Titanium coated with 2-decenoic analogs reduces bacterial and fungal biofilms.

AIMS: Due to antibiotic tolerance of microbes within biofilm, non-antibiotic methods for prevention and treatment of implant-related infections are preferable. The goal of this work is to evaluate a facile loading strategy for medium-chain fatty-acid signaling molecules 2-heptycyclopropane-1-carboxylic acid (2CP), cis-2-decenoic acid (C2DA), and trans-2-decenoic acid, which all act as diffusible signaling factors (DSFs), onto titanium surfaces for comparison of their antimicrobial efficacy. METHODS AND RESULTS: Titanium coupons were drop-coated with 0.75 mg of DSF in ethanol and dried. Surface characteristics and the presence of DSF were confirmed with Fourier Transform infrared spectroscopy, x-ray photoelectron spectroscopy, and water contact angle. Antimicrobial assays analyzing biofilm and planktonic Staphylococcus aureus, Escherichia coli, or Candida albicans viability showed that planktonic growth was reduced after 24-h incubation but only sustained through 72 h for S. aureus and C. albicans. Biofilm formation on the titanium coupons was also reduced for all strains at the 24-h time point, but not through 72 h for E. coli. Although ∼60% of the loaded DSF was released within the first 2 days, enough remained on the surface after 4 days of elution to significantly inhibit E. coli and C. albicans biofilm. Cytocompatibility evaluations with a fibroblast cell line showed that none of the DSF-loaded groups decreased viability, while C2DA and 2CP increased viability by up to 50%. CONCLUSIONS: In this study, we found that DSF-loaded titanium coupons can inhibit planktonic microbes and prevent biofilm attachment, without toxicity to mammalian cells.

A study of the interaction between TMAO and urea in water using NMR spectroscopy.

Trimethylamine N-oxide (TMAO) and urea are small organic biological molecules. While TMAO is known as a protective osmolyte that promotes the native form of biomolecules, urea is a denaturant. An understanding of the impact of TMAO and urea on water structure may aid in uncovering the molecular mechanisms that underlie this activity. Here we investigate binary solutions of TMAO-water, urea-water and ternary solutions of TMAO-urea-water using NMR spectroscopy at 300 K. An enhancement of the total hydrogen bonding in water was found upon the addition of TMAO and this effect was neutralised by a mole ratio of 1-part TMAO to 4-parts urea. Urea was found to have little effect on the strength of water's hydrogen bonding network and the dynamics of water molecules. Evidence was found for a weak interaction between TMAO and urea. Taken together, these results suggest that TMAO's function as a protective osmolyte, and its counteraction of urea, may be driven by the strength of its hydrogen bond interactions with water, and by a secondary reinforcement of water's own hydrogen bond network. They also suggest that the TMAO-urea complex forms through the donation of a hydrogen bond by urea.

Synthesis and Characterization of 2-Decenoic Acid Modified Chitosan for Infection Prevention and Tissue Engineering.

Chitosan nanofiber membranes are recognized as functional antimicrobial materials, as they can effectively provide a barrier that guides tissue growth and supports healing. Methods to stabilize nanofibers in aqueous solutions include acylation with fatty acids. Modification with fatty acids that also have antimicrobial and biofilm-resistant properties may be particularly beneficial in tissue regeneration applications. This study investigated the ability to customize the fatty acid attachment by acyl chlorides to include antimicrobial 2-decenoic acid. Synthesis of 2-decenoyl chloride was followed by acylation of electrospun chitosan membranes in pyridine. Physicochemical properties were characterized through scanning electron microscopy, FTIR, contact angle, and thermogravimetric analysis. The ability of membranes to resist biofilm formation by S. aureus and P. aeruginosa was evaluated by direct inoculation. Cytocompatibility was evaluated by adding membranes to cultures of NIH3T3 fibroblast cells. Acylation with chlorides stabilized nanofibers in aqueous media without significant swelling of fibers and increased hydrophobicity of the membranes. Acyl-modified membranes reduced both S. aureus and P.aeruginosa bacterial biofilm formation on membrane while also supporting fibroblast growth. Acylated chitosan membranes may be useful as wound dressings, guided regeneration scaffolds, local drug delivery, or filtration.

Protein Microgel-Stabilized Pickering Liquid Crystal Emulsions Undergo Analyte-Triggered Configurational Transition.

Herein, we report a novel approach that involves Pickering stabilization of micometer-sized liquid crystal (LC) droplets with biocompatible soft materials such as a whey protein microgel (WPM) to facilitate the analysis of analyte-induced configurational transition of the LC droplets. The WPM particles were able to irreversibly adsorb at the LC-water interface, and the resulting WPM-stabilized LC droplets possessed a remarkable stability against coalescence over time. Although the LC droplets were successfully protected by a continuous network of the WPM layer, the LC-water interface was still accessible for small molecules such as sodium dodecyl sulfate (SDS) that could diffuse through the meshes of the adsorbed WPM network or through the interfacial pores and induce an LC response. This approach was exploited to investigate the dynamic range of the WPM-stabilized LC droplet response to SDS. Nevertheless, the presence of the unadsorbed WPM in the aqueous medium reduced the access of SDS molecules to the LC droplets, thus suppressing the configuration transition. An improved LC response to SDS with a lower detection limit was achieved after washing off the unadsorbed WPM. Interestingly, the LC exhibited a detection limit as low as ∼0.85 mM for SDS within the initial WPM concentration ranging from 0.005 to 0.1 wt %. Furthermore, we demonstrate that the dose-response behavior was strongly influenced by the number of droplets exposed to the aqueous analytes and the type of surfactants such as anionic SDS, cationic dodecyltrimethylammonium bromide (DTAB), and nonionic tetra(ethylene glycol)monododecyl ether (C12E4). Thus, our results address key issues associated with the quantification of aqueous analytes and provide a promising colloidal platform toward the development of new classes of biocompatible LC droplet-based optical sensors.

Benchmarking GPCR homology model template selection in combination with de novo loop generation.

G protein-coupled receptors (GPCR) comprise the largest family of membrane proteins and are of considerable interest as targets for drug development. However, many GPCR structures remain unsolved. To address the structural ambiguity of these receptors, computational tools such as homology modeling and loop modeling are often employed to generate predictive receptor structures. Here we combined both methods to benchmark a protocol incorporating homology modeling based on a locally selected template and extracellular loop modeling that additionally evaluates the presence of template ligands during these modeling steps. Ligands were also docked using three docking methods and two pose selection methods to elucidate an optimal ligand pose selection method. Results suggest that local template-based homology models followed by loop modeling produce more accurate and predictive receptor models than models produced without loop modeling, with decreases in average receptor and ligand RMSD of 0.54 Å and 2.91 Å, respectively. Ligand docking results showcased the ability of MOE induced fit docking to produce ligand poses with atom root-mean-square deviation (RMSD) values at least 0.20 Å lower (on average) than the other two methods benchmarked in this study. In addition, pose selection methods (software-based scoring, ligand complementation) selected lower RMSD poses with MOE induced fit docking than either of the other methods (averaging at least 1.57 Å lower), indicating that MOE induced fit docking is most suited for docking into GPCR homology models in our hands. In addition, target receptor models produced with a template ligand present throughout the modeling process most often produced target ligand poses with RMSD values ≤ 4.5 Å and Tanimoto coefficients > 0.6 after selection based on ligand complementation than target receptor models produced in the absence of template ligands. Overall, the findings produced by this study support the use of local template homology modeling in combination with de novo ECL2 modeling in the presence of a ligand from the template crystal structure to generate GPCR models intended to study ligand binding interactions.

A benchmark study of loop modeling methods applied to G protein-coupled receptors.

G protein-coupled receptors (GPCR) are important drug discovery targets. Despite progress, many GPCR structures have not yet been solved. For these targets, comparative modeling is used in virtual ligand screening to prioritize experimental efforts. However, the structure of extracellular loop 2 (ECL2) is often poorly predicted. This is significant due to involvement of ECL2 in ligand binding for many Class A GPCR. Here we examine the performance of loop modeling protocols available in the Rosetta (cyclic coordinate descent [CCD], KIC with fragments [KICF] and next generation KIC [NGK]) and Molecular Operating Environment (MOE) software suites (de novo search). ECL2 from GPCR crystal structures served as the structure prediction targets and were divided into four sets depending on loop length. Results suggest that KICF and NGK sampled and scored more loop models with sub-angstrom and near-atomic accuracy than CCD or de novo search for loops of 24 or fewer residues. None of the methods were able to sample loop conformations with near-atomic accuracy for the longest targets ranging from 25 to 32 residues based on 1000 models generated. For these long loop targets, increased conformational sampling is necessary. The strongly conserved disulfide bond between Cys3.25 and Cys45.50 in ECL2 proved an effective filter. Setting an upper limit of 5.1 Å on the S-S distance improved the lowest RMSD model included in the top 10 scored structures in Groups 1-4 on average between 0.33 and 1.27 Å. Disulfide bond formation and geometry optimization of ECL2 provided an additional incremental benefit in structure quality.

Phospholipase D2-dependent inhibition of the nuclear hormone receptor PPARgamma by cyclic phosphatidic acid.

Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature's simplest phospholipids, is found in cells from slime mold to humans and has a largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus-coupled manner by phospholipase D2 (PLD2) and binds to and inhibits the nuclear hormone receptor PPARgamma with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARgamma target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant-negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARgamma inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARgamma, revealing that PPARgamma is regulated by endogenous agonists as well as by antagonists.

Discovery and synthetic optimization of a novel scaffold for hydrophobic tunnel-targeted autotaxin inhibition.

Autotaxin (ATX) is a ubiquitous ectoenzyme that hydrolyzes lysophosphatidylcholine (LPC) to form the bioactive lipid mediator lysophosphatidic acid (LPA). LPA activates specific G-protein coupled receptors to elicit downstream effects leading to cellular motility, survival, and invasion. Through these pathways, upregulation of ATX is linked to diseases such as cancer and cardiovascular disease. Recent crystal structures confirm that the catalytic domain of ATX contains multiple binding regions including a polar active site, hydrophobic tunnel, and a hydrophobic pocket. This finding is consistent with the promiscuous nature of ATX hydrolysis of multiple and diverse substrates and prior investigations of inhibitor impacts on ATX enzyme kinetics. The current study used virtual screening methods to guide experimental identification and characterization of inhibitors targeting the hydrophobic region of ATX. An initially discovered inhibitor, GRI392104 (IC50 4µM) was used as a lead for synthetic optimization. In total twelve newly synthesized inhibitors of ATX were more potent than GRI392104 and were selective for ATX as they had no effect on other LPC-specific NPP family members or on LPA1-5 GPCR.

Structure and catalytic function of sphingosine kinases: analysis by site-directed mutagenesis and enzyme kinetics.

Sphingosine kinases 1 and 2 (SK1 and SK2) generate the bioactive lipid mediator sphingosine 1-phosphate and as such play a significant role in cell fate and in human health and disease. Despite significant interest in and examination of the role played by SK enzymes in disease, comparatively little is currently known about the three-dimensional structure and catalytic mechanisms of these enzymes. To date, limited numbers of studies have used site directed mutagenesis and activity determinations to examine the roles of individual SK residues in substrate, calmodulin, and membrane binding, as well as activation via phosphorylation. Assays are currently available that allow for both single and bisubstrate kinetic analysis of mutant proteins that show normal, lowered and enhanced activity as compared to wild type controls. Additional studies will be required to build on this foundation to completely understand SK mediated substrate binding and phosphoryl group transfer. A deeper understanding of the SK catalytic mechanism, as well as SK interactions with potential small molecule inhibitors will be invaluable to the future design and identification of SK activity modulators as research tools and potential therapeutics. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Hits of a high-throughput screen identify the hydrophobic pocket of autotaxin/lysophospholipase D as an inhibitory surface.

Autotaxin (ATX), a lysophospholipase D, plays an important role in cancer invasion, metastasis, tumor progression, tumorigenesis, neuropathic pain, fibrotic diseases, cholestatic pruritus, lymphocyte homing, and thrombotic diseases by producing the lipid mediator lysophosphatidic acid (LPA). A high-throughput screen of ATX inhibition using the lysophosphatidylcholine-like substrate fluorogenic substrate 3 (FS-3) and ∼10,000 compounds from the University of Cincinnati Drug Discovery Center identified several small-molecule inhibitors with IC50 vales ranging from nanomolar to low micromolar. The pharmacology of the three most potent compounds: 918013 (1; 2,4-dichloro-N-(3-fluorophenyl)-5-(4-morpholinylsulfonyl) benzamide), 931126 (2; 4-oxo-4-{2-[(5-phenoxy-1H-indol-2-yl)carbonyl]hydrazino}-N-(4-phenylbutan-2-yl)butanamide), and 966791 (3; N-(2,6-dimethylphenyl)-2-[N-(2-furylmethyl)(4-(1,2,3,4-tetraazolyl)phenyl)carbonylamino]-2-(4-hydroxy-3-methoxyphenyl) acetamide), were further characterized in enzyme, cellular, and whole animal models. Compounds 1 and 2 were competitive inhibitors of ATX-mediated hydrolysis of the lysophospholipase substrate FS-3. In contrast, compound 3 was a competitive inhibitor of both FS-3 and the phosphodiesterase substrate p-nitrophenyl thymidine 5'-monophosphate. Computational docking and mutagenesis suggested that compounds 1 and 2 target the hydrophobic pocket, thereby blocking access to the active site of ATX. The potencies of compounds 1-3 were comparable to each other in each of the assays. All of these compounds significantly reduced invasion of A2058 human melanoma cells in vitro and the colonization of lung metastases by B16-F10 murine melanoma cells in C57BL/6 mice. The compounds had no agonist or antagonist effects on select LPA or sphingosine 1-phosphate receptors, nor did they inhibit nucleotide pyrophosphatase/phosphodiesterase (NPP) enzymes NPP6 and NPP7. These results identify the molecular surface of the hydrophobic pocket of ATX as a target-binding site for inhibitors of enzymatic activity.

Gold nanorods carrying paclitaxel for photothermal-chemotherapy of cancer.

Nanotechnology-based photothermal therapy has emerged as a promising treatment for cancer during the past decade. However, heterogeneous laser heating and limited light penetration can lead to incomplete tumor cell eradication. Here, we developed a method to overcome these limitations by combining chemotherapy with photothermal therapy using paclitaxel-loaded gold nanorods. Paclitaxel was loaded to gold nanorods with high density (2.0 × 10(4) paclitaxel per gold nanorod) via nonspecific adsorption, followed by stabilization with poly(ethylene glycol) linked with 11-mercaptoundecanoic acid. Paclitaxel was entrapped in the hydrophobic pocket of the polymeric monolayer on the surface of gold nanorods, which allows direct cellular delivery of the hydrophobic drugs via the lipophilic plasma membrane. Highly efficient drug release was demonstrated in a cell membrane mimicking two-phase solution. Combined photothermal therapy and chemotherapy with the paclitaxel-loaded gold nanorods was shown to be highly effective in killing head and neck cancer cells and lung cancer cells, superior to photothermal therapy or chemotherapy alone due to a synergistic effect. The paclitaxel-gold nanorod enabled photothermal chemotherapy has the potential of preventing tumor reoccurrence and metastasis and may have an important impact on the treatment of head and neck cancer and other malignancies in the clinic.

Autotaxin inhibition: development and application of computational tools to identify site-selective lead compounds.

Autotaxin (ATX) catalyzes the conversion of lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). Both ATX and LPA have been linked to pathophysiologies ranging from cancer to neuropathic pain. Inhibition of LPA production by ATX is therefore of therapeutic interest. Here we report the application of previously-developed, subsite-targeted pharmacophore models in a screening workflow that involves either docking or binary QSAR as secondary filters to identify ATX inhibitors from previously unreported structural types, four of which have sub-micromolar inhibition constants. Cell-based assays demonstrate that ATX inhibition and cytotoxicity structure-activity-relationships (SAR) exhibit selectivity cliffs, characterized by structurally similar compounds exhibiting similar biological activities with respect to ATX inhibition but very different biological activities with respect to cytotoxicity. Thus, general cytotoxicity should not be used as an early filter to eliminate candidate ATX inhibitor scaffolds from further SAR studies. Assays using two substrates of vastly different sizes demonstrate that the tools developed to identify compounds binding outside the central core of the active site did identify compounds acting at an allosteric site. In contrast, tools developed to identify active-site directed compounds did not identify active-site directed compounds. The stronger volume overlap imposed when selecting screening candidates expected to bind outside the active site is likely responsible for the stronger match between intended and actual target site.

Application of computational methods for class A GPCR Ligand discovery.

G protein-coupled receptors (GPCR) are integral membrane proteins of considerable interest as targets for drug development due to their role in transmitting cellular signals in a multitude of biological processes. Of the six classes categorizing GPCR (A, B, C, D, E, and F), class A contains the largest number of therapeutically relevant GPCR. Despite their importance as drug targets, many challenges exist for the discovery of novel class A GPCR ligands serving as drug precursors. Though knowledge of the structural and functional characteristics of GPCR has grown significantly over the past 20 years, a large portion of GPCR lack reported, experimentally determined structures. Furthermore, many GPCR have no known endogenous and/or synthetic ligands, limiting further exploration of their biochemical, cellular, and physiological roles. While many successes in GPCR ligand discovery have resulted from experimental high-throughput screening, computational methods have played an increasingly important role in GPCR ligand identification in the past decade. Here we discuss computational techniques applied to GPCR ligand discovery. This review summarizes class A GPCR structure/function and provides an overview of many obstacles currently faced in GPCR ligand discovery. Furthermore, we discuss applications and recent successes of computational techniques used to predict GPCR structure as well as present a summary of ligand- and structure-based methods used to identify potential GPCR ligands. Finally, we discuss computational hit list generation and refinement and provide comprehensive workflows for GPCR ligand identification.

Structure-based pharmacophore modeling 1. Automated random pharmacophore model generation.

Pharmacophores are three-dimensional arrangements of molecular features required for biological activity that are often used in virtual screening efforts to prioritize ligands for experimental testing. G protein-coupled receptors (GPCR) are integral membrane proteins of considerable interest as targets for ligand discovery and drug development. Ligand-based pharmacophore models can be constructed to identify structural commonalities between known bioactive ligands for targets including GPCR. However, structure-based pharmacophores (which only require an experimentally determined or modeled structure for a protein target) have gained more attention to aid in virtual screening efforts as the number of publicly available experimentally determined GPCR structures have increased (140 unique GPCR represented as of October 24, 2022). Thus, the goal of this study was to develop a method of structure-based pharmacophore model generation applicable to ligand discovery for GPCR that have few known ligands. Pharmacophore models were generated within the active sites of 8 class A GPCR crystal structures via automated annotation of 5 randomly selected functional group fragments to sample diverse combinations of pharmacophore features. Each of the 5000 generated pharmacophores was then used to search a database containing active and decoy/inactive compounds for 30 class A GPCR and scored using enrichment factor and goodness-of-hit metrics to assess performance. Application of this method to the set of 8 class A GPCR produced pharmacophore models possessing the theoretical maximum enrichment factor value in both resolved structures (8 of 8 cases) and homology models (7 of 8 cases), indicating that generated pharmacophore models can prove useful in the context of virtual screening.

Structure-based pharmacophore modeling 2. Developing a novel framework for structure-based pharmacophore model generation and selection.

Pharmacophore models are three-dimensional arrangements of molecular features required for biological activity that are used in ligand identification efforts for many biological targets, including G protein-coupled receptors (GPCR). Though GPCR are integral membrane proteins of considerable interest as targets for drug development, many of these receptors lack known ligands or experimentally determined structures necessary for ligand- or structure-based pharmacophore model generation, respectively. Thus, we here present a structure-based pharmacophore modeling approach that uses fragments placed with Multiple Copy Simultaneous Search (MCSS) to generate high-performing pharmacophore models in the context of experimentally determined, as well as modeled GPCR structures. Moreover, we have addressed the oft-neglected topic of pharmacophore model selection via development of a cluster-then-predict machine learning workflow. Herein score-based pharmacophore models were generated in experimentally determined and modeled structures of 13 class A GPCR and resulted in pharmacophore models exhibiting high enrichment factors when used to search a database containing 569 class A GPCR ligands. In addition, classification of pharmacophore models with the best performing cluster-then-predict logistic regression classifier resulted in positive predictive values (PPV) of 0.88 and 0.76 for selecting high enrichment pharmacophore models from among those generated in experimentally determined and modeled structures, respectively.

Cis-2-Decenoic Acid and Bupivacaine Delivered from Electrospun Chitosan Membranes Increase Cytokine Production in Dermal and Inflammatory Cell Lines.

Wound dressings serve to protect tissue from contamination, alleviate pain, and facilitate wound healing. The biopolymer chitosan is an exemplary choice in wound dressing material as it is biocompatible and has intrinsic antibacterial properties. Infection can be further prevented by loading dressings with cis-2-decenoic acid (C2DA), a non-antibiotic antimicrobial agent, as well as bupivacaine (BUP), a local anesthetic that also has antibacterial capabilities. This study utilized a series of assays to elucidate the responses of dermal cells to decanoic anhydride-modified electrospun chitosan membranes (DA-ESCMs) loaded with C2DA and/or BUP. Cytocompatibility studies determined the toxic loading ranges for C2DA, BUP, and combinations, revealing that higher concentrations (0.3 mg of C2DA and 1.0 mg of BUP) significantly decreased the viability of fibroblasts and keratinocytes. These high concentrations also inhibited collagen production by fibroblasts, with lower loading concentrations promoting collagen deposition. These findings provide insight into preliminary cellular responses to DA-ESCMs and can guide future research on their clinical application as wound dressings.

Building block aspect ratio controls assembly, architecture, and mechanics of synthetic and natural protein networks.

Fibrous networks constructed from high aspect ratio protein building blocks are ubiquitous in nature. Despite this ubiquity, the functional advantage of such building blocks over globular proteins is not understood. To answer this question, we engineered hydrogel network building blocks with varying numbers of protein L domains to control the aspect ratio. The mechanical and structural properties of photochemically crosslinked protein L networks were then characterised using shear rheology and small angle neutron scattering. We show that aspect ratio is a crucial property that defines network architecture and mechanics, by shifting the formation from translationally diffusion dominated to rotationally diffusion dominated. Additionally, we demonstrate that a similar transition is observed in the model living system: fibrin blood clot networks. The functional advantages of this transition are increased mechanical strength and the rapid assembly of homogenous networks above a critical protein concentration, crucial for in vivo biological processes such as blood clotting. In addition, manipulating aspect ratio also provides a parameter in the design of future bio-mimetic and bio-inspired materials.

Self-docking and cross-docking simulations of G protein-coupled receptor-ligand complexes: Impact of ligand type and receptor activation state.

G protein-coupled receptors (GPCR) are the largest family of cell surface receptors in vertebrates. Their abundance and role in nearly all physiological systems make GPCR the largest protein family targeted for development of pharmaceuticals. Ligand discovery aimed at identification of chemical tools and drug leads is aided by molecular docking simulations that allow critical analysis of the potential interactions between small molecules and proteins in resulting complexes. However, blind assessments of ligand pose quality and affinity prediction have thus far not provided broadly generalizable performance expectations for docking into experimentally-characterized GPCR targets. Likewise, the relative importance of receptor activation state and ligand function differences have also not been systematically assessed. This study compares performance when docking ligands of varied function into varied GPCR activation states in the absence of extensive resampling of the input GPCR structure, and only limited sidechain flexibility after ligand placement. Simulations were performed using 37 experimental structures of 11 Class A GPCR crystallized in multiple activation states (giving rise to 37 self-docking and 68 cross docking simulations). Our results show that one specific subset of cross-docking simulations gave results of similar quality to self-docking. Median ligand RMSD values for top-scored poses were 1.2 Å and 2.0 Å for self-docking and StateMatch/FunctionMatch cross-docking, respectively. The distributions of ligand RMSD values were not statistically different for these two conditions, according to a Kolmogorov-Smirnov test. Therefore, docking performance against GPCR targets can be estimated in advance based on docking target structure activation states, with higher accuracy expected when docking agonists into active state structures and inverse agonists or antagonists into inactive state structures. Receptor conformational sampling in advance of docking or receptor conformational adjustment after docking are more likely to produce substantial improvements for other pairings of receptor activation state and ligand function.

Polymorphism of a Highly Asymmetrical Triacylglycerol in Milk Fat: 1-Butyryl 2-Stearoyl 3-Palmitoyl-glycerol.

Milk fat has more than 200 triacylglycerols (TAGs), which play a pivotal role in its crystallization behavior. Asymmetrical TAGs containing short butyryl chains contribute to a significant portion of milk fat TAGs. This work aims to elucidate the crystallization behavior of asymmetrical milk fat TAGs by employing the pure compound of 1-butyryl 2-stearoyl 3-palmitoyl-glycerol (BuSP). The structural evolution of BuSP after being cooled down to 20 °C from the melt is evaluated by small- and wide-angle X-ray scattering (SAXS and WAXS) and differential scanning calorimetry (DSC). The temporal structural observation shows that BuSP crystallizes into the α-form with short and long spacings of 4.10 and 56.9 Å, respectively, during the first hour of isothermal hold at 20 °C. The polymorphic transformation of the α to ß' phase occurred after 4 h of isothermal hold, and the ß'- to α-form fraction ratio was about 70:30 at the end of the isothermal experiment (18 h). Pure ß'-form X-ray patterns are obtained from the BuSP powder with short spacings of 4.33, 4.14, and 3.80 Å, while the long spacing of 51.2 Å depicts a three-chain-length lamellar structure with a tilt angle of 32°. Corresponding DSC measurements display that BuSP crystallizes from the melt at 29.1 °C, whereas the melting of α- and ß'-forms was recorded at 30.3 and 47.8 °C, respectively. In the absence of the ß-form, the ß'-polymorph is the most stable observed form in BuSP. This work exemplarily explains the crystallization behavior of asymmetrical milk fat TAGs and thus provides new insights into their role in overall milk fat crystallization.

The phospholipase A1 activity of lysophospholipase A-I links platelet activation to LPA production during blood coagulation.

Platelet activation initiates an upsurge in polyunsaturated (18:2 and 20:4) lysophosphatidic acid (LPA) production. The biochemical pathway(s) responsible for LPA production during blood clotting are not yet fully understood. Here we describe the purification of a phospholipase A(1) (PLA(1)) from thrombin-activated human platelets using sequential chromatographic steps followed by fluorophosphonate (FP)-biotin affinity labeling and proteomics characterization that identified acyl-protein thioesterase 1 (APT1), also known as lysophospholipase A-I (LYPLA-I; accession code O75608) as a novel PLA(1). Addition of this recombinant PLA(1) significantly increased the production of sn-2-esterified polyunsaturated LPCs and the corresponding LPAs in plasma. We examined the regioisomeric preference of lysophospholipase D/autotaxin (ATX), which is the subsequent step in LPA production. To prevent acyl migration, ether-linked regioisomers of oleyl-sn-glycero-3-phosphocholine (lyso-PAF) were synthesized. ATX preferred the sn-1 to the sn-2 regioisomer of lyso-PAF. We propose the following LPA production pathway in blood: 1) Activated platelets release PLA(1); 2) PLA(1) generates a pool of sn-2 lysophospholipids; 3) These newly generated sn-2 lysophospholipids undergo acyl migration to yield sn-1 lysophospholipids, which are the preferred substrates of ATX; and 4) ATX cleaves the sn-1 lysophospholipids to generate sn-1 LPA species containing predominantly 18:2 and 20:4 fatty acids.