Systematic evaluation of lecithin:cholesterol acyltransferase binding sites in apolipoproteins via peptide based nanodiscs: regulatory role of charged residues at positions 4 and 7.

Lecithin:cholesterol acyltransferase (LCAT) exhibits α-activity on high-density and ß-activity on low-density lipoproteins. However, the molecular determinants governing LCAT activation by different apolipoproteins remain elusive. Uncovering these determinants would offer the opportunity to design and explore advanced therapies against dyslipidemias. Here, we have conducted coarse-grained and all-atom molecular dynamics simulations of LCAT with nanodiscs made with α-helical amphiphilic peptides either derived from apolipoproteins A1 and E (apoA1 and apoE) or apoA1 mimetic peptide 22A that was optimized to activate LCAT. This study aims to explore what drives the binding of peptides to our previously identified interaction site in LCAT. We hypothesized that this approach could be used to screen for binding sites of LCAT in different apolipoproteins and would provide insights to differently localized LCAT activities. Our screening approach was able to discriminate apoA1 helixes 4, 6, and 7 as key contributors to the interaction with LCAT supporting the previous research data. The simulations provided detailed molecular determinants driving the interaction with LCAT: the formation of hydrogen bonds or salt bridges between peptides E4 or D4 and LCAT S236 or K238 residues. Additionally, salt bridging between R7 and D73 was observed, depending on the availability of R7. Expanding our investigation to diverse plasma proteins, we detected novel LCAT binding helixes in apoL1, apoB100, and serum amyloid A. Our findings suggest that the same binding determinants, involving E4 or D4 -S236 and R7-D73 interactions, influence LCAT ß-activity on low-density lipoproteins, where apoE and or apoB100 are hypothesized to interact with LCAT.

Far-red light-triggered cargo release from liposomes b ound to a photosensitizer-cellulose nanofiber hydrogel.

In our research we used the anionic nanofibrillar cellulose (ANFC) as a platform for far-red light-induced release of cargo from liposomes. In contrast to previous works, where photosensitizers are usually in the liposomal bilayers, we used a cellulose-binding dye. Our phthalocyanine derivative has been shown to bind very strongly to cellulose and cellulose nanofiber hydrogels, allowing us to place it outside of the liposomes. Both the sensitizer and cationic liposomes bind strongly to the ANFC after mixing, making the system easy to fabricate. Upon light activation, the photosensitizer generates reactive oxygen species (ROS) within the ANFC hydrogel, where the reactive oxygen species oxidize unsaturated lipids in the liposomal membrane, which makes the liposomes more permeable, resulting in on-demand cargo release. We were able to achieve ca. 70 % release of model hydrophilic cargo molecule calcein from the hydrogels with a relatively low dose of light (262 J/cm2) while employing the straightforward fabrication techniques. Our system was remarkably responsive to the far-red light (730 nm), enabling deep tissue penetration. Therefore, this very promising novel cellulose-immobilized photosensitizer liposomal platform could be used as a controlled drug delivery system, which can have applications in externally activated coatings or implants.

Development of Robust Cationic Light-Activated Thermosensitive Liposomes: Choosing the Right Lipids.

Extensive research has been conducted on cationic light-activated thermosensitive liposomes (CLTSLs) as a means for site-specific and controlled drug release; however, less attention has been given to the stability of these nanoparticles. Selecting the appropriate lipids is crucial for the development of a stable and responsive system. In this study, we investigated the impact of various lipids on the physical properties of cationic light-activated liposomes. Incorporating poly(ethylene glycol) PEG molecules resulted in uniform liposomes with low polydispersity index, while the addition of unsaturated lipid (DOTAP) resulted in extremely leaky liposomes, with almost 80% release in just 10 min of incubation at body temperature. Conversely, the inclusion of cholesterol in the formulation increased liposome stability too much and decreased their sensitivity to stimuli-responsive release, with only 14% release after 2 min of light exposure. To achieve stable and functional CLTSL, we substituted an equivalent amount of unsaturated lipid with a saturated lipid (DPTAP), resulting in stable liposomes at body temperature that were highly responsive to light, releasing 90% of their content in 10 s of light exposure. We also conducted two atomistic molecular dynamics simulations using lipid compositions with saturated and unsaturated lipids to investigate the effect of lipid composition on the dynamical properties of the liposomal lipid bilayer. Our findings suggest that the nature of lipids used to prepare liposomes significantly affects their properties, especially when the drug loading needs to be stable but triggered drug release properties are required at the same time. Selecting the appropriate lipids in the right amount is therefore essential for the preparation of liposomes with desirable properties.

Expanding the Paradigm of Structure-Based Drug Design: Molecular Dynamics Simulations Support the Development of New Pyridine-Based Protein Kinase C-Targeted Agonists.

Protein kinase C (PKC) modulators hold therapeutic potential for various diseases, including cancer, heart failure, and Alzheimer's disease. Targeting the C1 domain of PKC represents a promising strategy; the available protein structures warrant the design of PKC-targeted ligands via a structure-based approach. However, the PKC C1 domain penetrates the lipid membrane during binding, complicating the design of drug candidates. The standard docking-scoring approach for PKC lacks information regarding the dynamics and the membrane environment. Molecular dynamics (MD) simulations with PKC, ligands, and membranes have been used to address these shortcomings. Previously, we observed that less computationally intensive simulations of just ligand-membrane interactions may help elucidate C1 domain-binding prospects. Here, we present the design, synthesis, and biological evaluation of new pyridine-based PKC agonists implementing an enhanced workflow with ligand-membrane MD simulations. This workflow holds promise to expand the approach in drug design for ligands targeted to weakly membrane-associated proteins.

Rational design of a polysaccharide-based viral mimicry nanocomplex for potent gene silencing in inflammatory tissues.

Rational design and fabrication of small interfering RNA (siRNA) delivery system with simple production scheme, specific targeting capability, responsiveness to endogenous stimuli and potential multi-functionalities remains technically challenging. Herein, we screen and design a virus-mimicking polysaccharide nanocomplex that shows specific gene delivery capability in a selective subset of leukocytes. A virus-inspired poly (alkyl methacrylate-co-methacrylic acid) fragment was conjugated on barley ß-glucans (EEPG) to endow the nanocomplex with pH-dependent endosomal membrane destabilization capabilities, as confirmed both biologically and computationally. siRNA loaded EEPG nanocomplex is feasibly fabricated in a single-step manner, which exhibit efficient gene silencing efficacy towards Dectin-1+ monocytes/macrophages. The inherent targeting affinity and feasible gene silencing potency of EEPG nanocomplex are investigated in three independent murine inflammation models, including myocardial infarction, lung fibrosis and acute liver damage. Significant enhanced accumulation level of EEPG nanocomplex is observed in cardiac lesion site, indicating its exclusive targeting capability for ischemic heart diseases. As a proof of concept, siTGF-ß based gene therapy is confirmed in murine model with heart fibrosis. Overall, our findings suggest the designed EEPG nanocomplex is favorable for siRNA delivery, which might have translational potential as a versatile platform in inflammation-related diseases.

Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids.

The mechanistic details behind the activation of lecithin-cholesterol acyltransferase (LCAT) by apolipoprotein A-I (apoA-I) and its mimetic peptides are still enigmatic. Resolving the fundamental principles behind LCAT activation will facilitate the design of advanced HDL-mimetic therapeutic nanodiscs for LCAT deficiencies and coronary heart disease and for several targeted drug delivery applications. Here, we have combined coarse-grained molecular dynamics simulations with complementary experiments to gain mechanistic insight into how apoA-Imimetic peptide 22A and its variants tune LCAT activity in peptide-lipid nanodiscs. Our results highlight that peptide 22A forms transient antiparallel dimers in the rim of nanodiscs. The dimerization tendency considerably decreases with the removal of C-terminal lysine K22, which has also been shown to reduce the cholesterol esterification activity of LCAT. In addition, our simulations revealed that LCAT prefers to localize to the rim of nanodiscs in a manner that shields the membrane-binding domain (MBD), αA-αA', and the lid amino acids from the water phase, following previous experimental evidence. Meanwhile, the location and conformation of LCAT in the rim of nanodiscs are spatially more restricted when the active site covering the lid of LCAT is in the open form. The average location and spatial dimensions of LCAT in its open form were highly compatible with the electron microscopy images. All peptide 22A variants studied here had a specific interaction site in the open LCAT structure flanked by the lid and MBD domain. The bound peptides showed different tendencies to form antiparallel dimers and, interestingly, the temporal binding site occupancies of the peptide variants affected their in vitro ability to promote LCAT-mediated cholesterol esterification.

Insights into the behavior of unsaturated diacylglycerols in mixed lipid bilayers in relation to protein kinase C activation-A molecular dynamics simulation study.

The lipid second messenger diacylglycerol (DAG) is known for its involvement in many types of cellular signaling, especially as an endogenous agonist for protein kinase C (PKC). Evidence has emerged that the degree of saturation of the DAG molecules can affect PKC activation. DAG molecules with different acyl chain saturation have not only been observed to induce varying extents of PKC activation, but also to express selectivity towards different PKC isozymes. Both qualities are important for precise therapeutic activation of PKC; understanding DAG behavior at the molecular level in different environments has much potential in the development of drugs to target PKC. We used molecular dynamics simulations to study the behavior of two different unsaturated DAG species in lipid environments with varying degrees of unsaturation. We focus on phosphatidylethanolamine (PE) instead of phosphatidylcholine (PC) to more accurately model the relevant biomembranes. The effect of cholesterol (CHOL) on these systems was also explored. We found that both high level of unsaturation in the acyl chains of the DAG species and presence of CHOL in the surrounding membrane increase DAG molecule availability at the lipid-water interface. This can partially explain the previously observed differences in PKC activation strength and specificity, the complete mechanism is, however, likely to be more complex. Our simulations coupled with the current understanding of lipids highlight the need for more simulations of biologically accurate lipid environments in order to determine the correct correlations between molecular mechanisms and biological behavior when studying PKC activation.

Mechanistic Insight into How PEGylation Reduces the Efficacy of pH-Sensitive Liposomes from Molecular Dynamics Simulations.

Liposome-based drug delivery systems composed of DOPE stabilized with cholesteryl hemisuccinate (CHMS) have been proposed as a drug delivery mechanism with pH-triggered release as the anionic form (CHSa) is protonated (CHS) at reduced pH; PEGylation is known to decrease this pH sensitivity. In this manuscript, we set out to use molecular dynamics (MD) simulations with a model with all-atom resolution to provide insight into why incorporation of poly(ethyleneglycol) (PEG) into DOPE-CHMS liposomes reduces their pH sensitivity; we also address two additional questions: (1) How CHSa stabilizes DOPE bilayers into a lamellar conformation at a physiological pH of 7.4? and (2) how the change from CHSa to CHS at acidic pH triggers the destabilization of DOPE bilayers? We found that (A) CHSa stabilizes the DOPE lipid membrane by increasing the hydrophilicity of the bilayer surface, (B) when CHSa changes to CHS by pH reduction, DOPE bilayers are destabilized due to a reduction in bilayer hydrophilicity and a reduction in the area per lipid, and (C) PEG stabilizes DOPE bilayers into the lamellar phase, thus reducing the pH sensitivity of the liposomes by increasing the area per lipid through penetration into the bilayer, which is our main focus.

Positive allosteric modulators of lecithin: Cholesterol acyltransferase adjust the orientation of the membrane-binding domain and alter its spatial free energy profile.

Lecithin:cholesterol acyltransferase protein (LCAT) promotes the esterification reaction between cholesterol and phospholipid-derived acyl chains. Positive allosteric modulators have been developed to treat LCAT deficiencies and, plausibly, also cardiovascular diseases in the future. The mechanism of action of these compounds is poorly understood. Here computational docking and atomistic molecular dynamics simulations were utilized to study the interactions between LCAT and the activating compounds. Results indicate that all drugs bind to the allosteric binding pocket in the membrane-binding domain in a similar fashion. The presence of the compounds in the allosteric site results in a distinct spatial orientation and sampling of the membrane-binding domain (MBD). The MBD's different spatial arrangement plausibly affects the lid's movement from closed to open state and vice versa, as suggested by steered molecular dynamics simulations.

Molecular Insights on Successful Reconstitution of Freeze-Dried Nanofibrillated Cellulose Hydrogel.

The diversity and safety of nanofibrillated cellulose (NFC) hydrogels have gained a vast amount of interest at the pharmaceutical site in recent years. Moreover, this biomaterial has a high potential to be utilized as a protective matrix during the freeze-drying of heat-sensitive pharmaceuticals and biologics to increase their properties for long-term storing at room temperature and transportation. Since freeze-drying and subsequent reconstitution have not been optimized for this biomaterial, we must find a wider understanding of the process itself as well as the molecular level interactions between the NFC hydrogel and the most suitable lyoprotectants. Herein we optimized the reconstitution of the freeze-dried NFC hydrogel by considering critical quality attributes required to ensure the success of the process and gained insights of the obtained experimental data by simulating the effects of the used lyoprotectants on water and NFC. We discovered the correlation between the measured characteristics and molecular dynamics simulations and obtained successful freeze-drying and subsequent reconstitution of NFC hydrogel with the presence of 300 mM of sucrose. These findings demonstrated the possibility of using the simulations together with the experimental measurements to obtain a more comprehensive way to design a successful freeze-drying process, which could be utilized in future pharmaceutical applications.

Rigorous Computational Study Reveals What Docking Overlooks: Double Trouble from Membrane Association in Protein Kinase C Modulators.

Increasing protein kinase C (PKC) activity is of potential therapeutic value. Its activation involves an interaction between the C1 domain and diacylglycerol (DAG) at intracellular membrane surfaces; DAG mimetics hold promise as new drugs. We previously developed the isophthalate derivative HMI-1a3, an effective but highly lipophilic (clogP = 6.46) DAG mimetic. Although a less lipophilic pyrimidine analog, PYR-1gP (clogP = 3.30), gave positive results in computational docking, it unexpectedly presented greatly diminished binding to PKC in vitro. Through more rigorous computational molecular modeling, we reveal that, unlike HMI-1a3, PYR-1gP forms an intramolecular hydrogen bond, which both obstructs binding and reorients PYR-1gP in the membrane in a fashion that prevents it from correctly accessing the PKC C1 domain. Our results highlight the great value of molecular dynamics simulations as a key component for the drug design process of ligands targeting weakly membrane-associated proteins, where simulation in the relevant membrane environment is crucial for obtaining biologically applicable results.

Insight into the antimicrobial mechanism of action of ß2,2-amino acid derivatives from molecular dynamics simulation: Dancing the can-can at the membrane surface.

The development of antimicrobial agents that target and selectively disrupt biofilms is a pressing issue since, so far, no antibiotics have been developed that achieve this effectively. Previous experimental work has found a promising set of antibacterial peptides: ß2,2-amino acid derivatives, relatively small molecules with common structural elements composed of a polar head group and two non-polar hydrocarbon arms. In order to develop insight into possible mechanisms of action of these novel antibacterial agents, we have performed an in silico investigation of four leading ß2,2-amino acid derivatives, interacting with models of both bacterial (target) and eukaryotic (host) membranes, using molecular dynamics simulation with a model with all-atom resolution. We found an unexpected result that could shed light on the mechanism of action of these antimicrobial agents: the molecules assume a conformation where one of the hydrophobic arms is directed downward into the membrane core while the other is directed upwards, out of the membrane and exposed above the position of the membrane headgroups; we dubbed this conformation the "can-can pose". Intriguingly, the can-can pose was most closely linked to the choice of headgroup. Also, the compound previously found to be most effective against biofilms displayed the strongest extent of this behavior and, additionally, this behavior was more pronounced for this compound in the bacterial than in the eukaryotic membrane. We hypothesize that adopting the can-can pose could possibly disrupt the protective peptidoglycan macronet found on the exterior of the bacterial membrane.

Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides.

LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.

Interactions between Chloramphenicol, Carrier Polymers, and Bacteria-Implications for Designing Electrospun Drug Delivery Systems Countering Wound Infection.

Antibacterial drug-loaded electrospun nano- and microfibrous dressings are of major interest as novel topical drug delivery systems in wound care. In this study, chloramphenicol (CAM)-loaded polycaprolactone (PCL) and PCL/poly(ethylene oxide) (PEO) fiber mats were electrospun and characterized in terms of morphology, drug distribution, physicochemical properties, drug release, swelling, cytotoxicity, and antibacterial activity. Computational modeling together with physicochemical analysis helped to elucidate possible interactions between the drug and carrier polymers. Strong interactions between PCL and CAM together with hydrophobicity of the system resulted in much slower drug release compared to the hydrophilic ternary system of PCL/PEO/CAM. Cytotoxicity studies confirmed safety of the fiber mats to murine NIH 3T3 cells. Disc diffusion assay demonstrated that both fast and slow release fiber mats reached effective concentrations and had similar antibacterial activity. A biofilm formation assay revealed that both blank matrices are good substrates for the bacterial attachment and formation of biofilm. Importantly, prolonged release of CAM from drug-loaded fibers helps to avoid biofilm formation onto the dressing and hence avoids the treatment failure.

The biophysical properties of plasmalogens originating from their unique molecular architecture.

Plasmalogens are a unique class of phospholipids that are present in many organisms. Their presence in cell membranes has intrigued researchers for decades due to their unique molecular structure, namely the vinyl-ether bond at the sn-1 position, and their association with brain related disorders. Apparently, based on their amount in the cell membranes, their function is to provide exclusive structural and dynamical properties to these complex molecular assemblies. Yet, many of their physiological roles manifested through their biophysical properties have been challenging to identify. In this review, the biophysical properties of plasmalogens are discussed and compared to other lipid species. The role of plasmalogens is examined in the context of cell membrane function, and some future directions are given.

Oligomerization Alters Binding Affinity Between Amyloid Beta and a Modulator of Peptide Aggregation.

The soluble oligomeric form of the amyloid beta (Aß) peptide is the major causative agent in the molecular pathogenesis of Alzheimer's disease (AD). We have previously developed a pyrroline-nitroxyl fluorene compound (SLF) that blocks the toxicity of Aß. Here we introduce the multi-parametric surface plasmon resonance (MP-SPR) approach to quantify SLF binding and effect on the self-association of the peptide via a label-free, real-time approach. Kinetic analysis of SLF binding to Aß and measurements of layer thickness alterations inform on the mechanism underlying the ability of SLF to inhibit Aß toxicity and its progression towards larger oligomeric assemblies. Depending on the oligomeric state of Aß, distinct binding affinities for SLF are revealed. The Aß monomer and dimer uniquely possess sub-nanomolar affinity for SLF via a non-specific mode of binding. SLF binding is weaker in oligomeric Aß, which displays an affinity for SLF on the order of 100 µM. To complement these experiments we carried out molecular docking and molecular dynamics simulations to explore how SLF interacts with the Aß peptide. The MP-SPR results together with in silico modeling provide affinity data for the SLF-Aß interaction and allow us to develop a new general method for examining protein aggregation.

A computational approach for studying antibody-antigen interactions without prior structural information: the anti-testosterone binding antibody as a case study.

Given the increasing exploitation of antibodies in different contexts such as molecular diagnostics and therapeutics, it would be beneficial to unravel the atomistic level properties of antibody-antigen complexes with the help of computational modeling. Thus, here we have studied the feasibility of computational tools to gather atomic scale information regarding the antibody-antigen complexes solely starting from an amino acid sequence. First, we constructed a homology model for the anti-testosterone binding antibody based on the knowledge based classification of complementary determining regions (CDRs) and implicit solvent molecular dynamics simulations. To further examine whether the generated homology model is suitable for studying antibody-antigen interactions, docking calculations were carried out followed by binding free-energy simulations. Our results indicate that with the antibody modeling approach presented here it is possible to construct accurate homology models for antibodies which correctly describes the antibody-antigen interactions, and produces absolute binding free-energies that are comparable with experimental values. In addition, our simulations suggest that the conformations of complementary determining regions (CDRs) may considerably change from the X-ray configuration upon solvation. In conclusion, here we have introduced an antibody modeling workflow that can be used in studying the interactions between antibody and antigen solely based on an amino acid sequence, which in turn provides novel opportunities to tune the properties of antibodies in different applications. Proteins 2017; 85:322-331. © 2016 Wiley Periodicals, Inc.

The biophysical properties of ethanolamine plasmalogens revealed by atomistic molecular dynamics simulations.

Given the importance of plasmalogens in cellular membranes and neurodegenerative diseases, a better understanding of how plasmalogens affect the lipid membrane properties is needed. Here we carried out molecular dynamics simulations to study a lipid membrane comprised of ethanolamine plasmalogens (PE-plasmalogens). We compared the results to the PE-diacyl counterpart and palmitoyl-oleyl-phosphatidylcholine (POPC) bilayers. Results show that PE-plasmalogens form more compressed, thicker, and rigid lipid bilayers in comparison with the PE-diacyl and POPC membranes. The results also point out that the vinyl-ether linkage increases the ordering of sn-1 chain substantially and the ordering of the sn-2 chain to a minor extent. Further, the vinyl-ether linkage changes the orientation of the lipid head group, but it does not cause changes in the head group and glycerol backbone tilt angles with respect to the bilayer normal. The vinyl-ether linkage also packs the proximal regions of the sn-1 and sn-2 chains more closely together which also decreases the distance between the rest of the sn-1 and sn-2 chains.

Biogenesis of Nascent High Density Lipoprotein Particles.

In this issue of Structure, Segrest et al. (2015) present a novel picture for the ABCA1-mediated lipid loading of lipid-poor apoA-I.

How anacetrapib inhibits the activity of the cholesteryl ester transfer protein? Perspective through atomistic simulations.

Cholesteryl ester transfer protein (CETP) mediates the reciprocal transfer of neutral lipids (cholesteryl esters, triglycerides) and phospholipids between different lipoprotein fractions in human blood plasma. A novel molecular agent known as anacetrapib has been shown to inhibit CETP activity and thereby raise high density lipoprotein (HDL)-cholesterol and decrease low density lipoprotein (LDL)-cholesterol, thus rendering CETP inhibition an attractive target to prevent and treat the development of various cardiovascular diseases. Our objective in this work is to use atomistic molecular dynamics simulations to shed light on the inhibitory mechanism of anacetrapib and unlock the interactions between the drug and CETP. The results show an evident affinity of anacetrapib towards the concave surface of CETP, and especially towards the region of the N-terminal tunnel opening. The primary binding site of anacetrapib turns out to reside in the tunnel inside CETP, near the residues surrounding the N-terminal opening. Free energy calculations show that when anacetrapib resides in this area, it hinders the ability of cholesteryl ester to diffuse out from CETP. The simulations further bring out the ability of anacetrapib to regulate the structure-function relationships of phospholipids and helix X, the latter representing the structural region of CETP important to the process of neutral lipid exchange with lipoproteins. Altogether, the simulations propose CETP inhibition to be realized when anacetrapib is transferred into the lipid binding pocket. The novel insight gained in this study has potential use in the development of new molecular agents capable of preventing the progression of cardiovascular diseases.