Machine learning-based prediction of drug and ligand binding in BCL-2 variants through molecular dynamics.

Venetoclax is a BH3 (BCL-2 Homology 3) mimetic used to treat leukemia and lymphoma by inhibiting the anti-apoptotic BCL-2 protein thereby promoting apoptosis of cancerous cells. Acquired resistance to Venetoclax via specific variants in BCL-2 is a major problem for the successful treatment of cancer patients. Replica exchange molecular dynamics (REMD) simulations combined with machine learning were used to define the average structure of variants in aqueous solution to predict changes in drug and ligand binding in BCL-2 variants. The variant structures all show shifts in residue positions that occlude the binding groove, and these are the primary contributors to drug resistance. Correspondingly, we established a method that can predict the severity of a variant as measured by the inhibitory constant (Ki) of Venetoclax by measuring the structure deviations to the binding cleft. In addition, we also applied machine learning to the phi and psi angles of the amino acid backbone to the ensemble of conformations that demonstrated a generalizable method for drug resistant predictions of BCL-2 proteins that elucidates changes where detailed understanding of the structure-function relationship is less clear.

T lymphocytes from malignant hyperthermia-susceptible mice display aberrations in intracellular calcium signaling and mitochondrial function.

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca2+ signaling and mitochondrial function in skeletal muscle and causes malignant hyperthermia in humans and mice under triggering conditions. We investigated whether T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca2+ concentration ([Ca2+]i), Ca2+ release from the store, store-operated Ca2+ entry (SOCE), and mitochondrial inner membrane potential (ΔΨm) compared with T lymphocytes from wild-type mice (WT T cells). We explored whether these variables can be used to distinguish between T cells with normal and altered RyR1 genotype. HET and WT T cells were isolated from spleen and lymph nodes and activated in vitro using phytohemagglutinin P. [Ca2+]i and ΔΨm dynamics were examined using Fura 2 and tetramethylrhodamine methyl ester fluorescent dyes, respectively. Activated HET T cells displayed elevated resting [Ca2+]i, diminished responses to Ca2+ mobilization with thapsigargin, and decreased rate of [Ca2+]i elevation in response to SOCE compared with WT T cells. Pretreatment of HET T cells with ryanodine or dantrolene sodium reduced disparities in the resting [Ca2+]i and ability of thapsigargin to mobilize Ca2+ between HET and WT T cells. While SOCE elicited dissipation of the ΔΨm in WT T cells, it produced ΔΨm hyperpolarization in HET T cells. When used as the classification variable, the amplitude of thapsigargin-induced Ca2+ transient showed the best promise in predicting the presence of RyR1-p.R163C mutation. Other significant variables identified by machine learning analysis were the ratio of resting cytosolic Ca2+ level to the amplitude of thapsigargin-induced Ca2+ transient and an integral of changes in ΔΨm in response to SOCE. Our study demonstrated that gain-of-function mutation in RyR1 significantly affects Ca2+ signaling and mitochondrial fiction in T lymphocytes, which suggests that this mutation may cause altered immune responses in its carrier. Our data link the RyR1-p.R163C mutation, which causes inherited skeletal muscle diseases, to deregulation of Ca2+ signaling and mitochondrial function in immune T cells and establish proof-of-principle for in vitro T cell-based diagnostic assay for hereditary RyR1 hyperfunction.

Ionotropic and Metabotropic Mechanisms of Allosteric Modulation of α7 Nicotinic Receptor Intracellular Calcium.

The pharmacological targeting of the α7 nicotinic acetylcholine receptor (α7) is a promising strategy in the development of new drugs for neurologic diseases. Because α7 receptors regulate cellular calcium, we investigated how the prototypical type II-positive allosteric modulator PNU120596 affects α7-mediated calcium signaling. Live imaging experiments show that PNU120596 augments ryanodine receptor-driven calcium-induced calcium release (CICR), inositol-induced calcium release (IICR), and phospholipase C activation by the α7 receptor. Both influx of calcium through the α7 nicotinic acetylcholine receptor (nAChR) channel as well as the binding of intracellular G proteins were involved in the effect of PNU120596 on intracellular calcium. This is evidenced by the findings that chelation of extracellular calcium, expression of α7D44A or α7345-348A mutant subunits, or blockade of calcium store release compromised the ability of PNU120596 to increase intracellular calcium transients generated by α7 ligand activation. Spatiotemporal stochastic modeling of calcium transient responses corroborates these results and indicates that α7 receptor activation enables calcium microdomains locally and to lesser extent in the distant cytosol. From the model, allosteric modulation of the receptor activates CICR locally via ryanodine receptors and augments IICR through enhanced calcium influx due to prolonged α7 nAChR opening. These findings provide a new mechanistic framework for understanding the effect of α7 receptor allosteric modulation on both local and global calcium dynamics.

Effect of Ca2+ on cardiac mitochondrial energy production is modulated by Na+ and H+ dynamics.

The energy production of mitochondria in heart increases during exercise. Several works have suggested that calcium acts at multiple control points to activate net ATP production in what is termed "parallel activation". To study this, a computational model of mitochondrial energy metabolism in the heart has been developed that integrates the Dudycha-Jafri model for the tricarboxylic acid cycle with the Magnus-Keizer model for mitochondrial energy metabolism and calcium dynamics. The model improves upon the previous formulation by including an updated formulation for calcium dynamics, and new descriptions of sodium, hydrogen, phosphate, and ATP balance. To this end, it incorporates new formulations for the calcium uniporter, sodium-calcium exchange, sodium-hydrogen exchange, the F(1)F(0)-ATPase, and potassium-hydrogen exchange. The model simulates a wide range of experimental data, including steady-state and simulated pacing protocols. The model suggests that calcium is a potent activator of net ATP production and that as pacing increases energy production due to calcium goes up almost linearly. Furthermore, it suggests that during an extramitochondrial calcium transient, calcium entry and extrusion cause a transient depolarization that serve to increase NADH production by the tricarboxylic acid cycle and NADH consumption by the respiration driven proton pumps. The model suggests that activation of the F(1)F(0)-ATPase by calcium is essential to increase ATP production. In mitochondria very close to the release sites, the depolarization is more severe causing a temporary loss of ATP production. However, due to the short duration of the depolarization the net ATP production is also increased.

Mitochondrial calcium signaling and energy metabolism.

A computational model of energy metabolism in the mammalian ventricular myocyte is developed to study the effect of cytosolic calcium (Ca(2+)) transients on adenosine triphosphate (ATP) production. The model couples the Jafri-Dudycha model for tricarboxylic acid cycle regulation to a modified version of the Magnus-Keizer model for the mitochondria. The fluxes associated with Ca(2+) uptake and efflux (i.e., the Ca(2+) uniporter and Na(+)-Ca(2+) exchanger) and the F(1)F(0)-ATPase were modified to better model heart mitochondria. Simulations were performed at steady state and with Ca(2+) transients at various pacing frequencies generated by the Rice-Jafri-Winslow model for the guinea pig ventricular myocyte. The effects of the Ca(2+) transients for mitochondria both adjacent to the dyadic space and in the bulk myoplasm were studied. The model shows that Ca(2+) activation of both the tricarboxylic acid cycle and the F(1)F(0)-ATPase are necessary to produce increases in ATP production. The model also shows that in mitochondria located near the subspace, the large Ca(2+) transients can depolarize the mitochondrial membrane potential sufficiently to cause a transient decline in ATP production. However, this transient is of short duration, minimizing its impact on overall ATP production.