Cathepsins are cysteine proteases primarily found in lysosomes. Some cathepsins are specifically expressed in certain tissues, while others are present in almost all tissues. Cathepsin V is found mainly in the thymus, testicles, and cornea, but is also found in the brain, skin, lungs, kidneys, esophagus, and gastrointestinal tissues. Increased expression and excessive activity of cathepsin V are associated with inflammatory diseases, autoimmune disorders, various types of cancer, and lung diseases. The enzyme participates in the formation of neuropeptide Y, which is involved in the proliferation of neural stem cells, the release of neurotransmitters, and neuroprotection. The most studied inhibitor of cathepsin V is a covalent inhibitor E-64. Research is focused on the discovery of new, noncovalent and nonpeptidic cathepsin V inhibitors that would regulate the immune response with higher selectivity and, consequently, fewer adverse effects.
As part of our master's thesis, we designed and synthesized inhibitors with a 1-(naphthalen-1-ylmethyl)urea scaffold based on a known, noncovalent cathepsin V inhibitor, compound A,. The inhibition of cathepsin V and the structurally related cathepsin L was determined in an in vitro biochemical assay. We found that N-(naphthalen-2-yl)urea 1 was the most potent inhibitor of cathepsin V; however, it also inhibited cathepsin L and is therefore a nonselective inhibitor. By changing the position of the substituent on the naphthalene ring, we obtained a weaker cathepsin V inhibitor, N-(naphthalen-1-yl)urea 2, which nevertheless still inhibited cathepsin L. Compound 7 differs from compound A in the substitution pattern on the central piperidine: compound 7 is 1,3-disubstituted piperidine, whereas compound A is 1,4-disubstituted piperidine; however, compound 7 did not inhibit neither cathepsin V nor cathepsin L. Derivatives 13–15 inhibited only cathepsin L. Although molecular docking did not provide results sufficient to predict structure–activity relationships for cathepsin V inhibitors, it indicated that binding of compound A or its analogues to the enzyme’s active site requires the formation of two hydrogen bonds with Gly68 and Asp162. These interactions are formed by the central urea or carbamate moiety. The naphthalene moiety engages in π-π interactions with Phe69, while the carbamate or amide group at the opposite end of the molecule forms water-mediated hydrogen bonds with His163 or Gln19, respectively. Notably, even minor structural modifications that do not prevent active-site binding lead to a loss of activity, suggesting that the actual binding conformation may differ from the predicted one or that an alternative binding site is involved.
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