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Vanderbilt Team Uses Enzymatic, Cell-Based Assays to Find Phospholipase D Inhibitors


Investigators at Vanderbilt University Medical Center have developed what they claim to be the first isoform-selective phospholipase D inhibitors, a new class of potential antimetastatic agents.

The researchers used a screening approach that evaluates the effects of small molecules in both in vitro enzymatic assays and cell-based assays to compare the PLD-inhibiting activities of existing compounds as well as a library of new compounds generated in the laboratory, a Vanderbilt official told CBA News this week.

To arrive at their results, the researchers screened existing cell lines and developed a new cell line, in order to obtain cell-based systems that provided PLD1- and PLD2-selective responses, respectively, said Alex Brown, a professor of pharmacology, biochemistry, and chemistry at Vanderbilt University School of Medicine.

“It has been a long-term goal of the field to develop good, specific, small molecule inhibitors” of phospholipase, according to Brown, who said he has been working on phospholipase D for the past 15 years. “There have been some PLD inhibitors identified in the past, but the majority of them worked indirectly, and those that worked directly appeared to have a lot of off-target effects.”

Brown said he and his team decided to develop an inhibitor with isoenzyme specificity because two isoforms of PLD, known as PLD1 and PLD2, are the major ones found in human cells.

“In order to do that, we approached the problem by developing an assay system in parallel, where we would look at activity both in cells and using recombinantly expressed PLD1- and PLD 2-based protein in an in vitro assay,” he said.

“We feel for a number of reasons that this parallel approach is quite important, because it both reveals isoform selectivity of the compounds screened, and helps us to identify whether or not [a compound] has a direct or indirect effect on phospholipase,” he added.

Brown said that the Vanderbilt researchers did an “extensive” search of available cell lines to see if they could identify a cell-based system that had separate PLD1- and PLD2-responding cells.

“To our surprise, many cell types are an integration or mixture of both isoforms,” said Brown, adding that his team was also able to distinguish PLD1 from PLD2 responses.

To observe PLD1 response, the researchers used non-small lung cancer Calu-1 cells that, when stimulated with phorbol esters, gave an essentially pure PLD1 response. Other reasons for choosing Calu-1 cells are that they are easy to grow, and are very tolerant.

“We know that [Calu-1 cells give a pure PLD1 response] from having knocked down the message for PLD1 versus PLD2 with RNAi approaches,” said Brown. Only the PLD1-interfering siRNA had an impact on the catalytic activity in the Calu-1 cells.

He added that his team found “other cells with a predominantly PLD1 response,” but that the Calu-1-phorbol ester response “was really one of the purest PLD1 responses we identified.”

PLD2 “was a little harder,” said Brown, whose group found most conventional cellular responses, including some that were reported as being PLD2, to really be mixtures of the two isoenzymes.

“That led to us constructing within the lab a PLD2-overexpressing cell in the HEK293Trex system,” said Brown.

As before, the Vanderbilt team used siRNA to validate that this system only interfered with siRNA against PLD2. “That was the cell-based system in which we screened all of our new compounds. We did it as an initial screen at a moderate and high dose of inhibitor,” said Brown.

The team performed its initial screen in the cell-based assay and generated concentration-response curves for PLD inhibitors whose cellular IC50 values ranged from 1 nM to greater than 20 µM. Of the 30 compounds tested, 21 were found to be potent in Calu-1 cells and 15 were potent in HEK293 cells. Compounds considered potent had IC50 values that were less than 1 µM.

The researchers followed the cell-based assays with in vitro reconstituted-protein biochemical assays, and Brown said that he and his colleagues felt that the combination of these two types of assays “was really important to the development of these compounds.”

Brown said his group’s study represents the first time in which a series of PLD inhibitors was described along with the assay system used to identify them.

“Previous work was vague and nondescript, shall we say, in terms of approaches,” Brown said. “Part of what is novel about this work is actually the assay system.”

Brown said his group is continuing to refine and develop more selective inhibitors for the two phospholipase isoforms because “we think that in terms of eventually developing therapeutics, it is important to have highly specific PLD1 versus PLD2 inhibitors.”

According to Brown, a cell or an organism may better tolerate having a single isoform inhibited compared to having two or more inhibited.

His team also researches liponomics, or lipid-based mass spectrometry, and uses these compounds to explore what other intracellular lipid messengers are downstream of PLD activity.

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