Targeting E1 Activating Enzymes through Small-Molecule Inhibition

Abstract

The covalent attachment of proteins with ubiquitin and/or ubiquitin-like molecules (Ubls) is important for the maintenance of various critical biological pathways. Aberrant regulation of these protein labelling systems is often involved in the progression of diseases such as cancer and neurological disorders. To this end, targeting ubiquitin/Ubl labelling has emerged as attractive avenue in the discovery of novel drug candidates.

Ubiquitin/Ubls are attached to protein substrates via a cascade of three enzymes: an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase. The main goal of this thesis is to target specific E1 enzymes with small molecule biological tools to investigate the role of these labelling systems in disease progression. First, a peptidomimetic strategy was used to target the E1 enzyme responsible for priming the Ubl SUMO, SAE. An Alanine-scan of peptides which mimic the C-terminus of SUMO proteins were used to understand which functional groups are necessary for disrupting SUMO-SAE interactions. This work concluded that mimicking the C-terminus of SUMO may not be sufficient to inhibit SAE enzymatic activity through a SUMO-competitive manner.

When targeting UBA5, the E1 enzyme responsible for activating UFM1, unique structural features of this E1 were used to develop a UBA5-selective inhibitor. A thorough investigation of the mechanism by which the lead compound, 5C-Zn, elicits its activity revealed that Zn²⁺ is the active inhibitory species which acts through a time-dependent and irreversible mechanism. Finally, some of the Cu²⁺-metallated complexes from the preliminary library screen which were inactive against the human UBA5 were proven efficacious against UBA5 in Leishmanial donovani. Current research efforts now focus on determining whether the inhibitory activity is a result of the metal-coordinated complexes or Cu²⁺, as observed with 5C-Zn and Zn²⁺.