A chemical that could potentially be used in eye drops to reverse cataracts, the leading cause of blindness, has been identified by a team of scientists from UC San Francisco (UCSF), the University of Michigan (U-M), and Washington University in St. Louis (WUSTL).
Identified as a “priority eye disease” by the World Health Organization, cataracts — caused when the lenses of the eyes lose their transparency — affect more than 20 million people worldwide. Although cataracts can be successfully removed with surgery, this approach is expensive, and most individuals blinded by severe cataracts in developing countries go untreated.
Reported November 5 in Science, the newly identified compound is the first that is soluble enough to potentially form the basis of a practical eye-drop medication for cataracts.
The research group used a method known as high-throughput differential scanning fluorimetry, or HT-DSF, in which proteins emit light when they reach their melting point. At the U-M Life Sciences Institute’s Center for Chemical Genomics, the team used HT-DSF to apply heat to amyloids while applying thousands of chemical compounds.
Because the melting point of amyloids is higher than that of normal crystallins, the team focused on finding chemicals that that lowered the melting point of crystallin amyloids to the normal, healthy range.
The group began with 2,450 compounds, eventually zeroing in on 12 that are members of a chemical class known as sterols. One of these, known as lanosterol, was shown to reverse cataracts in a June paper in Nature, but because lanosterol has limited solubility the group who published that study had to inject the compound into the eye for it to exert its effects.
Using lanosterol and other sterols as a clue, Gestwicki and his group assembled and tested 32 additional sterols, and eventually settled on one, which they call “compound 29,” as the most likely candidate that would be sufficiently soluble to be used in cataract-dissolving eye drops.
In laboratory dish tests, the team confirmed that compound 29 significantly stabilized crystallins and prevented them from forming amyloids. They also found that compound 29 dissolved amyloids that had already formed. Through these experiments, said Gestwicki, “we are starting to understand the mechanism in detail. We know where compound 29 binds, and we are beginning to know exactly what it’s doing.”
The team next tested compound 29 in an eye-drop formulation in mice carrying mutations that make them predisposed to cataracts. In experiments conducted with Usha P. Andley, PhD, professor of ophthalmology and visual sciences at WUSTL School of Medicine, they found that the drops partially restored transparency to mouse lenses affected by cataracts, as measured by a slit-lamp test of the sort used by ophthalmologists to measure cataracts in humans.
Similar results were seen when compound 29 eye drops were applied in mice that naturally developed age-related cataracts, and also when the compound was applied to human lens tissue affected by cataracts that had been removed during surgery.
Gestwicki cautions that slit-lamp measures of lens transparency used in the research are not a direct measure of visual acuity, and that only clinical trials in humans can establish the value of compound 29 as a cataract treatment. He has licensed the compound from U-M, however, and Makley, a former graduate student and postdoctoral fellow in the Gestwicki laboratory, is founder and chief scientific officer of ViewPoint Therapeutics, a company that is actively developing compound 29 for human use.
Dogs are also prone to developing cataracts. Half of all dogs have cataracts by nine years of age, and virtually all dogs develop them later in life. An effective eye-drop medication could potentially benefit about 70 million affected pet dogs in the United States.
Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins.
Cataracts are the most common cause of vision loss, especially in our ever-increasing elderly population. Cataracts arise when crystallin, a major protein component of the eye lens, begins to aggregate, which causes the lens to become cloudy. Makley et al. explored whether small molecules that reverse this aggregation might have therapeutic potential for treating cataracts, which normally require surgery (see the Perspective by Quinlan). They used a screening method that monitors the effect of ligands on temperature-dependent protein unfolding and identified several compounds that bind and stabilize the soluble form of crystallin. In proof-of-concept studies, one of these compounds improved lens transparency in mice.
Abstract – Lanosterol reverses protein aggregation in cataracts
The human lens is comprised largely of crystallin proteins assembled into a highly ordered, interactive macro-structure essential for lens transparency and refractive index. Any disruption of intra- or inter-protein interactions will alter this delicate structure, exposing hydrophobic surfaces, with consequent protein aggregation and cataract formation. Cataracts are the most common cause of blindness worldwide, affecting tens of millions of people1, and currently the only treatment is surgical removal of cataractous lenses. The precise mechanisms by which lens proteins both prevent aggregation and maintain lens transparency are largely unknown. Lanosterol is an amphipathic molecule enriched in the lens. It is synthesized by lanosterol synthase (LSS) in a key cyclization reaction of a cholesterol synthesis pathway. Here we identify two distinct homozygous LSS missense mutations (W581R and G588S) in two families with extensive congenital cataracts. Both of these mutations affect highly conserved amino acid residues and impair key catalytic functions of LSS. Engineered expression of wild-type, but not mutant, LSS prevents intracellular protein aggregation of various cataract-causing mutant crystallins. Treatment by lanosterol, but not cholesterol, significantly decreased preformed protein aggregates both in vitro and in cell-transfection experiments. We further show that lanosterol treatment could reduce cataract severity and increase transparency in dissected rabbit cataractous lenses in vitro and cataract severity in vivo in dogs. Our study identifies lanosterol as a key molecule in the prevention of lens protein aggregation and points to a novel strategy for
cataract prevention and treatment.