Novel, Nonpeptidic Cyanamides as Potent and Reversible Inhibitors of Human Cathepsins K and L

Abstract

Compounds containing a 1-cyanopyrrolidinyl ring were identified as potent and reversible inhibitors of cathepsins K and L. The original lead compound 1 inhibits cathepsins K and L with IC(50) values of 0. 37 and 0.45 M, respectively. Modification of compound 1 by replacement of the quinoline moiety led to the synthesis of N-(1-cyano-3-pyrrolidinyl)benzenesulfonamide (2). Compound 2 was found to be a potent inhibitor of cathepsins K and L with a K(i) value of 50 nM for cathepsin K. Replacement of the 1-cyanopyrrolidine of compound 2 by a 1-cyanoazetidine increased the potency of the inhibitor by 10-fold. This increase in potency is probably due to an enhanced chemical reactivity of the compound toward the thiolate of the active site of the enzyme. This is demonstrated when the assay is performed in the presence of glutathione at pH 7.0 which favors the formation of a GSH thiolate anion. Under these assay conditions, there is a loss of potency in the 1-cyanoazetidine series due to the formation of an inactive complex between the GSH thiolate and the 1-cyanoazetidine inhibitors. 1-Cyanopyrrolidinyl inhibitors exhibited time-dependent inhibition which allowed us to determine the association and dissociation rate constants with human cathepsin K. The kinetic data obtained showed that the increase of potency observed between different 1-cyanopyrrolidinyl inhibitors is due to an increase of k(on) values and that the association of the compound with the enzyme fits an apparent one-step mechanism. (13)C NMR experiments performed with the enzyme papain showed that compound 2 forms a covalent isothiourea ester adduct with the enzyme. As predicted by the kinetic analysis, the addition of the irreversible inhibitor E64 to the enzyme-cyanopyrrolidinyl complex totally abolished the signal of the isothiourea bond as observed by (13)C NMR, thereby demonstrating that the formation of the covalent bond with the active site cysteine residue is reversible. Finally, compound 2 inhibits bone resorption in an in vitro assay involving rabbit osteoclasts and bovine bone with an IC(50) value of 0.7 M. 1-Cyanopyrrolidine represents a new class of nonpeptidic compounds that inhibit cathepsin K and L activity and proteolysis of bone collagen.

Peptidomimetic 2-cyanopyrrolidines as potent selective cathepsin L inhibitors

“…While the electrophilic warhead binds reversibly or irreversibly with the enzyme to cause its inhibition, hydrogen bonding and hydrophobic interactions of the peptide backbone and attached side chains to the enzyme determine its selectivity towards a specific enzyme. Nonpeptidic N-cyanopyrrolidines [37] have been reported earlier as potent inhibitors of various cathepsins. Introduction of peptidic chains in such scaffolds has led to the development of compounds with major differences in inhibitory potencies and selectivities towards four different cathepsins (B, K, L and S) [38].…”

Novel Peptidyl Aryl Vinyl Sulfones as Highly Potent and Selective Inhibitors of Cathepsins L and B

“…hyde, [13] an aldehyde-hydroxamic acid combination, [14] nonpeptidic cyanamide, [15] dipeptidyl nitrile [16] or b-lactam penam or oxapenam compounds. [2] All of these are selective cathepsin L inhibitors, except the dipeptidyl nitriles, which show higher affinity for cathepsin B. Irreversible inhibitors include E-64 analogues [2] (mainly cathepsin B and L inhibitors) and alkyl vinyl sulfones (cathepsin B, L, O2, and S inhibitors).…”

Inhibition of Ulmus davidiana Planch (Ulmaceae) on bone resorption mediated by processing of Cathepsin K in cultured mouse osteoclasts

“…Cathepsin (Cat) L, B and K are involved in this process of bone resorption (Falgueyret et al, 2001). Of them, Cat K is the key enzyme involved in the resorptive process (Votta et al, 1997) and Cat K belongs to the papain superfamily of lysosomal cysteine proteases.…”