Design, synthesis and application of peptide mimetics as photoactivatable inhibitors for proteases
In this work, a general strategy was explored for the construction of selective chemical probes targeting proteases. There was a need for this, because up until now, a whole range of chemical probes have been designed for all different kinds of proteases. However, the synthetic strategies to obtain them differ greatly. The design of the probes pursued here exploited the substrate specificity of proteases and included the preferred amino acid residues around the scissile peptide bond in the structure. The scissile peptide bond itself was replaced with an uncleavable mimic. This way, the selectivity of the protease for its substrate is retained within the probe while it cannot be processed. Two mimics were discussed in this work: (1) a reduced amide, which is similar in size but differs in the electronic properties and conformational flexibility. (2) A triazole retains the dipole moment of a peptide bond, but is larger in size and conformationally more restricted. Both of these mimics can be conveniently synthesized on solid support after preparation of certain amino acid derivatives as building blocks in solution. The synthetic procedures to obtain these building blocks have been optimized to be compatible with most amino acids. In addition to these specificity elements, crosslinkers were introduced for covalent linkage to the protease. Lastly, an N-terminal alkyne handle would allow detection of the generated complex, allowing their use as chemical probes. The unexpected finding of photoactivatable, reversible inhbibitors for the soluble cysteine protease caspase-3 is presented using the described approach. In contrast to the expectations, no covalent complex between the synthesized compounds and caspase-3 could originally be detected. However, they still served as inhibitors, but only after activation of their crosslinkers. It also turned out that the reduced amide inhibitors displayed a much higher potency compared to their triazole counterparts. Additionally, it appeared that in some cases a covalent complex was formed after all, but it could only be detected with SDS-PAGE and without denaturing the evaluated samples. The complex was detected only for the reduced amide inhibitors, and within this group only for inhibitors containing a benzophenone or furan crosslinker. This suggested a reversible mechanism of inhibition, in which the irreversible crosslinking plays no direct role. This was confirmed in experiments in which the inhibitor and crosslinker (benzophenone), were not part of the same molecule, but where inhibition was still observed upon crosslinker activation. The nature of the active species was still elusive at this point, as the original inhibitor structure was not expected to be strong enough to outcompete the covalent activity-based probe during the competitive ABPP experiments that were used to evaluate the potency. As such, a small structural modification of the inhibitors was proposed upon crosslinker activation, namely the oxidation of the reduced amide to the corresponding imine. After hydrolysis by solvent water molecules, this would generate a highly potent C-terminal peptide aldehyde. This hypothesis seemed plausible, as the highest potencies were observed for the furan inhibitors, where activation of the crosslinker relies on oxidation, and the benzophenone inhibitors, which have previously been reported as photosensitizers in the oxidation of amines. The formation of the peptide aldehyde was ultimately proven in experiments with sodium bisulfite, which can form adducts with aldehydes, rendering them unable to inhibit. After treating the activated inhibitor with sodium bisulfite, complete recovery of the activity of caspase-3 was observed, confirming the presence of an aldehyde as the active inhibitor species. Aside from caspase-3, inhibitors containing a benzophenone crosslinker were also synthesized for the intramembrane serine protease GlpG. Similar to the results for caspase-3, there was no detection of a covalent complex despite complete inhibition of GlpG. Although this suggests a similar mechanism of inhibition, this has not yet been confirmed, and further experiments, similar to the ones described for the caspase-3 inhibitors are required. The most important ones will be the sodium bisulfite experiment and the synthesis of GlpG inhibitors with different or without crosslinkers to compare their potency. The unexpected finding of photo-induced oxidation of caspase-3 targeting inhibitors that can lead to the formation of a potent peptide aldehyde is reported. In the future, this strategy could be expanded to other proteases, producing selective inhibitors for many proteases of different classes. Because of the inhibitor design, proteases could be selectively targeted in complex systems, where they could be inhibited at a specific time and location. As a result, this type of inhibitors may be used in the photopharmacological study of protease function.
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