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MALDI-Based Method May Reduce Cost of Rx-Screening Assays, Speed Drug Development

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This story originally ran on Oct. 6.

By Tony Fong

Researchers from the University of Cincinnati and MDS Analytical Technologies have used mass spectrometry to develop a high-throughput screening method for drug discovery they say can be more precise and cost-effective than existing techniques.

The technique is based on a MALDI triple-quadrupole platform and exploits the selective multiple-reaction monitoring transition features of the platform. By doing so, the new method is able to lower the cost of high-throughput screening for drug compounds to pennies per well from as much as $1 per well currently, Ken Greis, associate professor of cancer and cell biology and director of proteomics and mass spectrometry at the University of Cincinnati College of Medicine, told ProteoMonitor recently.

A study detailing the method was published Sep. 15 in the online edition of Rapid Communications in Mass Spectrometry.

In the paper, Greis and his co-authors said that drug discovery typically begins with a validated target enzyme "with the initial goal of finding appropriate molecular scaffolds with inhibitory activity via high-throughput screening." The scaffolds are then subsequently used for lead compound optimization, and "ideally for the development of a safe and effective therapeutic compound."

The most common methods of high-throughput screening have been fluorescence- and chemiluminescence-based approaches. Such approaches, Greis said, have been "very successful" because the same reagents can be used for many different enzymes.

But that same characteristic also creates a risk for interference.

"When one's evaluating a compound repository for inhibitors, you often have a series of compounds that will fluoresce themselves," Greis said. "If they fluoresce, they're going to give you a false signal. Alternatively … there are compounds that inhibit the fluorescent properties, or what's called quenching fluorescence, [that] also give false read-outs."

Another problem is in the way the assays get generalized so that the reagents work for a wide range of enzymes. Such assays are called coupled assays: "You have a product being formed from your enzyme reaction but that's not what actually triggers the fluorescence," Greis said. "That product gets converted to another enzyme to another product through another enzyme to another product that then can be fluoresced.”

This series of enzyme step, or coupled assays, ultimately results in a read-out. "The problem is any compound that interferes with any of those steps along the way also gives you false read-outs," which tend to be false positives, he added.

But by using mass spectrometry to measure enzyme activity, Greis and his colleagues are able to get a direct read-out, "so a mass spectrometer effectively can give you a quantitation and a mass of a compound."

By taking a ratio of the substrate being converted to a product — the essence of an enzyme assay, Greis said — and measuring that directly on a mass spec, there is no interference either from quenching or auto-fluorescence.

"And what we've found thus far is we've not seen any false positive read-outs. If we get a compound that shows that it's active, even in single-point assays, it's been demonstrated that it's a dose-dependent inhibitor."

And because the method uses native peptides or small-molecule substrates, the method can be done for "at most, pennies per sample well," Greis said. By comparison, fluorescent and chemiluminescence reagents cost between 50 cents to $1 per well.

"So if you run a million compounds, you can run up a half-million dollars of reagents costs right away, whereas the label-free read-out is going to cost you maybe a couple thousand dollars for the reagents," he said. "That's a mass spec advantage."

A prior study by researchers in China had demonstrated the utility of a MALDI-Fourier transform mass spectrometer for high-throughput screening of small-molecule substrate/product conversion.

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In their work, though, Greis and his co-researchers wanted to extend the application to a small-molecule, non-peptide substrate to demonstrate the flexibility and technical range of their method. While a large number of therapeutic targets, including kinases and phosphatases, contain peptide substrates, some important targets don't, such as HMG CoA reductase, the target of statin drugs, and AChE, a target for neurodegenerative therapeutics.

The researchers chose AChE because of its long history of enzyme assay development, including colorimetric assays, pH-change assays, and most recently aggregation-induced fluorescence assays and mass-spec assays.

Speed is of the Essence

They also chose a MALDI platform, rather than an electrospray platform, because of the higher speed that can be achieved on the MALDI. Most enzymatic reactions contain salts that can interfere with mass spectrometry. An ESI platform requires a desalting step, which limits the throughput to five to 10 seconds per sample. A MALDI-based approach skips the desalting step, however, because the technology is less sensitive to salts.

"Essentially all that we do is run the enzyme reaction on a 384-well format," Greis said. "We transfer all at once into a matrix plate mix and onto our MALDI target plate."

Because there are no cleanup steps on the MALDI triple-quad, samples can be scanned at up to three samples per second, he said.

Greis acknowledged that the MALDI technology, especially the MALDI triple-quad, is not a popular tool for drug discovery. In his opinion, that's because drug-discovery researchers were trained on electrospray mass specs and are comfortable with them.

"To then move them into a MALDI platform that they don't understand, they've got a bias that it can't be quantitative, and all these sorts of things from earlier studies using MALDI-based approaches that have been demonstrated time and time again to not be true anymore — I think there's a cultural thing," he said.

A criticism of a MALDI approach is that while it works well for peptide substrate screening, it doesn't for small-molecule substrate products because of matrix interference in the low mass range.

"And we show very directly … that by taking advantage of the transition," a chemical fragmentation that is diagnostic of a substrate or product "that one can do in a triple-quad, that matrix interference completely goes away," Greis said.

The researchers tested their method by screening a library of 1,008 structurally diverse compounds across 384-well microtiter plates as an example of a single-dose primary screen, and reported that all known AChE inhibitors resulted in complete inhibition of enzyme activity, as expected. The hits were then validated "by demonstrating concentration-dependent inhibition and the rank order of inhibitory potency in hit follow-up assays," they said in their study.

The technique they've developed can also be used on a simple MALDI instrument, though it works best for peptide substrate enzymes. With low molecular-weight enzymes, sensitivity can be an order of magnitude lower on a simple MALDI "because you'd have to be using enough enzyme substrate product to see your substrate products down in those low mass ranges in amongst all of the matrix peaks," Greis said.

Also, Greis said there will be enzymes — such as fatty acids and long-chain hydrocarbons —that will not be amenable to a MALDI-based approach.

"The fact of the matter is that any mass spectrometry-based technique is only as good as the molecule that it's trying to evaluate," he said. "We have to be able to ionize the substrate and/or the product to be able to measure and quantify it."

In ongoing work, he and his team members are developing multiplex assays. The typical screening approach is to take a target enzyme and pass the whole repository across it to look for inhibitors, and then validate the inhibitors. The next therapeutic target is then set up and the process is repeated.

With a mass spec-based approach, "as long as your enzymes reactions are compatible … you can run multiple enzymes in one pot and pass your repository against it once and get hits for all those different enzymes," Greis said.

In conferences, Greis and his colleagues have presented proof-of-concept studies that show that "this in fact works quite well using a kinase and acetylcholinesterase or a kinase with a protease all in the same part," he said. "We've shown that we can get selective inhibitors for each of them individually without interference in the multiplex format."

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