Discovery route: Path to potential diabetes drugs began with a simple question

More than 12 years of research led Anath Shalev, M.D. (right, with Junqin Chen, Ph.D.) from a basic discovery to the first human trial of a new type of diabetes drug.

In 2002, diabetes researcher Anath Shalev, M.D., asked a basic question: What gene in the insulin-producing islets of the human pancreas is most turned on by high levels of glucose, a hallmark of diabetes?

The answer has led the UAB endocrinologist to discover new cellular pathways in beta cells of the islets, pathways that are a key to diabetes progression or protection. Those discoveries have now opened the door to the first human trial of a potential diabetes drug with a mode of action different from any current diabetes treatment. (Learn more about the trial, which will begin in early 2015, in this story.)

Anath Shalev explains verapamil's protective effects against diabetes, and a new human clinical trial of the drug at UAB, in this video.

Beta cells are critical in type 1 and type 2 diabetes. In both diseases, the cells are lost gradually due to programmed cell death (apoptosis); but the trigger for that programmed death was unknown. The loss of beta cells contributes to the progression of diabetes, a growing worldwide epidemic that affects more than 20 million people in the United States, making it the seventh leading cause of death and the source of complications like blindness and more than 40,000 lower limb amputations a year.

From Molecular Mechanisms to New Treatments

The beta-cell gene that responded to the high glucose in Shalev’s 2002 experiment produces TXNIP (pronounced "ticks-nip"), a protein normally involved in controlling oxygen radicals in many types of cells but never known to be important in beta-cell biology. Its response to glucose was intriguing because TXNIP (thioredoxin-interacting protein) was already recognized as a regulator of thioredoxin. Overexpression of thioredoxin had previously been shown to prevent experimentally induced diabetes by inhibiting the programmed death of islet beta cells. Since TXNIP inhibits thioredoxin, and because Shalev had discovered that islet TXNIP was highly regulated by glucose, Shalev realized that TXNIP might have major implications for beta-cell biology.

What does it take to go from a basic microarray gene discovery to a human trial of a completely novel drug to treat diabetes?

A dozen years of elegant research unraveling the control and function of a protein called TXNIP.

Over the next dozen years, Shalev — who left the University of Wisconsin–Madison to head the UAB Comprehensive Diabetes Center in 2010 — set out to reveal how TXNIP acts in cells at the molecular level, knowing that an understanding of those molecular mechanisms might point to possible new diabetes treatments. The payoff has been substantial: Using cell cultures, mouse models and pancreatic islets isolated from humans, the Shalev lab team has shown that manipulating TXNIP levels up or down in beta cells could exacerbate or protect against experimental diabetes.

Details about the research journey show the incremental steps that basic science takes, and how those connected steps sometimes lead to potential clinical impacts.

Controlling TXNIP to Treat Diabetes

In 2005, the Shalev lab team found that beta-cell TXNIP levels are higher in mouse diabetes models, and that experimentally increasing TXNIP levels in rat beta cells in vitro led to increased programmed cell death, by means of a well-known trigger signal of apoptosis. The Shalev team also found that sugars in general, whether metabolized or not, turn the TXNIP gene on. This clue led them to a newly identified carbohydrate response element (ChoRE) in the TXNIP promoter that acts as a regulator of TXNIP.

In 2008, the Shalev lab developed mice that had little or no TXNIP in their beta cells. These lower levels protected against experimental diabetes. The team also discovered that the lower levels sent a known signal that inhibited mitochondrial beta-cell death. Shalev wrote, “These results suggest that lowering beta-cell TXNIP production could serve as a novel strategy for the treatment of type 1 and type 2 diabetes by promoting endogenous beta-cell survival.”

An Approved Drug Offers Protection

In 2012, the Shalev group tested an already approved oral drug that they had earlier found to reduce levels of TXNIP in heart cells. The drug — verapamil — is a calcium channel blocker used primarily to treat high blood pressure, but also to treat migraine headaches. Shalev’s team found that exposing in vitro beta cells or isolated human islets to verapamil reduced TXNIP expression, and halted programmed apoptotic death of beta cells. Furthermore, mice that were fed verapamil in their drinking water were protected from experimentally induced diabetes, and verapamil rescued mice that already had diabetes. The verapamil mice had lower TXNIP levels and less programmed beta-cell death, as well as better levels of insulin

"I actually went down to the mouse house to see if the mice were getting diabetes," Shalev told The Birmingham News in 2012. When she found normal glucose levels, "We were dancing."

In those studies, the group also revealed how verapamil lowers TXNIP — the decreased intracellular level of calcium ions caused by verapamil led to phosphorylation of the ChoRE binding protein that normally responds to glucose to control TXNIP transcription at the ChoRE. This phosphorylation prevented the binding protein from entering the beta-cell nucleus and interacting with the TXNIP gene. Shalev noted that these verapamil results identified, for the first time, “… an effective pharmacological means … to inhibit pancreatic beta-cell expression of proapoptotic TXNIP, enhance beta-cell survival and function, and thereby prevent and even improve overt diabetes and shed light on the mechanisms involved.”

Another Role for TXNIP, Another Drug Target?

In 2013, TXNIP was shown to play another crucial role in beta-cell biology when the Shalev laboratory team discovered that high levels of TXNIP directly blocked insulin production in beta cells, acting through a newly identified pathway. TXNIP, they found, induced a microRNA called miR-204, which in turn down-regulated the MAFA transcription factor involved in promoting transcription of the insulin gene.

This means that miR-204 may offer another target for a future RNA drug, an area that is currently also being actively pursued by the Shalev lab. MicroRNAs, with 20 to 24 noncoding nucleotides, have rapidly gained prominence as regulators of gene expression in health and disease. Researchers are beginning to explore whether silencing targeted microRNAs may lead to a treatment for cancers or other diseases.

TXNIP's Vicious Cycle

This year Shalev reported that TXNIP — surprisingly — can induce its own transcription. Her UAB research team found that TXNIP does this by affecting the same ChoRE binding protein (ChREBP) that was previously found to be key in the response to the drug verapamil. The researchers experimentally elevated TXNIP levels in beta cells and found this caused decreased phosphorylation of ChREBP, which led to its increased entry into the nucleus and its increased binding to the TXNIP promoter to boost transcription. This creates a harmful positive-feedback loop.

"These findings support the notion,” Shalev wrote in this 2014 paper, “that TXNIP levels rise over time, not only as a result of elevated blood glucose levels and/or endoplasmic reticulum stress, but also as part of a vicious cycle by which increased TXNIP levels lead to more TXNIP expression and thereby amplify the associated detrimental effects on beta-cell biology including oxidative stress, inflammation, and ultimately beta-cell death and disease progression.”

First Human Trial

Get a quick overview of the science behind UAB's verapamil
trial in this animation

The story doesn’t end here. Shalev’s long trail of laboratory research has now led to the first human trial to see if verapamil has an effect in patients who have developed type 1 diabetes within the previous three months. Adult volunteers, ages 19-45, will be treated with verapamil or a placebo for one year, as their insulin and blood glucose levels are continuously monitored. The three-year, $2.2 million trial will be conducted by the UAB Comprehensive Diabetes Center with funding from JDRF, the largest charitable supporter of type 1 diabetes research.

Meanwhile, a UAB partnership with the Southern Research Institute — called the Alabama Drug Discovery Alliance — is already working to develop small therapeutic molecules that mimic the diabetes-protecting effect produced by verapamil and inhibit TXNIP, but have a greater selectivity and efficacy. [Learn more about this work, and other high-potential projects in the Alabama Drug Discovery Alliance, in a new feature from UAB Magazine.]

So Shalev’s simple question — what gene in insulin-producing beta cells is most turned on by glucose? — has thus led the research out of her laboratory to possible new drugs, acting against a novel target to alleviate or reverse diabetes.

— Jeff Hansen

Using magnets to find new drugs: Inside UAB's high-field nuclear magnetic resonance facility

Most high-end lab equipment is inaccessible to the public eye, but one of UAB's most powerful drug-discovery tools is clearly visible from the Campus Green. The Central Alabama High Field Nuclear Magnetic Resonance Facility occupies a gleaming ground-floor space in the Chemistry Building. Its massive magnets give researchers invaluable insight into disease-causing proteins — and the data they need to find new ways to stop them.

The cover story of the latest issue of UAB Magazine features the Alabama Drug Discovery Alliance, a partnership between UAB and Southern Research Institute that aims to accelerate high-potential discoveries from the lab to patient-ready treatments. One key tool in that process is the Central Alabama High Field Nuclear Magnetic Resonance Facility, which opened in 2013. The Mix takes a closer look in this new feature.

Spin This Way

Each of the facility's NMR machines specializes in a different type of job, but the basic functioning is the same, explains NMR director N. Rama Krishna, Ph.D., UAB professor in the Department of Biochemistry and Molecular Genetics. The machines generate strong magnetic fields that polarize the tiny magnets in the nuclei of hydrogen atoms. “Then, using radiofrequency pulses, you can count all of the individual hydrogen atoms in a sample, which tells you what amino acids are present and how they are arranged in space,” Krishna says. And that’s precisely the information you need to create a detailed picture of a protein’s structure.

Mapping a protein's structure is crucial to understanding its function — and to finding ways to alter that function to treat disease. For instance, locating suitable "binding pockets" on a protein linked to brain cancer tells medicinal chemists how to design a drug to block (or enhance) that protein. "That's why NMR is one of the most versatile tools for drug-discovery research," Krishna says.

The bigger your magnet, the better images you can get. The centerpiece of the NMR facility is an 850 MHz Bruker BioSpin model, one of the largest in the South, which allows scientists to analyze structural data on even the largest proteins.

(Story continues after graphic)

Building a Better Drug

The 850 MHz machine can also accelerate the drug-discovery process "by allowing researchers to rapidly test new compounds they've developed in the lab," Krishna adds. Using a technique called saturation transfer difference NMR (STD-NMR), Krishna and his team can register the minute changes in signals from hydrogen atoms that occur when a compound binds to a protein. It would be nearly impossible to capture this interaction directly, he points out, because "it may last only a few microseconds." With STD-NMR, researchers can screen a number of potential drugs at once, then focus on the ones that show signs of binding to the target protein.

UAB's Rama Krishna and scientists from Southern Research
Institute have collaborated in developing a novel high-field
NMR-based protocol for determining the binding of
allosteric ligands to target proteins. They used the kinesin-5
protein Eg5 (a cancer target) and its inhibitor monastrol
 as an example (see above) for this protocol.

Using other techniques, researchers can analyze the disease-causing interaction between two proteins, and then find the right location to dock an inhibitor that would prevent the proteins from coming together. Or they could do the opposite, in an approach dubbed “fragment-based discovery” — using NMR data to identify two compounds that bind close together on a protein and “cross link” them to significantly improve their binding.

Krishna uses these techniques in his own National Cancer Institute-funded research to find new treatments for pancreatic cancer. Other UAB investigators are using the NMR facility to further their drug-discovery efforts in Parkinson's disease, brain tumors, breast cancer, heart disease, HIV and more. And as word of these capabilities has spread, researchers at institutions across the South have begun sending in samples to the NMR facility for evaluation.

Early Warning Signs

NMR is useful for many applications beyond drug discovery, Krishna adds. The facility's 600 MHz machine specializes in a hot area of medicine known as metabolomics, which studies the way the body processes everything from food to medicines.

"If you are taking a drug that is toxic to the liver, the body will generate some small molecules — known as metabolites — associated with liver damage,” Krishna explains. "We can detect these molecules in the NMR spectra of biofluids such as urine and blood plasma and say, 'Aha, after this patient started taking the drug, we can see an increase in these signals, so something is going wrong." That can warn researchers of side effects from new drug treatments "long before there is any major problem," Krishna says.

"The range of applications in this facility is amazing," adds Krishna. "It is a unique platform for everything from basic science to translational research.”

UAB research rides into space on a Dragon

In 1992, UAB's Larry DeLucas, O.D., Ph.D., went into space aboard the Space Shuttle Columbia to conduct protein crystal growth experiments in orbit. Imaging protein crystals has great potential for drug discovery efforts; a good picture of a protein's structure can provide invaluable information to scientists looking for new ways to alter that protein in order to treat disease. The problem is, Earth's gravity interferes with protein formation. The gravity-free environment of space is much more conducive to crystal formation.

Today, DeLucas is director of the UAB Center for Biophysical Sciences and Engineering and principal investigator on a $6 million project to demonstrate the scientific and commercial potential of protein crystallization. (Learn more in this story from UAB News.)

Nearly 100 difficult-to-image proteins will soar into orbit on the SpaceX Dragon spacecraft.

In the meantime, check out the Dragon in this SpaceX video:

Microbe-made molecules may be future drugs

Our ancestors first “invited in” gut bugs 450 million years ago because it let them harness bacterial enzymes to get more energy from more kinds of food. Today, microbes contribute 360 times as many genes responsible for the human ability to convert food into energy as human genes themselves. Complex microbial communities occupy our skin, nose and mouth as well, and humans and their bugs may have become a single super-organism.

The subject made national news in June when the Human Microbiome Project, NIH-funded effort to catalog the mix of bugs living on and in Americans, reported its first results. With the typical set of bugs now outlined, researchers are searching for the bug profiles that correlate with diseases, including cancer.

Against this backdrop, the UAB Comprehensive Cancer Center chose "cancer and the microbiome" as the theme for its recent research retreat. The Mix interviewed several retreat presenters, and is featuring the chats as a podcast series.

Our guest for this last podcast in the series is James Versalovic, M.D., Ph.D., professor in the Department of Pathology and Immunology at Baylor College of Medicine. We talked about how new understanding of the mechanistic details behind human cell/microbial crosstalk may lead to new treatments.  

 

Show notes for the podcast

2:08 Different sites in the body play host to entirely different complex communities of bacteria and other microbes. 

2:45 The line is blurry between microbial cells and human cells because they constantly "talk" as they work together to do so many jobs in the human body. 

3:33  This conversation is really an exchange of biochemical signals, some of them carried by small molecules produced by microbes, the subject of Dr.Versalovic's presentation at the UAB retreat. Microbial small molecules were first studied because they interact with our immune system to cause inflammation.  More broadly, evidence is emerging that human organs evolved in such close cooperation with microbe-made molecules that such molecules have become critical to the ability of several organs to function.

4:34 As a baby is born, all the tools are in place for his or her immune system to develop, but those tools are not trained yet to work in the real world. Exposure to many bugs starts at birth, and in fact, the mother's bugs help to determine the baby's mix of bug species. 

4:48 One might think the most important lessons learned by a baby's immune cells are about which invading organisms to attack and destroy to protect the body from infection. In fact, much of the education is about tolerance. The cells develop in the presence of many helpful bugs, and learn not to become activated to easily to cause unwanted inflammation. A mature system only loses its cool when faced with a considerable threat. 

6:17  Just like some people who are quick to anger, some people happen to have a labile immune systems that too often and in the wrong context becomes activated. Not having had the proper education, such oversensitive system can lead to systemic autoimmune, allergic and inflammatory conditions like inflammatory bowel disease. 

7:25 Babies' microbiomes are getting off to different starts in life based on whether they are delivered vaginally versus through C-section.  A C-section baby is more likely to start with bacteria from a mother's skin, where the kid born via "natural childbirth" starts with the mother's gut bugs in his or her gut. Over time the babies' bodies compensate but there could be long-term consequences. 

9:09  Normally, the microbiome helps to keep the immune system in check, so that it is not constantly overreacting to cause systemic inflammation. Over time though, things like diet, obesity or smoking, perhaps a bad infection, may alter this balance.

10:25 A goal of Dr. Versalovic's effort to understand how microbial small molecules signal to the immune system may inform efforts to design drugs that calm down the immune system the same way a healthy microbiome does. Researcher may be able to synthesize compounds made by bacteria, or compounds in the diet changed by gut bacteria, which improve organ function. 

11:40 We feed our microbiome when we feed ourselves, so it pays to chose your diet carefully. As we understand it better, we will have better idea of how the molecules making up food interact with various microbial species to impact health and disease. 

12:25 The compounds produced by interactions between the gut microbiome and food may be affecting physiology throughout the body, including in the brain, where early work has tied diet-driven changes in the gut microbiome to behavioral changes. 

15:03 Dr. Versalovic recommends that students and researchers interested in finding out more about the microbiome visit the Human Microbiome Project's DACC site.

Please click on the following links to listen to the other podcasts in this series. 

Gut bugs drive cancer risk?

Massive computing power reveals gut bug/cancer links

Next gen sequencing reveals bug-driven cancer risk

Gut bugs and estrogen-related cancer

UAB team sets sights on neuroprotection

Neurological diseases are notoriously complex, and drugs have not improved significantly in decades. The main drug treatment for Parkinson's disease, L-DOPA, was first approved for use in 1970. It temporarily staves off symptoms but can itself cause heart arrhythmias, stomach bleeding and hallucinations. Patients with Parkinson's die at twice the rate of those without the disease.

For these reasons, researchers have been urgently seeking for years to understand Parkinson's to the point where they can begin to design drugs that go beyond symptom relief to counter the inflammation and nerve cell death at the disease's root. A team of researchers from the University of Alabama at Birmingham gave a presentation today at Neuroscience 2012, the annual meeting of the Society for Neuroscience in New Orleans,  in which they revealed that they may be approaching that point. The researchers have designed a set of experimental drugs called LRRK2 inhibitors that show evidence of protecting nerve cells, at least in the rodent and cell culture studies they have carried out so far, which are meant to approximate human disease.

But these are still just models, and therein lies the problem. Despite the excitement among researchers, when should patients begin to raise their expectations?

The UAB research team, and the field of neurology in general, is excited just to have identified an enzyme like LRRK2 against which they can design drugs that could reverse underlying disease processes. That would be a first for any neurodegenerative disease. Along with evidence that LRRK2 plays a crucial role in the mechanisms of Parkinson’s disease, it is the same kind of enzyme (although not the same one) that has been successfully targeted by existing cancer treatments, including Herceptin. On the other hand, the UAB team's LRRK2 inhibitors are still years away from human clinical trials. They must pass several basic tests (e.g. toxicology tests) before even being considered for human trials, and a great many drug candidates fail at this stage.

Perhaps the best we can do is to set down the facts, and offer just enough hope while avoiding hype.

The Mix sat down with Andrew West, Ph.D., associate professor in the Department of Neurology within the UAB School of Medicine, who gave the presentation today at Neuroscience 2012. We wanted his take on what has been accomplished so far, and on what lies ahead. Also please take a look at our related press release on his meeting presentation.


 
Show notes for the interview:

1:00 Patients are surprised to hear that there is today no treatment that reverses the underlying disease processes related to Parkinson's disease, and that the focus for decades has been on symptom relief only.

1:37 The other surprise facing newly diagnosed patients is that most of the treatments in use today were developed at least 50 years ago, so it's frustrating for them to learn about how limited their options are.

1:48 Traditionally, researchers have seen the death of nerve cells that make dopamine, the signaling chemical that contributes to our ability to control our movements, as the relentlessly progressive disease process underlying Parkinson's disease. This would explain how the disease, as it gets worse, eventually overwhelms older drugs that seek to relieve symptoms by replacing lost dopamine.

2:07  In recent years, however, the field has learned that although loss of dopaminergic neurons is important, disease processes may well affect pathways beyond dopamine.

2:48 In 2004, population studies found genetic mutations in the gene for an enzymne called LRRK2
in families at greater risk for an inherited form of late onset Parkinson's disease. The mutation most closely associated with the disease makes LRRK2 slightly over-active. The idea is to dial LRRK2 back with drugs.  The question still to be answered is whether or not LRRK2 represents a key controller of Parkinson's severity in all patients with the disease, including those that develop it for reasons unknown in their sixties.   

4:09  While there are still years to go before LRRK2 inhibitors could become available to patients, West says the field is further along in the process of developing a specific target to design drugs against than many Parkinson's researchers ever thought would happen.  

5:05 One of the challenges in neurodegenerative disorders is that humans may be the only creature to get certain diseases of the brain. And yet, to test whether an experimental drug is worthy of human trials, you need to try it first in animal models that mimic the human condition. West says the field is now making progress on creating such models, which may quicken the pace toward human studies. 

6:04  When it comes to developing drugs in the face of stricter regulations, industry and academia have learned in recent years to do more experiments on drug candidates early on, before research teams even apply for permission to start a clinical trial. West's team is repeating its experiments right now to be sure of its data, and to ensure that the team's would-be drug has strong effects in a model that mimics human disease. 

7:17  LRRK2 gets researchers excited because it is rare to find enzymes that are both proven to have a role in a disease of the brain, and that are structured such that a drug can change their action. LRRK2 is the same kind of enzyme (although not the same one) that has been safely and potently targeted by existing treatments for other diseases, including the cancer drugs Herceptin, Tarceva and Erbitux.

8:49 Inherited forms of disease can hide from evolution if they start late in life. They do not keep anyone from reproducing so there is no evolutionary pressure to weed them out of the gene pool.  

10:47 Researchers cannot differentiate between the symptoms of inherited Parkinson's disease linked to a LRRK2 mutation and symptoms in those who develop PD late in life for reasons unknown. That creates at least the possibility that LRRK2 may have a role in all of PD and that a drug fine-tuning LRRK2 could be helpful in all cases. West says he was shocked when it came to light that a disease as complicated as inherited Parkinson's could be caused by a mutation in a single gene.

12:00 Along with whatever is triggering Parkinson's disease, the idea has emerged in the field that the body's reaction to that trigger, the response of the immune system, may be making the disease worse by causing inflammation. LRRK2 may be a critical switch to deciding whether or not inflammation makes the disease worse. 

13:46 Getting a drug into clinical trials today requires a massive investment, so it does not pay to enter clinical trials prematurely. A rushed trial that fails because of poor design can result in a "black eye" for that drug target, making it harder to find funding for related research projects after that.  

15:22 West's team has been working with LRRK2 for several years now, and has been refining  proposed drug candidates that inhibit it. Their latest lead drug candidate overcomes many of the limitations of earlier generations of proposed drugs. It is capable of having its effect in the brain, and targets only LRRK2, and not any of the hundreds of enzymes it might interact with to cause side effects. 

16:32 West recommends that those interested in Parkinson's disease and related research look up the relevant webpage from the National Institute of Neurological Disorders and Stroke. Also very helpful are the websites for the Michael J. Fox Foundation, the Parkinson's Disease Foundation and the American Parkinson's Disease Association.