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Kinetic Characterization and Molecular Docking of a Novel, Potent, and Selective Slow-Binding Inhibitor of Human Cathepsin L

Institute for Medicine and Engineering (P.P.S., M.P.B., J.E.P., H.J., A.D.N., S.L.D.) and the Department of Chemistry (M.C.M., D.M.H., B.S.C., A.B.S.), Penn Center for Molecular Discovery, University of Pennsylvania, Philadelphia, Pennsylvania; and University of Pittsburgh Drug Discovery Institute, Pittsburgh, Pennsylvania (H.J.G., E.R.S.)

Received February 25, 2008; accepted April 10, 2008

	Abstract

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A novel small molecule thiocarbazate (PubChem SID 26681509), a potent inhibitor of human cathepsin L (EC 3.4.22.1 [EC] 5) with an IC₅₀ of 56 nM, was developed after a 57,821-compound screen of the National Institutes of Health Molecular Libraries Small Molecule Repository. After a 4-h preincubation with cathepsin L, this compound became even more potent, demonstrating an IC₅₀ of 1.0 nM. The thiocarbazate was determined to be a slow-binding and slowly reversible competitive inhibitor. Through a transient kinetic analysis for single-step reversibility, inhibition rate constants were k_on = 24,000 M^-1s^-1 and k_off = 2.2 x 10^-5 s^-1 (K_i = 0.89 nM). Molecular docking studies were undertaken using the experimentally derived X-ray crystal structure of papain/CLIK-148 (1cvz. pdb). These studies revealed critical hydrogen bonding patterns of the thiocarbazate with key active site residues in papain. The thiocarbazate displayed 7- to 151-fold greater selectivity toward cathepsin L than papain and cathepsins B, K, V, and S with no activity against cathepsin G. The inhibitor demonstrated a lack of toxicity in human aortic endothelial cells and zebrafish. In addition, the thiocarbazate inhibited in vitro propagation of malaria parasite Plasmodium falciparum with an IC₅₀ of 15.4 µM and inhibited Leishmania major with an IC₅₀ of 12.5 µM.

As part of the National Institutes of Health Molecular Libraries Screening Centers Network, the Penn Center for Molecular Discovery performs high-throughput screens against various targets, deposits data into PubChem, and develops small molecule probes. A high-throughput screen of 57,821 compounds from the National Institutes of Health Molecular Libraries Small Molecule Repository (BioFocus DPI, San Francisco, CA) was performed against human cathepsin L. The average Z' factor for the screen was 0.73, indicating good plate uniformity throughout the run (Zhang et al., 1999). Results of this screen are available on PubChem as BioAssay #460 (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=460).

View larger version (24K): [in this window] [in a new window]   Fig. 1. A, oxadiazole SID 861540, the major constituent of the most potent hit from the high-throughput screen. B, thiocarbazate SID 26681509, the Boc-protected S-enantiomer of the ring-opened by-product of the original high-throughput screen hit. C, activity of SID 26681509 against human cathepsin L after preincubation with the enzyme target for 0 h (), 1 h (), 2 h (), and 4 h ().

View larger version (23K): [in this window] [in a new window]   Fig. 2. A, dilution protocol for determination of reversibility. Cathepsin L at 100-fold its final assay concentration (870 ng/ml) and inhibitor at 10-fold its IC50 after 1-h preincubation (75 nM) were combined and incubated for 1 h at room temperature at 2 µl. This mixture was then diluted 100-fold with assay buffer containing 1 µM Z-Phe-Arg-AMC. A rapidly reversible inhibitor should dissociate from the enzyme to restore greater than approximately 90% of enzymatic activity. B, reversibility data for SID 26681509 after 0 min (), 15 min (), 1 h (), and 4 h () preincubation with cathepsin L and upon 100-fold dilution into assay buffer containing Z-Phe-Arg-AMC. A full enzyme-substrate reaction without inhibitor () served as a positive control. By comparing initial substrate conversion rates of the control and 1-h preincubation study, it can be seen that only 11% enzymatic activity is restored after 6000 s, making SID 26681509 a slowly reversible inhibitor of human cathepsin L. C, reaction progress curve with 4-h preincubation of cathepsin L and SID 26681509. This upward curvature demonstrates that the enzyme was recovering and that the reaction was reversible. The rate of AMC release after 8820 s was 4.7 times greater than the initial release rate.

	Materials and Methods

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Cathepsin L Assay Optimization. The cathepsin L assay was carried out using 1 µM Z-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC; Sigma, St. Louis, MO) and 8.7 ng/ml human liver cathepsin L (Calbiochem, San Diego, CA) in 100-µl reactions (96-well plate). Assay buffer consisted of 20 mM sodium acetate, 1 mM EDTA, and 5 mM cysteine, pH 5.5. Cathepsin L was incubated in assay buffer for 30 min before dispensing into wells to allow for efficient reduction of the active site cysteine required for full enzymatic activity. AMC dilution controls were performed and no inner filter effect quenching was observed at fluorophore concentrations as high as 50 µM.

IC₅₀ Determination. SID 26681509 was synthesized as described previously (Myers et al., 2008). A 16-point, 2-fold serial dilution dose response of SID 26681509 was performed to determine its relative potency against cathepsin L. Each well of a 96-well assay plate (Corning Life Sciences, Acton, MA) contained 38 µl of water and 2 µl of inhibitor (ranging from 2.5 mM to 76 nM) in dimethyl sulfoxide (DMSO). Positive and negative controls were present to serve as internal controls into which 2 µl of DMSO was transferred in place of the inhibitor. Ten microliters of 10 µM Z-Phe-Arg-AMC in 5x concentrated assay buffer and 50 µl of 17.4 ng/ml cathepsin L in assay buffer were added sequentially to initiate the proteolytic reaction. A total of 50 µl of assay buffer was dispensed in place of enzyme into negative control wells. This resulted in a final dose response concentration range from 50 µM to 1.5 nM inhibitor (2% DMSO) in a 100-µl final reaction volume. The fluorescence intensity of each well of the assay plates was monitored on a microplate reader (excitation at 355 nm; emission at 460 nm; Envision; PerkinElmer Life and Analytical Sciences, Waltham, MA) to measure the AMC released by the enzyme-catalyzed hydrolysis of Z-Phe-Arg-AMC. Data were scaled using internal controls and fit to a four-parameter logistic model (IDBS XLfit eq. 205; Guildford, Surrey, UK) to obtain IC₅₀ values in triplicate.

View larger version (23K): [in this window] [in a new window]   Fig. 3. A, single-step mechanism for simple, reversible, slow binding inhibition governed by kinetic constants kon and koff. B, Km and kcat determination for human cathepsin L enzymatic reaction with Z-Phe-Arg-AMC substrate. Km and kcat were determined to be 0.77 µM and 1.5 s-1, respectively.

View larger version (39K): [in this window] [in a new window]   Fig. 4. A, ordinary differential equations governing the single-step mechanism of inhibition shown in Fig. 3A. B, reaction progress curves () are shown for 8.7 ng/ml human cathepsin L enzyme and 1 µM Z-Phe-Arg-AMC substrate with varying concentrations of SID 26681509 inhibitor. They have been fit to a five-parameter inhibition kinetic model () using APPSPACK optimization software with a linear least-squares objective function.

Reversibility. To test the reversibility of SID 26681509, cathepsin L at 100-fold its final assay concentration (870 ng/ml) and inhibitor at 10-fold its IC₅₀ after 1 h preincubation were combined and incubated for 1 h at room temperature at 2 µl. This mixture was then diluted 100-fold in a Corning 96-well plate with assay buffer containing 1 µM Z-Phe-Arg-AMC to a final volume of 200 µl. A rapidly reversible inhibitor should dissociate from the enzyme to restore greater than approximately 90% of enzymatic activity (Fig. 2A). Fluorescence intensities of the 200-µl reaction wells were monitored continuously for AMC hydrolysis on the Envision plate reader.

Data Fitting. In the kinetic simulations, the concentrations of chemical species ([E], [S], [I], [P], [ES], [EI]) over time were calculated using a system of ordinary differential equations for each reaction step (Fig. 4A). Progress curves for each inhibitor concentration were fit to a five-parameter (k₁, k_-1, k_on, k_off, k_cat) kinetic inhibition model using APPSPACK optimization software. APPSPACK is a generic solver for linearly constrained optimization problems (Griffin and Kolda, 2006).

Selectivity. SID 26681509 was assayed for inhibition against papain and cathepsins B, G, K, S, and V. Papain from Carica papaya (11 ng/ml; Calbiochem), human cathepsin K (35 ng/ml; Calbiochem), human spleen cathepsin S (40 ng/ml; Calbiochem), and human cathepsin V (39 ng/ml; Calbiochem) were assayed using Z-Phe-Arg-AMC substrate at 20, 20, 15, and 1 µM, respectively. Human liver cathepsin B (65 ng/ml; Calbiochem) was assayed using 15 µM Z-Arg-Arg-AMC substrate (Bachem, King of Prussia, PA). Human neutrophil cathepsin G (4.2 µg/ml; Calbiochem) was assayed using 15 µM Suc-Ala-Ala-Pro-Phe-AMC substrate (Sigma). All reactions were performed in 20 mM sodium acetate buffer containing 5 mM cysteine and 1 mM EDTA, pH 5.5. Reaction progress was monitored using the Envision microplate reader. IC₅₀ values were measured in triplicate.

Cytotoxicity. Human aortic endothelial cells were seeded in a 384-well white sterile tissue culture-treated microplate (Corning) at 1000 cells/25 µl/well. The plate was centrifuged and incubated at 37°C for 24 h. SID 26681509 and doxorubicin positive control were then serially diluted in EGM-2 endothelial cell media (Lonza Walkersville, Inc., Walkersville, MD). Five microliters each of these serial dilutions were added to the cells in triplicate, resulting in final concentrations of compound from 100 µM to 156 nM (0.17% DMSO). The plate was centrifuged and incubated at 37°C for 24 h. Thirty microliters of CellTiter-Glo (Promega, Madison, WI) were added to each well and centrifuged. After 10 min, luminescence was measured using the Envision microplate reader.

Malaria Assay. Eight 2-fold serial dilutions of SID 26681509 in RPMI 1640 media (Invitrogen, Carlsbad, CA) containing L-glutamine, 50 mg/l hypoxanthine, 6 g/l HEPES, 0.5% Albumax II bovine serum (Invitrogen), 0.225% sodium bicarbonate, and 1 µg/ml gentamicin were performed in Corning microplates. Adding 30 µl of red blood cells infected with synchronized ring stage luciferase-expressing Plasmodium falciparum parasites at 0.5% parasitemia and 4% hematocrit to 10 µl of compound resulted in final concentrations tested of 50 µM to 1.5 nM. In addition, 30 µl of normal red blood cells and 30 µl of infected red blood cells were added to two control columns containing 10 µl of media. The plates were incubated at 37°C in a 92% humidity chamber with 5% CO₂, 5%O₂, and 90% N₂ for 48 h to allow for two cycles of red blood cell rupture and invasion to take place. Forty microliters BrightGlo (Promega) were added to each well and centrifuged. After 5 min, luminescence was measured using the Envision microplate reader.

Leishmaniasis Assay. Five thousand Leishmania major promastigotes were plated in each well of a 384-well microtiter plate in a 20-µl volume of promastigote growth medium. Promastigotes were treated with a concentration range from 0 to 50 µM SID 26681509 for 44 h. Five microliters of Cell-Titer-Blue were added to each well and incubated for 4 h. Relative fluorescence units (A₅₆₀/A₅₉₀) were captured on a SpectraMax M5 microtiter plate reader. DMSO concentrations were held constant at 0.5%.

	Results

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Kinetic Characterization. With immediate mixing of enzyme, substrate, and inhibitor (no preincubation of enzyme and inhibitor), SID 26681509 was found to inhibit human cathepsin L with an IC₅₀ of 56 ± 4 nM. After preincubation with enzyme for 1, 2, and 4 h before substrate addition at t = 0, SID 26681509 displayed increasing potency, with IC₅₀ values falling to 7.5 ± 1.0, 4.2 ± 0.6, and 1.0 ± 0.5 nM, respectively, demonstrating a slow onset of inhibition against the target enzyme (Fig. 1C).

The mechanism of inhibition, to determine whether the compound acted as a rapidly reversible, slowly reversible, or irreversible inhibitor, was evaluated using a preincubation/dilution assay (Copeland, 2005). By preincubating human cathepsin L and the compound for 1 h at 10-fold its IC₅₀ after 1 h of preincubation (75 nM), a condition was created in which >90% of the enzyme should have been in an enzyme-inhibitor complex (Fig. 2A). Upon 100-fold dilution of the 1 h preincubated mixture of cathepsin L and the inhibitor into assay buffer containing 1 µM Z-Phe-Arg-AMC substrate, approximately 11% enzymatic activity was returned after 6000 s into the reaction, by comparison of the substrate conversion rates of the preincubated and uninhibited reactions (Fig. 2B). For the 4-h preincubated enzyme-inhibitor reaction condition (Fig. 2C), 99.8% of the reaction was inhibited immediately after addition of substrate because almost all the enzyme was bound to the small-molecule inhibitor SID 26681509. After 8820 s, the rate of product formation for the 4-h preincubated reaction was 4.7 times greater than the initial rate of product formation, showing that the inhibitor was being released from the enzyme-inhibitor complex and enzymatic activity was indeed recovering. Therefore, SID 26681509 was determined to be a very slowly reversible inhibitor of human cathepsin L.

Nonlinear Regression of Transient Kinetics. For human cathepsin L cleavage of Z-Phe-Arg-AMC, K_m and k_cat were determined through initial rate analysis to be 0.77 µM and 1.5 s^-1, respectively (Fig. 3B). A nonlinear regression for transient dynamics was conducted based on the reaction scheme shown in Fig. 3A. Here, the values of k₁, k_-1, k_on, and k_off are explicitly estimated rather than combined into the equilibrium parameters, K_m and K_i, estimated by traditional kinetic analyses. The best fit parameters were k₁ = 2.3 x 10⁶ M^-1s^-1, k_-1 = 0.30 s^-1, k_cat = 4.0 s^-1, k_on = 24,000 M^-1s^-1, and k_off = 2.2 x 10^-5 s^-1 (Fig. 4B). The regressed K_i = 0.89 nM was quite consistent with the measured IC₅₀ = 1.0 ± 0.5 nM obtained after 4 h preincubation of human cathepsin L with SID 26681509. To explore alternate models for inhibition, the data were fit to models for irreversible inhibitor binding ([E]+[I][EI]); two-step inhibitor binding ([E]+[I][EI]₁[EI]₂), where a weak enzyme-inhibitor encounter complex is formed before the formation of a more tightly bound enzyme-inhibitor complex; and uncompetitive inhibitor binding ([ES]+[I][ESI]), where inhibitor binds only to the enzyme-substrate complex. These models failed to reproduce the data as well as the five-parameter model described above for reversible, single-step competitive inhibition.

View larger version (21K): [in this window] [in a new window]   Fig. 5. A, IC50 against P. falciparum was determined to be 15.4 ± 0.6 µM. B, IC50 against L. major was determined to be 12.5 ± 0.6 µM.

Selectivity against Papain and Cathepsins B, G, K, S, and V. SID 26681509 was tested for inhibitory activity against papain and human cathepsins B, G, K, L, S, and V (Table 1) with no preincubation of enzyme and inhibitor. IC₅₀ values were calculated at time points of 10, 30, 60, and 90 min. The selectivity indexes of SID 26681509 (a ratio of the IC₅₀ against the assayed protease divided by the IC₅₀ against cathepsin L) ranged from 7 to 151 for the various papain-like cysteine proteases (Table 2). SID 26681509 inhibited papain and cathepsins B, K, S, and V with IC₅₀ values, determined after 1 h, ranging from 618 nM to 8.442 µM. As expected, SID 26681509 showed no inhibitory activity against the serine protease cathepsin G. The IC₅₀ values systematically decreased with time for each protease, demonstrating the slow-binding nature of the small molecule inhibitor. The qualitative order of the selectivity index is fairly insensitive to when the measurement was taken; however, the weak trends observed in the selectivity index data probably reflect the relative rates of slowly reversible inhibition of the enzyme. Thus, it seems that the slowly reversible reaction proceeds faster for cathepsins V and S than for cathepsin L, whereas it proceeds more slowly for papain.

Biological Assays. SID 26681509 was found to be non-toxic to human aortic endothelial cells at 100 µM. The inhibitor also demonstrated a lack of toxicity to zebrafish in a live organism assay at 100 µM. SID 26681509 was active in an in vitro propagation assay against P. falciparum with an IC₅₀ of 15.4 ± 0.6 µM (Fig. 5A). In addition, the thiocarbazate inhibitor was toxic toward L. major promastigotes with an IC₅₀ of 12.5 ± 0.6 µM (Fig. 5B).

Molecular Docking of SID 26681509 in Papain. The cocrystal structure of CLIK-148 bound to papain (1cvz.pdb) (Katunuma et al., 1999; Tsuge et al., 1999) was used as a model to study hydrogen bonding and hydrophobic interactions of the thiocarbazate inhibitor SID 26681509 within the cysteine protease binding site. The chemical structure of CLIK-148 is depicted in Fig. 6A. Other researchers have used papain to design highly specific cathepsin inhibitors and CLIK-148 directly inhibits cathepsin L (LaLonde et al., 1998; Katunuma et al., 1999; Tsuge et al., 1999). An effort to construct a cathepsin L homology model based on the coordinates of 1cvz.pdb led to inconclusive docking results, particularly with respect to the arrangement of critical hydrophobic groups in the S2 subsite of the enzyme binding pocket [Molecular Operating Environment (MOE) software; Chemical Computing Group, Montreal, ON, Canada]. The cocrystal structure coordinates of human cathepsin L complexed with the small molecule inhibitor E-64 (Fujishima et al., 1997) would have been preferred; however, they were not publicly available. Most of the residues within the catalytic binding site are conserved between papain and cathepsin L, including those in papain that make direct hydrogen bonding contacts to the CLIK-148 inhibitor: Gln19, Cys25, Gly66, Asp158, and Trp177. Because cathepsin L is most homologous to papain within the papain superfamily of cysteine proteases, the high-resolution (1.7 Å) structure of papain/CLIK-148 served as an excellent starting point for studying small molecule inhibitors of cathepsin L.

View larger version (30K): [in this window] [in a new window]   Fig. 6. A, CLIK-148, a cathepsin L-specific inhibitor (Katunuma et al., 1999; Tsuge et al., 1999). B, overlay of papain/CLIK-148 crystal structure (1cvz.pdb, green) with independently docked epoxide ring-opened form of CLIK-148 (gray). The XP Glide score for the top-scoring pose was -9.27 kcal/mol.

To prepare this system for docking, the covalent bond between CLIK-148 and papain was broken, and the epoxide ring-opened form of CLIK-148 was independently docked into papain. The highest scoring pose for CLIK-148 obtained from this docking study overlaid very well with the experimentally derived bound inhibitor CLIK-148 (Fig. 6B). The XP Glide score for CLIK-148 in papain was -9.27 kcal/mol. With this validation, we studied the interaction of SID 26681509 with papain.

SID 26681509 was prepared for docking using LigPrep software (Schrödinger LLC, New York, NY). The highest scoring pose of SID 26681509 had an excellent score of -9.04 kcal/mol. This score was very close to the XP Glide score obtained for independently docked CLIK-148 in papain. In addition, many of the residues that made contacts between CLIK-148 and papain were also involved in making contacts between SID 26681509 and papain (Fig. 7A). The backbone NH hydrogens of Gln19 and Cys25 made direct hydrogen bonding contacts to the thiocarbazate carbonyl oxygen of SID 26681509, the backbone NH hydrogen of Gly66 made a hydrogen bond to the acyl hydrazine CO oxygen of the ligand, the backbone carbonyl oxygen of Asp158 was involved in a hydrogen bonding network to both a hydrazine NH and an amide NH of SID 26681509, and finally, the Trp177 side chain NH formed a hydrogen bond to an amide carbonyl oxygen of SID 26681509. In addition, the 2-ethylanilide group of SID 26681509 made a large hydrophobic contact with the aromatic side chain of Trp177; Trp177 is located in the prime region of the enzyme binding pocket (S1' subsite). The indole group of SID 26681509 occupies the S2 subsite of the enzyme binding pocket. When the docking poses of CLIK-148 and SID 26681509 were overlaid (Fig. 7B), SID 26681509 looked remarkably like the epoxide ring-opened form of CLIK-148. This overlay illustrated that both inhibitors maintain the same critical distances between the two carbonyl groups that are disposed in a 1,4 relationship to each other. The intramolecular distance between the 1,4-dicarbonyl in both CLIK-148 and SID 26681509 was approximately 4.80 to 4.92 Å.

View larger version (75K): [in this window] [in a new window]   Fig. 7. A, hydrogen bonding interactions between SID 26681509 and papain involve catalytic residues Gln19, Cys25, Gly66, Asp158, and Trp177. The distance between the Cys25 sulfur atom and the thiocarbazate carbonyl carbon is 3.287 Å. This is the carbon likely to undergo nucleophilic attack by the sulfur. B, overlay of papain/CLIK-148 (light green) with computational model of SID 26681509 in papain (tan). There are striking similarities between the binding interactions of the two inhibitors.

Finally, the active site Cys25 sulfur was in close proximity (3.287 Å) to the carbonyl carbon of the thiocarbazate. Although the contribution from covalent bonding between this carbon and sulfur cannot be directly assessed through our docking studies, the molecule sits in the proper orientation to achieve this covalent binding interaction (Fig. 7A). As indicated above, however, we lack compelling evidence for covalent binding between the enzyme and SID 26681509.

	Discussion

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Our kinetic analyses demonstrated that SID 26681509 was a highly potent and selective competitive inhibitor of human cathepsin L with slow binding and slow reversibility kinetics. Molecular docking of SID 26681509 into the papain crystal structure revealed hydrophobic interactions within the S2 and S1' subsites and hydrogen bonding interactions with Gln19, Cys25, Gly66, Asp158, and Trp177. These interactions share a high degree of similarity with the papain/CLIK-148 complex (Katunuma et al., 1999; Tsuge et al., 1999).

With the exception of cathepsin K, the range of selectivity indexes of SID 26681509 decreased as the percentage identity to cathepsin L increased. This may be due to the strong P2 preference of cathepsin K for proline (Choe et al., 2006). It is interesting to note that SID 26681509 is 7 to 11 times more potent against cathepsin L than against cathepsin V. Cathepsin V, which is sometimes referred to as cathepsin L2, shares 78% amino acid sequence identity with cathepsin L and has been shown to compensate for the role of cathepsin L in epidermal homeostasis and hair follicle morphogenesis of knockout mice (Reinheckel et al., 2001; Nägler and Menard, 2003; Hagemann et al., 2004).

Using the papain/CLIK-148 coordinate system, we were able to independently dock SID 26681509 into the binding site of papain. This led to the conclusion that SID 26681509 seemed to bind to papain in a manner similar to CLIK-148. Five residues that are conserved between papain and cathepsin L made direct contacts to both inhibitors. In addition, a highly hydrophobic/aromatic site involving Trp177 interacted with the hydrophobic 2-ethylanilide group of SID 26681509.

The fact that SID 26681509 inhibited both malaria and leishmaniasis suggests that it acts in a cellular system requiring transit across lipid membranes. However, the micromolar potency, as opposed to subnanomolar potency against purified human cathepsin L, was not surprising because 1) we have no measure of the internal concentration of inhibitor achieved in these organisms and 2) the active site geometries of their cathepsin L-like cysteine proteases might differ from that of the human enzyme. Further investigations of SID 26681509 and related analogs against purified cathepsin L-like enzymes such as falcipain, congopain, cruzipain, T. gondii cathepsin L, histolysain, and rhodesain are warranted based on the findings of this study. The thiocarbazate scaffold can be readily derivatized, introducing functional groups to occupy specific binding sites in a variety of cysteine proteases, and thus holds promise as a general scaffold for the design of specific cysteine protease inhibitors.

	Acknowledgements

 
We thank Professor Michael A. Pack (University of Pennsylvania School of Medicine) for testing SID 26681509 for zebrafish toxicity. We also thank Claire Darling and Professor Doron C. Greenbaum (University of Pennsylvania School of Medicine) for assistance in testing SID 26681509 against P. falciparum.

	Footnotes

 
Financial support for this work was provided by National Institutes of Health grants 5U54-HG003915-02 and 5U54-HG003915-03).

ABBREVIATIONS: LC-MS, liquid chromatography-mass spectrometry; SID, substance identifier; SID 861540, 2-[[5-[(1S)-1-amino-2-(1H-indol-3-yl)ethyl]-1,3,4-oxadiazol-2-yl]sulfanyl]-N-(2-ethylphenyl)acetamide hydrochloride; SID 26681509, S-[2-[(2-ethylphenyl)amino]-2-oxoethyl] [2-[(2S)-3-(1H-indol-3-yl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoyl]hydrazinyl]methanethioate; AMC, 7-amido-4-methylcoumarin; DMSO, dimethyl sulfoxide; Z-, N-benzyloxycarbonyl-; E-64, 1-[L-N-(trans-epoxysuccinyl)leucyl]amino-4-guanidinobutane.

Address correspondence to: Scott L. Diamond, Penn Center for Molecular Discovery, University of Pennsylvania, 1024 Vagelos Laboratories, Philadelphia, PA 19104-6383. E-mail: sld{at}seas.upenn.edu

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