Dipeptide-derived Alkynes as Novel Irreversible Inhibitors of Cathepsin B

Until recently, alkynes were considered bioinert. Thus, they are popular reaction partners in bioorthogonal click reactions in vitro and in vivo. Despite the virtual chemical inertness of the alkyne moiety, two research groups observed the irreversible inhibition of a cysteine protease by an alkyne functionalised substrate derivative: both EKKEBUS et al. and SOMMER et al. independently described the unexpected inactivation of de-ubiquitinating enzymes by propargylated ubiquitin or ubiquitin-like modifiers bearing propargylamine in place of C-terminal glycine [1, 2]. We intended to harness that finding for the design of inhibitor-based probes for the imaging of tumour-associated cysteine proteases.
Cysteine cathepsins play an important role in tumour progression. In particular, cathepsin B is involved in a variety of tumour progression-related processes and an elevated extracellular levels are linked to increased malignancy and poor prognosis [3]. Therefore, this enzyme represents a promising target for the therapy and imaging of tumours.
GREENSPAN et al. reported a potent and highly selective, dipeptidyl nitrile-based cathepsin B inhibitor (N-[2-[(3-Carboxyphenyl)methoxy]-1(S)-cyanoethyl]-3-methyl-N²-(2,4-difluorobenzoyl)-L-phenylalaninamide) [4]. Based on that lead compound, cathepsin B-targeting dipeptide alkynes were designed by isoelectronic replacement of the nitrile nitrogen atom by by a methine group and consecutive variation of the 2,4-difluorobenzoyl and (3-carboxybenzyl)oxymethyl residue. Formation of the C-C triple bond by reaction of the corresponding open-chain serine-derived aldehyde with the Bestmann-Ohira reagent was accompanied by partial enantiomerisation. Therefore, the synthesis was performed via Garner’s aldehyde, which accounted for high stereochemical purity of the final compounds. The inhibitory potential was investigated against cathepsins B, S, L and K. The most potent compound exhibited irreversible inhibition of cathepsin B with an inactivation constant (k_inact/K_I=771 M^-1s^-1). Values for cathepsins L, S and K were significantly lower; no irreverisible ihibition was observed for cathepsin K. In addition, inhibition of cathepsin B activity in human glioblastoma cell lysates and living cells has been demonstrated. Based on these promising results, dipeptidyl alkynes have the potential to become a valuable tool for imaging due to the expected low activity towards other cysteine proteases. In further studies, selected inhibitors for cathepsin B will be labelled with suitable radionuclides to obtain an inhibitor-based probe directed towards cathepsin B.

[1] Ekkebus et al., J. Am. Chem. Soc., 2013, 135, 2867-2870
[2] Sommer et al., Bioorg. Med. Chem., 2013, 21, 2511-2517
[3] Löser and Pietzsch, Front. Chem., 2015, 3:37
[4] Greenspan et al., J. Med. Chem., 2001, 44, 4524-4534.