Synthesis and application of probes for serine and cysteine hydrolases and the polyamine transport system

Activity-based probes (ABPs) are small molecular tools that allow the detection and monitoring of enzyme activity by covalently binding onto the active site of the enzyme. They consist of a warhead that binds the enzyme, a recognition element that alters the selectivity of the probe and a detection tag that allows the visualization, quantification or purification of probe-labeled enzymes. ABPs are applied to elucidate enzyme function, enzyme localization and the involvement of enzymes in health and disease. They can furthermore be used as screening tools for drug candidates.

To monitor the activity a single enzyme, the selectivity of an ABP has to be steered towards this target. This often requires a difficult and time-consuming synthetic process, and as a result, selective ABPs have only been developed for relatively few enzymes expressed in human cells. One enzyme family of which only a few members have selective ABPs are the serine hydrolases. To speed up the process of ABP development for serine hydrolases, solid-phase synthesis procedures that allow a fast synthesis of six classes of serine-reactive probes were developed in Chapter III. The application of these procedures is shown by the combinatorial on-resin synthesis of a library of ABPs. These probes bear a recognition element made up of an aromatic moiety and a piperidine/piperazine ring, and a serine reactive warhead. Two probes with a triazole urea warhead were found to be reactive and selective for acyl-protein thioesterases 1 and 2 (APT-1/2) in a screening on serine hydrolases present in mouse brain.

Chapter IV describes the use of the solid-phase synthesis procedures established in Chapter III to synthesize a library of triazole urea ABPs, based on the APT-1/2 reactive ABPs found in the previous chapter. In this library, changes were made in the recognition elements of the probes to drive their reactivity towards one of the APT-isoforms and creating isoform-selective ABPs. Although this yielded no selective probes, several probes with a higher reactivity for both APT-isoforms than the original probes were discovered.

In Chapter V, the development of ABPs for the SARS-CoV-2 main protease (Mpro) is described. Mpro is a cysteine protease that is the key player in the proteolytic maturation of the non-structural proteins of the SARS-CoV-2 virus by cleavage of its polyproteins. It has gained a lot of attention as a potential target for antiviral drugs to combat the Covid-19 pandemic. In this chapter, azapeptidic ABPs for Mpro are synthesized that can be fully made on resin. The recognition element of these probes consists of the preferred P1, P2 and P3 substrates of Mpro, of which the P1 residue is an aza-amino acid. The ABPs are capped with a cysteine-reactive warhead on the P1 aza-residue. A probe with a chloroacetamide warhead was found to be very potent in labelling Mpro and to have a very low detection limit. This probe could furthermore inhibit cell death in SARS-CoV-2 infected cells and can potentially be used for labeling Mpro in live cells.

The mammalian polyamine transport system was a mystery at the start of this research project, as no proteins had been identified as polyamine transporters. Chapter VI describes the synthesis of azide-functionalized polyamines, which were converted to fluorescent probes through “click” chemistry with an alkyne-BODIPY fluorophore. Through a SAR study with polyamine probes with different lengths, different positionings of the fluorophore and a different amount of positive charges, it was found that probes in which the fluorophore is attached to an outer amine of the polyamine get taken best into cells. These polyamine probes have since been used by other research groups in the identification of polyamine transporters.

In summary, this thesis describes the synthesis and validation of probes that target APT-1/2, SARS-CoV-2 Mpro and the polyamine transport system. These probes can be synthesized efficiently via solid-phase synthesis or click chemistry. The developed solid-phase synthesis procedures are applicable for the synthesis of probes for other targets as well.