Unveiling (−)‐Englerin A as a Modulator of L‐Type Calcium Channels

Abstract The voltage‐dependent L‐type Ca2+channel was identified as a macromolecular target for (−)‐englerin A. This finding was reached by using an unprecedented ligand‐based prediction platform and the natural product piperlongumine as a pharmacophore probe. (−)‐Englerin A features high substructure dissimilarity to known ligands for voltage‐dependent Ca2+ channels, selective binding affinity for the dihydropyridine site, and potent modulation of calcium signaling in muscle cells and vascular tissue. The observed activity was rationalized at the atomic level by molecular dynamics simulations. Experimental confirmation of this hitherto unknown macromolecular target expands the bioactivity space for this natural product and corroborates the effectiveness of chemocentric computational methods for prioritizing target‐based screens and identifying binding counterparts of complex natural products.

S3 conjugate gradient method. [9] The system was then heated from 0 K to 100 K using Langevin dynamics [10] for 5 ps at constant volume, with weak restraints on the lipid (force constant 10 kcal mol #1 Å #2 ). Following this, the volume was allowed to change freely and the temperature increased to 300K with a Langevin collision frequency of !=1.0 ps #1 , and anisotropic Berendsen regulation [11] (1 atm) with a time constant of 2 ps for 100 ps. The same weak restraint of 10 kcal mol #1 Å #2 was maintained on the lipid molecules. Constant pressure and constant temperature (NPT) runs were performed on the different systems using the AMBER 14. Bonds involving hydrogen were constrained using the SHAKE algorithm, allowing a 2 fs time step. Structural data was recorded every 10 ps. PME was used to treat all electrostatic interactions with a real space cutoff of 10 Å. A long-range analytical dispersion correction was applied to the energy and pressure. All simulations were performed at constant pressure of 1 atm and constant target temperature. Temperature was controlled by the Langevin thermostat, with a collision frequency of !=1.0 ps #1 . Pressure was regulated by the anisotropic Berendsen method (1 atm) with a pressure relaxation time of 1.0 ps.
Each system was simulated for 125 ns.

Nearest neighbour and Principal Component Analysis
L-type channel modulators (IC 50 < 50 $M) were collected from ChEMBL v20. Extended connectivity fingerprints, radius 4 (ECFP4), and Tanimoto indices were calculated with RDKit native nodes implemented in KNIME (www.knime.org). RDKit native nodes were employed to calculate topological descriptors, including molecular weight, logP, number of hydrogen bond donors / acceptors, fraction of sp 3 bonds, number of rings, and atom count. Data was submitted to Principal Component Analysis (PCA) and PC1 vs. PC2 plotted in GraphPad Prism.

Dynamic light scattering
Particle size distributions were measured at a wavelength of 633 nm and a scattering angle of 173° with linear spacing of the correlation time in a Malvern ZetaSizer ZSP. Data was collected at 25 ºC and at a voltage of 150 V prior to analysis by a digital autocorrelation software (Zetasizer Software).
The effective diameter was intensity-based calculated. Average values are reported for the zeta potentials (n = 3).
Differentiation was initiated in the same medium except for 1% FBS and 10nM retinoic acid for 5 days as previously described. [16] 1.4.2 Intracellular calcium (Ca 2+ i ) measurement in H9C2 rat cardiomyocytes Intracellular calcium measurements were performed with Fura-2 AM (Life Technologies) and modified from previously described studies. [17] In short, 1 hour before the measurement, the H9C2 medium was changed to calcium recording buffer (CRB) (125 mM NaCl, 2 mM

Contractility assay in tissue
The assay was performed at Cerep, SA (Celle l'Evescault, France) on a fee-for-service basis according to the procedure of Okamiya et al. [18] Rings of rat thoracic aorta denuded of endothelium were suspended in 20 mL organ baths filled with an oxygenated (95% O 2 and 5% CO 2 ) and pre- methysergide (1 $M) were also present throughout the experiments to block the "-adrenergic, #adrenergic, histamine H 1 , muscarinic and 5-HT 2 receptors, respectively. The tissues were connected to force transducers for isometric tension recordings. They were stretched to a resting tension of 2 g, and then allowed to equilibrate for 60 minutes during which time they were washed repeatedly and the tension readjusted. The experiments were carried out using semi-automated isolated organ systems possessing eight organ baths, with multichannel data acquisition. The parameter measured is the maximum change in tension induced by each compound concentration.
Two independent measurements were conducted.

Evaluation of agonist activity
The tissues were exposed to a high K + solution (60 mM KCl) to verify responsiveness and to obtain a control contractile response. Following washings and recovery of the basal tension, the tissues were exposed to increasing concentrations of the test compound or KCl. The concentrations were added cumulatively and each was left in contact with the tissues until a stable response was obtained or for a maximum of 30 minutes. If an agonist-like response (contraction) was obtained, the reference antagonist nitrendipine (0.03 $M) was tested against the highest concentration of the compound to confirm the involvement of the L-type Ca 2+ channels in this response.

Evaluation of antagonist activity
The tissues were exposed to a high K + solution (60 mM KCl) to obtain a control contractile response. After stabilization of the KCl-induced response, the tissues were exposed to increasing concentrations of the test compound or the reference antagonist nitrendipine. The concentrations were added cumulatively and each was left in contact with the tissues until a stable response was obtained or for a maximum of 30 minutes. An inhibition of the KCl-induced response by the test compound indicates an antagonist activity at the L-type Ca 2+ channels.

Target predictions
No targets were predicted for (-)-EA using SuperPred. Table S2. SPiDER target predictions for (-)-EA.    Natural product-inspired chemical matter [20] shares divergent topological properties to most of bioactive chemical entities.