Inhibitory Mechanism of the Isoflavone Derivative Genistein in the Human Ca_V3.3 Channel

Abstract

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, Ca V 3.1, Ca V 3.2, and Ca V 3.3, 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 Ca V 3.3 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 Ca V 3.3 channel. Starting from different molecular poses, we carried out all-atom molecular dynamics (MD) simulations to identify the interacting determinants for the Ca V 3.3/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 Ca V 3.1/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 Ca V 3.3/genistein complex formation and propose that genistein directly blocks the function of the human Ca V 3.3 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.

Theoretical Study of the Structural Stability, Chemical Reactivity, and Protein Interaction for NMP Compounds as Modulators of the Endocannabinoid System

“…Further, in the recently solved structure of the cryo-EM Ca v 3.1 channel with the Z944 ligand, not only a similar binding site was identified for the blocker, but also the ligand displayed interactions with equivalent hydrophobic residues and the T921 polar residue (T586 in Ca v 3.2 described above) [26]. Lastly, by integrating electrophysiological and computational techniques, a similar pose for the inhibitory action of genistein in the human Ca v 3.3 calcium channel was identified [30]. Figure 7f shows a diagram of the arrangement of the segments for the four domains that form the Ca v 3.2 channel.…”

“…The structure of the Ca v 3.2 channel was obtained by homology modeling using the reported methodology [30]. The amino acid sequence for the Ca v 3.2 was obtained from the UNIPROT database with accession code O95180|CAC1H_Human.…”

“…The ligand Gasteiger charges and hydrogen atoms added in the receptor were calculated with Autodock tools 1.5.7rc1 [47], generating PDBQT files for both the ligand and the receptor. The rigid blind docking method was used to find the best ligand-receptor poses, and literature reporting important binding sites on related proteins was used as reference of criteria to discriminate the best poses [26][27][28][29][30]. The ligand-receptor prediction was made using a grid dimension space delimited by a 90 Å × 110 Å × 110 Å box located in center defined by the coordinates: x(21.647), y(22.126), and z(4.242), that contained the entire receptor structure.…”

Conceptual DFT, QTAIM, and Molecular Docking Approaches to Characterize the T-Type Calcium Channel Blocker Anandamide

“…Also, in a recent study, the Ca v 3.1/Z944 complex was solved by cryo-EM ( Zhao et al, 2019 ), where common hydrophobic interacting residues stabilize the ligand binding pose, including the residue T921 (which is equivalent to T586 of the Ca v 3.2 located in the pore-blocking site). This ligand binding site was proposed also for the genistein/Ca v 3.3 complex using a combination of in vitro and in silico technics for the study ( Rangel-Galván et al, 2021 ). Another recent study mapping the binding site of the Ca v 3.1 channel with a small cyclic peptide PnCs1 using docking and molecular dynamics showed a pocket located mainly in the pore region and including some fenestration ( Depuydt et al, 2021 ).…”

Interacting binding insights and conformational consequences of the differential activity of cannabidiol with two endocannabinoid-activated G-protein-coupled receptors

“…To provide the interacting ligand binding determinants of the protein systems (CBD/GPR55, ML186/GPR55, CBD/CB 1 , and AM251/CB 1 ) and the concomitant conformational consequences, we performed all-atom molecular dynamics (MD) simulations in each of the four protein complexes. Similar general preparation for the systems has been previously used for GPCRs ( Perez-Aguilar et al, 2014 ; Perez-Aguilar et al, 2019 ; Dror et al, 2011 ; Lee and Lyman, 2012 ; Méndez-Luna et al, 2015 ) and other membrane proteins ( Rangel-Galván et al, 2021 ). Briefly, using the structural information from the docking calculations as the initial complex structure, hydrogen atoms were included representing the most probable protonation states for all the amino acids residues at neutral pH.…”