Progress with proteins

TSRI teams report on work that could boost anticancer efforts and drug development

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LA JOLLA, Calif.—Researchers at The Scripps Research Institute (TSRI) have recently announced two important advances in understanding how drugs can better target proteins responsible for cancer growth. One group of researchers has identified a protein which has a role in launching cancer cells; another has found a technique to better identify drug candidates that bind to specific proteins. Both of these studies seem to tap into veins rich with potential for further research and therapies.
The protein GlyRS was first isolated in the 1960s, and has long been known to create proteins to help cells function and grow. In a study published in Nature Structural & Molecular Biology, TSRI researchers have shown that GlyRS can also help to further modify proteins in a way that launches cancer growth—making it a “double agent.”
The trouble comes at the intersection of GlyRS with p27, a protein that regulates the cell cycle and stops potential cancer growth. Too much GlyRS, the researchers found, may lead to too little p27. According to a press release, the team discovered that “GlyRS creates a protective shield around a modifier protein, called NEDD8, and safely ‘chaperones’ it to meet its target protein, called cullin. With NEDD8 in place, cullin is activated to degrade p27.… When GlyRS levels increase, too much p27 gets degraded and cells multiply unchecked.”
Since GlyRS, again acting as double agent, also supplies cancers with the proteins they need to keep growing, this process is even more dangerous. “Ultimately, both functions are linked to cell proliferation and tumorigenesis,” said TSRI Prof. Xiang-Lei Yang, who led the study. TSRI Research Associate Zhongying Mo analyzed data from a breast cancer tissue database and found that patients with increased GlyRS had higher mortality.
The team believes that it may be possible in the future to use GlyRS as a marker to determine how quickly a cancer may be likely to progress. Researchers plan to examine how the protein functions in diseases other than breast cancer, such as colorectal cancer, and whether a drug may be developed to inhibit the protein. The team hopes to identify small molecules to target GlyRS and simultaneously inhibit neddylation and protein synthesis within the next two years, according to Yang.
Separately in the proteomics space, a Nature paper reports how TSRI researchers revealed a new technique that may unlock the potential to target new classes of proteins for research and drug therapies. This technique involves the discovery of ligands for many proteins which had hitherto not been thought to bind well to small molecules that can be used as probes determine the functions of their protein targets and can serve as starting compounds for the development of drugs.
Researchers have for some time been looking for a better way to find small-molecule ligands and determine to which classes of proteins they are likely to bind. Since pharmaceutical companies are unlikely to develop drug therapies to target certain proteins unless they can tell that those proteins are likely to have those ligands, only about 600 of the roughly 20,000 human proteins have been targeted successful by FDA-approved drugs. The best method had been to employ algorithms to identify classes of proteins which might be “ligandable.” “There really hasn’t been a way to determine empirically, rather than theoretically, what fraction of the human proteome can be targeted by small molecules,” said principal investigator Benjamin F. Cravatt.
Starting with their own experience in legacy cysteine profiling, says Keriann M. Backus, a research associate in the Cravatt lab and first author of the study with TSRI professional scientific collaborator Bruno Correia, the team took note of success that other researchers had in using fragment molecules to explore protein ligandability. These fragments, about half the size of the molecules in pill-based drugs, had the advantage of forming covalent bonds, which proved both more efficient, as they required a smaller selection of compounds for a thorough search, and provided a significant boost in the potency of the bonds.
Backus relays that the team believes that fragment-based ligand discovery (FBLD) is an powerful tool for the exploration of chemical space and the development of new chemical probes. “However,” she explains, “all FBLD efforts prior to our study had required large amounts of recombinant purified protein, a requirement which precluded inhibitor screening of a large fraction of the human proteome. We thought that the combination of our cysteine profiling technology with cysteine reactive fragments would allow us for the first time to conduct fragment screening in native biological systems, thus for the first time accessing many proteins with small molecules, including many proteins that were thought to be ‘undruggable.’“
The team developed a screening system to apply the covalent-bonding fragment molecules individually to entire collections of proteins expressed in human cells, a method that can also be used with living cells in a dish. Using this approach, researchers can detect and identify which small-molecule fragments have bound covalently to which proteins and which sites on the proteins are responsible for binding.
Scientists applied a small library of cysteine-reactive fragments to the proteins found in two types of human cancer cells, and found that the fragments successfully “liganded” more than 750 different cysteines found on more than 600 distinct proteins—more than 20 percent of all the proteins assayed. About 85 percent of the newly liganded proteins were not listed in a standard database of proteins with known small-molecule ligands, and were therefore thought “undruggable.”
The ligands discovered for enzymes IDH1 and IDH2 turned out to block the activity of the normal versions of the enzymes as well as the mutant versions implicated in many cancers. The team also showed that one of its identified ligands inhibits the activities of caspase-8 and caspase-10, two enzymes that help switch on apoptosis, which is thought to contribute to brain damage after strokes and brain injuries and to neurodegenerative diseases such as Huntington’s disease and Alzheimer’s disease, but which might also, in its absence, allow cancers and autoimmune diseases to flourish. Cravatt’s team found that the anti-caspase ligand they discovered works by binding the precursor forms of caspase-8 and -10. They were able to discover new details of how caspase-8 and -10 promote apoptosis in human T cells by chemically modifying the ligand into one that selectively binds just the precursor of caspase-8, and using their two ligands as probes.
Backus relates that the team is currently working to expand this technology to other amino acids and trying to improve the potency and selectivity of some of their lead compounds.

Experience plays huge role in early stages of brain circuit development
LA JOLLA, Calif.—A healthy brain has just the right ratio of cells that enhance signals (excitatory neurons) and cells that tone down signals (inhibitory neurons). These two sets of neurons start out looking exactly the same, but questions remain as to what determines their roles. A new study in animal models from The Scripps Research Institute (TSRI) suggests that stimulation from the outside world guides these neurons’ early development so that inhibitory neurons split into two different types of neurons, each with a different job in the brain. This adds another level of complexity and regulation to this circuitry.
If these findings hold true in humans, they could provide insight into how brain circuits develop and how future therapeutics might better treat neurological disorders such as autism, schizophrenia and depression.
“The function of inhibitory neurons in developing circuits is defined at earlier stages of development than previously thought—and it’s defined, at least partly, by the responses of the neurons to sensory input,” said study senior author Hollis Cline, chair of the Department of Molecular and Cellular Neuroscience and director of the Dorris Neuroscience Center at TSRI.
The research team looked for biochemical signatures and other markers that distinguish excitatory and inhibitory neurons, but found no known markers at this early stage, leading them to conclude that visual stimulation might be triggering the expression of certain genes that make the neuron types different.
In other words, some neurons might not be pre-programmed to have a certain function—their experiences might instead determine how they develop.
“The big surprise was that neurons that look very similar have opposite plasticity responses to experience,” said TSRI Senior Research Associate Hai-yan He, first author of the study.
The researchers suggest that one type of inhibitory neuron inhibits the other inhibitors, adding a second layer of control to this complicated system and keeping the overall circuit in balance. It will be important to consider both subtypes of inhibitory neurons when developing new therapies for neurological disorders, He said. If scientists develop a treatment to boost the response of all inhibitory neurons, for example, they could inadvertently send the system further out of balance.

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