Estimation of activation energy for electroporation and pore growth rate in liquid crystalline and gel phases of lipid bilayers using molecular dynamics simulations.

Molecular dynamics simulations of electroporation in POPC and DPPC lipid bilayers have been carried out at different temperatures ranging from 230 K to 350 K for varying electric fields. The dynamics of pore formation, including threshold field, pore initiation time, pore growth rate, and pore closure rate after the field is switched off, was studied in both the gel and liquid crystalline (Lα) phases of the bilayers. Using an Arrhenius model of pore initiation kinetics, the activation energy for pore opening was estimated to be 25.6 kJ mol(-1) and 32.6 kJ mol(-1) in the Lα phase of POPC and DPPC lipids respectively at a field strength of 0.32 V nm(-1). The activation energy decreases to 24.2 kJ mol(-1) and 23.7 kJ mol(-1) respectively at a higher field strength of 1.1 V nm(-1). At temperatures below the melting point, the activation energy in the gel phase of POPC and DPPC increases to 28.8 kJ mol(-1) and 34.4 kJ mol(-1) respectively at the same field of 1.1 V nm(-1). The pore closing time was found to be higher in the gel than in the Lα phase. The pore growth rate increases linearly with temperature and quadratically with field, consistent with viscosity limited growth models.

A new microscopic insight into membrane penetration and reorganization by PETIM dendrimers.

Dendrimers are highly branched polymeric nanoparticles whose structure and topology, largely, have determined their efficacy in a wide range of studies performed so far. An area of immense interest is their potential as drug and gene delivery vectors. Realizing this potential, depending on the nature of cell surface-dendrimer interactions, here we report controlled model membrane penetration and reorganization, using a model supported lipid bilayer and poly(ether imine) (PETIM) dendrimers of two generations. By systematically varying the areal density of the lipid bilayers, we provide a microscopic insight, through a combination of high resolution scattering, atomic force microscopy and atomistic molecular dynamics simulations, into the mechanism of PETIM dendrimer membrane penetration, pore formation and membrane re-organization induced by such interactions. Our work represents the first systematic observation of a regular barrel-like membrane spanning pore formation by dendrimers, tunable through lipid bilayer packing, without membrane disruption.

In silico energetic and molecular dynamic simulations studies demonstrate potential effect of the point mutations with implications for protein engineering in BDNF.

Protein engineering by directed evolution is time-consuming. Hence, in silico techniques like FoldX-Yasara for ∆∆G calculation, and SNPeffect for predicting propensity for aggregation, amyloid formation, and chaperone binding are employed to design proteins. Here, we used in silico techniques to engineer BDNF-NTF3 interaction and validated it using mutations with known functional implications for NGF dimer. The structures of three mutants representing a positive, negative, or neutral ∆∆G involving two interface residues in BDNF and two mutations representing a neutral and positive ∆∆G in NGF, which is aligned with BDNF, were selected for molecular dynamics (MD) simulation. Our MD results conclude that the secondary structure of individual protomers of the positive and negative mutants displayed a similar or different conformation from the NTF3 monomer, respectively. The positive mutants showed fewer hydrophobic interactions and higher hydrogen bonds compared to the wild-type, negative, and neutral mutants with similar SASA, suggesting solvent-mediated disruption of hydrogen-bonded interactions. Similar results were obtained for mutations with known functional implications for NGF and BDNF. The results suggest that mutations with known effects in homologous proteins could help in validation, and in silico directed evolution experiments could be a viable alternative to the experimental technique used for protein engineering.

Synthesis, computational docking and molecular dynamics studies of a new class of spiroquinoxalinopyrrolidine embedded chromanone hybrids as potent anti-cholinesterase agents.

Novel structurally intriguing heterocycles embedded with spiropyrrolidine, quinoxaline and chromanone units were synthesized in good yields using a [Bmim]Br accelerated multicomponent reaction strategy. The key step of the reaction is 1,3-dipolar cycloaddition involving highly functionalized dipolarophile, viz. 3-benzylidenechroman-4-one, to afford spiroquinoxalinopyrrolidine embedded chromanone hybrid heterocycles. The formation of spiro products occurs via two C-C, two N-C and one C-N bonds possessing four adjoining stereogenic centers, two of which are spiro carbons. The newly synthesized spiro compounds showed potent acetylcholinesterase and butyrylcholinesterase inhibitory activities. Moreover, compounds with fluorine displayed the highest AChE (3.20 ± 0.16 µM) and BChE (18.14 ± 0.06 µM) inhibitory activities. Further, docking studies, followed by all-atom molecular dynamics, showed results that are consistent with in vitro experimental findings. Although docking scores for the synthesized derivatives were higher than those of the standard drug, MD MMPBSA results showed better binding of synthesized derivatives (-93.5 ± 11.9 kcal mol-1) compared to the standard drug galantamine (-66.2 ± 12.3 kcal mol-1) for AChE but exhibited similar values (-98.1 ± 11.2 and -97.9 ± 11.5 kcal mol-1) for BChE. These differences observed in drug binding with AChE/BChE are consistent with RMSD, RMSF, LIG plots, and FEL structural analysis. Taken together, these derivatives could be potential candidates as inhibitors of AChE and BChE.

Synthesis, topology, molecular docking and dynamics studies of o-phenylenediamine derivative.

The N, N'-(1,2-phenylene) bis (1- (4- chlorophenyl) methanimine) (CS4) was synthesized and characterized by infrared (IR), absorption (UV-vis) and NMR (1H and 13C) spectral analyses. The structural parameters, vibrational frequencies, potential energy and the distribution analysis (PED) were calculated by using DFT with the basis set of B3LYP/cc-pVDZ and these spectral values were compared to the experimental values. HOMO and LUMO studied were performed in order to understand the stability and biological activity of the compound. The most reactive sites on the compound were investigated by utilizing MEP energy surface and Fukui function descriptor with the natural population analysis (NPA) of the charges. The study of the natural bond orbitals (NBO) reveals the delocalization of the intramolecular interaction of the charges in the compound. Additionally, topological investigations (ELF, LOL), determination of thermodynamic parameters and noncovalent interaction (NCI) study by using topology (RDG) analysis were also carried out. Finally, the molecular docking and molecular dynamics simulations was carried out by examining against glycosylphosphatidylinositol phospholipase D inhibitor receptor for distinct protein targets (3MZG).Communicated by Ramaswamy H. Sarma.

Computational analysis of sodium-dependent phosphate transporter SLC20A1/PiT1 gene identifies missense variations C573F, and T58A as high-risk deleterious SNPs.

SLC20A1/PiT1 is a sodium-dependent inorganic phosphate transporter, initially recognized as the retroviral receptor for Gibbon Ape Leukemia Virus in humans. SNPs in SLC20A1 is associated with Combined Pituitary Hormone Deficiency and Sodium Lithium Counter transport. Using in silico techniques, we have screened the nsSNPs for their deleterious effect on the structure and function of SLC20A1. Screening with sequence and structure-based tools on 430 nsSNPs, filtered 17 nsSNPs which are deleterious. To evaluate the role of these SNPs, protein modeling and MD simulations were performed. A comparative analysis of model generated with SWISS-MODEL and AlphaFold shows that many residues are in the disallowed region of Ramachandran plot. Since SWISS-MODEL structure has a 25-residue deletion, the AlphaFold structure was used to perform MD simulation for equilibration and structure refinement. Further, to understand perturbation of energetics, we performed in silico mutagenesis and ΔΔG calculation using FoldX on MD refined structures, which yielded SNPs that are neutral (3), destabilizing (12) and stabilizing (2) on protein structure. Furthermore, to elucidate the impact of SNPs on structure, we performed MD simulations to discern the changes in RMSD, Rg, RMSF and LigPlot of interacting residues. RMSF profiles of representative SNPs revealed that A114V (neutral) and T58A (positive) were more flexible & C573F (negative) was more rigid compared to wild type, which is also reflected in the changes in number of local interacting residues in LigPlot and ΔΔG. Taken together, our results show that SNPs can lead to structural perturbations and impact the function of SLC20A1 with potential implications for disease.Communicated by Ramaswamy H. Sarma.

Hydrophobic Gating and 1/f Noise of the Anthrax Toxin Channel.

"Pink" or 1/f noise is a natural phenomenon omnipresent in physics, economics, astrophysics, biology, and even music and languages. In electrophysiology, the stochastic activity of a number of biological ion channels and artificial nanopores could be characterized by current noise with a 1/f power spectral density. In the anthrax toxin channel (PA63), it appears as fast voltage-independent current interruptions between conducting and nonconducting states. This behavior hampers potential development of PA63 as an ion-channel biosensor. On the bright side, the PA63 flickering represents a mesmerizing phenomenon to investigate. Notably, similar 1/f fluctuations are observed in the channel-forming components of clostridial binary C2 and iota toxins, which share functional and structural similarities with the anthrax toxin channel. Similar to PA63, they are evolved to translocate the enzymatic components of the toxins into the cytosol. Here, using high-resolution single-channel lipid bilayer experiments and all-atom molecular dynamic simulations, we suggest that the 1/f noise in PA63 occurs as a result of "hydrophobic gating" at the ϕ-clamp region, the phenomenon earlier observed in several water-filled channels "fastened" inside by the hydrophobic belts. The ϕ-clamp is a narrow "hydrophobic ring" in the PA63 lumen formed by seven or eight phenylalanine residues at position 427, conserved in the C2 and iota toxin channels, which catalyzes protein translocation. Notably, the 1/f noise remains undetected in the F427A PA63 mutant. This finding can elucidate the functional purpose of 1/f noise and its possible role in the transport of the enzymatic components of binary toxins.

Translocation of Bioactive Molecules through Carbon Nanotubes Embedded in the Lipid Membrane.

One of the major challenges of nanomedicine and gene therapy is the effective translocation of drugs and genes across cell membranes. In this study, we describe a systematic procedure that could be useful for efficient drug and gene delivery into the cell. Using fully atomistic molecular dynamics (MD) simulations, we show that molecules of various shapes, sizes, and chemistries can be spontaneously encapsulated in a single-walled carbon nanotube (SWCNT) embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer, as we have exemplified with dendrimers, asiRNA, ssDNA, and ubiquitin protein. We compute the free energy gain by the molecules upon their entry inside the SWCNT channel to quantify the stability of these molecules inside the channel as well as to understand the spontaneity of the process. The free energy profiles suggest that all molecules can enter the channel without facing any energy barrier but experience a strong energy barrier (≫kBT) to translocate across the channel. We propose a theoretical model for the estimation of encapsulation and translocation times of the molecules. Whereas the model predicts the encapsulation time to be of the order of few nanoseconds, which match reasonably well with those obtained from the simulations, it predicts the translocation time to be astronomically large for each molecule considered in this study. This eliminates the possibility of passive diffusion of the molecules through the CNT-nanopore spanning across the membrane. To counter this, we put forward a mechanical method of ejecting the encapsulated molecules by pushing them with other free-floating SWCNTs of diameter smaller than the pore diameter. The feasibility of the proposed method is also demonstrated by performing MD simulations. The generic strategy described here should work for other molecules as well and hence could be potentially useful for drug- and gene-delivery applications.

Dendrimer Interactions with Lipid Bilayer: Comparison of Force Field and Effect of Implicit vs Explicit Solvation.

The understanding of dendrimer interactions with cell membranes has great importance in drug/gene delivery based therapeutics. Although molecular simulations have been used to understand the nature of dendrimer interactions with lipid membranes, its dependency on available force field parameters is poorly understood. In this study, we have carried out fully atomistic molecular dynamics (MD) simulations of a protonated G3 poly(amido amine) (PAMAM) dendrimer-dimyristoylphosphatidylcholine (DMPC) lipid bilayer complex using three different force fields (FFs) namely, CHARMM, GAFF, and GROMOS in the presence of explicit water to understand the structure of the lipid-dendrimer complex and nature of their interaction. CHARMM and GAFF dendrimers initially in contact with the lipid head groups were found to move away from the lipid bilayer during the course of simulation; however, the dendrimer remained strongly bound to the lipid head groups with the GROMOS FF. Potential of the mean force (PMF) computations of the dendrimer along the bilayer normal showed a repulsive barrier (∼20 kcal/mol) between dendrimer and lipid bilayer in the case of CHARMM and GAFF force fields. In contrast, an attractive interaction (∼40 kcal/mol) is obtained with the GROMOS force field, consistent with experimental observations of membrane binding observed with lower generation G3 PAMAM dendrimers. This difference with the GROMOS dendrimer is attributed to the strong dendrimer-lipid interaction and lowered surface hydration of the dendrimer. Assessing the role of solvent, we find that the CHARMM and GAFF dendrimers strongly bind to the lipid bilayer with an implicit solvent (Generalized Born) model, whereas binding is not observed with explicit water (TIP3P). The opposing nature of dendrimer-membrane interactions in the presence of explicit and implicit solvents demonstrates that hydration effects play an important role in modulating the dendrimer-lipid interaction warranting a case for refinement of the existing dendrimer/lipid force fields.

pH controlled gating of toxic protein pores by dendrimers.

Designing effective nanoscale blockers for membrane inserted pores formed by pore forming toxins, which are expressed by several virulent bacterial strains, on a target cell membrane is a challenging and active area of research. Here we demonstrate that PAMAM dendrimers can act as effective pH controlled gating devices once the pore has been formed. We have used fully atomistic molecular dynamics (MD) simulations to characterize the cytolysin A (ClyA) protein pores modified with fifth generation (G5) PAMAM dendrimers. Our results show that the PAMAM dendrimer, in either its protonated (P) or non-protonated (NP) states can spontaneously enter the protein lumen. Protonated dendrimers interact strongly with the negatively charged protein pore lumen. As a consequence, P dendrimers assume a more expanded configuration efficiently blocking the pore when compared with the more compact configuration adopted by the neutral NP dendrimers creating a greater void space for the passage of water and ions. To quantify the effective blockage of the protein pore, we have calculated the pore conductance as well as the residence times by applying a weak force on the ions/water. Ionic currents are reduced by 91% for the P dendrimers and 31% for the NP dendrimers. The preferential binding of Cl(-) counter ions to the P dendrimer creates a zone of high Cl(-) concentration in the vicinity of the internalized dendrimer and a high concentration of K(+) ions in the transmembrane region of the pore lumen. In addition to steric effects, this induced charge segregation for the P dendrimer effectively blocks ionic transport through the pore. Our investigation shows that the bio-compatible PAMAM dendrimers can potentially be used to develop therapeutic protocols based on the pH sensitive gating of pores formed by pore forming toxins to mitigate bacterial infections.

Molecular Dynamics Study of the Structure, Flexibility, and Hydrophilicity of PETIM Dendrimers: A Comparison with PAMAM Dendrimers.

A new class of dendrimers, the poly(propyl ether imine) (PETIM) dendrimer, has been shown to be a novel hyperbranched polymer having potential applications as a drug delivery vehicle. Structure and dynamics of the amine terminated PETIM dendrimer and their changes with respect to the dendrimer generation are poorly understood. Since most drugs are hydrophobic in nature, the extent of hydrophobicity of the dendrimer core is related to its drug encapsulation and retention efficacy. In this study, we carry out fully atomistic molecular dynamics (MD) simulations to characterize the structure of PETIM (G2-G6) dendrimers in salt solution as a function of dendrimer generation at different protonation levels. Structural properties such as radius of gyration (Rg), radial density distribution, aspect ratio, and asphericity are calculated. In order to assess the hydrophilicity of the dendrimer, we compute the number of bound water molecules in the interior of dendrimer as well as the number of dendrimer-water hydrogen bonds. We conclude that PETIM dendrimers have relatively greater hydrophobicity and flexibility when compared with their extensively investigated PAMAM counterparts. Hence PETIM dendrimers are expected to have stronger interactions with lipid membranes as well as improved drug encapsulation and retention properties when compared with PAMAM dendrimers. We compute the root-mean-square fluctuation of dendrimers as well as their entropy to quantify the flexibility of the dendrimer. Finally we note that structural and solvation properties computed using force field parameters derived based on the CHARMM general purpose force field were in good quantitative agreement with those obtained using the generalized Amber force field (GAFF).