Phospholipid-mediated signaling pathways control a myriad of physiological processes including various aspects of brain function. Among the phospholipid enzyme families, phospholipase D (PLD) is emerging as a key player in regulating phospholipid metabolism, and a newly appreciated therapeutic target for Alzheimer's disease (AD), stroke, and other brain disorders (Oliveira and Di Paolo, 2010). PLD catalyzes the conversion of phosphatidylcholine to the lipid second messenger phosphatidic acid and choline. Two mammalian isoforms of conventional PLDs have been identified, PLD1 and PLD2, which share 53% sequence identity and are subject to different regulatory mechanisms. Previous research relied on the overexpression of either catalytically active or inactive forms of either PLD1 or PLD2 in cells, or employed siRNA for the individual isoforms in an effort to discern discrete roles for PLD1 and PLD2 in brain disorders (Oliveira and Di Paolo, 2010). In 2010, PLD1−/− and PLD2−/− mice developed via gene targeting were reported, clearly defining, nonoverlapping roles, and therapeutic potential for both PLD1 and PLD2 in the pathogenesis of AD. From overexpression and biochemical studies, it has been shown that PLD1 (but not PLD2) regulates the trafficking of APP and the assembly of the γ-secretase complex via a direct interaction with PS1 (Cai et al, 2006). In 2010, PLD2−/− mice provided the first in vivo evidence implicating PLD in AD. Here, PLD2 was shown to be required for the synaptotoxic action of Aβ, and that PLD2 ablation rescues memory deficits and engenders synaptic protection in SwAPP mice, despite a high Aβ load (Oliveira et al, 2010). Also in 2010, PLD1−/− mice were shown to display impaired αIIbβ3 intergrin activation and defective glycoprotein 1b-dependent aggregate formation, leading to protection from thrombosis and ischemic brain injury without increasing bleeding time (Elvers et al, 2010). Historically, few small molecule tools existed to study PLD function, and none of the inhibitors displayed PLD isoform-selective inhibition. The classical biochemical approach relies on n-butanol, a small molecule that is not a PLD inhibitor, rather n-butanol blocks PLD-catalyzed phosphatidic acid production by competing with water as a nucleophile, thereby generating phosphatidylbutanol in a competitive transphosphatidylation reaction. A renaissance in the PLD inhibitor field began in 2007 when halopemide (1), a psychotropic agent discovered in the late 1970s, was shown to be a dual PLD1/2 inhibitor (Scott et al, 2009). More importantly, 1 has been in humans in five clinical trials and was shown to be safe and effective; thus inhibition of PLD with a small molecule is a viable therapeutic approach, a finding also noted in the PLD KO mice. Using 1 as a lead, a diversity-oriented synthesis campaign was pursued by the Brown and Lindsley labs, where ∼1000 analogs of 1 were synthesized and evaluated in cell-based and biochemical PLD1 and PLD2 assays (Scott et al, 2009). From this effort, isoform-selective PLD1 (2) and PLD2 (3) inhibitors were developed with low nanomolar potencies, unprecedented PLD isoform selectivity and DMPK profiles to allow in vivo target validation studies to be pursued (Lavieri et al, 2010; Figure 1).

Figure 1
figure 1

Evolution of small molecule, isoform-selective PLD inhibitors.

PowerPoint slide