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Combating Central Nervous System Disorders with Novel Screening Approaches, Collaborations

New treatments for central nervous system (CNS) disorders are urgently needed, yet many companies have scaled back their work in this area, citing “high costs, lengthy development times and low success rates,” according to an editorial in a new Journal of Biomolecular Screening (JBS) Special Issue on Innovative Screening Methodologies to Identify New Compounds for the Treatment of Central Nervous System Disorders. Novel screening approaches could help turn the tide.

“The complexity of the CNS and difficulties in understanding the molecular and cellular mechanisms by which current treatments exert their effects are among the overall challenges in identifying new candidates for Alzheimer’s, Parkinson’s disease, mood and anxiety disorders and other neurological disorders that affect millions of people worldwide,” says JBS Editor-in-Chief Robert M. Campbell, Ph.D.

For drug discovery in particular, challenges include a paucity of valid animal models for new targets and mechanisms, and lack of target-engagement biomarkers, according to an editorial in the JBS Special Issue by Kevin Burris of Lilly Research Laboratories, Indianapolis, IN, and Steven Dworetzky of Knopp Biosciences, Pittsburgh, PA. Research groups from around the world are rising to these challenges, using innovative phenotypic- and target-based methodologies to identify relevant candidate CNS compounds.

Teaming Up to Identify a7-Nicotinic Acetylcholine Receptor Modulators

Jeroen Kool of Vrije Universiteit Amsterdam, The Netherlands, and colleagues have developed a rapid, cost-effective screening technique for identifying bioactive compounds in natural extracts and other complex mixtures. Kool, an analytical chemist, emphasizes, “All of our projects start with researchers from other fields. They tell us specifically what they’re looking for, and also let us know from the beginning what the difficulties will be, and what analyses or results they would need.”

Kool’s job is to develop techniques that can overcome those difficulties and efficiently screen extracts for the specified target. “Essentially, I combine analytical chemistry, separation science and mass spectrometry to find bioactives, and correlate bioactivity with identity,” he explains.

For the study reported in the special issue, “At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor,” Kool collaborated with scientists at the university’s neurobiology campus to identify bioactives that target the alpha7-nicotinic acetylcholine receptor (a7-nAChR), a ligand-gated ion channel expressed in different regions of the CNS. Receptor malfunctions are associated with Alzheimer’s disease, epilepsy and schizophrenia.

Often, the problem with working with ion channels in cellular bioassays is the need to overexpress them, Kool observes. In contrast to G-protein-coupled receptors, where only one receptor is overexpressed, “with ion channels, you have to overexpress different subunits to get heteromeric ion channels, and you don’t necessarily get the correct order of assembly of a pentamer, for example, that you’re working with.”

Assaying ion channels is also difficult, Kool says, “because they react very quickly. You get a fast onset, fast desensitization—all kinds of different responses. So, you cannot just start the bioassay, incubate for an hour and then start measuring every well in a 96- or 384-well plate for example. You have to add your ligands and start measuring immediately, because ion channels start signaling within a few seconds to a few minutes, then the signal goes down, and the measurement is done. There’s very little time to get the assay readout, and preferably the readout is done in time.”

“We also encounter problems if we don’t have the correct oxygen and CO2 concentrations, because that can cause slight shifts in the signal over time,” says Kool.

Systems such as FLIPR (Fluorometric Imaging Plate Readers) can overcome these difficulties, he acknowledges, but Kool’s lab does not have this technology currently. “In academia, we usually can’t just buy expensive equipment to solve problems for every study we decide to do. We work on many different types of projects. We’re not in a lab that will work on ion channels for the next 10 years, which would make the investment worthwhile.”

“Right now, for example, I’m working on e. coli, so we had to buy an e. coli cell culturing facilities and readout apparatus. In a year, I may need a fluorescent polarization assay or a luminescence assay, and so on,” Kool explains. “So our group has to collaborate with other labs and develop some clever techniques to deal with problems that would otherwise be solved by running fully automated systems.”

For the search for bioactives in mixtures targeting the a7-nAChR, Kool came up with “a very simple trick” to overcome many of the difficulties associated with traditional ion channel screening methods. He combines liquid chromatography (LC) coupled via a T-split to both an at-line calcium-flux assay and, separately, to high-resolution mass spectrometry (MS), which is done in parallel.

“Bioactivity can be assessed after LC separation, while parallel MS and nanofractionation enables compound identification,” Kool says. Nanofractionation is a rapid method that allows the research team to quickly remove compounds that are not bioactive, “leaving only one or two possible candidates for bioactivity. By contrast, with normal bioassay-guided fractionation, it can take weeks or months to identify bioactive compounds.”

Kool and his colleagues tested the approach by screening the hallucinogenic mushroom Psilocybe Mckennaii. They analyzed the crude mushroom extract using both reversed-phase and hydrophilic interaction LC. By matching retention times and peak shapes of the identified bioactives with data from the parallel MS measurements, they could quickly pinpoint masses corresponding to the bioactives.

“We’ve also analyzed snake venom using this approach, and more recently, for cardiac treatments, we used it to identify inhibitors of thrombin and Factor Xa in various mixtures,” notes Kool, who developed a similar “trick” for fractionating after gas chromatography. 

Scientists can also nanofractionate in their own lab, according to Kool. When one of the fractionation robots in his lab developed problems, he used “a cell phone as a stop watch and some PEEK tubing to collect the nanofractions in well plates” along with his LC system. “Trying this approach costs just one hour of your time per run,” he says. “You use your hands to become your own manual nanofractionator—and I can tell you how.”

Kool invites readers who want to learn more about the at-line screening approach or manual nanofractionation to e-mail him at

Teaming Up to Identify NMDAR Modulators

In another report, “An Integrated Approach for Screening and Identification of Positive Allosteric Modulators (PAMs) of N-Methyl-D-Aspartate Receptors” (NMDARs), Timothy Spicer of The Scripps Research Institute, Jupiter, FL, is collaborating with Eli Lilly and Company. Hypofunctioning NMDARs have been implicated in schizophrenia and Alzheimer’s disease, among other neurological disorders. In the special issue article, Spicer and colleagues describe the approach that led to the selection of NMDAR-PAMS for further study as potential candidates for treatment of cognitive impairment.

The multi-site collaboration started when Lilly approached Scripps “looking to progress an assay through our chemical library, which contains 650,000 compounds, as well as some of their own libraries,” Spicer explains. Such collaborations make sense, he says, when pharmas have already tested all or most of their libraries and want to extend their coverage in the hope of discovering additional potential candidates.

Since the NMDAR is a ligand-gated ion channel, Spicer, like Kool, had to deal with the challenges inherent in working with ion channels. Specifically for high-throughput (HTS) and ultra-high-throughput screening, “detection formats for ion channels are limited,” Spicer says. “In order to get right down to the discrete biology, you have to use patch clamp technology, and that puts you into non-1536-well plate format. If your chemical library is set up in 1536-well format—as ours is at Scripps and at lots of large pharmas as well—it’s difficult to try to deconvolve backwards to a 384-well format.”

Instead, the team did an initial HTS based on a 1536-well formatted assay at Scripps, then confirmed the HTS hits using a lower throughput patch clamp assay at Lilly. This approach added “another level of instrumentation and another level of medicinal chemistry support to the screen,” Spicer says.

The team screened more than 810,000 compounds, which yielded 864 NMDAR-PAMs. Subsequent analyses identified several novel chemical series, six of which were selected and analyzed for pharmacological properties, subtype selectivity, mode of action and activity at native NMDARs.

In addition, the Scripps team has embarked on a U.S. National Institutes of Health-funded project on synaptogenesis, which involves running high-content screens of primary neurons with a local research group, and they have a number of other CNS-related collaborations in place.

More Work Underway

The JBS special issue highlights other strategies to hone in on promising candidates as starting points for CNS drug discovery efforts. Whereas Kool and Spicer addressed methods to screen ligand-gated ion channels, Michael Finley and colleagues at Merck describe a screen of voltage-gated sodium channel antagonists to identify selective antagonists of NaV1.7, a target for the treatment of neuropathic pain. Using a cell-based membrane potential dye FLIPR assay, the team developed an HTS screen for both NaV1.7 and cardiac NaV1.5 channels. Subsequent analyses were able to differentiate compounds with NaV1.7 pharmacological selectivity over NaV1.5.

A KNIME-Based Analysis of the Zebrafish Photomotor Response Clusters the Phenotypes of 14 Classes of Neuroactive Molecules” by Daniëlle Copmanns of the University of Leuven, Belgium, and colleagues describes an academic collaboration that used an open source analytical platform, KNIME, to develop an automated analysis workflow based on the photomotor response (PMR) of zebrafish embryos. The PMR “is reported as a robust behavior that is useful for high-throughput neuroactive drug discovery and mechanism prediction,” according to the team, which made their workflow freely accessible on the KNIME Public Example server (050_Applications/050021_PMR Analysis).

The novel behavioral data analysis paradigm was able to identify phenotypes associated with different classes of neuroactive molecules. The results “suggest the utility of PMR for mechanism prediction for adrenergics, dopaminergics, serotonergics, metabotropic glutamatergics, opioids and ion channel ligands,” they conclude.

Laura Vela and colleagues at GSK provide another example of the utility of a phenotypic screen – “Discovery of Enhancers of the Secretion of Leukemia Inhibitory Factor for the Treatment of Multiple Sclerosis.” As part of a project aimed at finding treatments for multiple sclerosis, the researchers sought to identify CNS-permeable and orally available small molecules that enhance production of leukemia inhibitory factor (LIF), a cytokine involved in neuroinflammation. Five chemical series of compounds and a few single compounds were selected for further progression.

Learn More in the June 2016 JBS Special Issue

In addition to the contributions discussed in this article, the JBS Special Issue on Innovative Screening Methodologies to Identify New Compounds for the Treatment of Central Nervous System Disorders features other original reports that exemplify the efforts of labs worldwide to address the need for new treatments for these debilitating disorders.

June 6, 2016