The LilrB2 receptor on the surface of neurons binds Aβ and is thought to mediate some of its toxicity. Researchers led by Lin Jiang and David Eisenberg at the University of California, Los Angeles, now detail the part of Aβ involved, and the specific LilrB2 pocket it fits. As reported in the October 8 Nature Chemistry, designing a molecule that competes for the pocket blocks Aβ toxicity in neurons. "It's nice that they could figure out how the molecules fit, create a small molecule to disrupt that interaction, and decrease toxicity,” wrote David Teplow, now professor emeritus at UCLA, who was not involved in the work. “This appears to be a ‘complete story.’”
- Scientists resolved the crystal structure of Aβ-binding receptor LilrB2.
- They found a binding pocket and complementary region on Aβ.
- A compound binds the pocket, blocking Aβ and protecting neurons.
“This work validates the structural reality of a true interaction between the LilrB2 receptor D1-D2 domain and β-amyloid,” said Carla Shatz, Stanford University, California, who originally discovered that Aβ binds the receptor (Kim et al., 2013). Shatz found LilrB2 signaling was altered in the brains of patients with Alzheimer’s disease. Genetically knocking out the mouse equivalent, PirB, saved APP transgenic mice from Aβ-induced memory problems and protected cultured cortical neurons from Aβ toxicity, giving credence to the idea that the Aβ-LilrB2 interaction could make a good therapeutic target.
To design an inhibitor, first author Qin Cao and colleagues had to determine exactly where Aβ bound on the extracellular side of the receptor. They used a 200-residue stretch of the extracellular domains D1 and D2, where Shatz reported that Aβ binds. They narrowed down binding to a six-amino-acid stretch between amino acids 16 and 21 of Aβ—KLVFFA. Oligomers, but not monomers, of this peptide bound the receptor, suggesting this region of Aβ binds in oligomeric form as well. Since oligomers of Aβ are heterogeneous and transient, Cao used a more stable tandem repeat of the KLVFFA fragment to study binding.
Crystal Structure. A pocket between extracellular domains D1 and D2 of LilrB2 holds four benzamidines (Ben 1 to 4), similar to the phenylalanine side chains of Aβ. [Courtesy of Cao et al., 2018. Nature.]
The group then grew crystals of D1D2 in the presence of the Aβ tandem. X-ray analysis of those revealed a pocket, nestled in between D1 and D2, that was the right size, shape, and electrostatic environment for Aβ. It bound two benzamidines, added for crystal optimization, which mimicked two phenylalanines lying close to one other, as they would in a tandem repeat version of KLVFFA. One at a time, the authors mutated a few amino acids lining those pockets and found that for each, less than half the Aβ bound there compared with wild-type domains. This gave them more confidence that they had hit on the right binding site. They confirmed these binding predictions by computationally modeling interactions between the LilrB2 protein and Aβ.
Cao then searched a library of 32,000 small molecules—some approved as drugs, some currently in clinical trials, and some natural compounds already tested in animals and humans—to find compounds that could inhibit the interaction between LilrB2 and Aβ and might be safe for use in people. He selected 12 based on three-dimensional structure and probability of fitting the Aβ-binding pocket of D1D2. The researchers then narrowed this down to the six that best bound LilrB2 and prevented Aβ binding in vitro.
In HEK293T cells expressing full-length LilrB2, 10μM of each inhibitor reduced Aβ binding by 50 to 70 percent. Computational software predicted that one of these inhibitors, ALI6, best bound LilrB2 and elbowed out Aβ. In cultured mouse neurons, ALI6 reduced Aβ binding by 60 percent, but no more, suggesting the peptide attaches to other receptors on the neuronal surface as well. ALI6 is also known as fluspirilene, and is an antipsychotic drug used to treat chronic schizophrenia.
In HEK293T cells and cultured mouse neurons, ALI6 decreased Aβ-induced cell death by 90 and 66 percent, respectively. It completely blocked the downstream dephosphorylation of the actin-depolymerizing factor, cofilin, which results from LilrB2-Aβ binding. Shatz had reported that cofilin dephosphorylation caused synapse loss.
Together the results imply that inhibiting the LilrB2-Aβ interaction could prevent some of the toxic effects of Aβ and stave off damage to neurons. The authors acknowledge that the high concentrations of ALI6 used in this study would be hard to achieve in vivo. Liang proposed optimizing a compound to have higher affinity for the receptor. He plans to test ALI6 in transgenic mouse models of AD to see if it can protect them from Aβ-caused toxicity or symptoms.
“In the future it will be very exciting to see if inhibiting the Aβ interaction with LilrB2 in the brain curtails synaptotoxic signaling, leading to improved synaptic plasticity, memory, and cognitive function,” wrote Iryna Benilova, University College London, to Alzforum.
“The authors are quite properly cautious about the long-term therapeutic implications since their molecule is a long way from being a drug,” wrote Gregory Petsko, Weill Cornell Medicine, New York, to Alzforum. “Perhaps the approach taken in this paper will, if not lead to a single curative or preventive agent, eventually produce one that may have value in combination with other treatments.”—Gwyneth Dickey Zakaib
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