Alkyne-based cysteine cathepsin inhibitors as basis for PET tracer development

Among the intertwined processes leading to cancer progression, protease activity plays an important role. Various attempts to develop molecular imaging probes have been made, as such probes can allow functional imaging and thus improve the understanding of tumour progression mechanisms and enable personalised cancer treatment. PET and SPECT tracers are particularly suitable for such applications. However, novel tracers have to overcome challenges such as stability, target efficiency and off-target effects.
Multiple members of the cathepsin family have been demonstrated to be involved in tumour invasion, metastasis, and angiogenesis. Especially high expression levels of the cysteine cathepsins B, K, L, S, and X are correlated with an increased metastatic potential and poor prognosis [1]. Due to their high expression in a multitude of tumours, those enzymes represent promising targets for the therapy and imaging of tumours.
Despite being virtually chemically inert, alkynes were shown to be able to irreversibly inhibit cysteine proteases: 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 [2, 3]. We aimed to take advantage of those findings for designing alkyne-based cysteine cathepsin inhibitors suitable for radiolabelling with PET nuclides. The probes thus obtained would irreversibly bind to the target molecule without showing indiscriminate thiol reactivity.
Based on a potent, highly selective dipeptidyl nitrile-based cathepsin B inhibitor reported by GREENSPAN et al. (left structure) [4], we designed dipeptide alkynes by isoelectronic replacement of the nitrile nitrogen atom by a methine group. To avoid partial enantiomerisation during the formation of the C-C triple bond as observed for the open-chain serine-derived alkyne, the synthesis was performed via Garner’s aldehyde. This ensured high stereochemical purity of the final compounds. The inhibitory potential was investigated against cathepsin B, S, L and K. To optimise the inhibitory potential and selectivity, we consecutively varied all moieties attached to the dipeptidic scaffold.
We identified potent alkyne-based inhibitors for all tested cathepsins, with inactivation constants (k_inact/K_I) up to 10133 M^-1s^-1 and distinct selectivity profiles. We demonstrated irreversibility in a “jump-dilution” experiment and inhibitor reactivity in cell lysates and on living cells was exemplarily verified for cathepsin B. During our research, MONS et al. successfully demonstrated irreversible cathepsin K inhibition by alkyne-based small molecule inhibitors with no indiscriminate thiol reactivity [5], which indicates the viability of our concept.
Among the tested inhibitors we identified two promising radiotracer candidates which are selective for cathepsin S and L. We successfully radiolabelled the cathepsin S-selective inhibitor with N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). Radiopharmacological characterisation of the activity-based probe obtained by that approach is in progress.

[1] Löser, R; Pietzsch, J.: Front. Chem., 2015, 3: 37.
[2] Ekkebus et al.: J. Am. Chem. Soc., 2013, 135(8): 2867-2870.
[3] Sommer et al.: Bioorg. Med. Chem., 2013, 21(9): 2511-2517.
[4] Greenspan et al.: J. Med. Chem., 2001, 44(26): 4524-4534.
[5] Mons et al.: J. Am. Soc. 2019, 141(8): 3507-3514.