Cataracts affect about 50% of individuals over the age of 70 and are a common cause of blindness worldwide. Many people eventually require surgery to remove cataracts because there are currently no non-surgical treatments available.
Cataracts develop in the eye lens when proteins called crystallins become damaged and fold in an abnormal manner. They then start to aggregate and form amyloid-like fibers that create a barrier to light.
Two of the major crystallin proteins in the lens are αA-crystallin (cryAA) and αB-crystallin (cryAB). These proteins are molecular chaperones that maintain the solubility of other proteins in the lens such as the β-crystallins and the γ-crystallins. The role of molecular chaperones is to stabilize unfolded proteins, unfold them for degradation or for translocation across the cell membrane and/ or help refold the proteins and correctly assemble them.
Researchers are currently trying to develop so called pharmaceutical chaperones (CPS) for cryAB, but since this protein has no natural substrates that would serve as a starting point for drug design, no PC has yet been identified. As with some other disease-associated proteins, this means cryAb is often thought of as an “undruggable” target.
Previous studies of hereditary cataract have shown that a lens mutation called R120G destabilizes cryAB by disrupting ionic interactions that usually stabilize the protein. This not only reduces chaperone activity in the lens, but the unstable cryAB starts to form amyloid-like aggregates.
Makley Leah and colleagues from Michigan University in the US have recently used a technique called differential scanning fluorimetry (DSF) to measure the apparent melting transition (Tm) of wild-type cryAB and the more heat resistant R120G cryAB. They came to the conclusion that ligands that can lower the Tm of R120G cryAB may be potential PCs.
After screening a collection of compounds known to be active in various different
assays, they identified one, 5-cholesten-3b,25-diol, that directly interacted with R120G cryAB and lowered its apparent Tm by 2˚C. In addition, it could lower the apparent Tm of three cryAA mutants. The researchers then performed 15N–heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance experiments, using the Bruker Avance III 500 MHz spectrometer. This showed that the compound bound to the crystallin domain at the dimer interface. Having used NMR to locate this specific interaction, the researchers went on to perform docking experiments. These showed that the compound fitted into a groove that lies between two protomers, suggesting that it may stabilize the native state of R120G cryAB.
In vitro studies showed partial suppression and even reversal of amyloid formation when the compound was added to R120G cryAB. Furthermore, studies of heterozygous R120G cryAB and heterozygous R49C cryAA knock-in mice with hereditary cataracts showed that, after two weeks of treatment, the compound significantly improved lens opacity in both groups of mice. It also improved lens transparency in wild-type mice that had spontaneous age-associated cataracts.
Finally, adding the compound to lens material from patients with grade 1 to 4 cataracts increased the amount of soluble protein by 18%.
Makley and team say the findings point to a promising new approach to treating cataracts and also suggest that high throughput DSF may be suitable for identifying PCs for other targets that, until now, have been considered “undruggable.”
Reference
Makley L, et al. Pharmacological chaperone for a-crystallin partially restores transparency in cataract models. Science 2013;350:674–677