Plant cyclotides protect against β-amyloid toxicity in transgenic Caenorhabditis elegans

Neha Kalmankar’s interview with Bio Patrika hosting “Vigyaan Patrika”, a series of author interviews. Neha is a PhD student under the supervision of Dr. Radhika Venkatesan and Prof. R. Sowdhamini at the National Centre for Biological Sciences, Bengaluru. She completed her Bachelors in Engineering degree in Biotechnology at Sir. M. Visvesvaraya Institute of Technology, Bengaluru. Soon after completing herundergraduate degree, she joined Prof. P. Balaram’s lab at the Indian Institute of Science (IISc) as a JRF, exploring the computational aspects of protein sequence, structure & function. Her PhD topic includes biochemical characterization, conformational analysis and therapeutic application of plant cyclic peptides – “cyclotides”. She has worked towards isolating and sequencing cyclotides from different tissues of Clitoria ternatea using chromatographic, mass-spectrometric and transcriptomic techniques. She has further investigated the therapeutic potential of cyclotides, specifically as agents against neurodegenerative disorders. When she is not working in the lab, she enjoys travelling, exploring different cultures, cuisines and languages. She also enjoys painting and exploring creative arts in her free time. Here, Neha talks about her work on “Disulfide-rich cyclic peptides from Clitoria ternatea protect against β‑amyloid toxicity and oxidative stress in transgenic Caenorhabditis elegans” published in Journal of Medicinal Chemistry (2021).

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How would you explain your paper’s key results to the non-scientific community?
We have all heard of Alzheimer’s disease (AD). Alzheimer’s is a chronic and progressive neurodegenerative disorder that causes the brain to shrink and brain cells to slowly die. It is the most common cause of dementia and cognitive impairment, constituting 70-80% of all dementia cases. So far, there is no cure for AD but existing treatment protocols can help slow the progression and can improve the quality of life of those affected. Our goal was to discover drug candidates that can help cure or halt AD progression. Neurons in the human brain synthesize a protein called amyloid-beta (Aβ). In an Alzheimer’s patient, such proteins undergo aggregation and form large deposits called Aβ plaques, thereby impairing important tasks of the neurons. As a part of my PhD thesis topic, I have been working with the Clitoria ternatea (butterfly pea) plant also commonly known in India as Shankhapushi, Aparajita, Sangu poo, etc. Anecdotal evidence testify this plant for its medicinal values such as nootropic, memory enhancer, enhancing cognitive impairment etc, but more commonly it used as fodder and forage crop, and for ornamental purposes due to its twinning growth and vivid blue and white flowers. During the course of my research, I isolated certain peptides from this plant that are cyclic, disulfide-rich, ultra-stable and structurally very interesting molecules. These disulfide-rich macrocyclic peptides are emerging as versatile scaffolds, in both fields of agriculture and medicine. This potential is mainly due to their structural stability and range of bioactivities they display. What we went on to test was the effects of these peptides in slowing the progression of AD neurotoxicity, specifically by inhibiting the Aβ aggregation. We soon found out that these peptides were able to inhibit these small toxic aggregates. We checked this activity by performing multiple assays and in all cases, these peptides show high activity against neurotoxicity caused by Aβ aggregation. We demonstrated the activity of these bioactive peptides by feeding Caenorhabditis elegans or tiny free-living soil worms, a model organism for neurodegenerative diseases.

Schematic

Our study shows that naturally-occurring cyclic peptides have immense potential as drug leads for understanding Aβ fibril aggregation and as a treatment option for AD.

What are the possible consequences of these findings for your research area?

Alzheimer’s disease is an overwhelmingly debilitating disease affecting the patient, their families and caregivers. As it is an age-related disorder, it is particularly difficult to manage those with this disease as there is a continuous decline in thinking, behavioural and social skills that essentially affects the person’s ability to function independently. Majority of the strategies to combat AD have focused on inhibition of amyloid aggregation and/or elimination of aggregated amyloid peptides. But despite the numerous medications currently in use for AD, studies investigating potential inhibitors of Aβ, they offer only symptomatic benefits in mild-to-moderate patients and come with considerable side effects. Our work in identifying cyclotides as a novel drug lead for Aβ inhibition is promising because these peptides are from natural sources and peptide therapeutics is advancing as peptides have few advantages over small molecules due to their low toxicity, non-immunogenicity, better bioavailability and convenient production. Our study shows that naturally-occurring cyclic peptides have immense potential as drug leads for understanding Aβ fibril aggregation and as a treatment option for AD.

What was the exciting moment (eureka moment) during your research?

When I was performing experiments and the study was constantly evolving, I was faced with many ups and downs. I would say there was never one eureka moment but several exciting moments that made me ecstatic and motivated me to proceed further. One such moment was when I fed cyclotides to C. elegans worms, which previously exhibited cognitive and movement deficits, suddenly their motility started improving. When I observed these worms under the microscope, the number of fluorescent amyloid deposits were also greatly reduced! To visualize the worms behaving more coherently and showing improved movements with your own eyes, was quite exciting. This meant what I had initially hypothesized, in reality had actually worked!

What do you hope to do next?

I hope that our work gets the desired attention from the scientific community and drug development industries. Thereafter, cyclotides can be tested further and approved for pre-clinical trials for the treatment of AD. Since many age-related diseases are associated with the accumulation of toxic proteins, the research could also lead to treatment options for several other diseases like Parkinson’s, Huntington’s, etc. What I have not yet mentioned, is that we also found cyclotides to be potent antioxidants. Oxidative stress is a common related process in pathogenesis of AD. Both the attributes i.e. inhibitor of amyloid fibrils and antioxidant, are nascent discoveries in the cyclotide field and I hope to take these preliminary findings further and test their efficacy in comparison with existing drugs in the market. Additionally, I wish to identify which particular cyclotide is responsible for the bioactivities shown. As we used mixtures, it is imperative to find out at least the top 5 best cyclotide molecules showing the maximum activity. As a long-term goal, I aim to establish my own research lab in India. My approach will be to combine basic and applied science in the field of translational research for the benefit of society.

Where do you seek scientific inspiration?

My doctoral thesis advisor and venerable supervisor Prof. P. Balaram (IISc), has sparked a great interest in me to continue my research career. He has been patient with me, provided me with excellent scientific training and transferred the know-hows to me. My scientific inspiration comes from him. If one day I can become half the scientist as him, I will consider myself fortunate.

How do you intend to help Indian science improve?

Indian science has greatly improved in the last few decades I would say, but still there is much more to achieve and overcome. One of the challenges in conducting good science in India is that most individuals have to constantly compete with very talented members, all for the same position and funds. Many such able persons unfortunately miss out on acquiring desired funds and their research ideas get impeded. If I become a mentor and can have a research lab of my own, instead of competing with everyone I wish to collaborate with labs that have potential but for various reasons have not been able to come up in the scientific arena. This will not just allow me to widen my own expertise but help the Indian scientific community to collectively grow and someday reach its pinnacle.

Reference

Kalmankar NV, Hari H, Sowdhamini R, Venkatesan R. Disulfide-Rich Cyclic Peptides from Clitoria ternatea Protect against β-Amyloid Toxicity and Oxidative Stress in Transgenic Caenorhabditis elegans. J Med Chem. 2021 Jun 10;64(11):7422-7433. doi: 10.1021/acs.jmedchem.1c00033.

Prof. Venkatesan R’s Lab website: ilab.ncbs.res.in

Prof. Sowdhamini R’s lab website: https://www.ncbs.res.in/faculty/mini

Edited by: Kshipra S. Kapoor

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