Cathepsin B Inhibitors: Combining Dipeptide Nitriles with an Occluding Loop Recognition Element by Click Chemistry

Abstract

An active site mapping of human cathepsin B with dipeptide nitrile inhibitors was performed for a combinatorial approach by introducing several points of diversity and stepwise optimizing the inhibitor structure. To address the occluding loop of cathepsin B by a carboxylate moiety, click chemistry to generate linker-connected molecules was applied. Inhibitor 17 exhibited K i values of 41.3 nM, 27.3 nM, or 19.2 nM, depending on the substrate and pH of the assay. Kinetic data were discussed with respect to the conformational selection and induced fit models. KEYWORDS:Copper-catalyzed azide−alkyne cycloaddition, cysteine proteases, human cathepsin B, nitrile inhibitors C athepsin B is a papain-like cysteine protease that is ubiquitously expressed and involved in various physiological processes. 1 In nontumor cells, it is localized within endosomes/lysosomes, while in transformed cells it adopts a peripheral distribution in the cytoplasm and is, moreover, associated with the plasma membrane. 2,3 At the surface of invasive tumor cells, cathepsin B was found to bind to annexin II or caveolin-1, the structural protein of caveolae. Caveolae serve as a platform for a proteolytic cascade for the degradation of the extracellular matrix (ECM), executed by several proteases, including cathepsin B. 2−4 Cathepsin B affects the ECM either directly by extracellular proteolytic degradation of its components or indirectly via activation of other proteases. Partially degraded ECM components can be internalized to intracellular endosomal/lysosomal vesicles where the proteolysis is continued. This ECM breakdown remodels the tumor environment, promotes tumor invasion, and enables angiogenesis and metastasis. Cathepsin B also contributes to angiogenesis by degrading metalloprotease inhibitors and releasing growth factors that are bound to ECM components. 2,5,6 The crucial roles of cathepsin B at multiple points of the tumor development have been established in several in vitro and in vivo models and cathepsin B was proposed to be a prognostic marker in patients with various types of cancer. 1,2,7 Cathepsin B shows endopeptidase and exopeptidase activity, a unique feature for cysteine cathepsins, arousing from a flexible structural element, referred to as the occluding loop. The occluding loop is a 19−20 amino acid section that blocks the active site cleft at the end of the primed site. This event leaves only space for two amino acid residues of the substrate C-terminal of the scissile bond, that is, in the P1′ and P2′ positions. In this socalled closed conformation, two salt bridges, His110-Asp22 and Arg116-Asp224, hold the occluding loop over the primed subsites of the substrate binding cleft, preventing extended binding of large endopeptidase substrates and conferring an exopeptidase activity to cathepsin B. Mutations of His110 and Asp22 or the deletion of 12 amino acids of the occluding loop to enforce an open conformation reduced the exopeptidase activity of cathepsin B in favor of its endopeptidase activity. 8,9 His110...

Mapping the S1 and S1’ subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents

“…In P2 position, the transformation of the leucine moiety to phenylalanine or its derivatives resulted in a loss in affinity up to one log unit for Cz. A similar replacement led to a gain in affinity towards CatL and CatB, and it is consistent with previously reported data [31]. For compound 11 , as for compound 9 , the stereochemistry of the substituent in P1 was vitally for the bimolecular recognition process.…”

“…It is not trivial to achieve a significant selectivity for Cz inhibitors (Δp K i > 1.0) over mammalian CPs due to their high structural similarity of the active site. Undeniably, CatB has a different mode of binding due to the larger S2 and S3 pockets [31]. Compounds 14 and 67 displayed a significant selectivity toward CatL and CatS, respectively.…”

Mapping the S1 and S1’ subsites of cysteine proteases with new dipeptidyl nitrile inhibitors as trypanocidal agents

“…At position P2, the transformation of the leucine moiety into tryptophan, phenylalanine, or derivatives thereof resulted in an affinity loss of up to one log unit for Cz. A similar replacement led to a gain in affinity towards CatL and CatB, and it is consistent with previously reported data [33]. For compound 11, as for compound 9, the stereochemistry of the substituent in P1 was vitally for the bimolecular recognition process.…”

“…Pairwise plots for the selectivity towards Cz in relation to other human cathepsins are provided in Fig 11. It is not trivial to achieve a significant selectivity for Cz inhibitors (ΔpK i > 1.0) over mammalian CPs due to their high structural similarity of the active site. Undeniably, CatB has a different mode of binding due to the larger S2 and S3 pockets [33]. Compounds 15 (pK i of 7.8 for Cz) and 67 (pK i of 7.1) displayed a significant selectivity toward CatL (pK i of https://doi.org/10.1371/journal.pntd.0007755.g010 6.6) and CatS (pK i of 5.0), respectively.…”

Addition of 2-(ethylamino)acetonitrile group to nitroxoline results in significantly improved anti-tumor activity in vitro and in vivo

“…Several groups of cathepsin B inhibitors have been identified. The majority are covalent inhibitors that contain a peptidyl backbone with an electrophilic warhead that reacts with the active site cysteine residue, either reversibly or irreversibly [ 2 , 21 , 22 ]. However, due to their low bioavailability and off-target side effects, none of these compounds can be translated into clinical practice [ 2 ].…”