Design and synthesis of cathepsin E small molecule inhibitors and PROteolysis TArgeting Chimeras (PROTACs) to modulate neuroinflammation in Alzheimer’s disease

Alzheimer’s disease (AD) is a multifactorial neurodegenerative disorder for which current therapies are limited. Beyond amyloid-β and tau pathology, sustained neuroinflammation contributes to synaptic dysfunction and neurodegeneration. In this inflammatory context, neutrophils have been implicated in worsening pathology and cognitive decline, while cathepsin E (CTSE), an immune-cell-enriched aspartic protease linked to inflammatory polarization, represents a promising target for modulating neutrophil-driven neuroinflammation. This thesis aimed to develop anti-neuroinflammatory CTSE-directed small molecules by combining two complementary strategies: (i) small-molecule inhibition and (ii) targeted protein degradation (TPD) using proteolysis-targeting chimeras (PROTACs). In Chapter 2, ritonavir was selected as a CTSE-directed repurposed starting point to generate a library of 34 inhibitor analogues. Its hydroxyethylene transition-state mimetic core was retained, while the terminal region was diversified, to explore structure–activity relationships (SAR) and early drug-like properties. Biological evaluation combined: CTSE enzymatic screening, which identified several analogues with low-nanomolar IC₅₀ values, selectivity profiling against cathepsin D (CTSD), and preliminary efficacy assessment in a neuron–neutrophil co-culture model. Computational studies supported SAR interpretation and revealed a CTSE-specific negatively charged region that could be further exploited for the design of improved inhibitors. In Chapter 3, CTSE-directed PROTACs were designed by functionalizing the primary amino group of des-ritonavir, incorporating thalidomide-based CRBN recruiters, modular linker variations, yielding 12 degraders and 4 matched negative controls. The resulting library was preliminary evaluated for CTSE engagement and plasma stability, and a neuron-neutrophil co-culture assay showed effectiveness, warranting further investigation of CTSE degradation in neutrophils. In Chapter 4, an orthogonal structural–biophysical toolbox was developed and applied to interrogate ternary complex formation in a TPD molecular-glue system, generating a transferable methodology to support future CTSE degrader development. Overall, this thesis provides the first integrated CTSE-focused chemical toolbox comprising both inhibitors and PROTACs, establishing a foundation for CTSE-directed anti-neuroinflammatory strategies in AD.