug structures . Generally, in cancer lesions, caspase 3 is not active until it is activated by various antitumor stimuli (chemotherapy, radiation, etc).…”

Section: Peptide Prodrugsmentioning

confidence: 99%

Protease‐activated prodrugs: strategies, challenges, and future directions

Poręba

2020

The FEBS Journal

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Proteases play critical roles in virtually all biological processes, including proliferation, cell death and survival, protein turnover, and migration. However, when dysregulated, these enzymes contribute to the progression of multiple diseases, with cancer, neurodegenerative disorders, inflammation, and blood disorders being the most prominent examples. For a long time, disease-associated proteases have been used for the activation of various prodrugs due to their well-characterized catalytic activity and ability to selectively cleave only those substrates that strictly correspond with their active site architecture. To date, versatile peptide sequences that are cleaved by proteases in a site-specific manner have been utilized as bioactive linkers for the targeted delivery of multiple types of cargo, including fluorescent dyes, photosensitizers, cytotoxic drugs, antibiotics, and pro-antibodies. This platform is highly adaptive, as multiple protease-labile conjugates have already been developed, some of which are currently in clinical use for cancer treatment. In this review, recent advancements in the development of novel protease-cleavable linkers for selective drug delivery are described. Moreover, the current limitations regarding the selectivity of linkers are Abbreviations AAC, antibody-antibiotic conjugate; Ab, antibody; ACC, 7-amino-4-carbamoylmethylcoumarin; ADAM, a disintegrin and metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; ADC, antibody-drug conjugate; aPSs, activatable photosensitizers; BHQ-3, black hole quencher 3; Boc, tert-butyloxycarbonyl group; BT20, a type of mammary gland carcinoma; Cas9, CRISPR-associated protein 9;

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Small Molecule Active Site Directed Tools for Studying Human Caspases

Cited by 75 publications

References 432 publications

Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

Noninvasive optical detection of Granzyme B from natural killer cells using enzyme-activated fluorogenic probes

Non-steroidal Anti-inflammatory Drugs Are Caspase Inhibitors

Protease‐activated prodrugs: strategies, challenges, and future directions

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