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Posts Tagged ‘optogenetics’


Targeting Neuronal Cell Growth

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Remote Mind Control

Using chemogenetic tools to spur the brain into action

By Kelly Rae Chi | November 1, 2015     http://www.the-scientist.com//?articles.view/articleNo/44321/title/Remote-Mind-Control/

http://www.the-scientist.com/November2015/LT_2_opener.jpg

A MATTER OF TIME: Optogenetics methods, which work on the millisecond timescale, allow for the finest level of temporal control over neuron excitation and inhibition. The chemogenetic tools, DREADDs and PSAMs-PSEMs, are ideal for the study of longer-lasting behaviors such as appetite, thirst, or anxiety because they work over a scale of the minutes-to-hours. The receptors are incorporated into specific neurons or cells using viruses. Ligands—CNO or salvinorin B (for DREADD receptors) or PSEMs—are administered via injection or drinking water. Both receptors and ligands are orthogonal, meaning they do not bind to anything else in the body.
REDRAWN WITH PERMISSION OF SCOTT STERNSON FROM SCIENCE, 333:1292, 2011; NEUROENDOCRINOLOGY, 100:98, 2014

In a pharmacology lab at the University of North Carolina at Chapel Hill, doctoral student Reid Olsen, working with brain tissue harvested from a mouse just a few hours earlier, readies half a dozen dime-size slices for live calcium imaging. This mouse’s brain contains a genetically engineered receptor that Olsen has targeted to cells thought to control the making of new neurons in adult mice. He is about to use a synthetic drug to activate this receptor in the tissue. When it indeed works—just as he has predicted—he turns his attention to attempting to stimulate neurogenesis in a freely moving mouse that has the same engineered receptors in its brain.

Less than a decade ago, such precise control over neuronal activity in a dish, let alone in a living brain, was impossible. The drugs available to repress neurons or encourage them to fire would produce off-target effects or eliminate cell populations indiscriminately.

Working in the lab of Juan Song, Olsen is using a “designer receptor exclusively activated by a designer drug,” or DREADD. These modified G protein–coupled receptors (GPCRs) are usually either virally administered or bred into animals, then activated by a specific ligand that’s either injected or taken orally. Both the receptor and the ligand are designed to be orthogonal, effectively meaning they bind to each other but to nothing else.

Along with DREADDs, recently developed orthogonal ligand-gated ion channels called “pharmacologically selective actuator molecules” and “pharmacologically selective effector molecules” (PSAMs-PSEMs), are allowing researchers to dial up or dial down neuronal activity in living animals, with the goal of clarifying the brain wiring that controls appetite, thirst, anxiety, and many other behaviors.

Along with DREADDs, recently developed orthogonal ligand-gated ion channels called “pharmacologically selective actuator molecules” and “pharmacologically selective effector molecules” (PSAMs-PSEMs), are allowing researchers to dial up or dial down neuronal activity in living animals, with the goal of clarifying the brain wiring that controls appetite, thirst, anxiety, and many other behaviors.

“[DREADDs and PSAMs–PSEMs] are completely complementary methods, and they can in principle be used together in the same animal.—Scott Sternson, HHMI Janelia Research Campus”

These are not the only so-called chemical genetic, or “chemogenetic,” tools for controlling cells. Orthogonal kinases have been used in the brain to deduce the mechanisms underlying epilepsy, memory, and neuronal development. And inducible genetic systems, now in wide use for two decades—for example, tetracycline-dependent transcriptional promoters—are incredibly powerful for expressing specific genes at a particular point in an animal’s development, says Bruce Conklin, senior investigator at the University of California, San Francisco–affiliated Gladstone Institute of

Cardiovascular Disease, whose group pioneered the development of engineered GPCRs. At the other extreme of temporal control from inducible genetic systems, optogenetics—a set of methods that use light to activate genetically encoded opsins—is widely used for controlling brain cells on the millisecond time scale in vivo.

As tools, DREADDs and PSAMs-PSEMs allow control of neuronal activity over a middle ground—from minutes to hours. “These are the time scales that are most useful, in my opinion, for neurobiology experiments,” says Scott Sternson of the Howard Hughes Medical Institute’s Janelia Research Campus in Ashburn, Virginia, who has developed PSAMs-PSEMs but who also regularly uses DREADDs and optogenetics. (For a review, see Ann Rev Neurosci, 37:387-407, 2014.)

The Scientist talked to developers about the basics behind DREADDs and PSAMs-PSEMs. Here’s what they said.

DREADDs and PSAMs-PSEMs: A history

In 1991, scientists showed that engineering orthogonal GPCRs was possible, and first iterations of such tools, dubbed “receptor activated solely by a synthetic ligand”  or RASSLs, came onto the scene in 1998.

Bryan Roth, of the UNC School of Medicine, made the second generation of RASSLs, which he called DREADDs, using an engineered muscarinic GPCR and, importantly, a ligand that was chemically inert (PNAS, 104:5163-68, 2007). Since the publication of that first paper on DREADDs, hundreds of labs have administered them in vivo, Roth says. This chemical genetic technique has the advantage of being easier to implement and less invasive than optogenetics, he adds.

Ligand-gated ion channel–based chemical genetic tools have their own history, but were not used in vivo in animals until 2011, when Sternson developed PSAMs-PSEMs. The researchers mutated ligand-binding domains and mixed and matched them to different ion-pore domains. But they altered the receptors and their ligands further so that they don’t interact with anything in the body. “As I was thinking about that system, I imagined I would want it to have easily optimizable, nontoxic ligands and the ability to tune the ion[-pore] or ligand-binding domain easily,” Sternson says.

DREADDs and PSAM-PSEM combinations in action

There are five different classes of DREADDs available, each designed for a different purpose:

  • hM3Dq raises calcium levels in a cell, causing burst firing;
  • hM4Di lowers cAMP and the activation of a particular potassium channel, causing neuronal silencing; also inhibits presynaptic neurotransmitter release;
  • GsD enhances cAMP, causing modulation signaling;
  • Rq(R165L) enhances arrestin signaling, a specific pathway that has been linked to the mechanisms of psychoactive drugs;
  • κ-opioid receptor DREADD or  KORD quiets neurons and also inhibits presynaptic neurotransmitter release.

The synthetic ligand for each of the first four DREADDs is clozapine-N-oxide (CNO), whereas KORDs are activated by salvinorin B. That means combining DREADDs is now possible: Roth’s group showed recently that they could insert the hM3Dq and KORD to be able to activate and silence the same neurons (Neuron, 86:936-46, 2015).

Roth’s lab has also made light-activated (photocaged) CNO, which allows for more-precise control over the timing of DREADD receptor activation. He has not published any papers using this yet, but will provide the caged ligand to interested researchers upon request. To make use of photocaged CNO, however, you will need to surgically implant an optic cable to provide light to the brain region of interest. If you’re going to go to the trouble, you might consider optogenetics, Roth adds.

Scientists have paired different PSAMs with various ion channels and PSEMs in order to control neurons. Among the most popular:

  • PSAML141F, Y115F– 5HT3 HC is activated by the ligand PSEM89S, allowing cations to flow into the cell and boost excitability;
  • PSAML141F, Y115F – GlyR is activated by the ligand PSEM89S, silencing neurons;
  • PSAMQ79G, L141S-nAChR V13 is activated by the ligand PSEM9S, enhancing calcium signaling. (Because there are two different PSEM ligands, PSAMs-PSEMs can also be combined in the same animal.)

When to opt for optogenetics

The single biggest consideration in your choice of, and among, these technologies is the temporal control needed for your experiment. Optogenetics, for example, offers the finest level of control, on the order of milliseconds to seconds. If you’re examining decision-making behaviors, for example, then optogenetics (or electrical stimulation) is for you.

If you’re studying behaviors—such as eating or drinking—or physiological changes that occur over minutes to hours, then either optogenetics, chemogenetics, or both might work.

For long-lasting behaviors being measured over the course of hours to days, a chemogenetic approach such as DREADDs or PSAMs-PSEMs is the clear winner. The ligands linger longer than short light pulses and can even be dissolved into the animals’ drinking water. The chemogenetic approach is also superior for investigating larger swaths of brain, which are challenging to illuminate using optogenetics methods, Roth says.

Researchers have also successfully combined the approaches, typically using an optogenetic approach to turn on neurons and chemogenetic approaches to switch them off in the same animal. The inhibitory DREADD hM4Di targets presynaptic terminals, which could be especially helpful if you’re investigating a region of the brain where long-range projection neurons terminate.

Use DREADDs or PSAMs-PSEMs first?

TWOFERS: In 2015, researchers announced a new DREADD: KORD. Because it is activated by a different ligand than the one for previously developed DREADDs, the engineered receptors can now be combined in the same animal. BRYAN ROTH

Researchers tend to start with DREADDs simply because they have been around longer, Sternson says. But some will turn to PSAMs if DREADDs have been ineffective. “Most cell types will respond to [DREADDs] as they’re supposed to, but not all,” Sternson says. That’s because DREADDs tap into a complex signaling pathway that eventually results in neuronal activation or silencing. In contrast, PSAMs work by controlling the gating of an ion channel. On the other hand, ligand-gated ion channels may affect some types of cells, such as developing neurons, differently than they do adult cells, says Olsen, who coauthored the Neuronpaper describing KORD, the newest DREADD. But in general, Sternson says, “they’re completely complementary methods, and they can in principle be used together in the same animal.”

Another consideration is that PSEMs tend to take slightly longer to work—15 minutes, compared with the DREADD ligand CNO, which takes 5–10 minutes. On the other hand, PSEMs tend to take less time to clear from the body, 1–2 hours (vs. about 2 hours for CNO). Salvinorin B, the ligand of the new KOR-based DREADD, works almost instantaneously, and the effects last less than an hour. Although these differences are minor, they may factor into your experiment.

Experimental procedure

The operational steps are similar for both tools. Most people inject viruses carrying the engineered receptors into the brain area of interest and wait two to three weeks for expression. They then administer the ligand and make their measurements.
If you’ve already performed stereotactically guided brain surgery, there’s nothing new to learn. For newcomers, a Journal of Visualized Experiments protocol describes the surgery and injection of the virally ferried chemogenetic tools (100:e52859, 2015), though it’s best to learn by shadowing someone with experience, Roth says.

Viral constructs for both DREADDs and PSAMs are available from Addgene. For DREADDs, the UNC Vector Core sells high-titer virus stocks. CNO is available for free or at a reduced price for NIH-funded investigators through the National Institute of Drug Abuse’s Drug Supply Program. For PSAMs, you make your own receptor-carrying virus. Sternson provides PSEMs to researchers for their pilot experiments, and they are available for purchase through Apex Scientific for about $15 for 10 mg, he says.

You don’t necessarily need to do surgery if you can afford mutant mice whose DREADDs are under the control of an inducible promoter, such as Cre. Such mice are available through Jackson Labs. In general, just be sure to use validated Cre driver lines, Roth says.

You should make sure the receptor is expressed and working in vitro before you move to whole animals. Expression of both DREADDs and PSAMs is linked to the translation of a fluorescent protein. On his blog, chemogenetic.blogspot.com, Roth gives more specific advice on immunofluorescent staining for visualization of DREADDs.

To make sure that the receptors are actually working involves more-detailed studies, such as the calcium imaging Olsen used to ascertain whether his activating DREADD responded to the ligand, or electrophysiological studies in slices, but the particulars depend on what mechanism your receptor-ligand uses.

“One thing that’s important to know when using these receptors is that they’re not completely off when expressed at high levels,” Conklin says, referring to DREADDs. “[Simply] by expressing them, one cannot be sure.” To get around potential abnormal background activity, you have to include a control without the receptor. Also, having a good label on the receptor is helpful. Using DREADDs in combination with an inducible transcription system, such as Cre, allows you to measure receptor expression before and after inducing it.

Future uses

Although DREADDs and PSAMs-PSEMs are proving to be useful research tools for cell biologists and neurobiologists, both Roth and Sternson are actively developing orthogonal systems for potential clinical use, either as gene-based therapies that would go directly into humans or to be used in stem cell–based therapies.

 

Chemogenetic tools to interrogate brain functions.

Annu Rev Neurosci. 2014;37:387-407. doi: 10.1146/annurev-neuro-071013-014048. Epub 2014 Jun 16.

Elucidating the roles of neuronal cell types for physiology and behavior is essential for understanding brain functions. Perturbation of neuron electrical activity can be used to probe the causal relationship between neuronal cell types and behavior. New genetically encoded neuron perturbation tools have been developed for remotely controlling neuron function using small molecules that activate engineered receptors that can be targeted to cell types using genetic methods. Here we describe recent progress for approaches using genetically engineered receptors that selectively interact with small molecules. Called “chemogenetics,” receptors with diverse cellular functions have been developed that facilitate the selective pharmacological control over a diverse range of cell-signaling processes, including electrical activity, for molecularly defined cell types. These tools have revealed remarkably specific behavioral physiological influences for molecularly defined cell types that are often intermingled with populations having different or even opposite functions.

 

A Method for Remotely Silencing Neural Activity in Rodents During Discrete Phases of Learning.

J Vis Exp. 2015 Jun 22;(100):e52859. doi: 10.3791/52859.

This protocol describes how to temporarily and remotely silence neuronal activity in discrete brain regions while animals are engaged in learning and memory tasks. The approach combines pharmacogenetics (Designer-Receptors-Exclusively-Activated-by-Designer-Drugs) with a behavioral paradigm (sensory preconditioning) that is designed to distinguish between different forms of learning. Specifically, viral-mediated delivery is used to express a genetically modified inhibitory G-protein coupled receptor (the Designer Receptor) into a discrete brain region in the rodent. Three weeks later, when designer receptor expression levels are high, a pharmacological agent (the Designer Drug) is administered systemically 30 min prior to a specific behavioral session. The drug has affinity for the designer receptor and thus results in inhibition of neurons that express the designer receptor, but is otherwise biologically inert. The brain region remains silenced for 2-5 hr (depending on the dose and route of administration). Upon completion of the behavioral paradigm, brain tissue is assessed for correct placement and receptor expression. This approach is particularly useful for determining the contribution of individual brain regions to specific components of behavior and can be used across any number of behavioral paradigms.
It is important to indicate that after the protein has being made it acts in fast form (milliseconds etc,) as protein do…
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Notable Awards – 2015

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Breakthrough Prizes Give Top Scientists the Rock Star Treatment

“By challenging conventional thinking and expanding knowledge over the long term, scientists can solve the biggest problems of our time,” Mr. Zuckerberg said in a statement. “The Breakthrough Prize honors achievements in science and math so we can encourage more pioneering research and celebrate scientists as the heroes they truly are.”

Left, Karl Deisseroth, Stanford School of Medicine; Edward S. Boyden of the McGovern Institute for Brain Research at M.I.T.CreditLeft, Winni Wintermeyer for The New York Times; Dominick Reuter/M.I.T. News

http://i1.nyt.com/images/2015/11/08/science/08breakthrough_comp_1/08breakthrough_comp_1-tmagArticle.jpg

Karl Deisseroth and Edward S. Boyden
Optogenetics

Karl Deisseroth, a professor at Stanford University and a Howard Hughes Medical Institute investigator, and Edward S. Boyden, a professor at the Massachusetts Institute of Technology, each received $3 million for their roles in the development of optogenetics, a technique that allows scientists to use light to turn neurons and groups of neurons on and off.

The technique is transforming the study of the brain because it allows scientists to test ideas about how the brain works. It has already been used to turn a kind of aggression on and off in flies, and thirst on and off in mice, pinpointing the brain cells involved.

The technique is universally praised, but the question of who will be recognized for its development is an issue for any prize committee. Dr. Boyden, Dr. Deisseroth and three other scientists published a paper in 2005that is recognized as a breakthrough. They demonstrated how to reliably control mammalian neurons with light, making widespread use of the technique inevitable.

Their paper built on earlier work, as much of science does. Opsins, light-sensitive chemicals that are crucial to optogenetics, have been studied since the 1970s. And the fact that optogenetics could be done was demonstrated in 2002.

In 2013, the European Brain Prize recognized six scientists for work on optogenetics, including Dr. Boyden and Dr. Deisseroth.

JAMES GORMAN

 

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John Hardy
Alzheimer’s research

Alzheimer’s disease was a complete mystery in the late 1980s. In autopsies, pathologists could see the ravages left in patients’ brains, but how and why did the process start? There were rare families in which the disease seemed to be inherited, though, and perhaps there was a gene mutation that might provide a clue to what goes awry. The problem was finding those families.

In the late 1980s, a woman who lived in Nottingham, England, contacted John Hardy at University College London and asked if he and his team wanted to study her family. Her father was one of 10 siblings, five of whom had developed Alzheimer’s disease, and she could trace the disease back for three generations. Their investigation led to the discovery of a gene mutation that, if inherited, always caused the disease. The gene was presenilin, and its protein was the amyloid precursor protein, or APP. Every person in that family who inherited the gene overproduced amyloid and got the disease. For the first time, scientists had a clue to what starts the horrendous destruction of brain cells in Alzheimer’s disease. And for the first time, by putting that gene mutation in mice, they could study Alzheimer’s in a lab animal, look for drugs to block the gene’s effects and finally use the tools of science to look for a cure.

GINA KOLATA

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Helen Hobbs
Cholesterol research

Helen Hobbs, a professor at the University of Texas Southwestern Medical Center and a Howard Hughes Medical Institute investigator, and her colleague Jonathan Cohen were intrigued when they read a short paper describing a French family with stunningly high levels of LDL cholesterol, the dangerous kind, and early deaths from heart attacks and strokes. The family members turned out to have a mutation in a gene, PCSK9, whose function was unknown. Dr. Hobbs and Dr. Cohen began to wonder: If too much PCSK9 caused heart disease, would people who made too little be protected? They scrutinized genetic data from a federal study and found that about 2.5 percent of blacks had a mutation that destroyed one copy of the gene; 3.2 percent of whites had a mutation that hobbled a copy of the gene but did not destroy it. In both cases, less PCSK9 was made and LDL levels were low. The people with the mutations seemed almost immune to heart disease, even if they had other risk factors like high blood pressure, smoking or diabetes.

What would happen if someone had both copies of PCSK9 destroyed? Dr. Hobbs found one young woman, an aerobics instructor, without PCSK9. She was healthy and fertile even though her LDL level was 14, lower than seemed possible (the average is 100). That discovery led to a race among drug companies to make cholesterol-lowering drugs that mimicked the effects of the PCSK9 mutations. The result is drugs that can make LDL levels plunge to the 30s, the 20s, even the teens. The first two such PCSK9 inhibitors were approved this year for people with high cholesterol levels who cannot get them down with statins and are at high risk of heart disease.

GINA KOLATA

 

TED Prize Goes to Archaeologist Who Combats Looting With Satellite Technology
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http://www.nytimes.com/2015/11/09/arts/international/ted-grant-goes-to-archaeologist-who-combats-looting-with-satellite-technology.html

 

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Recent Insights in Drug Development

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

A Better Class of Cancer Drugs
http://www.technologynetworks.com/medchem/news.aspx?ID=183124
An SDSU chemist has developed a technique to identify potential cancer drugs that are less likely to produce side effects.
A class of therapeutic drugs known as protein kinase inhibitors has in the past decade become a powerful weapon in the fight against various life-threatening diseases, including certain types of leukemia, lung cancer, kidney cancer and squamous cell cancer of the head and neck. One problem with these drugs, however, is that they often inhibit many different targets, which can lead to side effects and complications in therapeutic use. A recent study by San Diego State University chemist Jeffrey Gustafson has identified a new technique for improving the selectivity of these drugs and possibly decreasing unwanted side effects in the future.

Why are protein kinase–inhibiting drugs so unpredictable? The answer lies in their molecular makeup.

Many of these drug candidates possess examples of a phenomenon known as atropisomerism. To understand what this is, it’s helpful to understand a bit of the chemistry at work. Molecules can come in different forms that have exactly the same chemical formula and even the same bonds, just arranged differently. The different arrangements are mirror images of each other, with a left-handed and a right-handed arrangement. The molecules’ “handedness” is referred to as chirality. Atropisomerism is a form of chirality that arises when the spatial arrangement has a rotatable bond called an axis of chirality. Picture two non-identical paper snowflakes tethered together by a rigid stick.

Some axes of chirality are rigid, while others can freely spin about their axis. In the latter case, this means that at any given time, you could have one of two different “versions” of the same molecule.

Watershed treatment

As the name suggests, kinase inhibitors interrupt the function of kinases—a particular type of enzyme—and effectively shut down the activity of proteins that contribute to cancer.

“Kinase inhibition has been a watershed for cancer treatment,” said Gustafson, who attended SDSU as an undergraduate before earning his Ph.D. in organic chemistry from Yale University, then working there as a National Institutes of Health poctdoctoral fellow in chemical biology.

“However, it’s really hard to inhibit a single kinase,” he explained. “The majority of compounds identified inhibit not just one but many kinases, and that can lead to a number of side effects.”

Many kinase inhibitors possess axes of chirality that are freely spinning. The problem is that because you can’t control which “arrangement” of the molecule is present at a given time, the unwanted version could have unintended consequences.

In practice, this means that when medicinal chemists discover a promising kinase inhibitor that exists as two interchanging arrangements, they actually have two different inhibitors. Each one can have quite different biological effects, and it’s difficult to know which version of the molecule actually targets the right protein.

“I think this has really been under-recognized in the field,” Gustafson said. “The field needs strategies to weed out these side effects.”

Applying the brakes

So that’s what Gustafson did in a recently published study. He and his colleagues synthesized atropisomeric compounds known to target a particular family of kinases known as tyrosine kinases. To some of these compounds, the researchers added a single chlorine atom which effectively served as a brake to keep the atropisomer from spinning around, locking the molecule into either a right-handed or a left-handed version.

When the researchers screened both the modified and unmodified versions against their target kinases, they found major differences in which kinases the different versions inhibited. The unmodified compound was like a shotgun blast, inhibiting a broad range of kinases. But the locked-in right-handed and left-handed versions were choosier.

“Just by locking them into one or another atropisomeric configuration, not only were they more selective, but they  inhibited different kinases,” Gustafson explained.

If drug makers incorporated this technique into their early drug discovery process, he said, it would help identify which version of an atropisomeric compound actually targets the kinase they want to target, cutting the potential for side effects and helping to usher drugs past strict regulatory hurdles and into the hands of waiting patients.

 

Inroads Against Leukaemia
http://www.technologynetworks.com/medchem/news.aspx?ID=183594

 

Potential for halting disease in molecule isolated from sea sponges.
A molecule isolated from sea sponges and later synthesized in the lab can halt the growth of cancerous cells and could open the door to a new treatment for leukemia, according to a team of Harvard researchers and other collaborators led by Matthew Shair, a professor of chemistry and chemical biology.

“Once we learned this molecule, named cortistatin A, was very potent and selective in terms of inhibiting the growth of AML [acute myeloid leukemia] cells, we tested it in mouse models of AML and found that it was as efficacious as any other molecule we had seen, without having deleterious effects,” Shair said. “This suggests we have identified a promising new therapeutic approach.”

It’s one that could be available to test in patients relatively soon.

“We synthesized cortistatin A and we are working to develop novel therapeutics based on it by optimizing its drug-like properties,” Shair said. “Given the dearth of effective treatments for AML, we recognize the importance of advancing it toward clinical trials as quickly as possible.”

The drug-development process generally takes years, but Shair’s lab is very close to having what is known as a development candidate that could be taken into late-stage preclinical development and then clinical trials. An industrial partner will be needed to push the technology along that path and toward regulatory approval. Harvard’s Office of Technology Development (OTD) is engaged in advanced discussions to that end.

The molecule works, Shair explained, by inhibiting a pair of nearly identical kinases, called CDK8 and CDK19, that his research indicates play a key role in the growth of AML cells.

The kinases operate as part of a poorly understood, massive structure in the nucleus of cells called the mediator complex, which acts as a bridge between transcription factors and transcriptional machinery. Inhibiting these two specific kinases, Shair and colleagues found, doesn’t shut down all transcription, but instead has gene-specific effects.

“We treated AML cells with cortistatin A and measured the effects on gene expression,” Shair said. “One of the first surprises was that it’s affecting a very small number of genes — we thought it might be in the thousands, but it’s in the low hundreds.”

When Shair, Henry Pelish, a senior research associate in chemistry and chemical biology, and then-Ph.D. student Brian Liau looked closely at which genes were affected, they discovered many were associated with DNA regulatory elements known as “super-enhancers.”

“Humans have about 220 different types of cells in their body — they all have the same genome, but they have to form things like skin and bone and liver cells,” Shair explained. “In all cells, there are a relatively small number of DNA regulatory elements, called super-enhancers. These super-enhancers drive high expression of genes, many of which dictate cellular identity. A big part of cancer is a situation where that identity is lost, and the cells become poorly differentiated and are stuck in an almost stem-cell-like state.”

While a few potential cancer treatments have attacked the disease by down-regulating such cellular identity genes, Shair and colleagues were surprised to find that their molecule actually turned up the activity of those genes in AML cells.

“Before this paper, the thought was that cancer is ramping these genes up, keeping the cells in a hyper-proliferative state and affecting cell growth in that way,” Shair said. “But our molecule is saying that’s one part of the story, and in addition cancer is keeping the dosage of these genes in a narrow range. If it’s too low, the cells die. If they are pushed too high, as with cortistatin A, they return to their normal identity and stop growing.”

Shair’s lab became interested in the molecule several years ago, shortly after it was first isolated and described by other researchers. Early studies suggested it appeared to inhibit just a handful of kinases.

“We tested approximately 400 kinases, and found that it inhibits only CDK8 and CDK19 in cells, which makes it among the most selective kinase inhibitors identified to date,” Shair said. “Having compounds that precisely hit a specific target, like cortistatin A, can help reduce side effects and increase efficacy. In a way, it shatters a dogma because we thought it wasn’t possible for a molecule to be this selective and bind in a site common to all 500 human kinases, but this molecule does it, and it does it because of its 3-D structure. What’s interesting is that most kinase-inhibitor drugs do not have this type of 3-D structure. Nature is telling us that one way to achieve this level of specificity is to make molecules more like cortistatin A.”

Shair’s team successfully synthesized the molecule, which helped them study how it worked and why it affected the growth of a very specific type of cell. Later on, with funding and drug-development expertise provided by Harvard’s Blavatnik Biomedical Accelerator, Shair’s lab created a range of new molecules that may be better suited to clinical application.

“It’s a complex process to make [cortistatin A] — 32 chemical steps,” said Shair. “But we have been able to find less complex structures that act just like the natural compound, with better drug-like properties, and they can be made on a large scale and in about half as many steps.”

“Over the course of several years, we have watched this research progress from an intriguing discovery to a highly promising development candidate,” said Isaac Kohlberg, senior associate provost and chief technology development officer. “The latest results are a real testament to Matt’s ingenuity and dedication to addressing a very tough disease.”

While there is still much work to be done — in particular, to better understand how CDK8 and CDK19 regulate gene expression — the early results have been dramatic.

“This is the kind of thing you do science for,” Shair said, “the idea that once every 10 or 20 years you might find something this interesting, that sheds new light on important, difficult problems. This gives us an opportunity to generate a new understanding of cancer and also develop new therapeutics to treat it. We’re very excited and curious to see where it goes.”

 

Seeking A Better Way To Design Drugs

http://www.technologynetworks.com/medchem/news.aspx?ID=183338

NIH funds research at Worcester Polytechnic Institute to advance a new chemical process for more effective drug development and manufacturing.
The National Institutes of Health (NIH) has awarded $346,000 to Worcester Polytechnic Institute (WPI) for a three-year research project to advance development of a chemical process that could significantly improve the ability to design new pharmaceuticals and streamline the manufacturing of existing drugs.

Led by Marion Emmert, PhD, assistant professor of chemistry and biochemistry at WPI, the research program involves early-stage technology developed in her lab that may yield a more efficient and predictable method of bonding a vital class of structures called aromatic and benzylic amines to a drug molecule.

“Seven of the top 10 pharmaceuticals in use today have these substructures, because they are so effective at creating a biologically active compound,” Emmert said. “The current processes used to add these groups are indirect and not very efficient. So we asked ourselves, can we do it better? ”

For a drug to do its job in the body it must interact with a specific biological target and produce a therapeutic effect. First, the drug needs to physically attach or “bind” to the target, which is a specific part of a cell, protein, or molecule. As a result, designing a new drug is like crafting a three-dimensional jigsaw puzzle piece that fits precisely into an existing biological structure in the body. Aromatic and benzylic amines add properties to the drug that help it bind more efficiently to these biological structures.

Getting those aromatic and benzylic amines into the structure of a drug, however, is difficult. Traditionally, this requires a specialized chemical bond as precursor in a specific location of the drug’s molecular structure. “The current approach to making those bonds is indirect, requires several lengthy steps, and the outcome is not always precise or efficient,” Emmert said. “Only a small percentage of the bonds can be made in the proper place, and sometimes none at all.”

Emmert’s new approach uses novel reagents and metal catalysts to create a process that can attach amines directly, in the right place, every time. In early proof-of-principle experiments, Emmert has succeeded in making several amine bonds directly in one or two days, whereas the standard process can take two weeks with less accuracy. Over the next three years, with support from the NIH, Emmert’s team will continue to study the new catalytic processes in detail. They will also use the new process to synthesize Asacol, a common drug now in use for ulcerative colitis, and expect to significantly shorten its production.

“Some of our early data are promising, but we have a lot more work to do to understand the basic mechanisms involved in the new processes,” Emmert said. “We also have to adapt the process to molecules that could be used directly for drug development.”

 

Antiparasite Drug Developers Win Nobel

William Campbell, Satoshi Omura, and Youyou Tu have won this year’s Nobel Prize in Physiology or Medicine in recognition of their contributions to antiparasitic drug development.

By Karen Zusi and Tracy Vence | October 5, 2015

http://www.the-scientist.com//?articles.view/articleNo/44159/title/Antiparasite-Drug-Developers-Win-Nobel/

William Campbell, Satoshi Omura, and Youyou Tu have made significant contributions to treatments for river blindness, lymphatic filariasis, and malaria; today (October 5) these three scientists were jointly awarded the 2015 Nobel Prize in Physiology or Medicine in recognition of these advancements.

Tu is being recognized for her discoveries leading to the development of the antimalarial drug artemisinin. Campbell and Omura jointly received the other half of this year’s prize for their separate work leading to the discovery of the drug avermectin, which has been used to develop therapies for river blindness and lymphatic filariasis.

“These discoveries are now more than 30 years old,” David Conway, a professor of biology of the London School of Hygiene & Tropical Medicine, told The Scientist. “[These drugs] are still, today, the best two groups of compounds for antimalarial use, on the one hand, and antinematode worms and filariasis on the other.”

Omura, a Japanese microbiologist at Kitasato University in Tokyo, isolated strains of the soil bacteriaStreptomyces in a search for those with promising antibacterial activity. He eventually narrowed thousands of cultures down to 50.

Now research fellow emeritus at Drew University in New Jersey, Campbell spent much of his career at Merck, where he discovered effective antiparasitic properties in one of Omura’s cultures and purified the relevant compounds into avermectin (later refined into ivermectin).

“Bill Campbell is a wonderful scientist, a wonderful man, and a great mentor for undergraduate students,” said his colleague Roger Knowles, a professor of biology at Drew University. “His ability to speak about disease mechanisms and novel strategies to help [fight] these diseases. . . . that’s been a great boon to students.”

Tu began searching for a novel malaria treatment in the 1960s in traditional herbal medicine. She served as the head of Project 523, a program at the China Academy of Chinese Medical Sciences in Beijing aimed at finding new drugs for malaria. Tu successfully extracted a promising compound from the plant Artemisia annu that was highly effective against the malaria parasite. In recognition of her malaria research, Tu won a Lasker Award in 2011.

 

Optogenetics Advances in Monkeys

Researchers have selectively activated a specific neural pathway to manipulate a primate’s behavior.

By Kerry Grens | October 5, 2015

http://www.the-scientist.com//?articles.view/articleNo/44156/title/Optogenetics-Advances-in-Monkeys/

Scientists have used optogenetics to target a specific neural pathway in the brain of a macaque monkey and alter the animal’s behavior. As the authors reported in Nature Communications last month, such a feat had been accomplished only in rodents before.

Optogenetics relies on the insertion of a gene for a light-sensitive ion channel. When present in neurons, the channel can turn on or off the activity of a neuron, depending on the flavor of the channel. Previous attempts to use optogenetics in nonhuman primates affected brain regions more generally, rather than particular neural circuits. In this case, Masayuki Matsumoto of Kyoto University and colleagues delivered the channel’s gene specifically to one area of the monkey’s brain called the frontal eye field.

They found that not only did the neurons in this region respond to light shone on the brain, but the monkey’s behavior changed as well. The stimulation caused saccades—quick eye movements. “Our findings clearly demonstrate the causal relationship between the signals transmitted through the FEF-SC [frontal eye field-superior colliculus] pathway and saccadic eye movements,” Matsumoto and his colleagues wrote in their report.

“Over the decades, electrical microstimulation and pharmacological manipulation techniques have been used as tools to modulate neuronal activity in various brain regions, permitting investigators to establish causal links between neuronal activity and behaviours,” they continued. “These methodologies, however, cannot selectively target the activity (that is, the transmitted signal) of a particular pathway connecting two regions. The advent of pathway-selective optogenetic approaches has enabled investigators to overcome this issue in rodents and now, as we have demonstrated, in nonhuman primates.”

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