Phospholipase D3 (PLD3) is an emerging target in Alzheimer’s disease (AD) drug discovery, particularly for late-onset AD (the most common form of dementia). Certain PLD3 variants are linked to increased vulnerability to neurodegeneration and amyloid pathology, with recent studies establishing a direct association between the enzyme and the disease. PLD3 is a type II transmembrane protein with its soluble domain released after cleavage in acidic cellular compartments, while there is no indication of any membrane signal transduction abilities so far. Our research aims to shed light on PLD3 biology and provide the groundwork for the development of novel AD therapeutics. The soluble domain of human PLD3 was recombinantly expressed and purified, followed by extensive biochemical, biophysical, and functional characterization of the protein. These efforts revealed a dynamic equilibrium between monomers, dimers, and high order oligomers. Concentration, pH and post-translational modifications appeared to be critical factors in driving the preference for different species. Cryo-EM was used to study the observed oligomerization behavior. Enzymatic assays confirmed that our protein has 5’ exonuclease activity but no sign of phospholipase activity. Structural information we gathered from initial crystallographic experiments—where PLD3 crystals diffracted to 2.3 Å were used to initiate a structure-based drug discovery pipeline. We have used a combination of in silico compound screening and in vitro direct binding assays, together with X-ray crystallography, to validate the binding modes of the small molecule compounds to PLD3. Enzymatic assays were also used to explore any effects on PLD3 activity in the presence of confirmed binders. The functional and structural findings from our experiments provide an increased understanding of PLD3 biology and will aid the development of AD therapeutic agents.
Georgopoulou et al. (Sun,) studied this question.