Rajesh Bhardwaj’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Trained in Bioinformatics at the University of Hyderabad, Dr. Rajesh Bhardwaj, who hails from Haryana, switched gears to Biochemistry and conducted his doctoral research on physical coupling mechanisms of endoplasmic reticulum and plasma membrane at Heidelberg University, Germany. He is currently working as a senior research associate at the University of Bern, Switzerland. His work combines molecular biology and computational structural biology techniques with state-of-the-art high-throughput screening and electrophysiology. The goal is to understand different aspects of store-operated, calcium release-activated calcium (CRAC) channels and epithelial calcium channel TRPV6, such as the mechanisms that regulate their activation and inactivation, regulation by oxidative stress, and modulation by chemical compounds. Here, Dr. Rajesh talks about how ‘novel drugs block TRPV6 calcium channel like a natural plug’ and discuss his first author research article titled “Inactivation-mimicking block of the epithelial calcium channel TRPV6” published in Sci Adv. (2020).
How would you explain your paper’s key results to the non-scientific community?
When it comes to human physiology, not everyone understands the role of calcium as a “second messenger” carrying out vital functions. However, “bones” or “teeth” certainly rings a bell when one thinks of calcium. Indeed, around 99% of the body’s calcium is dedicated to building strong bones and teeth. Less than a fraction of calcium in its ionic form (Ca2+) flows through the cells. To prevent aggregation of proteins and nucleic acids as well as precipitation of phosphates and maintain the integrity of lipids, cells maintain very low amounts of Ca2+ inside themselves, which is nearly 10-20 thousand times lower than their outside environment. Now, Ca2+ cannot just freely cross through the dense lipid bilayer around a cell called the membrane, which separates the components of a cell from the outside environment as well from one another. Among various other proteins embedded in the cell membrane, TRPV6 is an important channel protein, four units of which assemble to form a pore or simply a tiny hole in the center of the TRPV6 channel assembly. This TRPV6 pore can be envisioned as a narrow tunnel with an entry point outside the cell and an exit point inside the cell. Calcium ions are permitted inside the cell through this TRPV6 tunnel, much like cars pass through a narrow tunnel. The illustration in Figure 1 shows the uptake of dietary Ca2+ through TRPV6 channels located in the membranes of epithelial cells that line the intestine walls. When 1,25-dihydroxy vitamin D acts in these cells at times of increased calcium demand, synthesis of both TRPV6 and a Ca2+-binding protein, calbindin-D9K, is activated to boost intestinal calcium absorption (Figure 1). In order to prevent the uncontrolled entry of Ca2+ through the TRPV6 channel, it must be inactivated. One of the natural mechanisms to inactivate the TRPV6 channel is through calmodulin (CaM) protein which functions as a Ca2+ sensor inside the cells. CaM senses the rising Ca2+ inside the cells as it undergoes a change in shape upon binding Ca2+ (Figure 1). CaM, in a manner analogous to traffic police preventing the entry of too many cars through the tunnel, blocks the pore of the TRPV6 channel from inside of the cells (Singh and coworkers, 2018), preventing further Ca2+ entry into the cells (Figure 2, panel A). Improper functioning or increased amounts of TRPV6 channels have been linked to the development of a number of cancers by disrupting the control of cell proliferation and cell death. TRPV6 thus forms a promising drug target candidate for cancer therapy (Stewart 2020).
At the University of Bern, Switzerland, the laboratory of Prof. Matthias A. Hediger teamed up with the laboratory of Prof. Jean Louis-Reymond and in the year 2015 identified a drug they called cis-22a, which very potently and specifically prevented the Ca2+ entry through TRPV6. More recently, we have generated advanced analogues of cis-22a with similar inhibiting potency but enhanced stability and lesser off-target effects (Cunha and coworkers, 2020). The mechanism of how cis-22a and its advanced derivatives blocked the TRPV6 channel remained unidentified, and to investigate that, we further collaborated with Prof. Alexander I. Sobolevsky at Columbia University, New York, USA, and Prof. Christoph Romanin at Johannes Kepler University Linz, Austria. Our recent publication in Science Advances nails down the mechanism of action of cis-22a and its modified variants on the TRPV6 channel using a combination of state-of-the-art techniques, namely, site-directed mutagenesis, fluorescence imaging plate reader assays, cryo-electron microscopy (cryo-EM), electrophysiology and molecular dynamics simulations (Bhardwaj and coworkers, 2020). Our work shows that the cis-22a family of chemical compounds directly plug the pore of the TRPV6 channel from inside of the cells in a similar manner to the natural inhibitor CaM (Figure 2). The substitution of a negatively charged amino acid near the exit of the TRPV6 pore to a positively charged amino acid (aspartic acid at position 580 to lysine) prevented the block of the TRPV6 channel by cis-22a. Notably, the cryo-EM structures representing distinct frozen states of the TRPV6 channel showed that cis-22a and its derivatives directly plug the pore of the TRPV6 channel from inside of the cells.
What are the possible consequences of these findings for your research area?
We reported the first-of-its-kind inhibitor of the TRPV6 channel, which directly blocks the channel’s pore. We showed that this blocking effect by cis-22a is similar to the natural inactivation mechanism of TRPV6 by Ca2+/CaM. Therefore, the cis-22a family of drugs might be less invasive and more effective with fewer adverse side effects. The knowledge on the mechanism of action of this drug that we provide makes it a valuable tool to further pharmacologically address new roles of TRPV6 in health and disease. Also, the structural information will help to further build on the drug scaffolds to improve their TRPV6 inhibiting potency.
“The knowledge on the mechanism of action of this drug that we provide makes it a valuable tool to further pharmacologically address new roles of TRPV6 in health and disease.”
What was the exciting moment (eureka moment) during your research?
The elucidation of the mechanism of action of cis-22a family of TRPV6 inhibitors was a massive team effort from four different laboratories, which required putting several pieces of the puzzle together and thinking from different perspectives of chemistry, biochemistry, cell physiology, structural and computational biology. Therefore, a collective eureka moment was realized only when we started to see the whole picture together with findings from different groups fitting to one another. For me, it was exhilarating to figure out that mutations of the amino acids in TRPV6 pore, which were structurally identified to interact with cis-22a, prevented the block of TRPV6 by cis-22a.
What do you hope to do next?
I did not mention it yet, but we also reported another binding site of cis-22a within the TRPV6 channel at a place remote from the pore region. This site was earlier shown by the Sobolevsky laboratory to also interact with membrane lipids regulating the function of TRPV6. When we mutated amino acids from this particular cis-22a binding site, it did not make the channel immune to block by cis-22a, but the mutant TRPV6 channel required a six times higher dose of the drug to exert the same blocking effect. We, therefore, suggested that binding of cis-22a in this non-pore pocket exerts a remote effect by altering the pore geometry. We aim to further understand the role of this second cis-22a binding pocket in the TRPV6 channel. Also, we hope to understand how different membrane lipids may regulate the activity of the TRPV6 channel. Finally, we would like to address the role of TRPV6-mediated Ca2+ entry in cancer cell proliferation using the cis-22a family of inhibitors. In another research project based on Ca2+ signaling, I aim to understand the activation mechanisms of CRAC channels. If interested, please watch my European Calcium Society research webinar: https://www.youtube.com/watch?v=dhCU62X0HPo
Where do you seek scientific inspiration?
My doctoral supervisor Dr. Matthias Seedorf showed all the patience and provided me excellent scientific training, and sparked a great interest in me to continue my research career. Prof. Hediger provided me the right platform with state-of-the-art training opportunities during my postdoctoral research work in his laboratory. I appreciate his critique, and I am grateful for his consistent encouragement during the last seven years. I look up to him for scientific advice during tough times. Apart from him, I seek inspiration from colleagues who are performing exceptionally well within our research group. Also, I am inspired by peers from the Ca2+ signaling field whom I have seen moving up on the academic ladder and finally becoming independent principal investigators. Encouragement, as well as my family’s belief in me, keeps my motivation high.
How do you intend to help Indian science improve?
If given a chance, I would be happy to take a faculty position in India and thus would be able to directly contribute to Indian science. I have actively contributed to setting up several international research collaborations in our laboratory, and I am open to collaborating in India, given I find common interests with colleagues back home. I look forward to attending future calcium meetings in India as well as networking with Indian scientists.
- Singh AK, McGoldrick LL, Twomey EC, Sobolevsky AI. Mechanism of calmodulin inactivation of the calcium-selective TRP channel TRPV6. Sci Adv. 2018.
- Stewart JM. TRPV6 as A Target for Cancer Therapy. J Cancer. 2020.
- Cunha MR, Bhardwaj R, Carrel AL, Lindinger S, Romanin C, Parise-Filho R, Hediger MA, Reymond JL. Natural product inspired optimization of a selective TRPV6 calcium channel inhibitor. RSC Med Chem. 2020.
- Bhardwaj R, Lindinger S, Neuberger A, Nadezhdin KD, Singh AK, Cunha MR, Derler I, Gyimesi G, Reymond JL, Hediger MA, Romanin C, Sobolevsky AI. Inactivation-mimicking block of the epithelial calcium channel TRPV6. Sci Adv. 2020. DOI: 10.1126/sciadv.abe1508.
Edited by: Manveen K. Sethi (Volunteer, Biopatrika)