Gal Bitan, Ph.D.
University of California, Los Angeles

Gal Bitan has been studying the problem of protein misfolding and aggregation since 1999. Together with Dr. David Teplow, Dr. Bitan introduced the use of novel photochemical protein cross-linking techniques for investigation of the assembly of amyloid β-protein (Aβ), which plays a central role in the pathologic mechanism underlying Alzheimer’s disease. His studies led to the discovery of one of the earliest Aβ oligomers in the assembly cascade, the paranucleus. Since joining the faculty of the Department of Neurology at UCLA, Dr. Bitan’s research has focused on investigation of molecules that can prevent and correct abnormal protein folding and aggregation, and development of these molecules for potential treatment of Alzheimer’s disease, Parkinson’s disease, and related diseases.

The Bitan group has discovered the anti-amyloid activity of molecular tweezers and has spearheaded the use of these compounds, particularly CLR01, as inhibitors and modulators as potential drugs against diseases caused by abnormal protein folding and aggregation.

After forming a collaboration with Drs. Klärner and Schrader, the early days of investigation focused on characterizing the effect of the molecular tweezers on the aggregation and toxicity of Aβ. Later, this investigation was expanded to other amyloidogenic proteins and concluded in a 2011 paper demonstrating the inhibitory effect of CLR01 on 10 different proteins. The study was led by Dr. Sharmistha Sinha, then postdoctoral fellow in the Bitan laboratory.

Together with another former postdoc, Dr. Panchanan Maiti, Dr. Sinha compared CLR01 to two compounds predicted to have similar effects, which have gone on to clinical trials – the sugar derivative scyllo-inositol, and the green-tea compound EGCG. CLR01 was found to have similar activity to EGCG and both compounds performed better than scyllo-inositol in the tests used (Sinha et al., 2012). NMR experiments by Chunyu Wang’s group showed that CLR01 bound to Aβ at the expected binding sites – the lysine and arginine residues, whereas no clear binding site was found for EGCG on Aβ, leaving the mechanism of action of EGCG unclear (for a detailed comparison of CLR01 and EGCG, see our recent review).

The main binding site of CLR01 is lysine residues, and the binding of CLR01 to lysines is predicted to disrupt molecular interactions that are important for the abnormal self-assembly of different proteins that leads to formation of toxic oligomers and aggregates. To test the importance of lysine residues in Aβ, Dr. Sinha studied Aβ derivatives in which each of the two lysines was substituted by an alanine (a small amino acid that cannot form the same types of molecular interactions as the larger lysine). She found not only that both the lysines were important mediators of the aggregation of Aβ, but also that one of them in particular was crucial for Aβ toxicity. When this particular lysine – at position 16 in the Aβ amino acid sequence – was replaced by alanine, the toxicity almost completely disappeared!

Although in advanced Alzheimer’s disease the brain suffers massive atrophy, the earliest stages of the disease are characterized by loss of synapses. At this stage, neurons are still alive, but the fail to communicate. This manifests as loss of tiny protrusions on the nerve cells, called dendritic spines, and decline in the electric activity of the affected neurons. Using high-resolution microscopy, Dr. Maiti showed that Aβ oligomers caused a sharp decline in the number of dendritic spines on neurons, and that this toxic effect of Aβ could be prevented by CLR01 (Figure 1). In addition, experiments by Claudio Grassi’s group showed that CLR01 prevented the decline in electrical activity by the neurons.

Figure 2. CLR01 prevents formation of α-synuclein fibrils

Aida Attar was a graduate student in the Bitan lab and led the study of CLR01’s effect in a mouse model of Alzheimer’s disease. The study was done in collaboration with Sally Frautschy’s group and showed that following treatment with a very low dose of CLR01 – just 40 micrograms per kilogram a day, there was a significant reduction in the brain load of amyloid plaques (Figure 3) and neurofibrillary tangles, the two hallmark pathological lesions in Alzheimer’s disease. The treatment also caused a significant reduction in brain inflammation, which is part of the disease process in Alzheimer’s. There were no side effects associated with the treatment (Attar et al., 2012).

Dr. Attar then tested the safety and pharmacology of CLR01. She found that the compound was safe in mice at doses 250-times higher than those that showed the therapeutic effect in the mouse model of Alzheimer’s. At that high dose, there were no behavioral or histological findings indicating toxicity. Blood analysis did not find any signs of toxicity. The only significant difference between the mice that received CLR01 and those that received placebo was approximately 40% reduction in blood cholesterol in the CLR01-treated mice.

Figure 1. CLR01 protects dendritic spines from the harmful effect of Aβ. Dr. Panchanan Maiti (top left) used fluorescence microscopy to visualize normal hipppocampal neurons (top right). Neurons treated with Aβ showed loss of spines (bottom left). Neurons treated with both Aβ and CLR01 were protected from the harmful effect of Aβ (bottom right).

The first demonstration of the therapeutic effect of CLR01 in a living organism was in a collaborative study with Jeff Bronstein’s group using zebrafish embryos genetically engineered to express the human protein α-synuclein, which aggregates and kills dopamine-producing cells in the brain of people who have Parkinson’s disease. Our in vitro characterization of the interaction between CLR01 and α-synuclein showed that CLR01 not only prevented aggregation of α-synuclein (Figure 2), but also dissociated pre-formed aggregates (Prabhudesai et al., 2012).

Figure 3. CLR01 promotes clearance of amyloid plaques from the brain