Noninvasive optical detection of granzyme B from natural killer cells with enzyme-activated fluorogenic probes

Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate. Natural killer (NK) cells are key innate immunity effectors that combat viral infections and control several cancer types. For their immune function, human NK cells rely largely on five different cytotoxic proteases, called granzymes (A/B/H/K/M). Granzyme B (GrB) initiates at least three distinct cell death pathways, but key aspects of its function remain unexplored because selective probes that detect its activity are currently lacking. In this study, we used a set of unnatural amino acids to fully map the substrate preferences of GrB, demonstrating previously unknown GrB substrate preferences. We then used these preferences to design substrate-based inhibitors and a GrB-activatable activity-based fluorogenic probe. We show that our GrB probes do not significantly react with caspases, making them ideal for in-depth analyses of GrB localization and function in cells. Using our quenched fluorescence substrate, we observed GrB within the cytotoxic granules of human YT cells. When used as cytotoxic effectors, YT cells loaded with GrB attacked MDA-MB-231 target cells, and active GrB influenced its target cell-killing efficiency. In summary, we have developed a set of molecular tools for investigating GrB function in NK cells and demonstrate noninvasive visual detection of GrB with an enzyme-activated fluorescent substrate. In the last two decades, significant advances in the understanding of natural killer (NK) cells have been made (1Caligiuri M.A. Human natural killer cells.Blood. 2008; 112 (18650461): 461-46910.1182/blood-2007-09-077438Crossref PubMed Scopus (1161) Google Scholar). These cytotoxic lymphocytes are key effectors of innate immunity and are involved in viral infection responses as well as controlling several types of tumors. The activation of these cells is initiated by major compatibility complex (MHC) class I protein loss in compromised cells (1Caligiuri M.A. Human natural killer cells.Blood. 2008; 112 (18650461): 461-46910.1182/blood-2007-09-077438Crossref PubMed Scopus (1161) Google Scholar). NK cell activation changes the balance between the activating and inhibiting receptors on cell surfaces. Activated cells rapidly and quickly secrete cytokines, tumor necrosis factor α and γ interferon, leading to subsequent stimulation of the immune system. Reciprocal interactions with dendritic cells, macrophage T cells, and endothelial cells also enhance the immune system response. To prevent autoimmune damage, the NK cell inhibitory receptors recognize MHC class I proteins and protect healthy host cells (2Vivier E. Tomasello E. Baratin M. Walzer T. Ugolini S. Functions of natural killer cells.Nat. Immunol. 2008; 9 (18425107): 503-51010.1038/ni1582Crossref PubMed Scopus (2080) Google Scholar). 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, or 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 (3Jenne D.E. Tschopp J. Granzymes: a family of serine proteases in granules of cytolytic T lymphocytes.Curr. Top. Microbiol. Immunol. 1989; 140 (2644074): 33-4710.1007/978-3-642-73911-8_4PubMed Google Scholar, 4Masson D. Tschopp J. A family of serine esterases in lytic granules of cytolytic T lymphocytes.Cell. 1987; 49 (3555842): 679-68510.1016/0092-8674(87)90544-7Abstract Full Text PDF PubMed Scopus (325) Google Scholar). These enzymes are not only stored within the cytotoxic granules of immune killer cells but also detected in primary breast carcinoma and in chondrocytes of articular cartilage (5Horiuchi K. Saito S. Sasaki R. Tomatsu T. Toyama Y. Expression of granzyme B in human articular chondrocytes.J. Rheumatol. 2003; 30 (12913938): 1799-1810PubMed Google Scholar). 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 (6Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. Nuclear transport of granzyme B (fragmentin-2). Dependence of perforin in vivo and cytosolic factors in vitro.J. Biol. Chem. 1996; 271 (8940058): 30781-3078910.1074/jbc.271.48.30781Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 7Pinkoski M.J. Hobman M. Heibein J.A. Tomaselli K. Li F. Seth P. Froelich C.J. Bleackley R.C. Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis.Blood. 1998; 92 (9680374): 1044-105410.1182/blood.V92.3.1044.415k12_1044_1054Crossref PubMed Google Scholar). The mechanism of GrB entering the target cell through perforin-formed pores in the plasma membrane is still unclear (8Voskoboinik I. Whisstock J.C. Trapani J.A. Perforin and granzymes: function, dysfunction and human pathology.Nat. Rev. Immunol. 2015; 15 (25998963): 388-40010.1038/nri3839Crossref PubMed Scopus (398) Google Scholar). 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 with the inhibitor of caspase-activated DNase (ICAD) BID, or 3) 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 (3Jenne D.E. Tschopp J. Granzymes: a family of serine proteases in granules of cytolytic T lymphocytes.Curr. Top. Microbiol. Immunol. 1989; 140 (2644074): 33-4710.1007/978-3-642-73911-8_4PubMed Google Scholar). 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 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 (caspase-8) (9Poreba M. Strozyk A. Salvesen G.S. Drag M. Caspase substrates and inhibitors.Cold Spring Harb. Perspect. Biol. 2013; 5 (23788633): a00868010.1101/cshperspect.a008680Crossref PubMed Scopus (116) Google Scholar), cleaving the same substrates at the same cleavage site (synthetic, IEPD tetrapeptide [10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar]; natural, caspase-3 or BID [11Andrade F. Roy S. Nicholson D. Thornberry N. Rosen A. Casciola-Rosen L. Granzyme B directly and efficiently cleaves several downstream caspase substrates: implications for CTL-induced apoptosis.Immunity. 1998; 8 (9586635): 451-46010.1016/S1074-7613(00)80550-6Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar]); therefore, the reliable detection of individual Grs with either chemical or antibody-related techniques remains challenging (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar, 12Zhang D. Beresford P.J. Greenberg A.H. Lieberman J. Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis.Proc. Natl. Acad. Sci. U S A. 2001; 98 (11331782): 5746-575110.1073/pnas.101329598Crossref PubMed Scopus (123) Google Scholar). To date, only a few groups have succeeded in making functional activity-based probes for granzymes, but the major issues in these studies are the lack of selectivity of these probes toward individual enzymes and the low kinetic parameters (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar, 13Mahrus S. Craik C.S. Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells.Chem. Biol. 2005; 12 (15911377): 567-57710.1016/j.chembiol.2005.03.006Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). For example, an activity-based probe for GrB developed using combinatorial chemistry resulted in an inactivation rate constant for the target activity-based probes (GrB) equal to kobs/I = 460 m−1 s−1 (13Mahrus S. Craik C.S. Selective chemical functional probes of granzymes A and B reveal granzyme B is a major effector of natural killer cell-mediated lysis of target cells.Chem. Biol. 2005; 12 (15911377): 567-57710.1016/j.chembiol.2005.03.006Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Here, we design and characterize a set of selective chemical probes, including a quenched fluorescence substrate for GrB imaging in live cells. The development of new chemical tools for investigating Grs allows the examination of the unexplored functions of these proteases and their localization. Our objective was to develop selective and potent substrates and probes for human granzyme B (GrB). Although the available GrB substrates are tetrapeptides, it was previously reported that lengthening the peptide substrate improves the substrate hydrolysis rate for some proteases (14Rut W. Drag M. Human 20S proteasome activity towards fluorogenic peptides of various chain lengths.Biol. Chem. 2016; 397 (27176742): 921-92610.1515/hsz-2016-0176Crossref PubMed Scopus (7) Google Scholar). To define the optimal peptide length for which GrB is most active, six fluorescent substrates were designed based on literature data. The S1 binding pocket of GrB has an unusual preference for aspartic acid (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar, 15Van de Craen M. Van den Brande I. Declercq W. Irmler M. Beyaert R. Tschopp J. Fiers W. Vandenabeele P. Cleavage of caspase family members by granzyme B: a comparative study in vitro.Eur. J. Immunol. 1997; 27 (9174624): 1296-129910.1002/eji.1830270535Crossref PubMed Scopus (46) Google Scholar); therefore, we incorporated Asp at P1, and we elongated the peptide up to six amino acids. The investigated peptides were Ac-AAIEPD-ACC, Ac-AIEPD-ACC, Ac-IEPD-ACC, Ac-EPD-ACC, Ac-PD-ACC, and Ac-D-ACC. We measured the activity of GrB against each of the new substrates (at equal concentrations) and observed that neither the tripeptide, the dipeptide, nor the single-amino-acid-based substrates were hydrolyzed. The longer tetra-, penta-, and hexapeptides were cleaved by GrB. The pentapeptide and hexapeptides were more efficiently cleaved by GrB than was the classic tetrapeptide substrate (Fig. 1A). Because there was no significant difference in the efficiency of the cleavage of the penta- and hexapeptides, we selected the pentapeptides as the most appropriate chain length for activity-based probes and substrates. To further explore the substrate specificity of GrB and allow better optimization of GrB substrates, we analyzed the S1–S5 pocket preferences using both combinatorial chemistry methods and the screening of defined peptides possessing natural and diverse unnatural amino acids (16Kasperkiewicz P. Poreba M. Snipas S.J. Parker H. Winterbourn C.C. Salvesen G.S. Drag M. Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling.Proc. Natl. Acad. Sci. U S A. 2014; 111 (24550277): 2518-252310.1073/pnas.1318548111Crossref PubMed Scopus (103) Google Scholar, 17Poreba M. Kasperkiewicz P. Snipas S.J. Fasci D. Salvesen G.S. Drag M. Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.Cell Death Differ. 2014; 21 (24832467): 1482-149210.1038/cdd.2014.64Crossref PubMed Scopus (54) Google Scholar). The GrB S1 pocket almost exclusively recognizes aspartic acid, and this feature is also seen in cysteine protease caspases (9Poreba M. Strozyk A. Salvesen G.S. Drag M. Caspase substrates and inhibitors.Cold Spring Harb. Perspect. Biol. 2013; 5 (23788633): a00868010.1101/cshperspect.a008680Crossref PubMed Scopus (116) Google Scholar, 10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar, 17Poreba M. Kasperkiewicz P. Snipas S.J. Fasci D. Salvesen G.S. Drag M. Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.Cell Death Differ. 2014; 21 (24832467): 1482-149210.1038/cdd.2014.64Crossref PubMed Scopus (54) Google Scholar). To reduce substrate cross-reactivity, especially with caspases, we tested whether any acidic and nonacidic modifications of natural amino acid residues are accommodated by the GrB S1 pocket. For this purpose, we designed and synthesized (using a solid-phase peptide synthesis method, see Fig. S1) a library of 95 defined peptides that share the same leading sequence and differ only by one amino acid residue at P1 (for the structures, see Fig. S2). ACC (7-amino-4-carbamoylmethylcoumarin) was applied as a fluorescent leaving group, allowing the substrate hydrolysis rate to be determined based on the increase in fluorescence. In the positions P4–P2, defined natural amino acid residues were incorporated based on literature data (Ac-Ile-Ser-Pro-P1-ACC), while the N termini of the peptide substrates were acetylated. We tested the activity of GrB against the new P1 library and determined that GrB almost exclusively requires aspartic acid in the S1 pocket (l-Asp, 100%); it can accommodate the methylated derivative (l-Asp [O-Me], 30%) and, less potently, tyrosine (l-Tyr) and its derivatives (<10%) (Fig. 1B and Fig. S3A). This confirms the literature data where, similar to caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar, 17Poreba M. Kasperkiewicz P. Snipas S.J. Fasci D. Salvesen G.S. Drag M. Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.Cell Death Differ. 2014; 21 (24832467): 1482-149210.1038/cdd.2014.64Crossref PubMed Scopus (54) Google Scholar), the GrB S1 pocket is essentially restricted to aspartic acid due to its interaction with the positively charged guanidine group of Arg-226. Additionally, the carboxyl group of Asp forms hydrogen bonds with three water molecules within the S1 subsite of GrB (18Rotonda J. Garcia-Calvo M. Bull H.G. Geissler W.M. McKeever B.M. Willoughby C.A. Thornberry N.A. Becker J.W. The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1.Chem. Biol. 2001; 8 (11325591): 357-36810.1016/S1074-5521(01)00018-7Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Not only the charge but also the shape of this pocket dictates amino acid binding, since only Asp or uncharged Asp derivatives with minimal modifications (Asp [O-Me]) can occupy this pocket, despite the lack of a negative charge, while more sizable Asp derivatives (Asp [O-Chx] and Asp [O-Bzl]) are not recognized by GrB, revealing the limited capacity of the S1 subsite and the involvement of additional interactions in peptide binding. The shape of Asp allows it to fit perfectly in the S1 pocket, and this is supported by the interaction between the guanidine group and the COOH group (for the P1 library screening, see Fig. S3). Although P1-Asp specificity distinguishes GrB from other granzymes, many GrB substrates are also recognized by caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar), so conventional strategies for enzyme activity analysis cannot be used to selectively monitor GrB activity in cells. Therefore, we sought a substrate sequence that distinguishes GrB activity from other proteases. To this end, we next determined the extended catalytic preferences of GrB at S4–S2 using a well-established HyCoSuL strategy (Hybrid Combinatorial Substrate Library) incorporating a wide range of different nonproteinogenic amino acids (16Kasperkiewicz P. Poreba M. Snipas S.J. Parker H. Winterbourn C.C. Salvesen G.S. Drag M. Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling.Proc. Natl. Acad. Sci. U S A. 2014; 111 (24550277): 2518-252310.1073/pnas.1318548111Crossref PubMed Scopus (103) Google Scholar, 17Poreba M. Kasperkiewicz P. Snipas S.J. Fasci D. Salvesen G.S. Drag M. Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.Cell Death Differ. 2014; 21 (24832467): 1482-149210.1038/cdd.2014.64Crossref PubMed Scopus (54) Google Scholar, 19Poreba M. Salvesen G.S. Drag M. Synthesis of a HyCoSuL peptide substrate library to dissect protease substrate specificity.Nat. Protoc. 2017; 12 (28933778): 2189-221410.1038/nprot.2017.091Crossref PubMed Scopus (39) Google Scholar) (Fig. S3). We observed that bulky hydrophobic proline derivatives, such as octahydroindolecarboxylic acid (l-Oic), 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and O-benzyl-l-hydroxyproline (l-Hyp [Bzl]) are strongly preferred by GrB in P2, while substrates with hydroxyproline (l-Hyp) bearing unprotected hydroxyl groups are not hydrolyzed. Additionally, bulky hydrophobic 6-benzyloxy-l-norleucine (l-Nle [O-Bzl]), benzyl-l-histidine (l-His [Bzl]), and benzyl-l-serine (l-Ser [Bzl]) were tolerated in the S2 pocket, indicating that amino acids with benzyl groups can be accommodated within this pocket (Fig. 1B and Fig. S3) and revealing that the S2 pocket is very capacious. We speculate that this pocket, filled by the flexible cyclohexane group of proline derivative l-Oic or the benzyl groups of l-Nle (O-Bzl) or l-Ser (Bzl), enables the amino acid residues to perfectly fill the S2 pocket. The HyCoSuL strategy also revealed that the S3 pocket of GrB has a broad substrate scope; however, it has strong preferences for glutamic acid (l-Glu, 100%), hydrophobic tyrosine bearing a benzyl group (l-Tyr [Bzl], 72%), and mono-oxidized methionine l-Met (O) (Fig. 1B and Fig. S3). The strong preference for an acidic amino acid is due to Asn-218 and Lys-192 in the S3 pocket of GrB, which interact with the side chain of l-Glu and stabilize the positively charged side chain of lysine (18Rotonda J. Garcia-Calvo M. Bull H.G. Geissler W.M. McKeever B.M. Willoughby C.A. Thornberry N.A. Becker J.W. The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1.Chem. Biol. 2001; 8 (11325591): 357-36810.1016/S1074-5521(01)00018-7Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The S3 pockets of all caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar) share GrB’s preferences for glutamic acid, confirming the structural similarity of these enzymes (17Poreba M. Kasperkiewicz P. Snipas S.J. Fasci D. Salvesen G.S. Drag M. Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.Cell Death Differ. 2014; 21 (24832467): 1482-149210.1038/cdd.2014.64Crossref PubMed Scopus (54) Google Scholar). Our results demonstrated that the S4 pocket also possesses broad substrate preferences. It can accommodate branched amino acids, such as isoleucine (l-Ile, 72%), valine (l-Val, 36%), or linear hydroxyl-l-norvaline (l-Hnv, 82%), as well as a proline derivative (l-Tic, 100%) and benzyloxymethyl-l-histidine (l-His [3-Bom], 82%) and some basic amino acids, such as 2,4-diaminobutyrylic acid (l-Dab, 39%), citrulline (l-Cit, 10%), and 1,3-diaminopropionic acid (l-Dap, 10%) (Fig. 1B and Fig. S3). According to Rotonda et al., this GrB pocket is a “shallow hydrophobic depression formed by aromatic rings” (Tyr-174 and Tyr-215) and the side chain of Leu-172 (18Rotonda J. Garcia-Calvo M. Bull H.G. Geissler W.M. McKeever B.M. Willoughby C.A. Thornberry N.A. Becker J.W. The three-dimensional structure of human granzyme B compared to caspase-3, key mediators of cell death with cleavage specificity for aspartic acid in P1.Chem. Biol. 2001; 8 (11325591): 357-36810.1016/S1074-5521(01)00018-7Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); therefore, there is not enough space for phenylalanine within this pocket. Our data from the HyCoSuL screening revealed that a bulkier amino acid (l-His [3-Bom]) was hydrolyzed by GrB (Fig. S3). To find the optimal amino acid for P5, we synthesized a combinatorial library of pentapeptides. For this purpose, based on the literature data and our results related to GrB specificity, we designed a library consisting of 1) defined amino acids at P1 (Asp) and P3 (Glu), 2) equimolar mixtures (X) at P2 and P4 (a mixture of natural amino acids with l-Nle replacing l-Met and l-Cys), and 3) one of the 174 defined amino acids at P5 (Ac-P5-X-Glu-X-Asp-ACC) (for the structures, see Fig. S2). GrB was tested against the library, and we observed that it displays no S5 substrate specificity and is capable of hydrolyzing most substrates regardless of the residue at this position; however, the addition of an extra amino acid to the substrate (P5) clearly leads to a dramatic increase in substrate hydrolysis (Fig. 1B–D). To validate the results of the HyCoSuL screening, we selected the most promising amino acid residues for the S4–S2 positions (P4, l-Tic, l-His [3-Bom], and l-Ile; P3, l-Glu and l-Tyr [Bzl]; and P2, l-His [Bzl], l-Oic, l-Tic, and l-Hyp [Bzl]) and synthesized eighteen different fluorogenic tetrapeptides using a previously described method (20Maly D.J. Leonetti F. Backes B.J. Dauber D.S. Harris J.L. Craik C.S. Ellman J.A. Expedient solid-phase synthesis of fluorogenic protease substrates using the 7-amino-4-carbamoylmethylcoumarin (ACC) fluorophore.J. Org. Chem. 2002; 67 (11856036): 910-91510.1021/jo016140oCrossref PubMed Scopus (117) Google Scholar). We then tested the activity of GrB on the new substrates and observed that peptides with Ile at P4 were exclusively hydrolyzed by GrB (TJ40–44), while substrates with other selected amino acids, such as Tic (TJ2, TJ4, TJ6, and TJ35–39) and l-His (3-Bom) (TJ3 and TJ30–34), were not recognized by GrB under these assay conditions. Additionally, we confirmed that sequences containing l-Nle (O-Bzl) or l-Oic at S2 were preferred by GrB, confirming the large size of S2 (Fig. S4A). With this in mind, we performed a detailed kinetic analysis of the most hydrolyzed and promising substrates, namely, TJ40 (Ac-Ile-Glu-Oic-Asp-ACC), TJ41 (Ac-Ile-Glu-Hyp [Bzl]-Asp-ACC), TJ42 (Ac-Ile-Glu-Tic-Asp-ACC), TJ43 (Ac-Ile-Glu-His [Bzl]-Asp-ACC), and TJ44 (Ac-Ile-Glu-Nle [O-Bzl]-Asp-ACC) (Fig. S4), and we compared those results with those of reference substrate TJ7 (Ac-Ile-Glu-Pro-Asp-ACC). Substrates TJ40, TJ43, and TJ44 were cleaved more rapidly by GrB than was TJ7, and their kinetic constants were kcat/Km = 379.09 m−1 s−1, 267.89 m−1 s−1, and 685.51 m−1 s−1, respectively, and that of the reference is kcat/Km = 76.95 m−1 s−1 (Fig. S4B). This was in agreement with our initial substrate screening (Fig. S4A). Since l-Oic and other proline derivatives are poorly or not recognized by caspases (10Thornberry N.A. Rano T.A. Peterson E.P. Rasper D.M. Timkey T. Garcia-Calvo M. Houtzager V.M. Nordstrom P.A. Roy S. Vaillancourt J.P. Chapman K.T. Nicholson D.W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.J. Biol. Chem. 1997; 272 (9218414): 17907-1791110.1074/jbc.272.29.17907Abstract Full Text Full Text PDF PubMed Scopus (1786) Google Scholar, 17Poreba M. Kasperkiewicz P. Snipas S.J. Fasci D. Salvesen G.S. Drag M. Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates.Cell Death Differ. 2014; 21 (24832467): 1482-149210.1038/cdd.2014.64Crossref PubMed Scopus (54) Google Scholar, 21Poreba M. Rut W. Groborz K. Snipas S.J. Salvesen G.S. Drag M. Potent and selective caspase-2 inhibitor prevents MDM-2 cleavage in reversine-treated colon cancer cells.Cell Death Differ. 2019; 26: 2695-270910.1038/s41418-019-0329-2Crossref PubMed Scopus (8) Google Scholar), we decided to use the sequence of substrate TJ40 as the core for our future substrates and probes. The optimization of the P4–P1 GrB peptide sequence resulted in a champion substrate that is well recognized by GrB and is less likely to show cross-reactivity with other granzymes and caspases. From the broad range of investigated amino acids, we selected the most promising structures (l-Lys [TFA], l-Nva, l-Ile, l-Phe [2-Cl], and l-hPhe), and we synthesized ten defined pentapeptide substrates, incorporating the sequences previously selected for P4–P1 (l-Ile-l-Glu-l-Oic/l-Nle [O-Bzl]-l-Asp). After an initial screening of GrB activities against the new substrates (TJ46–TJ56) (Fig. 1C), we selected substrates TJ47, TJ49, TJ52, and TJ55 as being hydrolyzed with the highest efficiency, and we studied their kinetics in detail (Fig. 1D). Substrate TJ49 possesses the lowest Km value (32.33 μm) and, at the same time, the highest kcat parameter (0.152 s−1), with 40% higher activity (kcat/Km of 4960 m−1 s−1) than that of the optimal tetrapeptide. Exchanging l-Nle (O-Bzl) at P2 (in TJ49) for l-Oic (affording TJ55) caused the enzyme activity to decrease by approximately a factor of four; however, we used this sequence in further analyses to minimize the cross-reactivity with the majority of caspases (17Poreba M. Kasperkiewicz P. Snipas S.J. Fasci D. Salvesen G.S. Drag M. Unnatural amino acids increase sensitivity and provide for t