RESEARCHERS DISCOVER POTENTIAL DRUG TARGET FOR EARLY ONSET OF GLAUCOMA

Using a novel high-throughput screening process, scientists have for the first time identified molecules with the potential to block the accumulation of a toxic eye protein that can lead to early onset of glaucoma. Researchers have implicated a mutant form of a protein called myocilin as a possible root cause of this increased eye pressure. Mutant myocilin is toxic to the cells in the part of the eye that regulates pressure. These genetically inherited mutants of myocilin clump together in the front of the eye, preventing fluid flow out of the eye, which then raises eye pressure. This cascade of events can lead to early onset-glaucoma, which affects several million people from childhood to age 35.

To find molecules that bind to mutant myocilin and block its aggregation, researchers designed a simple, high-throughput assay and then screened a library of compounds. They identified two molecules with potential for future drug development to treat early onset glaucoma.

"These are really the first potential drug targets for glaucoma," said Raquel Lieberman, an associate professor in the School of Chemistry and Biochemistry at the Georgia Institute of Technology in Atlanta, whose lab led the research.

Lieberman presented her findings on January 20 at the Society for Laboratory Automation and Screening conference in San Diego, Calif.

The study was published on Nov. 26, 2013, in the journal ACS Chemical Biology. The National Institutes of Health and the Pew Scholar in Biomedical Sciences program provided support for the research. The work was a collaboration involving Georgia Tech, Emory University and the University of South Florida.

At the heart of the study was an assay that Lieberman’s lab created to take advantage of the fundamental principles of ligand binding. In their assay, mutant myocilin is mixed with a fluorescent compound that emits more light when the protein is unwound. When a molecule from the library screen binds to myocilin, the pair becomes highly stable - tightly wound - and the fluorescent light emitted decreases. By measuring fluorescence, researchers were able to identify molecules that bound tightly to mutant myocilin.

The researchers then added these molecules to cultured human cells that were making the toxic aggregating myocilin. Treating the cells with the newly identified molecules blocked the aggregation and caused the mutated version of myocilin to be released from the cells, reducing toxicity.

"We found two molecules from that initial screen that bound to our protein and also inhibited the aggregation," Lieberman said. "When we saw that these compounds inhibited aggregation then we knew we were onto something good because aggregation underlies the pathogenesis of this form of glaucoma."

In a separate study, Lieberman's lab characterized the toxic myocilin aggregates. That study was published in December 2013 in the Journal of Molecular Biology. The study found that myocilin aggregates are similar to the protein deposits called amyloid, which are responsible for Alzheimer’s disease and other neurodegenerative diseases.

"In Alzheimer's disease, the deposits are extracellular and kill neurons. In glaucoma the aggregates are not directly killing neurons in the retina to cause vision loss, but they are cytotoxic in the pressure-regulating region of the eye," Lieberman said. "It's parallel to all these other amyloids that are out there in neurodegenerative disease."

The researchers are now focusing on mapping the structure of myocilin to learn more about what myocilin does and why it is in the eye in the first place.

"The underlying problem with myocilin is that for 14 years it has been studied and still nobody really knows what its biological role is inside the eye," Lieberman said.

CLEVER CHEMISTRY AND A NEW CLASS OF ANTIBIOTICS

As concerns about bacterial resistance to antibiotics grow, researchers are racing to find new kinds of drugs to replace ones that are no longer effective. One promising new class of molecules called acyldepsipeptides - ADEPs - kills bacteria in a way that no marketed antibacterial drug does - by altering the pathway through which cells rid themselves of harmful proteins.

Now, researchers from Brown University and the Massachusetts Institute of Technology have shown that giving the ADEPs more backbone can dramatically increase their biological potency. By modifying the structure of the ADEPs in ways that make them more rigid, the team prepared new ADEP analogs that are up to 1,200 times more potent than the naturally occurring molecule.

A paper describing the research was released on-line by the Journal of the American Chemical Society.

"The work is significant because we have outlined and validated a strategy for the enhancing the potency of this promising class of antibacterial drug leads," said Jason Sello, professor of chemistry at Brown and the paper's senior author. "The molecules that we have synthesized are among the most potent antibacterial agents ever reported in the literature."

ADEPs kill bacteria by a mechanism by that is distinct from all clinically available anti-bacterial drugs. They work by binding to a protein in bacterial cells that acts as a "cellular garbage disposal," as Sello describes it. This barrel-shaped protein, called ClpP, breaks down proteins that are misfolded or damaged and could be harmful to the cell. However, when ClpP is bound by an ADEP, it's no longer so selective about the proteins it degrades In essence, the binding by ADEP causes the garbage disposal to run amok and devour healthy proteins throughout the cell. For bacteria, a runaway ClpP is deadly.

ADEPs have been shown to kill bacteria that cause staph infections, some kinds of pneumonia, tuberculosis, and other types of infection in the lab. The molecules have also been reported to cure bacterial infections in mice and rats.

ADEPs were first discovered as naturally occurring compounds. Certain bacteria produce them for chemical defense. But for the last few years, scientists including Sello's group have been making synthetic ADEP analogs, in the hope of identifying compounds with potential as new drugs.

One approach the researchers thought might work involves making the ADEP molecule more rigid. Compared to the ClpP molecule to which it binds, the ADEP molecule is a bit "floppy," Sello said. "We often use the expression 'lock and key' to describe how a small molecule binds to a protein. One can imagine that it is easier to fit a rigid key into a lock rather than a floppy key. In the same sense, rigid molecules often bind to their protein targets more tightly."

Sello and his team synthesized several new ADEP molecules. They swapped out certain amino acids in the naturally occurring molecule with ones they thought might increase the molecule's rigidity. To find out if the new molecules were indeed more rigid, the team performed experiments that tested the strength of hydrogen bonds within the molecule. Stronger hydrogen bonds would indicate a more rigid molecule.

The researchers placed ADEP molecules in a solution rich in deuterium, a hydrogen atom that has an extra neutron. Over time, the deuterium atoms in the solution will swap places with the hydrogen atoms in the ADEP molecules. The deuterium swap happens more slowly, however, when hydrogen atoms are involved with strong bonds. So if the modified ADEPs exchanged deuterium more slowly, it would be an indication of strong bonds and a more rigid molecule.

The experiments showed that the modified ADEPs exchanged deuterium as much as 380 times more slowly than the natural molecule, a clear indication that the molecules were more rigid.

"It was exciting to see how rather simple modifications to the ADEP structure could affect their rigidity in such a profound manner," said Daniel Carney, a graduate student in Sello's group. "More importantly, the results were in line with our ADEP design principle. It is always rewarding when a sophisticated chemical theory can be applied and validated by laboratory experiments."

To follow up on the prediction that the rigid ADEPs would bind ClpP more tightly, Robert Sauer and Karl Schmitz at MIT measured the capacity of the ADEP analogs and the parent compound to produce the "runaway garbage disposal" phenomenon in solutions containing the ClpP protein. The experiments showed that the modified ADEPs produced the effect at much lower concentrations, indicating a higher binding efficiency. The results implied that the modified molecules were about seven times better than the standard ones at binding to ClpP.

The final step was testing whether the rigid ADEPs were better at killing bacteria in a test tube. Those tests showed that, compared to published reports for standard ADEPs, the modified compounds were much more potent against three different dangerous bacteria - 32 times more potent against S. aureus, 600 times more potent against E. faecalis, and 1,200 times more potent against S. pneumoniae.

Sello was a bit surprised by the dramatic increase in ADEP potency compared to the much more modest improvement in ClpP binding.

"We found that the most potent ADEP analog binds ClpP seven-fold better than the parent compound, yet it has 1,200-fold better antibacterial activity," Sello said. "We believe that some of the increase in potency may stem from the fact that the rigidified ADEPs bind ClpP more tightly and have an enhanced capacity to cross the cell membrane. The improved cell permeability of the ADEP analogs is consistent with reports in the literature that molecules with strong intramolecular hydrogen bonds are particularly good at penetrating cells."

Antibacterial Soaps Don’t Work and May Cause Harm Says the FDA

“Now a Days There is recently no evidence that they are any more effective at preventing illness than washing with plain soap and water.”
Anti-bacterial soaps make kill your hands of germs easy, right? Just a few squirts and you’re germ-free … well, at least that is what the makers of these soaps tell us.
According to the Food and Drug Administration (FDA), anti-bacterial soaps make some pretty lofty claims but may not be all that they are marketed to be. The federal agency that monitors the safety of food and drugs in our country released a statement noting that they have seen no evidence that antibacterial soaps perform better at arresting the spread of germs than non-antibacterial soaps that make no germ-fighting power claims. This release comes out of an ongoing review into the safety and efficacy of the active ingredients in the soap.
Dangers of Triclosan
Studies have been revealing the dangers of antibacterial soap for years now. In 2005, research found that the antibacterial agent triclosan reacts with chlorinated water to produce chloroform, a known carcinogen.
 FDA even published a draft stating that triclosan was “not generally recognized as safe and effective.”
Yet the ingredient is included in a wide range of consumer products, most commonly in soaps, but also in everything from toothpastes and cosmetics to kitchenware, apparel and even toys.
Just some of the other dangers of triclosan include:

• Muscle function impairment
• Contribution to heart disease and heart failure
• Alteration of levels of thyroid hormones and reproductive hormones like testosterone and estrogen
• Increased risk of infertility
• Early puberty

Even the typically conservative American Medical Association slammed antibacterial soaps years earlier, stating that there was “undisputed evidence that nothing works better when it comes to hand washing than plain soap and water, without the unnecessary toxic antibacterial chemicals.”
Despite the numerous studies and massive amount of evidence, in 2010 the FDA claimed that it “did not have sufficient safety evidence” to recommend changing consumer use of products that contain triclosan.
Earlier this year: After four decades since its first use, the FDA has decided they would make the determination as to whether or not antibacterial soaps, and specifically triclosan, are doing more harm than good. Government researchers stated that they plan to deliver a review this year of the chemical that has been used for cleaning kitchens and the human body.
A spokesperson for the FDA noted that it is now one of their highest priorities, but the fact that it has taken this long to review something so potentially harmful should make one take notice.
Now, They See It (or at least part of it)
In addition to the agency finding no super powers in antibacterial soaps they claim that they could even cause harm by increasing antibiotic resistance and disrupting hormones. Antibiotic-resistant diseases have greatly increased since the use of products with triclosan, posing an even a greater threat than some plagues.
Starting immediately, the FDA requires that antibacterial soap makers prove that their soaps have some clinical benefit that outweighs the risks of regular contact with antibiotics.
Janet Woodcock, director of the FDA Center for Drug Evaluation and Research, noted
“Due to consumers’ extensive exposure to the ingredients in antibacterial soaps, we believe there should be a clearly demonstrated benefit from using antibacterial soap to balance any potential risk.”
This new requirement comes on the heels of agency plans to buckle down on the use of antibiotics in industrial meat farming and is directly related to the escalation of antibiotic resistance.
The FDA wants to be sure that if Americans are going to spray, pump, wipe and squirt millions of gallons of antibacterial soap on their hands and body each day, that the benefits better outweigh the risks.
Strangely, Hospitals are Exempt
One strange twist to this new regulation is the fact that hospitals are exempt. The FDA explains this exemption by noting that there is a higher risk of disease being spread in a hospital than in other settings.
Their point, odd as it may be, is that these antibacterial soaps, which they claim do not work and can increase the risk of antibiotic resistance, deserve a place not among the healthy but among the sick.
According to the CDC, over one million Americans pick up infections at hospitals, medical offices, outpatient surgery centers and nursing homes every year. Approximately 100,000 of these people die as a result of their infections.
Just Wash the Old Fashioned Way
Often times, in our rush to save time, we compromise our health, as in the case of choosing fast and processed food over whole and nutritious food. Handwashing it seems is no different.
There is really no good replacement for a thorough hand wash using soap and warm water, even it it does take a little more time than a quick rinse with an antibacterial soap. Be sure to wash both the back and front of your hands, as well as between your fingers, for at least 20 seconds and rinse well. Parents can encourage their children to wash for as long as it takes them to sing their ABC’s.

WORLDS TINIEST DRUG CABINETS COULD BE ATTACHED TO CANCEROUS CELLS FOR LONG TERM TREATMENT

Reservoirs of pharmaceuticals could be manufactured to bind specifically to infected tissue such as cancer cells for slow, concentrated delivery of drug treatments, according to new research published in ACS Macro Letters. The findings, from the University of Copenhagen and the Institut Laue-Langevin (ILL), came as a result of neutron reflectometry studies at the world's leading neutron source in Grenoble, France. They could provide a way to reduce dosages and the frequency of injections administered to patients undergoing a wide variety of treatments, as well as minimising side effects of over-dosing.

The attachment of reservoirs of therapeutic drugs to cell membranes for slow diffusion and continuous delivery inside the cells is a major aim in drug R&D. A promising candidate for packaging and carrying concoctions of drugs is a group of self-assembled liquid crystalline particles. Composed of fatty molecules - phospholipids - and tree-like macromolecules called dendrimers, the particles form spontaneously and have the capacity to soak up and carry large quantities of drug molecules for prolonged diffusion. They are also known for their ability to bind to cellular membranes.

The first treatments using such particles are close to market through products incorporating a similar formulation called Cubosomes (cubic phase nanoparticles). Developed and commercialized by Swedish start-up Camarus Ab, its FluidCrystal® nanoparticles promise months of drug delivery from a single injection and the possibility of tuning the delivery to intervals of anything from once a day to once a month. However, a key requirement for optimal application of these formulations is a detailed understanding of how they interact with cellular membranes.

This was the focus of a collaboration between Dr Marité Cárdenas (Copenhagen) and Dr Richard Campbell and Dr Erik Watkins (ILL). In this experiment the team used neutrons to analyse the interaction of the liquid crystalline particles with a model cellular membrane whilst varying two parameters:

Gravity - to see how the interaction changed if the aggregates attacked the cell membrane from below as opposed to above

Electrostatics - to see how the balance between positive and negative charges of the aggregate and membrane affect the interaction

The team utilised a technique known as neutron reflectometry whereby beams of neutrons are skimmed off a surface. The reflectivity is measured and used to infer detailed information about the surface, including the thickness, detailed structure and composition of any layers beneath. These experiments were carried out on the FIGARO instrument at the ILL in Grenoble which offers unique reflection up vs. down modes that allowed the team to examine the top and bottom surfaces, alternating the samples on a two hourly basis during a 30 hour sampling period.

The interaction of the liquid crystalline particles with the membrane was shown to be driven by the charge on the model cell membrane. Subtle changes in the degree of negative charge on the membrane encouraged the tree-like dendrimer molecules to penetrate, allowing the rest of the molecule to bind to the surface, forming an attached reservoir. The sensitivity of the interaction to small changes in charge suggests that simple adjustments to the proportion of charged lipids and macromolecules could optimise this attachment. In the future this characteristic could also provide a mechanism to focus the treatment at targeted cells such as those infected by cancer, which are thought to be more negatively charge than healthy cells.

In terms of gravitational effects, the analysis also showed that aggregates interacted more strongly with membranes when located above the sample. Similar effects caused by differences in density and buoyancy of solutions are already exploited in some stomach treatments and the researchers would encourage future studies into how gravitational effects could be used to optimise these interactions for drug delivery.

"Cancerous cells have an imbalance that gives them a different molecular composition and overall different physical properties to normal healthy cells," explains Dr Cardenas. "Whilst all cells are negative, cancerous cells tend to be more negatively charged than healthy ones due to a different composition of fatty molecules on their surface. This is a property that we believe could be exploited in future research into delivery mechanisms involving the attachment of lamellar liquid crystalline particles. Our next step is to introduce the drug itself into the reservoirs and make sure it can move across the membrane. This work paves the way for cell tests and clinical trials in the future exploiting our methodology."

"Of course it's not new that particles in formulations can sink or float, but such dramatically different specific interactions of these nanocarriers with model membranes of different orientations took us completely by surprise" said Dr Campbell. "Very small sample volumes are often used in biomedical investigations so the effects of phase separation cannot be seen. Our findings suggest that laboratory researchers may need to re-evaluate the way in which they examine the effectiveness of newly developed formulations to account for strong gravitational effects."

Dr Watkins further commented: "This study is a perfect illustration of FIGARO’s unique capability to take data from above and below horizontal interfaces in the same experiment. Not only are neutrons uniquely sensitive to the lighter elements found in organic chemistry but the ability to take all the data at once in situ without disturbing the sample is vital. These biological samples are always subtlety changing throughout the time you are analysing them so it's vital that you can take this data as quickly as possible."