Optimization of an in Silico Protocol Using Probe Permeabilities to Identify Membrane Pan-Assay Interference Compounds.

Membrane pan-assay interference compounds (PAINS) are a class of molecules that interact nonspecifically with lipid bilayers and alter their physicochemical properties. An early identification of these compounds avoids chasing false leads and the needless waste of time and resources in drug discovery campaigns. In this work, we optimized an in silico protocol on the basis of umbrella sampling (US)/molecular dynamics (MD) simulations to discriminate between compounds with different membrane PAINS behavior. We showed that the method is quite sensitive to membrane thickness fluctuations, which was mitigated by changing the US reference position to the phosphate atoms of the closest interacting monolayer. The computational efficiency was improved further by decreasing the number of umbrellas and adjusting their strength and position in our US scheme. The inhomogeneous solubility-diffusion model (ISDM) used to calculate the membrane permeability coefficients confirmed that resveratrol and curcumin have distinct membrane PAINS characteristics and indicated a misclassification of nothofagin in a previous work. Overall, we have presented here a promising in silico protocol that can be adopted as a future reference method to identify membrane PAINS.

Effect of a pH Gradient on the Protonation States of Cytochrome c Oxidase: A Continuum Electrostatics Study.

Cytochrome c oxidase (CcO) couples the reduction of dioxygen to water with transmembrane proton pumping, which leads to the generation of an electrochemical gradient. In this study we analyze how one of the components of the electrochemical gradient, the difference in pH across the membrane, or ΔpH, influences the protonation states of residues in CcO. We modified our continuum electrostatics/Monte Carlo (CE/MC) method in order to include the ΔpH and applied it to the study of CcO, in what is, to our best knowledge, the first CE/MC study of CcO in the presence of a pH gradient. The inclusion of a transmembrane pH gradient allows for the identification of residues whose titration behavior depends on the pH on both sides of the membrane. Among the several residues with unusual titration profiles, three are well-known key residues in the proton transfer process of CcO: E286I, Y288I, and K362I. All three residues have been previously identified as being critical for the catalytic or proton pumping functions of CcO. Our results suggest that when the pH gradient increases, these residues may be part of a regulatory mechanism to stem the proton flow.

The anti-inflammatory action of the analgesic kyotorphin neuropeptide derivatives: insights of a lipid-mediated mechanism.

Recently, a designed class of efficient analgesic drugs derived from an endogenous neuropeptide, kyotorphin (KTP, Tyr-Arg) combining C-terminal amidation (KTP-NH2) and N-terminal conjugation to ibuprofen (Ib), IbKTP-NH2, was developed. The Ib moiety is an enhancer of KTP-NH2 analgesic action. In the present study, we have tested the hypothesis that KTP-NH2 is an enhancer of the Ib anti-inflammatory action. Moreover, the impact of the IbKTP-NH2 conjugation on microcirculation was also evaluated by a unified approach based on intravital microscopy in the murine cremasteric muscle. Our data show that KTP-NH2 and conjugates do not cause damage on microcirculatory environment and efficiently decrease the number of leukocyte rolling induced by lipopolysaccharide (LPS). Isothermal titration calorimetry showed that the drugs bind to LPS directly thus contributing to LPS aggregation and subsequent elimination. In a parallel study, molecular dynamics simulations and NMR data showed that the IbKTP-NH2 tandem adopts a preferential "stretched" conformation in lipid bilayers and micelles, with the simulations indicating that the Ib moiety is anchored in the hydrophobic core, which explains the improved partition of IbKTP-NH2 to membranes and the permeability of lipid bilayers to this conjugate relative to KTP-NH2. The ability to bind glycolipids concomitant to the anchoring in the lipid membranes through the Ib residue explains the analgesic potency of IbKTP-NH2 given the enriched glycocalyx of the blood-brain barrier cells. Accumulation of IbKTP-NH2 in the membrane favors both direct permeation and local interaction with putative receptors as the location of the KTP-NH2 residue of IbKTP-NH2 and free KTP-NH2 in lipid membranes is the same.

Constant-pH Molecular Dynamics Study of Kyotorphin in an Explicit Bilayer.

To our knowledge, we present the first constant-pH molecular dynamics study of the neuropeptide kyotorphin in the presence of an explicit lipid bilayer. The overall conformation freedom of the peptide was found to be affected by the interaction with the membrane, in accordance with previous results using different methodologies. Analysis of the interactions between the N-terminus amine group of the peptide and several lipid atoms shows that the membrane is able to stabilize both ionized and neutral forms of kyotorphin, resulting in a pKa value that is similar to the one obtained in water. This illustrates how a detailed molecular model of the membrane leads to rather different results than would be expected from simply regarding it as a low-dielectric slab.

The World of GPCR dimers - Mapping dopamine receptor D2 homodimers in different activation states and configuration arrangements.

G protein-coupled receptors (GPCRs) are known to dimerize, but the molecular and structural basis of GPCR dimers is not well understood. In this study, we developed a computational framework to generate models of symmetric and asymmetric GPCR dimers using different monomer activation states and identified their most likely interfaces with molecular details. We chose the dopamine receptor D2 (D2R) homodimer as a case study because of its biological relevance and the availability of structural information. Our results showed that transmembrane domains 4 and 5 (TM4 and TM5) are mostly found at the dimer interface of the D2R dimer and that these interfaces have a subset of key residues that are mostly nonpolar from TM4 and TM5, which was in line with experimental studies. In addition, TM2 and TM3 appear to be relevant for D2R dimers. In some cases, the inactive configuration is unaffected by the partnered protomer, whereas in others, the active protomer adopts the properties of an inactive receptor. Additionally, the ß-arrestin configuration displayed the properties of an active receptor in the absence of an agonist, suggesting that a switch to another meta-state during dimerization occurred. Our findings are consistent with the experimental data, and this method can be adapted to study heterodimers and potentially extended to include additional proteins such as G proteins or ß-arrestins. In summary, this approach provides insight into the impact of the conformational status of partnered protomers on the overall quaternary GPCR macromolecular structure and dynamics.

Identification of Pan-Assay INterference compoundS (PAINS) Using an MD-Based Protocol.

Pan-assay interference compounds (PAINS) are promiscuous molecules with multiple behaviors that interfere with assay readouts. Membrane PAINS are a subset of these compounds that influence the function of membrane proteins by nonspecifically perturbing the lipid membranes that surround them. Here, we describe a computational protocol to identify potential membrane PAINS molecules by calculating the effect that a given compound has on the bilayer deformation propensity.

New (Iso)quinolinyl-pyridine-2,6-dicarboxamide G-Quadruplex Stabilizers. A Structure-Activity Relationship Study.

G-quadruplex (G4)-interactive small molecules have a wide range of potential applications, not only as drugs, but also as sensors of quadruplex structures. The purpose of this work is the synthesis of analogues of the bis-methylquinolinium-pyridine-2,6-dicarboxamide G4 ligand 360A, to identify relevant structure-activity relationships to apply to the design of other G4-interactive small molecules bearing bis-quinoline or bis-isoquinoline moieties. Thermal denaturation experiments revealed that non-methylated derivatives with a relative 1,4 position between the amide linker and the nitrogen of the quinoline ring are moderate G4 stabilizers, with a preference for the hybrid h-Telo G4, a 21-nt sequence present in human telomeres. Insertion of a positive charge upon methylation of quinoline/isoquinoline nitrogen increases compounds' ability to selectively stabilize G4s compared to duplex DNA, with a preference for parallel structures. Among these, compounds having a relative 1,3-position between the charged methylquinolinium/isoquinolinium nitrogen and the amide linker are the best G4 stabilizers. More interestingly, these ligands showed different capacities to selectively block DNA polymerization in a PCR-stop assay and to induce G4 conformation switches of hybrid h-Telo G4. Molecular dynamic simulations with the parallel G4 formed by a 21-nt sequence present in k-RAS gene promoter, showed that the relative spatial orientation of the two methylated quinoline/isoquinoline rings determines the ligands mode and strength of binding to G4s.

Dynamical Rearrangement of Human Epidermal Growth Factor Receptor 2 upon Antibody Binding: Effects on the Dimerization.

Human epidermal growth factor 2 (HER2) is a ligand-free tyrosine kinase receptor of the HER family that is overexpressed in some of the most aggressive tumours. Although it is known that HER2 dimerization involves a specific region of its extracellular domain, the so-called "dimerization arm", the mechanism of dimerization inhibition remains uncertain. However, uncovering how antibody interactions lead to inhibition of HER2 dimerization is of key importance in understanding its role in tumour progression and therapy. Herein, we employed several computational modelling techniques for a molecular-level understanding of the interactions between HER and specific anti-HER2 antibodies, namely an antigen-binding (Fab) fragment (F0178) and a single-chain variable fragment from Trastuzumab (scFv). Specifically, we investigated the effects of antibody-HER2 interactions on the key residues of "dimerization arm" from molecular dynamics (MD) simulations of unbound HER (in a total of 1 µs), as well as ScFv:HER2 and F0178:HER2 complexes (for a total of 2.5 µs). A deep surface analysis of HER receptor revealed that the binding of specific anti-HER2 antibodies induced conformational changes both in the interfacial residues, which was expected, and in the ECDII (extracellular domain), in particular at the "dimerization arm", which is critical in establishing protein-protein interface (PPI) interactions. Our results support and advance the knowledge on the already described trastuzumab effect on blocking HER2 dimerization through synergistic inhibition and/or steric hindrance. Furthermore, our approach offers a new strategy for fine-tuning target activity through allosteric ligands.