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.

Rescue of Familial Lecithin:Cholesterol Acyltranferase Deficiency Mutations with an Allosteric Activator.

Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and α/ß-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme.

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.

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.

Gliflozins, sucrose and flavonoids are allosteric activators of lecithin-cholesterol acyltransferase.

Lecithin-cholesterol acyltransferase (LCAT) serves as a pivotal enzyme in preserving cholesterol homeostasis via reverse cholesterol transport, a process closely associated with the onset of atherosclerosis. Impaired LCAT function can lead to severe LCAT deficiency disorders for which no pharmacological treatment exists. LCAT-based therapies, such as small molecule positive allosteric modulators (PAMs), against LCAT deficiencies and atherosclerosis hold promise, although their efficacy against atherosclerosis remains challenging. Herein we utilized a quantitative in silico metric to predict the activity of novel PAMs and tested their potencies with in vitro enzymatic assays. As predicted, sodium-glucose cotransporter 2 (SGLT2) inhibitors (gliflozins), sucrose and flavonoids activate LCAT. This has intriguing implications for the mechanism of action of gliflozins, which are commonly used in the treatment of type 2 diabetes, and for the endogenous activation of LCAT. Our results underscore the potential of molecular dynamics simulations in rational drug design.

Freeze-drying-induced mutarotation of lactose detected by Raman spectroscopy.

Freeze-drying enables delicate, heat-sensitive biomaterials to be stored in a dry form even at room temperature. However, exposure to physicochemical stress induced by freeze-drying presents challenges for maintaining material characteristics and functionality upon reconstitution, for which reason excipients are required. Although wide variety of different excipients are available for pharmaceutical applications, their protective role in the freeze-drying is not yet fully understood. In this study our aim was to use molecular dynamics simulations to screen the properties of different sugars and amino acids, which could be combined with plant-based nanofibrillated cellulose (NFC) hydrogel to provide protective matrix system for future freeze-drying for pharmaceuticals and biologics. The changes in the NFC-based formulations before and after freeze-drying and reconstitution were evaluated using non-invasive Timegate PicoRaman spectroscopy and traditional characterization methods. We continued to the freeze-drying with the NFC hydrogel formulations including lactose with and without glycine, which showed the highest attraction preferences on NFC surface in silico. This formulation enabled successful freeze-drying and subsequent reconstitution with preserved physicochemical and rheological properties. Raman spectroscopy gave us insights of the molecular-level changes during freeze-drying, especially the mutarotation of lactose. This research showed the potential of integrating in silico screening and non-invasive spectroscopical method to design novel biomaterial-based formulations for freeze-drying. The research provided insights of the molecular-level interactions and orientational changes of the excipients, which might be crucial in future freeze-drying applications of pharmaceuticals and biologics.

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.