Constant-pH MD Simulations of an Oleic Acid Bilayer

Oleic acid is a simple molecule with an aliphatic chain and a carboxylic group whose ionization and, consequently, intermolecular interactions are strongly dependent on the solution pH. The titration curve of these molecules was already obtained using different experimental methods, which have shown the lipid bilayer assemblies to be stable between pH 7.0 and 9.0. In this work, we take advantage of our recent implementations of periodic boundary conditions in Poisson−Boltzmann calculations and ionic strength treatment in simulations of charged lipid bilayers, and we studied the ionization dependent behavior of an oleic acid bilayer using a new extension of the stochastic titration constant-pH MD method. With this new approach, we obtained titration curves that are in good agreement with the experimental data. Also, we were able to estimate the slope of the titration curve from charge fluctuations, which is an important test of thermodynamic consistency for the sampling in a constant-pH MD method. The simulations were performed for ionizations up to 50%, because an experimentally observed macroscopic transition to micelles occurs above this value. As previously seen for a binary mixture of a zwitterionic and an anionic lipid, we were able to reproduce experimental results with simulation boxes usually far from neutrality. This observation further supports the idea that a charged membrane strongly influences the ion distribution in its vicinity and that neutrality is achieved significantly far from the bilayer surface. The good results obtained with this extension of the stochastic titration constant-pH MD method strongly supports its usefulness to sample the coupling between configuration and protonation in these types of biophysical systems. This method stands now as a powerful tool to study more realistic lipid bilayers where pH can influence both the lipids and the solutes interacting with them

Reversibility of Prion Misfolding: Insights from Constant-pH Molecular Dynamics Simulations

The prion protein (PrP) is the cause of a group of diseases known as transmissible spongiform encephalopathies (TSEs). Creutzfeldt–Jakob disease and bovine spongiform encephalopathy are examples of TSEs. Although the normal form of PrP (PrP^C) is monomeric and soluble, it can misfold into a pathogenic form (PrP^Sc) that has a high content of β-structure and can aggregate forming amyloid fibrils. The mechanism of conversion of PrP^C into PrP^Sc is not known but different triggers have been proposed. It can be catalyzed by a PrP^Sc sample, or it can be induced by an external factor, such as low pH. The pH effect on the structure of PrP was recently studied by computational methods [Campos et al. <i>J. Phys. Chem. B</i> <b>2010</b>, <i>114</i>, 12692–12700], and an evident trend of loss of helical structure was observed with pH decrease, together with a gain of β-structures. In particular, one simulation at pH 2 showed an evident misfolding transition. The main goal of the present work was to study the effects of a change in pH to 7 in several transient conformations of this simulation, in order to draw some conclusions about the reversibility of PrP misfolding. Although the most significant effect caused by the change of pH to 7 was a global stabilization of the protein structure, we could also observe that some conformational transitions induced by pH 2 were reversible in many of our simulations, namely those started from the early moments of the misfolding transition. This observation is in good agreement with experiments showing that, even at pH as low as 1.7, it is possible to revert the misfolding process [Bjorndahl et al. <i>Biochemistry</i> <b>2011</b>, <i>50</i>, 1162–1173]

Protonation of DMPC in a Bilayer Environment Using a Linear Response Approximation

pH is a very important property, influencing all important biomolecules such as proteins, nucleic acids, and lipids. The effect of pH on proteins has been the subject of many computational works in recent years. However, the same has not been done for lipids, especially in their most biologically relevant environment: the bilayer. A reason for this is the inherent technical difficulty in dealing with this type of periodic systems. Here, we tackle this problem by developing a Poisson–Boltzmann-based method that takes in consideration the periodic boundary conditions of lipid bilayer patches. We used this approach with a linear response approximation to calculate the p<i>K</i>_a value of a DMPC molecule when diluted in zwitterionic lipids. Our results show that DMPC protonation only becomes relevant at quite low pH values (2–3). However, when it happens, it has a strong impact on lipid conformations, leading to significant heterogeneity in the membrane

p<i>K</i>_a Values of Titrable Amino Acids at the Water/Membrane Interface

Peptides and proteins protonation equilibrium is strongly influenced by its surrounding media. Remarkably, until now, there have been no quantitative and systematic studies reporting the p<i>K</i>_a shifts in the common titrable amino acids upon lipid membrane insertion. Here, we applied our recently developed CpHMD-L method to calculate the p<i>K</i>_a values of titrable amino acid residues incorporated in Ala-based pentapeptides at the water/membrane interface. We observed that membrane insertion leads to desolvation and a clear stabilization of the neutral forms, and we quantified the increases/decreases of the p<i>K</i>_a values in the anionic/cationic residues along the membrane normal. This work highlights the importance of properly modeling the protonation equilibrium in peptides and proteins interacting with membranes using molecular dynamics simulations

Constant-pH MD Simulations of DMPA/DMPC Lipid Bilayers

Current constant-pH molecular dynamics (CpHMD) simulations provide a proper treatment of pH effects on the structure and dynamics of soluble biomolecules like peptides and proteins. However, addressing such effects on lipid membrane assemblies has remained problematic until now, despite the important role played by lipid ionization at physiological pH in a plethora of biological processes. Modeling (de)­protonation events in these systems requires a proper consideration of the physicochemical features of the membrane environment, including a sound treatment of solution ions. Here, we apply our recent CpHMD-L method to the study of pH effects on a 25% DMPA/DMPC bilayer membrane model, closely reproducing the correct lipid phases of this system, namely, gel–fluid coexistence at pH 4 and a fluid phase at pH 7. A significant transition is observed for the membrane ionization and mechanical properties at physiological pH, providing a molecular basis for the well-established role of phosphatidic acid (PA) as a key player in the regulation of many cellular events. Also, as reported experimentally, we observed pH-induced PA–PA lipid aggregation at acidic pH. By including the titration of anionic phospholipids, the current methodology makes possible to simulate lipid bilayers with increased realism. To the best of our knowledge, this is the first simulation study dealing with a continuous phospholipid bilayer with pH titration of all constituent lipids

Treatment of Ionic Strength in Biomolecular Simulations of Charged Lipid Bilayers

Biological membranes are complex systems that have recently attracted a significant scientific interest. Due to the presence of many different anionic lipids, these membranes are usually negatively charged and sensitive to pH. The protonation states of lipids and the ion distribution close to the bilayer are two of the main challenges in biomolecular simulations of these systems. These two problems have been circumvented by using ionized (deprotonated) anionic lipids and enough counterions to preserve the electroneutrality. In this work, we propose a method based on the Poisson–Boltzmann equation to estimate the counterion and co-ion concentration close to a lipid bilayer that avoids the need for neutrality at this microscopic level. The estimated number of ions was tested in molecular dynamics simulations of a 25% DMPA/DMPC lipid bilayer at different ionization levels. Our results show that the system neutralization represents an overestimation of the number of counterions. Consequently, the resulting lipid bilayer becomes too ordered and practically insensitive to ionization. On the other hand, our proposed approach is able to correctly model the ionization dependent isothermal phase transition of the bilayer observed experimentally. Furthermore, our approach is not too computationally expensive and can easily be used to model diverse charged biomolecular systems in molecular dynamics simulations

Targeting Type 2 Diabetes with <i>C</i>‑Glucosyl Dihydrochalcones as Selective Sodium Glucose Co-Transporter 2 (SGLT2) Inhibitors: Synthesis and Biological Evaluation

Inhibiting glucose reabsorption by sodium glucose co-transporter proteins (SGLTs) in the kidneys is a relatively new strategy for treating type 2 diabetes. Selective inhibition of SGLT2 over SGLT1 is critical for minimizing adverse side effects associated with SGLT1 inhibition. A library of <i>C</i>-glucosyl dihydrochalcones and their dihydrochalcone and chalcone precursors was synthesized and tested as SGLT1/SGLT2 inhibitors using a cell-based fluorescence assay of glucose uptake. The most potent inhibitors of SGLT2 (IC₅₀ = 9–23 nM) were considerably weaker inhibitors of SGLT1 (IC₅₀ = 10–19 μM). They showed no effect on the sodium independent GLUT family of glucose transporters, and the most potent ones were not acutely toxic to cultured cells. The interaction of a <i>C</i>-glucosyl dihydrochalcone with a POPC membrane was modeled computationally, providing evidence that it is not a pan-assay interference compound. These results point toward the discovery of structures that are potent and highly selective inhibitors of SGLT2

Membrane-Induced p<i>K</i>_a Shifts in <i>wt</i>-pHLIP and Its L16H Variant

The pH (low) insertion peptides (pHLIPs) is a family of peptides that are able to insert into a lipid bilayer at acidic pH. The molecular mechanism of pHLIPs insertion, folding, and stability in the membrane at low pH is based on multiple protonation events, which are challenging to study at the molecular level. More specifically, the relation between the experimental p<i>K</i> of insertion (p<i>K</i>^exp) of pHLIPs and the p<i>K</i>_a of the key residues is yet to be clarified. We carried out a computational study, complemented with new experimental data, and established the influence of (de)­protonation of titrable residues on the stability of the peptide membrane-inserted state. Constant-pH molecular dynamics simulations were employed to calculate the p<i>K</i>_a values of these residues along the membrane normal. In the <i>wt</i>-pHLIP, we identified Asp14 as the key residue for the stability of the membrane-inserted state, and its p<i>K</i>_a value is strongly correlated with the experimental p<i>K</i>^exp measured in thermodynamics studies. Also, in order to narrow down the pH range at which pHLIP is stable in the membrane, we designed a new pHLIP variant, L16H, where Leu in the 16th position was replaced by a titrable His residue. Our results showed that the L16H variant undergoes two transitions. The calculated p<i>K</i>_a and experimentally observed p<i>K</i>^exp values are in good agreement. Two distinct p<i>K</i>^exp values delimit a pH range where the L16H peptide is stably inserted in the membrane, while, outside this range, the membrane-inserted state is destabilized and the peptide exits from the bilayer. pHLIP peptides have been successfully used to target cancer cells for the delivery of diagnostics and therapeutic agents to acidic tumors. The fine-tuning of the stability of the pHLIP inserted state and its restriction to a narrow well-defined pH range might allow the design of new peptides, able to discriminate between tissues with different extracellular pH values

Insights on the Mechanism of Action of INH‑C₁₀ as an Antitubercular Prodrug

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 <i>katG</i> 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 <i>N</i>′-acylated INH derivative with a long alkyl chain (INH-C₁₀) has been shown to be more effective than INH against the S315T variant of <i>Mycobacterium tuberculosis</i>, but the molecular details of this activity enhancement are still unknown. In this work, we show that INH <i>N</i>′-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 <i>in vivo</i> effectiveness of the compound. Restrained and unrestrained MD simulations of INH and its derivatives at the water/membrane interface were performed and showed a higher preference of INH-C₁₀ for the lipidic phase combined with a significantly higher membrane permeability rate (27.9 cm s^–1), compared with INH-C₂ or INH (3.8 and 1.3 cm s^–1, respectively). Thus, we propose that INH-C₁₀ is able to exhibit better minimum inhibitory concentration (MIC) values against certain variants because of its better ability to permeate through the lipid membrane, enhancing its availability inside the cell. MIC values of INH and INH-C₁₀ against two additional KatG mutations (S315N and D735A) revealed that some KatG variants are able to process INH faster than INH-C₁₀ into an effective antitubercular form (<i>wt</i> and S315N), while others show similar reaction rates (S315T and D735A). Altogether, our results highlight the potential of increased INH lipophilicity for treating INH-resistant strains

Wittig Reaction: Domino Olefination and Stereoselectivity DFT Study. Synthesis of the Miharamycins’ Bicyclic Sugar Moiety

2-<i>O</i>-Acyl protected-d-<i>ribo</i>-3-uloses reacted with [(ethoxycarbonyl)­methylene]­triphenylphosphorane in acetonitrile to afford regio- and stereoselectively 2-(<i>Z</i>)-alkenes in 10–60 min under microwave irradiation. This domino reaction is proposed to proceed via tautomerization of 3-ulose to enol, acyl migration, tautomerization to the 3-<i>O</i>-acyl-2-ulose, and Wittig reaction. Alternatively, in chloroform, regioselective 3-olefination of 2-<i>O</i>-pivaloyl-3-uloses gave (<i>E</i>)-alkenes, key precursors for the miharamycins’ bicyclic sugar moiety