Vesicle-like Nanocapsules Formed by Self-Assembly of Peptides with Oligoproline and -Leucine.

Since drug carriers are envisaged to be used in a wide variety of situations and environments, nanocarriers with diverse properties, such as biocompatibility, biodegradability, nonimmunogenicity, adequate particle size, robustness, and cell permeability, are required. Here, we report the construction of novel nanocapsules with the above-mentioned features by the self-assembly of peptides composed of oligoproline and oligoleucine (i.e., H-Pro10Leu4-NH2 and H-Pro10Leu6-NH2). The peptides self-organized via hydrogen bonds and hydrophobic interactions between oligoleucine moieties to form vesicle-like nanocapsules with cationic oligoproline exposed on the surface. The guest encapsulation experiments revealed that the nanocapsules were capable of uptake of both water-soluble and insoluble compounds. Furthermore, positively charged and/or oligoproline-based peptides are known to improve cell permeability and cellular uptake, suggesting that the peptide nanocapsules are good candidates for nanocarriers to complement liposomes and polymer micelles.

Conformational Preferences of the Non-Canonical Amino Acids (2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CH2F groups substitute specific methyl groups, can readily be probed by 19F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CH2F groups is unclear. We present high-resolution crystal structures of Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. Apart from the fluorinated leucine residues, the structures show complete structural conservation of the protein backbone and the amino acid side chains except for a single isoleucine side chain located next to a fluorine atom in the hydrophobic core of the protein. The carbon skeletons of the fluorinated leucine side chains are also mostly conserved. The CH2F groups show a strong preference for staggered rotamers and often appear locked into single rotamers. Substitution of leucine CH3 groups for CH2F groups is thus readily tolerated in the three-dimensional (3D) structure of a protein, and the rotation of CH2F groups can be halted at cryogenic temperatures.

(2S,4S)-5-Fluoroleucine, (2S,4R)-5-Fluoroleucine, and 5,5'-Difluoroleucine in Escherichia coli PpiB: Protein Production, 19F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect.

Global substitution of leucine for analogues containing CH2F instead of methyl groups delivers proteins with multiple sites for monitoring by 19F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa Escherichia coli peptidyl-prolyl cis-trans isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2S,4S)-5-fluoroleucine, (2S,4R)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein. 19F nuclear magnetic resonance (NMR) spectra showed large chemical shift dispersions between 6 and 17 ppm. The 19F chemical shifts correlate with the three-bond 1H-19F couplings (3JHF), providing the first experimental verification of the γ-gauche effect predicted by [Feeney, J. J. Am. Chem. Soc. 1996, 118, 8700-8706] and establishing the effect as the predominant determinant of the 19F chemical shifts of CH2F groups. Individual CH2F groups can be confined to single rotameric states by the protein environment, but most CH2F groups exchange between different rotamers at a rate that is fast on the NMR chemical shift scale. Interactions between fluorine atoms in 5,5'-difluoroleucine bias the CH2F rotamers in agreement with results obtained previously for 1,3-difluoropropane. The sensitivity of the 19F chemical shift to the rotameric state of the CH2F groups potentially renders them particularly sensitive for detecting allosteric effects.

Unravelling caramelization and Maillard reactions in glucose and glucose + leucine model cakes: Formation and degradation kinetics of volatile markers extracted during baking.

A large number of volatile compounds are formed during the baking of foods by reactions such as caramelization and Maillard reactions. Elucidating the reaction mechanisms may be useful to predict and control food quality. Ten reaction volatile markers were extracted during baking of solid model cakes implemented with known amounts of precursors (glucose with or without leucine) and then quantified by Thermal desorption-Gas chromatography-Mass spectrometry. The kinetic data showed that the level of air convection in the oven had no significant influence on the reaction rates. In contrast, increasing baking temperatures had a nonlinear accelerating impact on the generation of newly formed volatile compounds with a bell-shaped kinetic curve found for most of the markers at 200 °C. The presence of leucine triggered the activation of the Maillard and Strecker routes with a specific and very rapid formation of 3-Methylbutanal and pyrazines. A dynamic model was developed, combining evaporation flow rate and kinetic formation and consumption of reaction markers. It can be used to describe, for two furanic compounds of different volatilities, the vapor concentrations in the oven from the concentrations measured in the model cakes.

Stabilization of Prebiotic Vesicles by Peptides Depends on Sequence and Chirality: A Mechanism for Selection of Protocell-Associated Peptides.

Cells require oligonucleotides and polypeptides with specific, homochiral sequences to perform essential functions, but it is unclear how such oligomers were selected from random sequences at the origin of life. Cells were probably preceded by simple compartments such as fatty acid vesicles, and oligomers that increased the stability, growth, or division of vesicles could have thereby increased in frequency. We therefore tested whether prebiotic peptides alter the stability or growth of vesicles composed of a prebiotic fatty acid. We find that three of 15 dipeptides tested reduce salt-induced flocculation of vesicles. All three contain leucine, and increasing their length increases the efficacy. Also, leucine-leucine but not alanine-alanine increases the size of vesicles grown by multiple additions of micelles. In a molecular simulation, leucine-leucine docks to the membrane, with the side chains inserted into the hydrophobic core of the bilayer, while alanine-alanine fails to dock. Finally, the heterochiral forms of leucine-leucine, at a high concentration, rapidly shrink the vesicles and make them leakier and less stable to high pH than the homochiral forms do. Thus, prebiotic peptide-membrane interactions influence the flocculation, growth, size, leakiness, and pH stability of prebiotic vesicles, with differential effects due to sequence, length, and chirality. These differences could lead to a population of vesicles enriched for peptides with beneficial sequence and chirality, beginning selection for the functional oligomers that underpin life.

Genome Mining of a Fungal Polyketide Synthase-Nonribosomal Peptide Synthetase Hybrid Megasynthetase Pathway to Synthesize a Phytotoxic N-Acyl Amino Acid.

Guided by the retrobiosynthesis hypothesis, we characterized a fungal polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid megasynthetase pathway to generate 2-trans-4-trans-2-methylsorbyl-d-leucine (1), a polyketide amino acid conjugate that inhibits Arabidopsis root growth. The biosynthesis of 1 includes a PKS-NRPS enzyme to assemble an N-acyl amino alcohol intermediate, which is further oxidized to an N-acyl amino acid (NAAA), demonstrating a new biosynthetic logic for synthesizing NAAAs and expanding the chemical space of products encoded by fungal PKS-NRPS clusters.

Triple combination dry powder formulation of pretomanid, moxifloxacin, and pyrazinamide for treatment of multidrug-resistant tuberculosis.

Both latent and multidrug-resistant tuberculosis (TB) have been causing significant concern worldwide. A novel drug, pretomanid (PA-824), has shown a potent bactericidal effect against both active and latent forms of Mycobacterium tuberculosis (MTb) and a synergistic effect when combined with pyrazinamide and moxifloxacin. This study aimed to develop triple combination spray dried inhalable formulations composed of antitubercular drugs, pretomanid, moxifloxacin, and pyrazinamide (1:2:8 w/w/w), alone (PaMP) and in combination with an aerosolization enhancer, L-leucine (20 % w/w, PaMPL). The formulation PaMPL consisted of hollow, spherical, dimpled particles (<5 µm) and showed good aerosolization behaviour with a fine particle fraction of 70 %. Solid-state characterization of formulations with and without L-leucine confirmed the amorphous nature of moxifloxacin and pretomanid and the crystalline nature of pyrazinamide with polymorphic transformation after the spray drying process. Further, the X-ray photoelectron spectroscopic analysis revealed the predominant surface composition of L-leucine on PaMPL dry powder particles. The dose-response cytotoxicity results showed pyrazinamide and moxifloxacin were non-toxic in both A549 and Calu-3 cell lines up to 150 µg/mL. However, the cell viability gradually decreased to 50 % when the pretomanid concentration increased to 150 µg/mL. The in vitro efficacy studies demonstrated that the triple combination formulation had more prominent antibacterial activity with a minimum inhibitory concentration (MIC) of 1 µg/mL against the MTb H37Rv strain as compared to individual drugs. In conclusion, the triple combination of pretomanid, moxifloxacin, and pyrazinamide as an inhalable dry powder formulation will potentially improve treatment efficacy with fewer systemic side effects in patients suffering from latent and multidrug-resistant TB.

Leucine Motifs Stabilize Residual Helical Structure in Disordered Proteins.

Many examples are known of regions of intrinsically disordered proteins that fold into α-helices upon binding to their targets. These helical binding motifs (HBMs) can be partially helical also in the unbound state, and this so-called residual structure can affect binding affinity and kinetics. To investigate the underlying mechanisms governing the formation of residual helical structure, we assembled a dataset of experimental helix contents of 65 peptides containing HBM that fold-upon-binding. The average residual helicity is 17% and increases to 60% upon target binding. The helix contents of residual and target-bound structures do not correlate, however the relative location of helix elements in both states shows a strong overlap. Compared to the general disordered regions, HBMs are enriched in amino acids with high helix preference and these residues are typically involved in target binding, explaining the overlap in helix positions. In particular, we find that leucine residues and leucine motifs in HBMs are the major contributors to helix stabilization and target-binding. For the two model peptides, we show that substitution of leucine motifs to other hydrophobic residues (valine or isoleucine) leads to reduction of residual helicity, supporting the role of leucine as helix stabilizer. From the three hydrophobic residues only leucine can efficiently stabilize residual helical structure. We suggest that the high occurrence of leucine motifs and a general preference for leucine at binding interfaces in HBMs can be explained by its unique ability to stabilize helical elements.

13 Cß-Valine and 13 Cγ-Leucine Methine Labeling To Probe Protein Ligand Interaction.

Precise information regarding the interaction between proteins and ligands at molecular resolution is crucial for effectively guiding the optimization process from initial hits to lead compounds in early stages of drug development. In this study, we introduce a novel aliphatic side chain isotope-labeling scheme to directly probe interactions between ligands and aliphatic sidechains using NMR techniques. To demonstrate the applicability of this method, we selected a set of Brd4-BD1 binders and analyzed 1 H chemical shift perturbation resulting from CH-π interaction of Hß -Val and Hγ -Leu as CH donors with corresponding ligand aromatic moieties as π acceptors.

The role of leucine and isoleucine in tuning the hydropathy of class A GPCRs.

Leucine and Isoleucine are two amino acids that differ only by the positioning of one methyl group. This small difference can have important consequences in α-helices, as the ß-branching of Ile results in helix destabilization. We set out to investigate whether there are general trends for the occurrences of Leu and Ile residues in the structures and sequences of class A GPCRs (G protein-coupled receptors). GPCRs are integral membrane proteins in which α-helices span the plasma membrane seven times and which play a crucial role in signal transmission. We found that Leu side chains are generally more exposed at the protein surface than Ile side chains. We explored whether this difference might be attributed to different functions of the two amino acids and tested if Leu tunes the hydrophobicity of the transmembrane domain based on the Wimley-White whole-residue hydrophobicity scales. Leu content decreases the variation in hydropathy between receptors and correlates with the non-Leu receptor hydropathy. Both measures indicate that hydropathy is tuned by Leu. To test this idea further, we generated protein sequences with random amino acid compositions using a simple numerical model, in which hydropathy was tuned by adjusting the number of Leu residues. The model was able to replicate the observations made with class A GPCR sequences. We speculate that the hydropathy of transmembrane domains of class A GPCRs is tuned by Leu (and to some lesser degree by Lys and Val) to facilitate correct insertion into membranes and/or to stably anchor the receptors within membranes.

Developing a comprehensive solution aimed to disrupt LARS1/RagD protein-protein interaction.

Aminoacyl-tRNA synthetases are crucial enzymes involved in protein synthesis and various cellular physiological reactions. Aside from their standard role in linking amino acids to the corresponding tRNAs, they also impact protein homeostasis by controlling the level of soluble amino acids within the cell. For instance, leucyl-tRNA synthetase (LARS1) acts as a leucine sensor for the mammalian target of rapamycin complex 1 (mTORC1), and may also function as a probable GTPase-activating protein (GAP) for the RagD subunit of the heteromeric activator of mTORC1. In turn, mTORC1 regulates cellular processes, such as protein synthesis, autophagy, and cell growth, and is implicated in various human diseases including cancer, obesity, diabetes, and neurodegeneration. Hence, inhibitors of mTORC1 or a deregulated mTORC1 pathway may offer potential cancer therapies. In this study, we investigated the structural requirements for preventing the sensing and signal transmission from LARS to mTORC1. Building upon recent studies on mTORC1 regulation activation by leucine, we lay the foundation for the development of chemotherapeutic agents against mTORC1 that can overcome resistance to rapamycin. Using a combination of in-silico approaches to develop and validate an alternative interaction model, discussing its benefits and advancements. Finally, we identified a set of compounds ready for testing to prevent LARS1/RagD protein-protein interactions. We establish a basis for creating chemotherapeutic drugs targeting mTORC1, which can conquer resistance to rapamycin. We utilize in-silico methods to generate and confirm an alternative interaction model, outlining its advantages and improvements, and pinpoint a group of novel substances that can prevent LARS1/RagD interactions.Communicated by Ramaswamy H. Sarma.

Synthesis of 13C-methyl-labeled amino acids and their incorporation into proteins in mammalian cells.

Isotopic labeling of methyl-substituted proteinogenic amino acids with 13C has transformed applications of solution-based NMR spectroscopy and allowed the study of much larger and more complex proteins than previously possible with 15N labeling. Procedures are well-established for producing methyl-labeled proteins expressed in bacteria, with efficient incorporation of 13C-methyl labeled metabolic precursors to enable the isotopic labeling of Ile, Val, and Leu methyl groups. Recently, similar methodology has been applied to enable 13C-methyl labeling of Ile, Val, and Leu in yeast, extending the approach to proteins that do not readily fold when produced in bacteria. Mammalian or insect cells are nonetheless preferable for production of many human proteins, yet 13C-methyl labeling using similar metabolic precursors is not feasible as these cells lack the requisite biosynthetic machinery. Herein, we report versatile and high-yielding synthetic routes to 13C methyl-labeled amino acids based on palladium-catalyzed C(sp3)-H functionalization. We demonstrate the efficient incorporation of two of the synthesized amino acids, 13C-γ2-Ile and 13C-γ1,γ2-Val, into human receptor extracellular domains with multiple disulfides using suspension-cultured HEK293 cells. Production costs are reasonable, even at moderate expression levels of 2-3 mg purified protein per liter of medium, and the method can be extended to label other methyl groups, such as 13C-δ1-Ile and 13C-δ1,δ2-Leu. In summary, we demonstrate the cost-effective production of methyl-labeled proteins in mammalian cells by incorporation of 13C methyl-labeled amino acids generated de novo by a versatile synthetic route.

Improvement in Inhalation Properties of Theophylline and Levofloxacin by Co-Amorphization and Enhancement in Its Stability by Addition of Amino Acid as a Third Component.

Co-amorphous systems are amorphous formulations stabilized by the miscible dispersion of small molecules. This study aimed to design a stable co-amorphous system for the co-delivery of two drugs to the lungs as an inhaled formulation. Theophylline (THE) and levofloxacin (LEV) were used as model drugs for treating lung infection with inflammation. Leucine (LEU) or tryptophan (TRP) was employed as the third component to improve the inhalation properties. The co-amorphous system containing THE and LEV in an equal molar ratio was successfully prepared via spray drying where reduction of the particle size and change to the spherical morphology were observed. The addition of LEU or TRP at a one-tenth molar ratio to THE-LEV did not affect the formation of the co-amorphous system, but only TRP acted as an antiplasticizer. The Fourier transform infrared spectroscopy spectra revealed intermolecular interactions between THE and LEV in the co-amorphous system that were retained after the addition of LEU or TRP. The co-amorphous THE-LEV system exhibited better in vitro aerodynamic performance than a physical mixture of these compounds and permitted the simultaneous delivery of both drugs in various stages. The co-amorphous THE-LEV system crystallized at 40 °C, and this crystallization was not prevented by LEU. However, THE-LEV-TRP maintained its amorphous state for 1 month. Thus, TRP can act as a third component to improve the physical stability of the co-amorphous THE-LEV system, while maintaining the enhanced aerodynamic properties.

CHRISTMAS: Chiral Pair Isobaric Labeling Strategy for Multiplexed Absolute Quantitation of Enantiomeric Amino Acids.

Amino acids (AAs) in the d-form are involved in multiple pivotal neurological processes, although their l-enantiomers are most commonly found. Mass spectrometry-based analysis of low-abundance d-AAs has been hindered by challenging enantiomeric separation from l-AAs, low sensitivity for detection, and lack of suitable internal standards for accurate quantification. To address these critical gaps, N,N-dimethyl-l-leucine (l-DiLeu) tags are first validated as novel chiral derivatization reagents for chromatographic separation of 20 pairs of d/l-AAs, allowing the construction of a 4-plex isobaric labeling strategy for enantiomer-resolved quantification through single step tagging. Additionally, the creative design of N,N-dimethyl-d-leucine (d-DiLeu) reagents offers an alternative approach to generate analytically equivalent internal references of d-AAs using d-DiLeu-labeled l-AAs. By labeling cost-effective l-AA standards using paired d- and l-DiLeu, this approach not only enables absolute quantitation of both d-AAs and l-AAs from complex biological matrices with enhanced precision but also significantly boosts the combined signal intensities from all isobaric channels, greatly improving the detection and quantitation of low-abundance AAs, particularly d-AAs. We term this quantitative strategy CHRISTMAS, which stands for chiral pair isobaric labeling strategy for multiplexed absolute quantitation. Leveraging the ion mobility collision cross section (CCS) alignment, interferences from coeluting isomers/isobars are effectively filtered out to provide improved quantitative accuracy. From wild-type and Alzheimer's disease (AD) mouse brains, we successfully quantified 20 l-AAs and 5 d-AAs. The significant presence and differential trends of certain d-AAs compared to those of their l-counterparts provide valuable insights into the involvement of d-AAs in aging, AD progression, and neurodegeneration.

Hierarchy of the Components in Spray-Dried, Protein-Excipient Particles Using DNP-Enhanced NMR Spectroscopy.

Protein-based drugs are becoming increasingly important, but there are challenges associated with their formulation (for example, formulating stable inhalable aerosols while maintaining the proper long-term stability of the protein). Determining the morphology of multicomponent, protein-based drug formulations is particularly challenging. Here, we use dynamic nuclear polarization (DNP) solid-state NMR spectroscopy to determine the hierarchy of components within spray-dried particles containing protein, trehalose, leucine, and trileucine. DNP NMR was applied to these formulations to assess the localization of the components within the particles. We found a consistent scheme, where trehalose and the protein are co-located within the same phase in the core of the particles and leucine and trileucine are distributed in separate phases at the surface of the particles. The description of the hierarchy of the organic components determined by DNP NMR enables the rationalization of the performance of the formulation.

Amyloid Aggregation Is Potently Slowed Down by Osmolytes Due to Compaction of Partially Folded State.

Amyloid aggregation is a key process in amyloidoses and neurodegenerative diseases. Hydrophobicity is one of the major driving forces for this type of aggregation, as an increase in hydrophobicity generally correlates with aggregation susceptibility and rate. However, most experimental systems in vitro and prediction tools in silico neglect the contribution of protective osmolytes present in the cellular environment. Here, we assessed the role of hydrophobic mutations in amyloid aggregation in the presence of osmolytes. To achieve this goal, we used the model protein human muscle acylphosphatase (mAcP) and mutations to leucine that increased its hydrophobicity without affecting its thermodynamic stability. Osmolytes significantly slowed down the aggregation kinetics of the hydrophobic mutants, with an effect larger than that observed on the wild-type protein. The effect increased as the mutation site was closer to the middle of the protein sequence. We propose that the preferential exclusion of osmolytes from mutation-introduced hydrophobic side-chains quenches the aggregation potential of the ensemble of partially unfolded states of the protein by inducing its compaction and inhibiting its self-assembly with other proteins. Our results suggest that including the effect of the cellular environment in experimental setups and predictive softwares, for both mechanistic studies and drug design, is essential in order to obtain a more complete combination of the driving forces of amyloid aggregation.

Construction of metal-organic framework-based multienzyme system for L-tert-leucine production.

Chiral compounds are important drug intermediates that play a critical role in human life. Herein, we report a facile method to prepare multi-enzyme nano-devices with high catalytic activity and stability. The self-assemble molecular binders SpyCatcher and SpyTag were fused with leucine dehydrogenase and glucose dehydrogenase to produce sc-LeuDH (SpyCatcher-fused leucine dehydrogenase) and GDH-st (SpyTag-fused glucose dehydrogenase), respectively. After assembling, the cross-linked enzymes LeuDH-GDH were formed. The crosslinking enzyme has good pH stability and temperature stability. The coenzyme cycle constant of LeuDH-GDH was always higher than that of free double enzymes. The yield of L-tert-leucine synthesis by LeuDH-GDH was 0.47 times higher than that by free LeuDH and GDH. To further improve the enzyme performance, the cross-linked LeuDH-GDH was immobilized on zeolite imidazolate framework-8 (ZIF-8) via bionic mineralization, forming LeuDH-GDH @ZIF-8. The created co-immobilized enzymes showed even better pH stability and temperature stability than the cross-linked enzymes, and LeuDH-GDH@ZIF-8 retains 70% relative conversion rate in the first four reuses. In addition, the yield of LeuDH-GDH@ZIF-8 was 0.62 times higher than that of LeuDH-GDH, and 1.38 times higher than that of free double enzyme system. This work provides a novel method for developing multi-enzyme nano-device, and the ease of operation of this method is appealing for the construction of other multi-enzymes @MOF systems for the applications in the kinds of complex environment.

Spray dried progesterone formulations for carrier free dry powder inhalation.

Low oral absorption and extensive first pass metabolism of progesterone is reported for many oral formulations which warrants investigation into other routes of administration. It is the aim of this study to investigate the generation of inhaled formulations of progesterone though a spray drying approach with a focus on how spray drying impacts the physicochemical properties of progesterone. Formulations of progesterone with L-leucine and hydroxypropyl methylcellulose acetate succinate (HPMCAS) are reported to this aim. X-ray diffraction, spectroscopy and thermal analysis were used to characterise these formulations and confirmed that progesterone crystallises as the Form II polymorph during spray drying regardless of the solvent used. The resultant formulations showed higher aqueous solubility than progesterone Form I starting material and the addition of HPMCAS was shown to temporarily enable a supersaturated state. Thermal analysis was used to show that the Form II polymorph was sensitive to transformation to Form I during heating. The addition of L-leucine to the formulations reduced the temperature for the polymorphic transformation by âˆ¼ 10 °C. However, when HPMCAS was added to the formulation, the Form II polymorph was prevented from transforming to the Form I polymorph. Cascade impaction was used to determine the aerosol performance of the spray dried powders and showed promising lung deposition profiles (mass median aerodynamic diameter 5 µm) with significant variation depending on the organic solvent used and the ratio of organic to aqueous phase in the feedstock. However, further optimisation of formulations was required to direct more progesterone into the alveolar regions. The addition of HPMCAS was seen to increase the alveolar deposition and therefore formed a formulation with a lower fine particle fraction and mass median aerodynamic diameter. The most suitable formulation for inhalation was formed from a 50:50 acetone:water mixture and showed an ED, FPF and FPD of 81.7%, 44.5% and 7.3 mg respectively. Therefore, HPMCAS is suggested as a suitable excipient to increase solubility, prevent polymorphic transformation and improve inhalation properties of spray dried progesterone formulations. This study highlights the use of spray drying to form inhalable progesterone powders with higher solubility which may broaden the application of this medicine.

DiLeu Isobaric Labeling Coupled with Limited Proteolysis Mass Spectrometry for High-Throughput Profiling of Protein Structural Changes in Alzheimer's Disease.

High-throughput quantitative analysis of protein conformational changes has a profound impact on our understanding of the pathological mechanisms of Alzheimer's disease (AD). To establish an effective workflow enabling quantitative analysis of changes in protein conformation within multiple samples simultaneously, here we report the combination of N,N-dimethyl leucine (DiLeu) isobaric tag labeling with limited proteolysis mass spectrometry (DiLeu-LiP-MS) for high-throughput structural protein quantitation in serum samples collected from AD patients and control donors. Twenty-three proteins were discovered to undergo structural changes, mapping to 35 unique conformotypic peptides with significant changes between the AD group and the control group. Seven out of 23 proteins, including CO3, CO9, C4BPA, APOA1, APOA4, C1R, and APOA, exhibited a potential correlation with AD. Moreover, we found that complement proteins (e.g., CO3, CO9, and C4BPA) related to AD exhibited elevated levels in the AD group compared to those in the control group. These results provide evidence that the established DiLeu-LiP-MS method can be used for high-throughput structural protein quantitation, which also showed great potential in achieving large-scale and in-depth quantitative analysis of protein conformational changes in other biological systems.

Customizable resveratrol spray-dried micro-composites for inhalation as a promising contender for treatment of idiopathic pulmonary fibrosis.

The past decades have witnessed tremendous expansion in utilization of plant-derived medicines as resveratrol (RES) in treating several diseases like idiopathic pulmonary fibrosis (IPF). RES can exhibit its role in treating IPF via its outstanding antioxidant and anti-inflammatory activities. The goal of this work was to formulate RES-loaded spray-dried composite microparticles (SDCMs) suitable for pulmonary delivery via dry powder inhaler (DPI). They were prepared by spray drying of a previously prepared RES-loaded bovine serum albumin nanoparticles (BSA NPs) dispersion using different carriers. RES-loaded BSA NPs, prepared by the desolvation technique, acquired suitable particle size of 177.67 ± 0.95 nm and entrapment efficiency of 98.7 ± 0.35% with perfectly uniform size distribution and high stability. Considering the attributes of the pulmonary route, NPs were co-spray dried with compatible carriers viz. mannitol, dextran, trehalose, leucine, glycine, aspartic acid, and glutamic acid to fabricate SDCMs. All formulations showed suitable mass median aerodynamic diameter<5 µm; that is suitable for deep lung deposition. However, the best aerosolization behavior was attained from using leucine with fine particle fraction (FPF) of 75.74%, followed by glycine with FPF of 54.7%. Finally, a pharmacodynamic study was conducted on bleomycin-induced mice, and it strongly revealed the role of the optimized formulations in alleviating PF through suppressing the levels of hydroxyproline, tumor necrosis factor-α and matrix metalloproteinase-9 with obvious improvements in the treated lung histopathology. These findings indicate that in addition to leucine, the glycine amino acid, which is not commonly used yet, is very promising in the formulation of DPIs.