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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.

Tuberculosis remains one of the top causes of death worldwide, and combating its spread has been severely complicated by the emergence of drug-resistance mutations, highlighting the need for more effective drugs. Despite the resistance to isoniazid (INH) arising from mutations in the katG gene encoding the catalase-peroxidase KatG, most notably the S315T mutation, this compound is still one of the most powerful first-line antitubercular drugs, suggesting further pursuit of the development of tailored INH derivatives. The N′-acylated INH derivative with a long alkyl chain (INH-C10) has been shown to be more effective than INH against the S315T variant of Mycobacterium tuberculosis, but the molecular details of this activity enhancement are still unknown. In this work, we show that INH N′-acylation significantly reduces the rate of production of both isonicotinoyl radical and isonicotinyl–NAD by wild type KatG, but not by the S315T variant of KatG mirroring the in vivo effectiveness of the compound. Restrai...

Catechins are molecules with potential use in different pathologies such as diabetes and cancer, but their pharmaceutical applications are often hindered by their instability in the bloodstream. This issue can be circumvented using liposomes as their nanocarriers for in vivo delivery. In this work, we studied the molecular details of (-)-epigallocatechin-3-gallate (EGCG) interacting with 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) monolayer/bilayer systems to understand the catechin loading ability and liposome stability, using experimental and computational techniques. The molecular dynamics simulations show the EGCG molecules deep inside the lipid bilayer, positioned below the lipid ester groups, generating a concentration-dependent lipid condensation. This effect was also inferred from the surface pressure isotherms of DMPC monolayers. In the polarization-modulated infrared reflection absorption spectra assays, the predominant effect at higher concentrations of EGCG (e.g., 20 mol %) was an increase in lipid tail disorder. The steady-state fluorescence data confirmed this disordered state, indicating that the catechin-induced liposome aggregation outweighs the condensation effects. Therefore, by adding more than 10 mol % EGCG to the liposomes, a destabilization of the vesicles occurs with the ensuing release of entrapped catechins. The loading capacity for DMPC seems to be limited by its disordered lipid arrangements, typical of a fluid phase. To further increase the clinical usefulness of liposomes, lipid bilayers with more stable and organized assemblies should be employed to avoid aggregation at large concentrations of catechin.

Influenza virus is one of the most devastating human pathogens. In order to infect host cells, this virus fuses its membrane with the host membrane in a process mediated by the glycoprotein hemagglutinin. During fusion, the N-terminal region of hemagglutinin, which is known as the fusion peptide (FP), inserts into the host membrane, promoting lipid mixing between the viral and host membranes. Therefore, this peptide plays a key role in the fusion process, but the exact mechanism by which it promotes lipid mixing is still unclear. To shed light into this matter, we performed molecular dynamics (MD) simulations of the influenza FP in different environments (water, dodecylphosphocholine (DPC) micelles, and a dimyristoylphosphatidylcholine (DMPC) membrane). While in pure water the peptide lost its initial secondary structure, in simulations performed in the presence of DPC micelles it remained stable, in agreement with previous experimental observations. In simulations performed in the presence of a preassembled DMPC bilayer, the peptide became unstructured and was unable to insert into the membrane as a result of technical limitations of the method used. To overcome this problem, we used a self-assembly strategy, assembling the membrane together with the peptide. These simulations revealed that the peptide can adopt a membrane-spanning conformation, which had not been predicted by previous MD simulation studies. The peptide insertion had a strong effect on the membrane, lowering the bilayer thickness, disordering nearby lipids, and promoting lipid tail protrusion. These results contribute to a better understanding of the role of the FP in the fusion process.

During the infection process, the influenza fusion peptide (FP) inserts into the host membrane, playing a crucial role in the fusion process between the viral and host membranes. In this work we used a combination of simulation and experimental techniques to analyse the molecular details of this process, which are largely unknown. Although the FP structure has been obtained by NMR in detergent micelles, there is no atomic structure information in membranes. To answer this question, we performed bias-exchange metadynamics (BE-META) simulations, which showed that the lowest energy states of the membrane-inserted FP correspond to helical-hairpin conformations similar to that observed in micelles. BE-META simulations of the G1V, W14A, G12A/G13A and G4A/G8A/G16A/G20A mutants revealed that all the mutations affect the peptide’s free energy landscape. A FRET-based analysis showed that all the mutants had a reduced fusogenic activity relative to the WT, in particular the mutants G12A/G13A and G4A/G8A/G16A/G20A. According to our results, one of the major causes of the lower activity of these mutants is their lower membrane affinity, which results in a lower concentration of peptide in the bilayer. These findings contribute to a better understanding of the influenza fusion process and open new routes for future studies.

The emergence of an influenza pandemic is one of the biggest health threats of our time and there is an urgent need to develop vaccines and drugs against a broad spectrum of influenza viruses (IV). A promising strategy to combat IV is to inactivate the fusion process between the viral and host membranes, which is mediated by the surface protein hemagglutinin (HA). During this process, the N-terminal region of HA, known as fusion peptide (FP), inserts into the host membrane. Although it has been shown that the FP plays a crucial role in the fusion process, the molecular effect of the peptide remains unclear. To analyse the molecular determinants underlying the IV FP, we used state of the art simulation techniques, including metadynamics and constant pH molecular dynamics. The simulation results were combined spectroscopic methods to analyse the peptide's affinity for lipid membranes and its ability to promote lipid-mixing. This allowed us to obtain a detailed molecular characterization of the peptide's conformational properties and its effect on the host membrane. Our work also sheds light into the effect of mutations and external conditions, such as pH, on the FP activity. These results can be useful for the design of novel therapies against this devastating pathogen.

Like all enveloped viruses, influenza virus enters the host cell by fusing its membrane with the host membrane, in a process mediated by the hemagglutinin (HA) glycoprotein. HA is a homotrimeric protein comprised by a soluble part (which contains the receptor binding site and the fusion peptide) and a transmembrane (TM) peptide. The TM peptide attaches the protein to the viral membrane and is also thought to play a role in the fusion process. Although this peptide has been gaining considerable attention in recent years, its 3D structure and the molecular determinants of membrane insertion remain unknown.To analyze the structural determinants of membrane insertion of the TM peptide, we simulated this peptide in the presence of a DMPC bilayer [1]. We observed that the peptide adopts a mainly helical conformation and inserts in the membrane with a tilt angle of ∼64°. Simulations with mutant forms of the TM peptide revealed that mutations of Trp 24 and Tyr 5 found in the C-terminal and N-terminal regions, respectively, affect the helicity and consequently the peptide arrangement inside the membrane bilayer. Since HA is a trimer, we also performed simulations with three copies of the TM peptide embedded in a membrane. The simulations showed that the three peptides assemble in a triangular arrangement that approximately matches the positions where they should attach to the available crystallographic structure of the soluble part of HA.1. B. L. Victor, A. M. Baptista and C. M. Soares, J Chem Inf Model, 2012, 52, 3001-3012.

Membrane fusion is a process involved in a high range of biological functions, going from viral infections to neurotransmitter release. Fusogenic proteins increase the slow rate of fusion by coupling energetically downhill conformational changes of the protein to the kinetically unfavorable fusion of the membrane lipid bilayers. Hemagglutinin is an example of a fusogenic protein, which promotes the fusion of the membrane of the influenza virus with the membrane of the target cell. The N-terminus of the HA2 subunit of this protein contains a fusion domain described to act as a destabilizer of the target membrane bilayers, leading eventually to a full fusion of the two membranes. On the other hand, the C-terminus of the same subunit contains a helical transmembrane domain which was initially described to act as the anchor of the protein to the membrane of the virus. However, in recent years the study of this peptide segment has been gaining more attention since it has also been described to be involved in the membrane fusion process. Yet, the structural characterization of the interaction of such a protein domain with membrane lipids is still very limited. Therefore, in this work, we present a study of this transmembrane peptide domain in the presence of DMPC membrane bilayers, and we evaluate the effect of several mutations, and the effect of peptide oligomerization in this interaction process. Our results allowed us to identify and confirm amino acid residue motifs that seem to regulate the interaction between the segment peptide and membrane bilayers. Besides these sequence requirements, we have also identified length and tilt requirements that ultimately contribute to the hydrophobic matching between the peptide and the membrane. Additionally, we looked at the association of several transmembrane peptide segments and evaluated their direct interaction and stability inside a membrane bilayer. From our results we could conclude that three independent TM peptide segments arrange themselves in a parallel arrangement, very similarly to what is observed for the C-terminal regions of the hemagglutinin crystallographic structure of the protein, to where the segments are attached.

Influenza virus is responsible for worldwide outbreaks of flu, causing hundreds of thousands of deaths every year, which rise to millions in pandemic years. In order to infect the host cells, influenza virus promotes the fusion of the viral and host membranes. This process is mediated by hemagglutinin (HA), a glycoprotein located on the surface of the viral envelope. Influenza HA has a highly conserved N-terminal domain, comprising ∼20 residues, which inserts into the host membrane. This region is critical for destabilizing target membranes during the fusion process and is known as fusion peptide (FP).To elucidate the molecular determinants that lead to the destabilization of biological membranes by the FP, we used a molecular dynamics (MD) approach. We performed simulations with the wild type and four different FP mutants: G1V, W14A, G4A/G8A/G16A/G20A, and G12A/G13A, which are known or expected to have an impaired fusogenic activity. Our aim is to understand how these mutations affect the conformational distribution of the peptide and its ability to destabilize the membrane.Given that lipid membranes are very viscous, it is difficult to obtain reasonable sampling using standard MD. Therefore, in addition to standard MD simulations, we also performed bias-exchange metadynamics simulations, both in water and in a DMPC membrane. These simulations enable us to compare the energy landscapes of the wild type and mutant peptides and explain how these mutations affect the fusogenic ability of the peptide.

Heme-copper oxidases are membrane proteins found in the respiratory chain of aerobic organisms. They are the terminal electron acceptors coupling the translocation of protons across the membrane with the reduction of oxygen to water. Because the catalytic process occurs in the heme cofactors positioned well inside the protein matrix, proton channels must exist. However, due to the high structural divergence among this kind of proteins, the proton channels previously described are not necessarily conserved. In this work we modeled the structure of the quinol oxidase from Acidianus ambivalens using comparative modeling techniques for identifying proton channels. Additionally, given the high importance that water molecules may have in this process, we have developed a methodology, within the context of comparative modeling, to identify high water probability zones and to deconvolute them into chains of ordered water molecules. From our results, and from the existent information from other proteins from the same superfamily, we were able to suggest three possible proton channels: one K-, one D-, and one Q-spatial homologous proton channels. This methodology can be applied to other systems where water molecules are important for their biological function.