Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human CaV3.3 Channel.
Maricruz Rangel-GalvánAzahel RangelCatalina Romero-MéndezEliud Morales DávilaMaría Eugenia CastroNorma A CaballeroFrancisco J Meléndez BustamanteBrenda L Sanchez-GaytanUlises MezaJose Manuel Perez-AguilarPublished in: ACS chemical neuroscience (2021)
Regulation of cellular excitability and oscillatory behavior of resting membrane potential in nerve cells are largely mediated by the low-voltage activated T-type calcium channels. This calcium channel family is constituted by three isoforms, namely, CaV3.1, CaV3.2, and CaV3.3, that are largely distributed in the nervous system and other parts of the body. Dysfunction of T-type calcium channels is associated with a wide range of pathophysiologies including epilepsy, neuropathic pain, cardiac problems, and major depressive disorders. Due to their pharmacological relevance, finding molecular agents able to modulate the channel's function may provide therapeutic means to ameliorate their related disorders. Here we used electrophysiological experiments to show that genistein, a canonical tyrosine kinase inhibitor, reduces the activity of the human CaV3.3 channel in a concentration-dependent manner. The inhibitory effect of genistein is independent of tyrosine kinase modulation and does not affect the voltage-dependent gating of the channel. Subsequently, we used computational methods to identify plausible molecular poses for the interaction of genistein and the CaV3.3 channel. Starting from different molecular poses, we carried out all-atom molecular dynamics (MD) simulations to identify the interacting determinants for the CaV3.3/genistein complex formation. Our extensive (microsecond-length) simulations suggest specific binding interactions that seem to stabilize the protein/inhibitor complex. Furthermore, our results from the unbiased MD simulations are in good agreement with the recently solved cryoelectron microscopy structure of the CaV3.1/Z944 complex in terms of both the location of the ligand binding site and the role of several equivalent amino acid residues. Proposed interacting complex loci were subsequently tested and corroborated by electrophysiological experiments using another naturally occurring isoflavone derivative, daidzein. Thus, by using a combination of in vitro and in silico techniques, we have identified interacting determinants relevant to the CaV3.3/genistein complex formation and propose that genistein directly blocks the function of the human CaV3.3 channel as a result of such interaction. Specifically, we proposed that a combination of polar interactions involving the three hydroxyl groups of genistein and an aromatic interaction with the fused rings are the main binding interactions in the complex formation. Our results pave the way for the rational development of improved and novel low-voltage activated T-type calcium channel inhibitors.
Keyphrases
- molecular dynamics
- endothelial cells
- tyrosine kinase
- neuropathic pain
- amino acid
- density functional theory
- pluripotent stem cells
- mental health
- spinal cord
- oxidative stress
- bipolar disorder
- heart failure
- high resolution
- binding protein
- left ventricular
- mass spectrometry
- heart rate
- high throughput
- heart rate variability
- blood pressure
- molecular docking
- protein protein
- signaling pathway
- small molecule
- climate change
- single cell
- pi k akt
- atrial fibrillation
- transcription factor