Investigation of liquid chromatography-mass spectrometry analysis of a peptide aldehyde SJA6017 with identifying its hemiacetal, gem-diol, and enol ether.

The quantitative analysis of SJA6017, a peptide aldehyde inhibitor of calpain (Calpain Inhibitor VI), has encountered challenges in preclinical drug studies. The complex reverse-phase HPLC chromatographic behavior exhibits two peaks, each containing multiple species. An liquid chromatography-mass spectrometry (LC-MS/MS) study proposed an explanation for this phenomenon, caused by the amide aldehyde structure of SJA6017. Four chemical species corresponding to the two HPLC peaks have been identified as SJA6017 and its methyl hemiacetal, methyl enol ether, and gem-diol. In many instances of preclinical studies, methanol is favored as a substitute for DMSO. The hemiacetal is formed when the amide-activated peptide aldehyde reacts with methanol, which can then be further dehydrated in the mass spectrometer ion source under high temperature to form the methyl enol ether. The hemiacetal and gem-diol can also be decomposed to SJA6017 in the ion source. Additionally, the amide-activated peptide aldehyde can easily hydrate to the gem-diol of SJA6017 during sample incubation or sample preparation. The hemiacetal and gem-diol of SJA6017 are stable enough to have different retention times in the liquid chromatography, which explains why SJA6017 appears as two peaks, each containing multiple species. An LC-MS/MS tandem quadrupole mass spectrometer quantitative analysis method is proposed, enabling the analysis of these types of samples. This work serves as both an illustrative example and a cautionary note for mass analysis, sample incubations, and sample preparations involving compounds of peptide aldehyde, including similar aldehyde-containing metabolites, especially when methanol is present. This study provides the information needed to understand peptide aldehyde behavior at various steps of preclinical in vitro studies in the presence of methanol. It has assisted in the development of the SJA6017 bioanalysis method and will also aid in the development of bioanalysis methods for similar peptide aldehydes.

Enabling a novel solvent method on Albendazole solid dispersion to improve the in vivo bioavailability.

Albendazole, a vital medication endorsed by the World Health Organization for combating parasitic infections, encounters a challenge stemming from its low solubility, significantly impeding absorption and bioavailability. Albendazole has near-insolubility in most organic solvents, so the solid dispersions of albendazole were predominantly using the fusion method. However, the solvent method could offer the advantage of achieving molecular-level mixing homogeneity. In this investigation, we incorporated the pH adjustment to prepare albendazole solid dispersion using a solvent method, which utilizes trace amounts of HCl in methanol, yielding notably enhanced albendazole solubility. Subsequently, carriers such as PEG6000/Poloxamer 188 (PEG: polyethylene glycol) and PVP K30/Poloxamer 188 (PVP: polyvinylpyrrolidone) were employed to create albendazole solid dispersions. Comprehensive characterization through dissolution rate analysis, PXRD (Powder X-ray diffraction), SEM (Scanning electron microscopy), DSC (differential scanning calorimetry), and pharmacokinetic (PK) studies in mice and rats was conducted. The findings indicate that the solid dispersion effectively transforms the crystalline state of albendazole into an amorphous state, resulting in significantly enhanced in vivo absorption and a 5.9-fold increase in exposure. Besides, the exposure increased 1.64 times of commercial albendazole tablets. Notably, PEG6000/Poloxamer 188 and PVP K30/Poloxamer 188 solid dispersions exhibited superior dissolution rates and pharmacokinetic profiles compared to commercially available albendazole tablets.

Assessing Boron-Pleuromutilin AN11251 for the Development of Antibacterial Agents.

Pleuromutilins are a group of antibiotics derived from the naturally occurring compound. The recent approval of lefamulin for both intravenous and oral doses in humans to treat community-acquired bacterial pneumonia has prompted investigations in modifying the structure to broaden the antibacterial spectrum, enhance the activity, and improve the pharmacokinetic properties. AN11251 is a C(14)-functionalized pleuromutilin with a boron-containing heterocycle substructure. It was demonstrated to be an anti-Wolbachia agent with therapeutic potential for Onchocerciasis and lymphatic filariasis. Here, the in vitro and in vivo PK parameters of AN11251 were measured including PPB, intrinsic clearance, half-life, systemic clearance, and volume of distribution. The results indicate that the benzoxaborole-modified pleuromutilin possesses good ADME and PK properties. AN11251 has potent activities against the Gram-positive bacterial pathogens tested, including various drug-resistant strains, and against the slow-growing mycobacterial species. Finally, we employed PK/PD modeling to predict the human dose for treatment of disease caused by Wolbachia, Gram-positive bacteria, or Mycobacterium tuberculosis, which might facilitate the further development of AN11251.

Correction: Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

[This corrects the article DOI: 10.1039/D1SC02653D.].

Single-molecule FRET and conformational analysis of beta-arrestin-1 through genetic code expansion and a Se-click reaction.

Single-molecule Förster resonance energy transfer (smFRET) is a powerful tool for investigating the dynamic properties of biomacromolecules. However, the success of protein smFRET relies on the precise and efficient labeling of two or more fluorophores on the protein of interest (POI), which has remained highly challenging, particularly for large membrane protein complexes. Here, we demonstrate the site-selective incorporation of a novel unnatural amino acid (2-amino-3-(4-hydroselenophenyl) propanoic acid, SeF) through genetic expansion followed by a Se-click reaction to conjugate the Bodipy593 fluorophore on calmodulin (CaM) and ß-arrestin-1 (ßarr1). Using this strategy, we monitored the subtle but functionally important conformational change of ßarr1 upon activation by the G-protein coupled receptor (GPCR) through smFRET for the first time. Our new method has broad applications for the site-specific labeling and smFRET measurement of membrane protein complexes, and the elucidation of their dynamic properties such as transducer protein selection.

A Genetically Encoded Two-Dimensional Infrared Probe for Enzyme Active-Site Dynamics.

While two-dimensional infrared (2D-IR) spectroscopy is uniquely suitable for monitoring femtosecond (fs) to picosecond (ps) water dynamics around static protein structures, its utility for probing enzyme active-site dynamics is limited due to the lack of site-specific 2D-IR probes. We demonstrate the genetic incorporation of a novel 2D-IR probe, m-azido-L-tyrosine (N3Y) in the active-site of DddK, an iron-dependent enzyme that catalyzes the conversion of dimethylsulfoniopropionate to dimethylsulphide. Our results show that both the oxidation of active-site iron to FeIII , and the addition of denaturation reagents, result in significant decrease in enzyme activity and active-site water motion confinement. As tyrosine residues play important roles, including as general acids and bases, and electron transfer agents in many key enzymes, the genetically encoded 2D-IR probe N3Y should be broadly applicable to investigate how the enzyme active-site motions at the fs-ps time scale direct reaction pathways to facilitating specific chemical reactions.

Genetic Incorporation of Selenotyrosine Significantly Improves Enzymatic Activity of Agrobacterium radiobacter Phosphotriesterase.

Tyrosine plays important roles in many enzymes. To facilitate enzyme design, mechanistic studies and minimize structural perturbation in the active site, here we report the genetic incorporation of a novel unnatural amino acid selenotyrosine (SeHF), which has single-atom replacement in comparison to tyrosine. The arPTE-(Agrobacterium radiobacter Phosphotriesterase) Tyr309SeHF mutant exhibits a significant 12-fold increase in kcat and 3.2-fold enhancement in kcat /KM at pH 7.0. Molecular dynamics simulations show that the SeHF309 mutation results in a conformational switch which opens up the product release pocket and increases the product release rate, thereby elevating the overall enzyme activity. Significant improvement of the catalytic efficiency at neutral pH by single unnatural amino acid (UAA) mutation broadens the application of this enzyme, and provides valuable insights to the mechanism. Our method represents a new approach for designing enzymes with enhanced activity.

DeSiphering receptor core-induced and ligand-dependent conformational changes in arrestin via genetic encoded trimethylsilyl 1H-NMR probe.

Characterization of the dynamic conformational changes in membrane protein signaling complexes by nuclear magnetic resonance (NMR) spectroscopy remains challenging. Here we report the site-specific incorporation of 4-trimethylsilyl phenylalanine (TMSiPhe) into proteins, through genetic code expansion. Crystallographic analysis revealed structural changes that reshaped the TMSiPhe-specific amino-acyl tRNA synthetase active site to selectively accommodate the trimethylsilyl (TMSi) group. The unique up-field 1H-NMR chemical shift and the highly efficient incorporation of TMSiPhe enabled the characterization of multiple conformational states of a phospho-ß2 adrenergic receptor/ß-arrestin-1(ß-arr1) membrane protein signaling complex, using only 5 µM protein and 20 min of spectrum accumulation time. We further showed that extracellular ligands induced conformational changes located in the polar core or ERK interaction site of ß-arr1 via direct receptor transmembrane core interactions. These observations provided direct delineation and key mechanism insights that multiple receptor ligands were able to induce distinct functionally relevant conformational changes of arrestin.

Genetically Encoded Fluorescent Amino Acid for Monitoring Protein Interactions through FRET.

Förster resonance energy transfer (FRET) is a well-established method for studying macromolecular interactions and conformational changes within proteins. Such a method normally uses fluorescent proteins or chemical-labeling methods which are often only accessible to surface-exposed residues and risk-disturbing target protein structures. Here, we demonstrate that the genetic incorporation of a synthetic fluorescent amino acid, L-(7-hydroxycoumarin-4-yl) ethylglycine (Cou) and natural endogenous fluorophore Tryptophan (Trp) residues of a protein could serve as an efficient FRET pair to monitor protein interactions, using the signaling transducer ß-arrestin-1 as a model system. We used this technology to record the dynamic spectra in both binding and competition experiments of ß-arrestin-1, the contribution of each specific phosphate in ternary complex formation, in a rapid and efficient manner. The determined Kd value for the association between the active arrestin and Fab30 is 0.68 µM in the three-component interaction system. Moreover, we were able to determine the contributions of the site 3 phospho-site and the site 6 phospho-site binding, each contributing to the high affinity ternary complex assembly as 2.7 fold and 15.5 fold, respectively, which were never determined before. These results thus highlighted the potential usage of this new method in measurement of the allosteric-induced enhanced affinity with small amount proteins and in a fast manner and in a complex system. Collectively, our newly developed Trp:Cou FRET system based on genetic expansion technology has extended the molecular toolboxes available for biochemical and structural biology studies.

S-Click Reaction for Isotropic Orientation of Oxidases on Electrodes to Promote Electron Transfer at Low Potentials.

Electrochemical sensors are essential for point-of-care testing (POCT) and wearable sensing devices. Establishing an efficient electron transfer route between redox enzymes and electrodes is key for converting enzyme-catalyzed reactions into electrochemical signals, and for the development of robust, sensitive, and selective biosensors. We demonstrate that the site-specific incorporation of a novel synthetic amino acid (2-amino-3-(4-mercaptophenyl)propanoic acid) into redox enzymes, followed by an S-click reaction to wire the enzyme to the electrode, facilitates electron transfer. The fabricated biosensor demonstrated real-time and selective monitoring of tryptophan (Trp) in blood and sweat samples, with a linear range of 0.02-0.8 mm. Further developments along this route may result in dramatic expansion of portable electrochemical sensors for diverse health-determination molecules.

Rapid probing of sialylated glycoproteins in vitro and in vivo via metabolic oligosaccharide engineering of a minimal cyclopropene reporter.

ManNAc analogues are important chemical tools for probing sialylation dynamically via metabolic oligosaccharide engineering (MOE). The size of N-acyl and the nature of the chemical handle are two determinants of metabolic incorporation efficiency. We demonstrated a minimal, stable, bioorthogonal, and reactive N-Cp (N-(cycloprop-2-ene-1-ylcarbonyl)) group and the imaging of sialylated glycans using Ac4ManNCp in vitro and in vivo. The results revealed that the Cp group can efficiently be incorporated into the cellular sialic acid and detected rapidly by the reaction with FITC-Tz in different cells. The metabolic incorporation efficiency of non-cytotoxic Ac4ManNCp is not only superior to Ac4ManNMCp, but also superior to the widely-used Ac4ManNAz in some cell lines. Moreover, when Ac4ManNCp was administered to mice, a rapid and intense labelling of splenocytes as well as glycoproteins of sera and organs was observed. This is the first reported metabolic labelling of cyclopropene-modified sugars in vivo. Therefore, Ac4ManNCp is a powerful probe for efficient and rapid MOE and it may find wide applications in the labelling of glycans.

Enabling Wittig reaction on site-specific protein modification.

An efficient aqueous Wittig reaction was enabled on protein bioconjugation for the first time. By investigating the reaction on small molecules, peptides, and proteins, a site-specific reaction targeting "aldehyde tag" was presented. A variety of functional groups could be introduced into the protein of interest.