Phospholipase D (PLD) is an essential enzyme responsible for the production of the lipid second messenger phosphatidic acid. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. The lack of potent and isoform-selective inhibitors has limited progress in defining the cellular roles of PLD. We used a diversity-oriented synthetic approach and developed a library of PLD inhibitors with considerable pharmacological characterization. Here we report the rigorous evaluation of that library, which contains highly potent inhibitors, including the first isoform-selective PLD inhibitors. Specific members of this series inhibit isoforms with > 100-fold selectivity both in vitro and in cells. A subset of inhibitors was shown to block invasiveness in metastatic breast cancer models. These findings demonstrate the power of diversity-oriented synthesis combined with biochemical assays and mass spectrometric lipid profiling of cellular responses to develop the first isoform-selective PLD inhibitors—a new class of antimetastatic agents.
This Letter describes the synthesis and structure–activity-relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of alternative halogenated piperidinyl benzimidazolone privileged structures, in combination with a key (S)-methyl group, novel PLD inhibitors with low nM potency and unprecedented levels of PLD1 isoform selectivity (~1700-fold) over PLD2 were developed.
Phospholipase D (PLD) catalyzes the conversion of phosphatidylcholine to the lipid second messenger phosphatidic acid. Two mammalian isoforms of PLD have been identified, PLD1 and PLD2, which share 53% sequence identity and are subject to different regulatory mechanisms. Inhibition of PLD enzymatic activity leads to increased cancer cell apoptosis, decreased cancer cell invasion and decreased metastasis of cancer cells; therefore, the development of isoform-specific, PLD inhibitors is a novel approach for the treatment of cancer. Previously, we developed potent dual PLD1/PLD2, PLD1-specific (>1,700-fold selective) and moderately PLD2 preferring (>10-fold preferring) inhibitors. Here, we describe a matrix library strategy that afforded the most potent (PLD2 IC50 = 20 nM) and selective (75-fold selective versus PLD1) PLD2 inhibitor to date, N-(2(1-(3-fluorophenyl)-4-oxo-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)-2-naphthamide (22a), with an acceptable DMPK profile. Thus, these new isoform-selective PLD inhibitors will enable researchers to dissect the signaling roles and therapeutic potential of individual PLD isoforms to an unprecedented degree.
This Letter describes the synthesis and structure–activity relationships (SAR) of isoform-selective PLD inhibitors. By virtue of the installation of a 1,3,8-triazaspiro[4,5]decan-4-one privileged structure, PLD inhibitors with nanomolar potency and an unprecedented 40-fold selectivity for PLD2 over PLD1 were developed. Interestingly, SAR for this diverged from our earlier efforts, and dual PLD1/2 inhibitors were also discovered within this series.
Abstractα-Synuclein (αSyn) is a small cytosolic protein of unknown function, which is highly enriched in the brain. It is genetically linked to Parkinson's disease (PD) in that missense mutations or multiplication of the gene encoding αSyn cause early-onset familial PD. Furthermore, the neuropathological hallmarks of both sporadic and familial PD, Lewy bodies and Lewy neurites, contain insoluble aggregates of αSyn. Several studies have reported evidence that αSyn can inhibit phospholipase D (PLD), which hydrolyzes phosphatidylcholine to form phosphatidic acid and choline. Although various hypotheses exist regarding the roles of αSyn in health and disease, no other specific biochemical function for this protein has been reported to date. Because PLD inhibition could represent an important function of αSyn, we sought to extend existing reports on this interaction.Using purified proteins, we tested the ability of αSyn to inhibit PLD activity in cell-free assays. We also examined several cell lines and transfection conditions to assess whether αSyn inhibits endogenous or overexpressed PLD in cultured mammalian cells. In yeast, we extended our previous report of an interaction between αSyn and PLD-dependent phenotypes, for which PLD activity is absolutely necessary. Despite testing a range of experimental conditions, including those previously published, we observed no significant inhibition of PLD by αSyn in any of these systems. We propose that the previously reported effects of αSyn on PLD activity could be due to increased endoplasmic reticulum-related stress associated with αSyn overexpression in cells, but are not likely due to a specific and direct interaction between αSyn and PLD.Parkinson's disease (PD 1 ) is a debilitating neurodegenerative disorder that affects over one million people in the United States (for review, see (1)). Although more than 90% of PD cases are believed to occur sporadically, several genes are known to cause familial forms of PD. Among these, point mutations or increased dosage of the α-synuclein (αSyn) gene are known to cause rare cases of early-onset familial PD (2-7). The αSyn protein is also the major component of Lewy bodies and Lewy neurites, the insoluble aggregates that are neuropathological hallmarks of both familial and sporadic PD (8). Thus, dysregulation of αSyn at either the genetic or protein level can contribute to PD-type neurodegeneration.
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