IBNtxA, a morphine derivative, is 10-fold more potent and has a better safety profile than morphine. Animal studies indicate that the analgesic effect of IBNtxA appears to be mediated by the activation of truncated splice variants (6TM) of the Mu opioid receptor (MOR-1) where transmembrane helix 1 (TM1) is removed. Interestingly, morphine is unable to activate 6TM variants. To date, a high resolution structure of 6TM variants is missing, and the interaction of 6TM variants with IBNtxA and morphine remains elusive. In this study we used homology modeling, docking and molecular dynamics (MD) simulations to study a representative 6TM variant (G1) and a full-length 7TM variant of human MOR-1 in complex with IBNtxA and morphine respectively. The structural models of human G1 and 7TM were obtained by homology modeling using the X-ray solved crystal structure of the active mouse 7TM bound to an agonist BU72 (PDB id: ) as the template. Our 6000 ns MD data show that either TM1 truncation (i.e. from 7TM to 6TM) or ligand modification (i.e. from morphine to IBNtxA) alone causes the loss of key morphine-7TM interactions that are well-known to be required for MOR-1 activation. Receptor disruptions are mainly located at TMs 2, 3, 6 and 7 in comparison with the active crystal complex. However, when both perturbations occur in the 6TM-IBNtxA complex, the key ligand-receptor interactions and the receptor conformation are recovered to resemble those in the active 7TM-morphine complex. Our molecular switch analysis further explains well why morphine is not able to activate 6TM variants. The close resemblance between 6TM-IBTtxA and 7TM in complex with PZM21, a G-protein biased 7TM agonist, suggests the possible biased agonism of IBNtxA on G1, which is consistent with its reduced side effects.

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G-protein-coupled receptors (GPCRs) transmit signals into the cell in response to ligand binding at its extracellular domain, which is characterized by the coupling of agonist-induced receptor conformational change to guanine nucleotide (GDP) exchange with guanosine triphosphate on a heterotrimeric (αβγ) guanine nucleotide-binding protein (G-protein), leading to the activation of the G-protein. The signal transduction mechanisms have been widely researched in vivo and in silico. However, coordinated communication from stimulating ligands to the bound GDP still remains elusive. In the present study, we used microsecond (μS) molecular dynamic (MD) simulations to directly probe the communication from the β 2 adrenergic receptor (β 2 AR) with an agonist or an antagonist or no ligand to GDP bound to the open conformation of the Gα protein. Molecular mechanism-general Born surface area calculation results indicated either the agonist or the antagonist destabilized the binding between the receptor and the G-protein but the agonist caused a higher level of destabilization than the antagonist. This is consistent with the role of agonist in the activation of the G-protein. Interestingly, while GDP remained bound with the Gα-protein for the two inactive systems (antagonist-bound and apo form), GDP dissociated from the open conformation of the Gα protein for the agonist activated system. Data obtained from MD simulations indicated that the receptor and the Gα subunit play a big role in coordinated communication and nucleotide exchange. Based on residue interaction network analysis, we observed that engagement of agonist-bound β 2 AR with an α5 helix of G α is essential for the GDP release and the residues in the phosphate-binding loop, α1 helix, and α5 helix play very important roles in the GDP release. The insights on GPCR−G-protein communication will facilitate the rational design of agonists and antagonists that target both active and inactive GPCR binding pockets, leading to more precise drugs.

Amsacrine is an effective topoisomerase II enzyme inhibitor in acute lymphatic leukemia. Previous experimental studies have successfully identified two important mutations (R487K and E571K) conferring 100 and 25 fold resistance to Amsacrine respectively. Although the reduction of the cleavage ligand-DNA-protein ternary complex has been well thought as the major cause of drug resistance, the detailed energetic, structural and dynamic mechanisms remain to be elusive. In this study, we constructed human topoisomerase II alpha (hTop2α) homology model docked with Amsacrine based on crystal structure of human Top2β in complex with etoposide. This wild type complex was used to build the ternary complex with R487K and E571K mutants. Three 500ns molecular dynamics simulations were performed on complex systems of wild type and two mutants. The detailed energetic, structural and dynamic analysis were performed on the simulation data. Our binding data indicated a significant impairment of Amsacrine binding energy in the two mutants compared with the wild type. The order of weakening (R487K>E571K) was in agreement with the order of experimental drug resistance fold (R489K>E571K). Our binding energy decomposition further indicated that weakening of the ligand-protein interaction rather than the ligand-DNA interaction was the major contributor of the binding energy difference between R487K and E571K. In addition, key residues contributing to the binding energy (ΔG) or the decrease of the binding energy (ΔΔG) were identified through the energy decomposition analysis. The change in ligand binding pose, dynamics of protein, DNA and ligand upon the mutations were thoroughly analyzed and discussed. Deciphering the molecular basis of drug resistance is crucial to overcome drug resistance using rational drug design.

Morphine, activating Mu Opioid receptors (MOR-1), produces powerful and immediate analgesic effects. However, morphine use is limited by its high addiction tendency and other serious adverse effects. Recent studies have shown that IBNtxA, a morphine derivative, is 10-fold more potent and has a better safety profile than morphine. The animal studies indicated that the IBNtxA analgesics was from the activation of the truncated spice 6TM variants of MOR-1 in which TM1 is removed. Interestingly, IBNtxA was not able to activate the full length 7TM variants of MOR-1 and morphine was only able to activate 7TM variants but not 6TM variants. There is no high resolution structure of 6TM variants, and the activation mechanism of 6TM variants by IBNtxA remains to be elusive. In this study we used homology modeling, docking and molecular dynamics (MD) simulations to study a representative 6TM variant (G1) and the full length 7TM of human MOR-1 in complex with IBNtxA and morphine. The structure model of G1 was obtained by homology modeling based on X-ray solved crystal structure of active mouse MOR-1 bound to the agonist BU72 (PDB id: 5C1M). Our ms MD data shown that either TM1 truncation (from 7TM to 6TM) or ligand modification (from morphine to IBNtxA) alone caused the loss of key morphine-7TM interactions that are required for the receptor activation and the receptor conformation located at TM2, TM3, TM6 and TM7. However, when both perturbations occur in 6TM-IBNtxA complex, the key interactions and receptor active conformation were maintained. Our energetic, structural and dynamic data consistently supports our explanation.