Alzheimer’s disease is a neurodegenerative disorder that impairs cognitive abilities and memory in many people over the age of 60. However, current methods for detecting early manifestations of the disease through MRI, PET, and CT scans are complex, expensive, and often inconclusive.
“Our goal was to find a reliable, cost-effective solution,” says Debasis Das, Assistant Professor in the Department of Inorganic and Physical Chemistry (IPC) at the Indian Institute of Science (IISc). With this in mind, Das and Jagpreet Sidhu, a CV Raman postdoctoral fellow in IPC, have created a small molecular fluorogenic probe that identifies a specific enzyme linked to Alzheimer’s disease progression. This probe could easily be fashioned into a strip-based kit for on-site diagnosis.
“Fluorogenic probes become fluorescent after reacting with a target enzyme,” explains Das. “Our target enzyme is Acetylcholinesterase (AChE).” AChE has been identified as a potential biomarker for the disease, as studies have shown that AChE levels become imbalanced in the early stages of Alzheimer’s disease.
Brain cells or neurons release neurotransmitters, signaling molecules that tell other cells to carry out specific functions. Acetylcholine (ACh) is one such neurotransmitter, and its levels in our nervous system are tightly controlled by enzymes like AChE, which breaks it down into two parts: acetic acid and choline. AChE levels in the brain are often determined indirectly by measuring the levels of choline. Current approaches to AChE measurement are confounding because AChE has “sister enzymes” like butyrylcholinesterase and cholinesterase that also work on similar substrates to ACh, says Das.
The research team analyzed the crystal structures of AChE and its substrate ACh. To mimic ACh, the team developed a synthetic molecule. The probe they created has a quaternary ammonium group that specifically interacts with AChE and another that binds to the active site in AChE and is digested (like natural ACh), resulting in a fluorescent signal. The team modified the distance between the two elements to bind tightly to the enzyme. Sidhu, who is the first author of the study, says this is the first study to use a quaternary ammonium group.”We were able to attain specificity and selectivity,” he says. To check the probe’s ability to be digested by AChE, the team used commercially available AChE as well as AChE expressed in bacteria.
The team showed that the fluorogenic probe could penetrate brain cells cultured in the lab and fluoresce when in contact with AChE in collaboration with Deepak Saini’s lab at the Department of Developmental Biology and Genetics, IISc.
“We have a proof-of-concept and a lead,” says Das. “Our goal is to translate it into an Alzheimer’s disease model. For this, we need to modify the probe.” Currently, the probe is UV-active, which can be dangerous to tissues in high doses. “Modifications would result in the creation of near-infrared active probes, which would be safer for living cells and enable deep-tissue imaging. We are already quite close to doing this,” says Das.
In addition to Alzheimer’s disease, Sidhu says the probe may also be used to identify pesticide-related poisoning as compounds in certain pesticides can inhibit AChE.
More information:
Jagpreet S. Sidhu et al, Acetylcholine Structure-Based Small Activatable Fluorogenic Probe for Specific Detection of Acetylcholinesterase, Analytical Chemistry (2023). DOI: 10.1021/acs.analchem.3c00099
Citation:
Scientists create fluorogenic probe to detect enzyme linked to early stage of Alzheimer’s (2023, June 14)
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