We recently described the identification of a potent, non-peptide inhibitor of cathepsin L (1
, ), with an IC50
of 56 nM under defined conditions.11
This thiocarbazate contains a diacyl hydrazine functionality and a single stereogenic center. The most active congener proved to be the S
-enantiomer, with an IC50
of 56 nM. The R
, ), described here, was only modestly active against cathepsin L (IC50
= 33 μM). Molecular docking studies were initiated on both enantiomers, in order to probe the importance of the elements that contribute to binding. As a validation study, we first examined the non-covalent docking of the ring-opened epoxide form of CLIK-148 (3b
, ) in papain. Initially, the orientation of the water molecule near Asp 148 was not suitable for hydrogen bonding with the ligand or with Asp 148. Using the routines available in XP Glide, this water molecule was re-orientated so that adequate hydrogen bonding could be established (). Then, after breaking the covalent bond to Cys25 and re-docking CLIK-148 into the binding site, the experimentally derived binding mode was reproduced (). In this docking pose (orientation plus conformation), with a score of -9.27 kcal/mol, CLIK-148 hydrogen bonds to the protein through side chain and backbone atoms of Gly66, Cys25, Gln19, and Asp158. The pyridine group occupies a hydrophobic aromatic pocket in papain in the S1′ subsite near Trp177 and the phenylalanine of the ligand occupies the S2 subsite. Taken together, these features play a major role in positioning the ligand appropriately for covalent attachment to the protein. In the highest scoring pose, the distance between the Cys25 sulfur and the electrophilic carbon of the ligand (epoxide ring carbon) is about 3 Å.
Cathepsin L inhibitors 1 (S-enantiomer) and 2 (R-enantiomer).
CLIK-148 in epoxide form (3a) and epoxide ring-opened form (3b), and E-64 (4).
Figure 5 CLIK-148 docked to papain after breaking the covalent bond to Cys25 sulfur. The hydrogen bonding interactions (yellow dashed lines) between ligand and protein involve Gly66, Cys25, Gln19 and the backbone CO of Asp158; the aromatic/hydrophobic interactions (more ...)
Since the validation study revealed that XP Glide could accurately reproduce the experimentally derived binding mode of CLIK-148, thiocarbazate 1
was docked into the binding site of papain ( and ); the di-imide functionality in 1
was maintained in the anti orientation with respect to the geometry of the NH bonds (). The highest scoring pose obtained had a binding energy of -9.03 kcal/mol, very close to the energy value observed in our validation study with CLIK-148/papain (-9.27 kcal/mol). Furthermore, the orientations of the ligands (CLIK-148 and 1
) bound to papain were strikingly similar (). For the 1
/papain complex, Gln19, Cys25, Gly66, Asp158, and Trp177 all participated in hydrogen bonding interactions with the ligand (), as observed for CLIK-148/papain. In addition, both the pyridine group of CLIK-148 and the 2-ethylphenyl anilide group of 1
occupied the S1′ subsite containing Trp177. The indole group of 1
occupied the S2 subsite, near Ser 205 (papain residue), in a fashion similar to that of the phenylalanine group of CLIK-148. The Ser 205 residue in papain corresponds to the Ala 214 residue in cathepsin L; this is the only residue in the binding site of cathepsin L that is not identical to the aligned residue in papain. However, both Ala and Ser side chains are small enough to accommodate the bulky hydrophobic groups present in both inhibitors (phenylalanine in CLIK-148 and 2-ethylphenyl anilide in 1
so this difference would appear to be negligible. Both CLIK-148 and 1
fully occupy the S1′ and S2 subsites. The tert
-butoxy group of the NHBoc in compound 1
sits in the S3 subsite, occupying a large hydrophobic cleft. In addition, the amino acid-derived NH of inhibitor 1
hydrogen bonds to the backbone carbonyl of Asp 148, a conserved binding site residue. Inhibitors that span from the S to the S1′ subsites have high potency and selectivity toward cysteine proteases.15, 24
demonstrated selectivity towards cathepsin L versus other cysteine proteases, including cathepsins V, S, B, and K, with the greatest selectivity index observed for cathepsin K (150).23
The details of a structure activity relationship study for cathepsin L inhibition in the carbazate series are being published separately25
, but reveal that removal of the NHBoc group causes a dramatic loss of cathepsin L inhibitory activity. This single synthetic change to the carbazate scaffold of 1
removes both the potential for hydrogen bonding with Asp 148 and the hydrophobic contact of the tert
-butoxy group in the S3 subsite, resulting in a 400-fold reduction in cathepsin L inhibition.
Figure 6 a. Overlay of CLIK-148(light green carbons with heteroatom coloring) with compound 1 (tan carbons with heteroatom coloring) in the binding site of papain. b. Binding of 1 (S-enantiomer) to papain. Six critical hydrogen bonding interactions between the (more ...)
Figure 9 Compounds 1 (9a) and 2 (9b) bound to papain. The protein surface was calculated in Maestro (Schrodinger, Inc.) with molecular properties color-coded as follows: green: strongly hydrophobic; red: negatively charged (Asp158 residue); dark blue: positively (more ...)
As presented above, the R-enantiomer 2 had a cathepsin L inhibitory activity of only 33 μM. In an attempt to understand why this enantiomer was virtually inactive, we docked 2 into the binding site of papain. The highest scoring pose for 2 obtained from this XP Glide docking study (depicted in ) had a docking score of -7.0, two kcal/mol lower than the score for the S-enantioner 1. In addition, key hydrogen bonding and hydrophobic contacts that are established in the complexes of the active S-enantiomer (1) with papain, and in CLIK-148 with papain are completely disrupted for the R-enantiomer (2) in this binding site (, ). While the S-enantiomer 1 makes at least six hydrogen bonding contacts to the active site residues and large hydrophobic contacts within the S1′, S2 and S3 subsites (), the R-enantiomer forms only two hydrogen bonds to papain and does not occupy the S1′ subsite at all ( and ). This change in stereogenicity from S (1) to R (2) reveals a dramatic shifting of the 2-ethylphenyl anilide group out of this critical subsite in these docking studies (cf. ). The key hydrogen bond made between the NH of the Trp residue in the S-enantiomer 1 with Asp 148 is also absent in the binding of the R-enantiomer 2 (). In addition, the indole of 2 makes fewer significant hydrophobic contacts in the S2 subsite (near Ser 205) than the indole of 1. These observed differences in binding interactions between 1 and 2 and the corresponding difference in docking scores provide a cogent rationale for the observed decrease in the cathepsin L inhibitory activity of 2 vs. 1 of almost three orders of magnitude (). Also noteworthy is the increase in the distance of the electrophilic carbonyl carbon in 2 to the Cys25 to 4.4 Å, suggesting that covalent bond formation might be less favorable.
Compound 2 (R-enantiomer) in the binding site of papain. Only two hydrogen bonds are made between 2 and the binding site residues.
Figure 8 Overlay of 1 (S-enantiomer, green carbons with heteroatom coloring) with the significantly less active R-enantiomer (2, cyan carbons with heteroatom coloring) in the binding site of papain. The hydrogen bonds made between 1 and papain (yellow dashed lines) (more ...)
Cathepsin L inhibitory activities and Extra Precision (XP) Glide docking scores for compounds 1, 2, 5, and CLIK-148.
These mechanistic insights into the binding site interactions of 1 suggested additional room within the S1′ subsite, which led to the design of compound 5. This oxocarbazate analog of 1 was designed to contain the requisite S stereogenicity, an oxygen in place of the thiol ester sulfur, and a large hydrophobic/aromatic group (quinoline) to occupy further the S1′ subsite. When this virtual compound (5, ) was docked into the binding site of papain, the best pose obtained had a score of -10.00, a score improved by 1 kcal/mol compared to that of our initial lead 1 (-9.03 kcal/mol). This compound was synthesized and found to be more potent than 1, with an enzyme inhibitory activity of 7 nM against cathepsin L. In the highest scoring docking pose for this compound, three hydrogen bonds are formed between 5 and the protein; moreover, the tetrahydroquinoline group on the ligand occupies the large hydrophobic pocket with Trp177 in the S1′ subsite (). Changing the sulfur in 1 to an oxygen in 5 leads to a change in orientation of the ester bond, making a new interaction with His159 possible. This hydrogen bond is also observed in the binding of CLIK-148 to papain (). In both inhibitors (1 and 5), the carbazate carbonyl carbons are oriented for nucleophilic attack by Cys25, with the distances from the Cys sulfur to the carbonyl carbon in both ligands in the three angstrom range.
Compound 5 bound to papain with the tetrahydro-isoquinoline group fully occupying the S1′ subsite. The IC50 for cathepsin L inhibition is 7 nM.
The structure of pro-cathepsin L (1mhw.pdb) was also explored in molecular docking studies with ligand 1. However, only very poor XP Glide scores could be obtained from these studies. The two highest scoring docking poses for 1 in the binding site of the pro-cathepsin L structure had scores of 1.15 and 6.74 kcal/mol. When the interactions between 1 and the pro-cathepsin L structure were examined, severe steric clashes between the indole of the ligand and the Leu 69 side chain were observed (a distance of 0.61 angstroms between the ligand and the Leu side chain). This residue corresponds to Tyr 67 in papain. However, in 1mhw.pdb, the Leu 69 side chain is pointing into the binding site cavity, whereas in papain, the Tyr 67 side chain hydroxyl is 6.11 angstroms removed from any atom in 1, and no unfavorable contacts are observed. Further unfavorable contacts were also observed between ligand 1 and the backbone atoms surrounding the Cys 25 residue in the pro-cathepsin L structure, and only one hydrogen bond was observed between the ligand and the conserved binding site residues. A homology model of cathepsin L based on the coordinates of CLIK-148 bound to papain was also generated (MOE software, CCG, Inc.). Docking scores for 1 in the binding site of the resulting theoretical model were somewhat better than those obtained for the pro-cathepsin L structure (-3.82 and -2.40 kcal/mol for the two highest scoring poses of 1 bound to the model structure), but these scores were still unfavorable. Since significantly better scores were realized for ligand dockings of 1 with the papain structure than with either the pro-cathepsin L structure or the theoretical model, this experimentally-derived system (1cvz.pdb) was used directly for all docking studies of the carbazate ligands.
To compare our docking analysis with the kinetic behavior of compound 1
, we constructed a 5-parameter ODE model of reversible inhibitor binding and fit the model to reaction progress curves measured at various inhibitor concentrations (see Materials and Methods section). The best-fitting parameters were k1
= 2.3 μM-1
= 0.30 s-1
= 4.0 s-1
= 0.024 μM-1
, and koff
= 2.2 × 10-5
. Most notably, the rate of inhibitor dissociation (koff
) from cathepsin L was extremely slow, leading to a Ki
= 0.890 nM.23
Alternative reaction schemes for steady-state and irreversible inhibitor binding were also tested, but did not fit the data as well as the 5-parameter model. Taken together, the results from the docking and kinetic analyses suggest a covalent but slowly reversible mechanism of inhibition that is aided by strong non-covalent interactions.