Publications Repository - Helmholtz-Zentrum Dresden-Rossendorf

Even though the C-C triple bond is largely considered as a bioinert functional group, 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 ubiquitin or ubiquitin-like modifiers bearing propargylamine in place of C-terminal glycine by covalent targeting of the active-site cysteine residue [1, 2]. We intended to harness that finding for the design of inhibitor-based probes for the imaging of tumour-associated cysteine proteases.
All 11 human cysteine cathepsins have been linked to tumour progression. Especially high expression levels of the cathepsins B, K, L, S and X are correlated with an increased metastatic potential and poor prognosis. [3] Therefore, those enzymes represent promising targets for the therapy and imaging of tumours.
GREENSPAN et al. reported a potent, highly selective dipeptidyl nitrile-based cathepsin B inhibitor (1, structure shown above) [4]. Based on that lead compound, dipeptide alkynes were designed by isoelectronic replacement of the nitrile nitrogen atom by a methine group (2) and consecutive variation of the 2,4-difluorobenzoyl group and the amino acid-derived side chains. 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 to ensure high stereochemical purity of the final compounds.
By investigating the inhibitory potential against cathepsin B, S, L and K potent alkyne-based inhibitors were identified for all tested cathepsins, with second-order inactivation constants (k_inact/K_I) up to 10133 M^-1s^-1 and interesting selectivity profiles. Based on these promising results and considering their absent indiscriminate thiol reactivity, dipeptidyl alkynes have the potential to be translated into activity-based probes for molecular imaging in vivo. In further studies, selected inhibitors will be labelled with suitable radionuclides such as fluorine-18, which will in turn enable further pharmacological evaluations.
[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.