Formulation of Saxagliptin Oral Films: Optimization, Physicochemical Characterization, In-Vivo Assessment, and In-Vitro Real-Time Release Monitoring via a Novel Polyaniline Nanoparticles-Based Solid-Contact Screen Printed Ion-Selective Electrode.

Oral dispersible films have received broad interest due to fast drug absorption and no first-path metabolism, leading to high bioavailability and better patient compliance. Saxagliptin (SXG) is an antidiabetic drug that undergoes first-path metabolism, resulting in a less active metabolite, so the development of SXG oral dispersible films (SXG-ODFs) improves SXG bioavailability. The formula optimisation included a response surface experimental design and the impact of three formulation factors, the type and concentration of polymer and plasticiser concentration on in-vitro disintegration time and folding endurance. Two optimised SXG-ODFs prepared using either polyvinyl alcohol (PVA) or hydroxypropyl methylcellulose were investigated. SXG-ODFs prepared with PVA demonstrated a superior rapid disintegration time, ranging from 17 to 890 s, with the fastest disintegration time recorded at 17 s. These short durations can be attributed to the hydrophilic nature of PVA, facilitating rapid hydration and disintegration upon contact with saliva. Additionally, PVA-based films displayed remarkable folding endurance, surpassing 200 folds without rupture, indicating flexibility and stability. The high tensile strength of PVA-based films further underscores their robust mechanical properties, with tensile strength values reaching up to 4.53 MPa. SXG exhibits a UV absorption wavelength of around 212 nm, posing challenges for traditional quantitative spectrophotometric analysis, so a polyaniline nanoparticles-based solid-contact screen-printed ion-selective electrode (SP-ISE) was employed for the determination of SXG release profile effectively in comparison to HPLC. SP-ISE showed a better real-time release profile of SXG-ODFs, and the optimised formula showed lower blood glucose levels than commercial tablets.

Chiral zwitterionic stationary phases based on Cinchona alkaloids and dipeptides: Application in chiral separation of dipeptides under reversed phase conditions.

Chromatographic behavior of novel chiral stationary phases with bonded selectors based on Cinchona alkaloids modified with dipeptides was studied using dipeptides as probe molecules. Buffer-free and salt containing hydro-organic solutions were used as the mobile phases. The selectors exhibit pseudoenantiomeric behavior with respect to the L/D or LL/DD enantiomers and do not behave so with respect to the LD/DL enantiomers. The alkaloid part of the selectors is the driver of enantioselectivity, while the dipeptide substituent plays a modulating role. The quinidine-based selectors demonstrate stronger adsorption affinity and higher enantioselectivity as compared to the quinine-based selectors. The dipeptide analytes containing a glycyl fragment are weaker retained and their enantiomers are worse separated comparing to dipeptides with both units being larger amino acids. Moreover, a phenyl group in the structure of a dipeptide analyte facilitates enantioseparation. The effect of the mobile phase composition on retention depends on the hydrophobicity of an analyte. Hydrophobic dipeptides are better eluted by methanol-rich solvents, hydrophilic dipeptides are better eluted with water-rich solvents, and dipeptides with an intermediate hydrophobicity demonstrate a U-shaped or more complicated dependence of the retention factor on the percentage of methanol. Even a small buffer addition to the mobile phase decreases retention, but the ion-exchange mechanism was not confirmed. The effect of an electrolyte is rather due to the shielding of the charged groups of the selector reducing thereby electrostatic interaction between the selector and analyte. Efficiency of the novel columns is comparable to that of other brush-type chiral columns, the highest achieved number of the theoretical plates per 1 m varying between 30,000 and 40,000.

Deep learning path-like collective variable for enhanced sampling molecular dynamics.

Several enhanced sampling techniques rely on the definition of collective variables to effectively explore free energy landscapes. The existing variables that describe the progression along a reactive pathway offer an elegant solution but face a number of limitations. In this paper, we address these challenges by introducing a new path-like collective variable called the "deep-locally non-linear-embedding," which is inspired by principles of the locally linear embedding technique and is trained on a reactive trajectory. The variable mimics the ideal reaction coordinate by automatically generating a non-linear combination of features through a differentiable generalized autoencoder that combines a neural network with a continuous k-nearest neighbor selection. Among the key advantages of this method is its capability to automatically choose the metric for searching neighbors and to learn the path from state A to state B without the need to handpick landmarks a priori. We demonstrate the effectiveness of DeepLNE by showing that the progression along the path variable closely approximates the ideal reaction coordinate in toy models, such as the Müller-Brown potential and alanine dipeptide. Then, we use it in the molecular dynamics simulations of an RNA tetraloop, where we highlight its capability to accelerate transitions and estimate the free energy of folding.

Structure and inhibition of the human lysosomal transporter Sialin.

Sialin, a member of the solute carrier 17 (SLC17) transporter family, is unique in its ability to transport not only sialic acid using a pH-driven mechanism, but also transport mono and diacidic neurotransmitters, such as glutamate and N-acetylaspartylglutamate (NAAG), into synaptic vesicles via a membrane potential-driven mechanism. While most transporters utilize one of these mechanisms, the structural basis of how Sialin transports substrates using both remains unclear. Here, we present the cryogenic electron-microscopy structures of human Sialin: apo cytosol-open, apo lumen-open, NAAG-bound, and inhibitor-bound. Our structures show that a positively charged cytosol-open vestibule accommodates either NAAG or the Sialin inhibitor Fmoc-Leu-OH, while its luminal cavity potentially binds sialic acid. Moreover, functional analyses along with molecular dynamics simulations identify key residues in binding sialic acid and NAAG. Thus, our findings uncover the essential conformational states in NAAG and sialic acid transport, demonstrating a working model of SLC17 transporters.

Discovery of Glutamate Carboxypeptidase III Ligands to Compete the Uptake of [177Lu]Lu-PSMA-617 in Healthy Organs.

Prostate-specific membrane antigen (PSMA)-targeted radio ligand therapeutics (RLTs), such as [177Lu]Lu-PSMA-617 (Pluvicto), have been shown to accumulate in salivary glands and kidneys, potentially leading to undesired side effects. As unwanted accumulation in normal organs may derive from the cross-reactivity of PSMA ligands to glutamate carboxypeptidase III (GCPIII), it may be convenient to block this interaction with GCPIII-selective ligands. Parallel screening of a DNA-encoded chemical library (DEL) against GCPIII and PSMA allowed the identification of GCPIII binders. Structure-activity relationship (SAR) studies resulted in the identification of nanomolar GCPIII ligands with up to 1000-fold selectivity over PSMA. We studied the ability of GCPIII ligands to counteract the binding of [177Lu]Lu-PSMA-617 to human salivary glands by autoradiography and could demonstrate a partial radioprotection.

Contribution of amino acids in the active site of dipeptidyl peptidase 4 to the catalytic action of the enzyme.

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.

pH-sensitive release of nitric oxide gas using peptide-graphene co-assembled hybrid nanosheets.

Nitric oxide (NO) donating drugs such as organic nitrates have been used to treat cardiovascular diseases for more than a century. These donors primarily produce NO systemically. It is however sometimes desirable to control the amount, location, and time of NO delivery. We present the design of a novel pH-sensitive NO release system that is achieved by the synthesis of dipeptide diphenylalanine (FF) and graphene oxide (GO) co-assembled hybrid nanosheets (termed as FF@GO) through weak molecular interactions. These hybrid nanosheets were characterised by using X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, zeta potential measurements, X-ray photoelectron spectroscopy, scanning and transmission electron microscopies. The weak molecular interactions, which include electrostatic, hydrogen bonding and π-π stacking, are pH sensitive due to the presence of carboxylic acid and amine functionalities on GO and the dipeptide building blocks. Herein, we demonstrate that this formulation can be loaded with NO gas with the dipeptide acting as an arresting agent to inhibit NO burst release at neutral pH; however, at acidic pH it is capable of releasing NO at the rate of up to 0.6 µM per minute, comparable to the amount of NO produced by healthy endothelium. In conclusion, the innovative conjugation of dipeptide with graphene can store and release NO gas under physiologically relevant concentrations in a pH-responsive manner. pH responsive NO-releasing organic-inorganic nanohybrids may prove useful for the treatment of cardiovascular diseases and other pathologies.

Metal Cross-Linked Supramolecular Gel Noodles: Structural Insights and Antibacterial Assessment.

Achieving precise control over gelator alignment and morphology is crucial for crafting tailored materials and supramolecular structures with distinct properties. We successfully aligned the self-assembled micelles formed by a functionalized dipeptide 2NapFF into long 1-D "gel noodles" by cross-linking with divalent metal chlorides. We identify the most effective cross-linker for alignment, enhancing mechanical stability, and imparting functional properties. Our study shows that Group 2 metal ions are particularly suited for creating mechanically robust yet flexible gel noodles because of their ionic and nondirectional bonding with carboxylate groups. In contrast, the covalent nature and high directional bonds of d-block metal ions with carboxylates tend to disrupt the self-assembly of 2NapFF. Furthermore, the 2NapFF-Cu noodles demonstrated selective antibacterial activity, indicating that the potent antibacterial property of the copper(II) ion is preserved within the cross-linked system. By merging insights into molecular alignment, gel extrusion processing, and integrating specific functionalities, we illustrate how the versatility of dipeptide-based gels can be utilized in creating next-generation soft materials.

Quantifying Unbiased Conformational Ensembles from Biased Simulations Using ShapeGMM.

Quantifying the conformational ensembles of biomolecules is fundamental to describing mechanisms of processes such as protein folding, interconversion between folded states, ligand binding, and allosteric regulation. Accurate quantification of these ensembles remains a challenge for conventional molecular simulations of all but the simplest molecules due to insufficient sampling. Enhanced sampling approaches, such as metadynamics, were designed to overcome this challenge; however, the nonuniform frame weights that result from many of these approaches present an additional challenge to ensemble quantification techniques such as Markov State Modeling or structural clustering. Here, we present rigorous inclusion of nonuniform frame weights into a structural clustering method entitled shapeGMM. The result of frame-weighted shapeGMM is a high dimensional probability density and generative model for the unbiased system from which we can compute important thermodynamic properties such as relative free energies and configurational entropy. The accuracy of this approach is demonstrated by the quantitative agreement between GMMs computed by Hamiltonian reweighting and direct simulation of a coarse-grained helix model system. Furthermore, the relative free energy computed from a shapeGMM probability density of alanine dipeptide reweighted from a metadynamics simulation quantitatively reproduces the underlying free energy in the basins. Finally, the method identifies hidden structures along the actin globular to filamentous-like structural transition from a metadynamics simulation on a linear discriminant analysis coordinate trained on GMM states, illustrating how structural clustering of biased data can lead to biophysical insight. Combined, these results demonstrate that frame-weighted shapeGMM is a powerful approach to quantifying biomolecular ensembles from biased simulations.

Cyclodipeptide oxidase is an enzyme filament.

Modified cyclic dipeptides represent a widespread class of secondary metabolites with diverse pharmacological activities, including antibacterial, antifungal, and antitumor. Here, we report the structural characterization of the Streptomyces noursei enzyme AlbAB, a cyclodipeptide oxidase (CDO) carrying out α,ß-dehydrogenations during the biosynthesis of the antibiotic albonoursin. We show that AlbAB is a megadalton heterooligomeric enzyme filament containing covalently bound flavin mononucleotide cofactors. We highlight that AlbAB filaments consist of alternating dimers of AlbA and AlbB and that enzyme activity is crucially dependent on filament formation. We show that AlbA-AlbB interactions are highly conserved suggesting that other CDO-like enzymes are likely enzyme filaments. As CDOs have been employed in the structural diversification of cyclic dipeptides, our results will be useful for future applications of CDOs in biocatalysis and chemoenzymatic synthesis.

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.

Supramolecular Hydrogels and Water Channels of Differing Diameters from Dipeptide Isomers.

Dipeptides stereoisomers and regioisomers composed of norleucine (Nle) and phenylalanine (Phe) self-assemble into hydrogels under physiological conditions that are suitable for cell culture. The supramolecular behavior, however, differs as the packing modes comprise amphipathic layers or water channels, whose diameter is defined by either four or six dipeptide molecules. A variety of spectroscopy, microscopy, and synchrotron-radiation-based techniques unveil fine details of intermolecular interactions that pinpoint the relationship between the chemical structure and ability to form supramolecular architectures that define soft biomaterials.

Efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide analog potentiates the activity of 5-O-mycaminosyltylonolide for multi-drug resistant Pseudomonas aeruginosa.

The emergence and spread of antimicrobial resistance are global threats. Pseudomonas aeruginosa (P. aeruginosa) is responsible for a substantial proportion of this global health issue because of its intrinsic resistance to many antibiotics due to the impermeability of its outer membrane and its multidrug efflux pump systems. Therefore, therapeutic drugs are limited, and the development of new drugs is extremely challenging. As an alternative approach, we focused on a combinational treatment strategy and found that 5-O-mycaminosyltylonolide (OMT) showed potent antibacterial activity against P. aeruginosa in the presence of an efflux pump inhibitor, phenylalanine-arginine beta-naphthylamide (PAßN). In this report, we prepared a PAßN derivative and compared the potentiation activity of OMT by PAßNs against multidrug-resistant P. aeruginosa clinical isolates.

Novel Triazole-Containing "Dipeptides": Synthesis, Molecular Docking and Analgesic Activity Studies.

Dipeptides of a new structure based on ß-triazolalanines and (L)-α-amino acids were synthesized and optimal conditions were developed that ensure both chemical and optical purity of the final products. Molecular docking was carried out and possible intermolecular interactions of dipeptides with potential targets were established. Based on these studies, the analgesic property of chosen dipeptides was studied and it was found that some compounds possess revealed antinociceptive activity in the tail-flick test.

Cucurbit[8]uril Binds Nonterminal Dipeptide Sites with High Affinity and Induces a Type II ß-Turn.

In an effort to target polypeptides at nonterminal sites, we screened the binding of the synthetic receptor cucurbit[8]uril (Q8) to a small library of tetrapeptides, each containing a nonterminal dipeptide binding site. The resulting leads were characterized in detail using a combination of isothermal titration calorimetry, 1H NMR spectroscopy, electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS), and X-ray crystallography. The equilibrium dissociation constant values determined for the binding of Q8 to nonterminal dipeptide sites Lys-Phe (KF) and Phe-Lys (FK) were 60 and 86 nm, respectively. These are to the best of our knowledge the highest affinities reported to date for any synthetic receptor targeting a nonterminal site on an unmodified peptide. A 0.79 Å resolution crystal structure was obtained for the complex of Q8 with the peptide Gly-Gly-Leu-Tyr-Gly-Gly-Gly (GGLYGGG) and reveals structural details of the pair-inclusion motif. The molecular basis for recognition is established to be the inclusion of the side chains of Leu and Tyr residues, as well as an extensive network of hydrogen bonds between the peptide backbone, the carbonyl oxygens of Q8, and proximal water molecules. In addition, the crystal structure reveals that Q8 induces a type II ß-turn. The sequence-selectivity, high affinity, reversibility, and detailed structural characterization of this system should facilitate the development of applications involving ligand-induced polypeptide folding.

A virtual library of small molecules mimicking dipeptides.

Virtual combinatorial libraries are prevalent in drug discovery due to improvements in the prediction of synthetic reactions that can be performed. This has gone hand in hand with the development of virtual screening capabilities to effectively screen the large chemical spaces spanned by exhaustive enumeration of reaction products. In this study, we generated a small-molecule dipeptide mimic library to target proteins binding small peptides. The library was created based on the general idea of peptide synthesis, that is, amino acid mimics were reacted in silico to form the dipeptide mimics, yielding 2,036,819 unique compounds. After docking calculations, two compounds from the library were synthesized and tested against WD repeat-containing protein 5 (WDR5) and histamine receptors H1-H4 to evaluate whether these molecules are viable in assays. The compounds showed the highest potency at the histamine H3 receptor, with Ki values in the two-digit micromolar range.

Unlocking Amides: A General Method for the Self-Immolative Release of Amide-Containing Molecules.

The controlled liberation of molecules from a constructed framework is a subject of profound interest across various chemical fields. It allows for the masking of a molecule's properties and precise deployment upon a single controllable release event. While numerous methodologies have been developed for amines, alcohols, and thiols, approaches for utilising amides as payload-release handles are still in their early stages of development, despite the prevalence of amides in therapeutic compounds and materials. Herein, is presented a comprehensive strategy for the controlled and selective release of a diverse range of amides with stable linkers. The versatility of this approach is demonstrated by its successful application in the targeted release of various amide-containing drugs in their natural form via the use of commonly used trigger motifs, such as dipeptides or glycosides. As a proof of concept, the FDA-approved antibiotic linezolid has been successfully converted into a prodrug form and released selectively only in the presence of the trigger event. Significantly, in its prodrug state, no activity against Mycobacterium tuberculosis was exhibited. Linezolid's full potential was achieved only upon controlled release, where an equipotent efficacy to the free linezolid control was demonstrated, thus emphasising the immense potential of this method.

New origin of life theory may explain biomolecular handedness.

Experiments suggest chemical reaction rates explain how proteins came to be built from left-handed building blocks.

Interrupted Polonovski Strategy for the Synthesis of Functionalized Amino Acids and Peptides.

The α-functionalization of carbamate-protected hydroxylamine glycine derivatives, acting as imine surrogates via an interrupted Polonovski reaction, is described to access functionalized amino acid derivatives. The addition of C, N, O, and S nucleophiles was achieved in a one-pot procedure in 37% to 92% yield. This method could be extended to dipeptide derivatives for the functionalization of both the C-terminus and N-terminus.

The HIV capsid mimics karyopherin engagement of FG-nucleoporins.

HIV can infect non-dividing cells because the viral capsid can overcome the selective barrier of the nuclear pore complex and deliver the genome directly into the nucleus1,2. Remarkably, the intact HIV capsid is more than 1,000 times larger than the size limit prescribed by the diffusion barrier of the nuclear pore3. This barrier in the central channel of the nuclear pore is composed of intrinsically disordered nucleoporin domains enriched in phenylalanine-glycine (FG) dipeptides. Through multivalent FG interactions, cellular karyopherins and their bound cargoes solubilize in this phase to drive nucleocytoplasmic transport4. By performing an in vitro dissection of the nuclear pore complex, we show that a pocket on the surface of the HIV capsid similarly interacts with FG motifs from multiple nucleoporins and that this interaction licences capsids to penetrate FG-nucleoporin condensates. This karyopherin mimicry model addresses a key conceptual challenge for the role of the HIV capsid in nuclear entry and offers an explanation as to how an exogenous entity much larger than any known cellular cargo may be able to non-destructively breach the nuclear envelope.