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 hrs prior to substrate addition at t = 0, SID 26681509 displayed increasing potency with IC50 values falling to 7.5 ± 1.0 nM, 4.2 ± 0.6 nM, and 1.0 ± 0.5 nM, respectively, demonstrating a slow onset of inhibition against the target enzyme ().
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 hr at 10-fold its IC50
after 1 hr preincubation (75 nM), a condition is created whereby >90% of the enzyme should be in an enzyme-inhibitor complex (). Upon 100-fold dilution of the 1 hr 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 (). For the 4 hr preincubated enzyme-inhibitor reaction condition (), 99.8% of the reaction was inhibited immediately after addition of substrate due to almost all the enzyme being bound to small molecule inhibitor SID 26681509. After 8820 s, the rate of product formation for the 4 hr 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, Km and kcat were determined through initial rate analysis to be 0.77 μM and 1.5 s-1, respectively (). A nonlinear regression for transient dynamics was conducted based on the reaction scheme shown in . Here, the values of k1, k-1, kon, and koff are explicitly estimated rather than combined into the equilibrium parameters, Km and Ki, estimated by traditional kinetic analyses. The best fit parameters were k1 = 2.3 × 106 M-1s-1, k-1 = 0.30 s-1, kcat = 4.0 s-1, kon = 24,000 M-1s-1, and koff = 2.2 × 10-5 s-1 (). The regressed Ki = 0.89 nM was quite consistent with the measured IC50 = 1.0 ± 0.5 nM obtained after 4 hr 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 prior to 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.
Figure 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 (more ...)
Mechanism of reversibility
The return of activity shown in demonstrated that the thiocarbazate was a reversible inhibitor. Transient kinetic analyses () quantified the rate of reversibility. To investigate the mechanism of reversibility and the generation of a putative leaving group, a stoichiometric reaction between 4.5 μM cathepsin L and 4.5 μM SID 26681509 was analyzed by liquid chromatography-mass spectrometry [Shimadzu LC-MS/4.6 mm × 50 mm Premier C18 column, 1 mL/min and a step from 90:10 to 60:40 water:acetonitrile with 10 min hold time, mobile phase contained 0.05% formic acid]. A potential thiol leaving group formed by reaction of cathepsin L with the thiocarbazate carbonyl of SID 26681509 was synthesized (MW = 195) and was detectable on the LC-MS at a concentration of 100 nM with a retention time of 12.1 min (no suppression detected due to presence of human cathepsin L). However, this thiol leaving group was not detected by LC-MS after 6, 12, and 24 hrs incubation of human cathepsin L with SID 26681509. While this result argues against acylation of cathepsin L by the inhibitor, formation of a tetrahedral intermediate by attack of the active site Cys residue on the thiocarbazate carbonyl of SID 26681509 is not excluded. In fact, when the thiocarbazate sulfur in SID 26681509 is replaced by carbon, the resulting molecule is a much weaker inhibitor (IC50 > 50 μM, data not shown).
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 () with no preincubation of enzyme and inhibitor. IC50 values were calculated at time points of 10 min, 30 min, 60 min, 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 (). SID 26681509 inhibited papain and cathepsins B, K, S, and V with IC50 values determined after one hour 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 likely reflect the relative rates of slowly reversible inhibition of the enzyme. Thus, it would appear that the slowly reversible reaction proceeds faster for cathepsins V and S than for cathepsin L; whereas, it proceeds more slowly for papain.
IC50 values of SID 26681509 against papain and human cathepsins B, G, K, L, S, and V.
Selectivity indexes of SID 26681509 against papain and human cathepsins B, G, K, L, S, and V.
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 Plasmodium falciparum with an IC50 of 15.4 ± 0.6 μM (). Additionally, the thiocarbazate inhibitor was toxic toward Leishmania major promastigotes with an IC50 of 12.5 ± 0.6 μM ().
A. IC50 against Plasmodium falciparum was determined to be 15.4 ± 0.6 μM. B. IC50 against Leishmania major was determined to be 12.5 ± 0.6 μM.
Molecular docking of SID 26681509 in papain
The co-crystal 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 . Other researchers have used papain to design highly specific cathepsin inhibitors and CLIK-148 directly inhibits cathepsin L (Katunuma et al., 1999
; LaLonde et al., 1998
; 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.4
The co-crystal 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. Since 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.
Molecular docking studies of CLIK-148 and SID 26681509 in the binding site of papain were carried out using XP (extra precision) Glide software. Predictions of accurate binding modes have been accomplished with reasonable accuracy using XP Glide docking, resulting in computationally-derived protein/ligand complexes with adequate root mean square deviations from the known experimentally-derived co-crystal structure (Perola et al., 2004
). Our initial docking studies were conducted on the papain/CLIK-148 system in order to verify that XP Glide could reproduce the binding mode of CLIK-148.
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 (). 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. 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 (). 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 (), 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.
Figure 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 (more ...)
Finally, the active site Cys25 sulfur is in close proximity (3.289 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 (). As indicated above, however, we lack compelling evidence for covalent binding between the enzyme and SID 26681509.