Abstract
Granzymes are a family of 5 human serine proteases that are secreted from cytotoxic granules by T cells and Natural Killer cells. Granzyme B (Grnz B) induces apoptosis (programmed cell death) in pathogen-infected cells, such as virus-infected cells and cancer cells. Therefore it is a main effector of cancer cell killing by T cells and it can be a useful indicator to CAR T cell treatment response. These are cysteine proteases that are further down Grnz's B signalling cascade and are activated by it to induce apoptosis. Within this project, we design and synthesize novel Granzyme B inhibitors to obtain Structure-Activity-Relationship (SAR) data and with the aim of reducing the inhibitor's peptide character to overcome poor pharmacokinetics. In terms of selectivity, caspases and caspase-3 in particular are potential off-targets. The most potent and selective inhibitors are converted into a probe by introducing 18F or 68Ga to the N-terminal and assessed in vivo in tumor-bearing mice.
To synthesize novel Grnz B inhibitors, a previsously described potent tetrapeptide sequence was used as a reference, namely Ac-IE-indoline-D-CHO (ki = 13 nM, Fig. 1), and structural modifications were performed to obtain SAR data. By keeping the the main peptide sequence unchanged, the metabolically unstable aldehyde warhead was completely removed and the aspartic acid was changed to a tetrazole bioisostere (family 1). First, the optimum tetrazole position was explored, by adding an extra carbon in the P1 tetrazole chain. Next, smaller and larger analogues were explored (P1-P2, P1-P2-P3, P1-P4 and P1-P5) revealing that the former analogues showed no inhibition against Grnz B, whereas the latter were inhibiting it. Therefore, the P1 tetrazole was left unchanged and more full-length analogues were synthesized (P1-P4 and P1-P5. The SAR study showed that deviation from the initial P2-P4 core of the reference compound leads to complete loss of Grnz B inhibition, but changes in the P5 pocket are well tolerated. Biological evaluation of these compounds showed a μΜ range activity against Grnz B and no affinity towards caspase-3. To increase potency, we are now incorporating a warhead in the P1 position.
In the following year, compounds from family 3 will be synthesized and screened against Grnz B. If the inhibition is better than the previously synthesized compounds, a larger compound library will be synthesized to obtain SAR data and the most potent compounds will be screened against caspase-3 for selectivity. Simultaneously, our collaborators in LMB are working on crystalizing Grnz B with the most potent inhibitors synthesized so far, so that substantial SAR data will be obtained to synthesize more potent inhibitors (nM scale). Finally, the most potent inhibitors will be converted into a PET probe to move on to in vivo experiments.
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