Functional selectivity is a phenomenon observed in G protein-coupled receptors in which intermediate active-state conformations are stabilized by mutations or ligand binding, resulting in different sets of signaling pathways. Peptides capable of selectively activating β-arrestin, known as biased agonists, have already been characterized in vivo and could correspond to a new therapeutic approach for treatment of cardiovascular diseases. Despite the potential of biased agonism, the mechanism involved in selective signaling remains unclear. In this work, molecular dynamics simulations were employed to compare the conformational profile of the angiotensin II type 1 receptor (AT1R) crystal bound to angiotensin II, bound to the biased ligand TRV027, and in the apo form. Our results show that both ligands induce changes near the NPxxY motif in transmembrane domain 7 that are related to receptor activation. However, the biased ligand does not cause the rotamer toggle alternative positioning and displays an exclusive hydrogen-bonding pattern. Our work sheds light on the biased agonism mechanism and will help in the future design of novel biased agonists for AT1R.

AT1 is a G protein‐coupled receptor (GPCR), and the interaction with angiotensin II (AngII) causes vasoconstriction by Gq protein signaling. AT1 activation leads to receptor internalization by β‐arrestin, which can also act as a second messenger. Biased agonists are pharmacologically advantageous because they can antagonize the effects of Gq without interrupting β‐arrestin beneficial effects (cellular growth and survival), however, their binding mode remains unknown. Here, we investigated how biased agonists bind to AT1 using molecular docking and molecular dynamics simulations. AT1 homology model was built based on CXCR4 crystal structure and inserted in a lipid bilayer. The binding mode of five biased agonists (TRV027, TRV023, SI, SII and DVG) and 2 full agonists (AngII and SVdF) was compared. The analysis of interatomic distances showed that biased agonists interact with Trp84, Tyr87, Glu173, Asn174, Cys180 and Tyr292, while SVdF interacts with His183 and Gln187. Ser105, Arg167, Tyr184, Glu185 and Lys199 help positioning both biased and full agonists inside the receptor pocket, not affecting their activation mechanism. Because of the different binding mode, biased agonists cause conformational changes in AT1 that block Gq actions, still keeping β‐arrestin activated.

Heart Failure (HF) is a common syndrome with high morbimortality, being considered a serious public health problem. One of the therapeutic approaches for HF consists in the use of the sartan class, which are angiotensin II type 1 receptor (AT1R) antagonists. Recent studies have shown that a new class of compounds, known as biased agonists, is able to induce signaling via βarrestin without G-protein activation. This functional selectivity is particularly interesting since G-protein dependent signaling is responsible for cell death and cardiac tissue fibrosis, which leads to cardiac muscle hypertophy and HF progression. On the other hand, β-arrestin signaling is associated with cellular renewal and increased inotropism. In vivo studies suggests that biased agonists could correspond to a superior therapy over conventional angiotensin II type 1 receptor antagonists, which blocks cell signaling as a whole, however their peptidic structure restricts their use to intravenous administration. Moreover, the AT1R crystal structure determination holds great promise for more accurate molecular modeling studies. With that being said, the aim of this work was to plan and develop new non-peptidic biased agonists for ATR1 employing molecular modeling techniques and in vitro tests for hypothesis validation. Molecular dynamics (MD) simulations of the refined AT1R crystal (PDB ID: 4YAY) embedded in a lipid bilayer and molecular docking studies with angiotensin II (AngII) and TRV027 (biased agonist) were conducted. Selected docking poses from both ligands underwent complex MD simulations revealing differences between apo (ligand free) and holo (ligand in the binding site) systems. Our results suggest that TRV027 induces an exclusive hydrogen bond and secondary structure pattern, while AngII affects the hydrophobic pocket conformation, mainly Trp253. Based on the simulations, three pharmacophore models were created and used in virtual screenings in the ZINC15 database, resulting in the selection of five compounds that were tested in vitro. One of the compounds displayed affinity for AT1R and is a promising molecule. Nonetheless, it needs further pathway activation characterization in order to be a classified as a biased agonist. Furthermore, these results have contributed significantly for the proposition of new structures that could be hits with biased agonist activity for AT1R. Thus, for future works, we point out the necessity for synthesis and characterization of this new compounds.

Alkylphosphocholines (APC) are promising antitumor agents, which have the cellular membrane as primary target; however, red blood cell damage limits their wide therapeutic use. A variety of APC analogs has been synthesized and tested showing less hemolytic effect than the class prototype, Miltefosine (HePC). In this work, chemometric methods were applied to a set of 34 APC derivatives to identify the most relevant structural and molecular features of hemolytic activity. The APC derivatives were divided into three groups: (i) N-methylpiperidine and N-methylmorpholine derivatives with a long alkyl chain or flexible cyclopentadecyl rings, displaying a hemolytic rate of 17 %; (ii) adamantyl and cyclopentadecyl derivatives, showing an average hemolysis of 39 %; and, N,N,N-trimethylammonium, trans-N,N,N-trimethylcyclohexanamine, and trans-N,N,N-trimethylcyclopentanamine derivatives, whose average hemolysis was 41 %. The findings suggested that the presence of either bulky cationic head groups, or rings such as adamantyl and cyclohexyl, primarily increases the hemolysis of compounds with eleven atoms in the alkyl chain. Moreover, the macrocyclic cyclopentadecyl seems to be important to the hemolytic potential especially of compounds with five carbon atoms in the alkyl chain. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. Thus, in order to design more potent and less toxic APC antitumors, the reported structural/molecular patterns should not be included in their structure.

Cardiac muscle contraction is regulated by the calcium-binding component of the Troponin Complex, cardiac Troponin C (cTnC), in response to an increase in cytosolic calcium levels. Interaction with calcium is followed by a conformational change, which alters the interactions between troponin complex proteins and mediates the interaction between the thin and thick filaments. Mutations that affect the ability of cTnC to bind calcium are hypothesized to increase or decrease calcium affinity, and thereby predicted to induce hypertrophic or dilated cardiomyopathies, respectively. Cardiomyopathy-associated mutations in the N-Domain of cTnC were selected for this study. These mutations include A8V, L29Q, A31S, L48Q, and C84Y. Equilibrium molecular dynamics simulations were used to model the structural effects of these mutations, and their functional impact assessed using Potential of Mean Force (PMF) calculations, and Isothermal Titration Calorimetry (ITC). Very little structural variation was observed in the mutant protein models. Despite this, large differences in the free energy of calcium binding were observed as a function of these sequence substitutions. The results from ITC are, in some cases, at odds with those derived from MD simulations. These differences may be due to the effect of the mutations on the energetics of the transition between the open and closed conformation of the N-cTnC molecule that are not accessible to the timescales of the MD simulations, but will be readily measured by ITC. Taken together, these results demonstrate that mutations associated with Familial Hypertrophic Cardiomyopathy can act through the alterations in the thermodynamic properties of calcium binding and conformational changes.