Modifying the PSD-95 inhibitor NA-1 for brain delivery through proteolytic stabilization and conjugation to the potentially brain homing peptide BR1.

A stable all-D peptide ligand inspires broad-spectrum affinity and high performance in the purification of COVID-19 vaccines.

Amino acid conjugates are progressively becoming popular as a potent tactic to enhance the pharmacological efficacy of drugs, especially in the areas of cancer and antimicrobial therapy. By taking advantage of the intrinsic biological attributes of amino acids, their conjugates facilitate drug stability, selective accumulation, and enhanced therapeutic efficacies. In particular, the structural analogy of amino acids to physiological substrates enables these conjugates to use solute carrier transporters, commonly overexpressed in tumour cells, which allow for targeted and effective drug delivery. This review considers how amino acid properties like chirality, hydrophobicity and steric bulk can be modulated to maximize drug conjugates. We emphasize important design aspects, such as selection of linkers and coupling reagents, and how these have an impact on drug release and biodistribution. Specific focus is given to d-amino acid, which increases proteolytic stability and bioactivity for both anticancer and antimicrobial uses, and to l-amino acid, which is responsible for receptor recognition, metabolic compatibility and amino acid decorated nanoparticle formulation. The existing drawbacks of antibody-drug conjugates (ADCs) and peptide-drug conjugates (PDCs) are immunogenicity, enzymatic degradation and poor tissue penetration. Amino acid conjugates provide a strong rationale with higher chemical versatility and potential for better pharmacokinetics and less toxicity. By harnessing the insights from chemistry, transporter biology and therapeutic design, this review presents a strategy for the creation of next-generation amino acid conjugates that bridge molecular accuracy to clinical utility.

The distribution of D-amino acid in food and drug and their effects on mouse metabolism.

Elucidating the molecular mechanisms of the mongolian medicine Ana Gaqi Nari in gastric cancer treatment through a multi-omics-guided experimental validation.

D-p-hydroxyphenyl glycine (D-PHPG) is a D-amino acid used as an intermediate in the synthesis of semi-synthetic antibiotics. It is synthesized from hydantoin derivatives through two sequential enzymatic reactions involving D-hydantoinase (D-hase) and D-carbamoylase (D-case). Although whole-cell biocatalysis of D-PHPG is cost-effective, its efficiency suffers from transport obstacles, intracellular degradation, and limited substrate solubility. This study utilized a bacterial surface display system to express D-hase and D-case in Escherichia coli for D-PHPG production. Enzyme production optimization was carried out in two stages. Initially, key factors influencing cell density during co-culture were identified through culture media and fermentation parameters screening using the Plackett-Burman design, followed by optimization with the D-optimal method. Next, induction parameters were fine-tuned using response surface methodology. The optimal culture medium was found to contain glycerol (12g/L) and yeast extract (15g/L) under optimal induction conditions (0.17 mM IPTG, OD600 of 1.3, and 21°C). These conditions achieved an OD600 of 15.6, with expression levels of 20.18% for D-case and 20.82% for D-hase. Scaling up in a stirred tank bioreactor resulted in an OD600 of 32.15, with D-hase and D-case expression levels increasing to 25.8 and 24.2%, respectively, and enzymatic activities improving by 2.83 times for D-case and 3.42 times for D-hase. The optimized co-culture approach under optimized induction conditions achieved a conversion yield of 95% and a D-PHPG production yield of 90%. The study results showed that the suggested fermentation conditions will contribute to future scale-up studies aimed at improving enzyme activities for other surface protein production.

Amino acid racemases are pivotal for d-amino acid (DAA) biosynthesis with wide-ranging biotechnological applications, yet their industrial deployment is hindered by narrow substrate specificity and instability. Here, we report the discovery of Halocola ammonii gen. nov., sp. nov. DA487T, a novel taxon within the proposed family Halocolacceae fam. nov. (order Flavobacteriales), isolated from hypersaline sediments. Genomic analysis revealed a robust DAA metabolic network, including a putative broad-specificity racemase RacX. Biochemical characterization demonstrated RacX's exceptional catalytic efficiency (kcat/Km = 151.2 s-1 mM-1 for l-Lys, kcat/Km = 17.8 s-1 mM-1 for d-Lys) and broad substrate spectrum (15/17 tested l-amino acids). Homology modeling and mutagenesis identified Ala79 and Cys193 as putative catalytic residues, based on structural conservation with EcL-DER. Remarkably, the A79C variant enhanced the reverse reaction efficiency (d-Lys → l-Lys) by 44%, effectively shifting the enzyme's catalytic bias and the resulting steady-state ratio of enzyme-bound species. Computational docking suggested that Asn80, Thr81, Asn121, and Thr124 may modulate substrate binding, though experimental structural validation is required. The thermostability-lability tradeoff ([Formula: see text]) highlights targets for protein engineering. Our findings not only expand the phylogenetic diversity of microbial racemases but also identify a promising biocatalyst candidate for industrial DAA production.IMPORTANCEMicrobial adaptations to extreme environments serve as a valuable source of novel biocatalysts with potential for sustainable industrial applications. In this study, we characterized Halocola ammonii DA487ᵀ, a halophilic bacterium representing the novel family Halocolaceae within the order Flavobacteriales, and identified a broad-specificity amino acid racemase, RacX. RacX demonstrates exceptional catalytic efficiency (kcat/Km up to 151.2 s⁻¹ mM⁻¹ for l-Lys) across multiple amino acids and exhibits remarkable stability under neutral and alkaline conditions (pH 7.0-9.0)-properties intrinsically linked to its high-salt ecological niche. Unlike most known racemases from neutrophilic organisms, RacX originates from an understudied phylogenetic lineage and displays unique mechanistic features, including a strong innate bias toward d-amino acid (DAA) production that can be rationally reprogrammed via single-residue substitution (e.g., A79C). These functional and evolutionary insights, combined with its halotolerance and broad substrate scope, position RacX as a promising and engineerable biocatalyst for industrial processes requiring operation under high-salt or alkaline conditions, such as the synthesis of DAA precursors for antibiotics.

Delivery of therapeutic peptides and proteins to the cytosol is of great interest due to their ability to inhibit intracellular protein-protein interactions, which are mostly deemed undruggable by small molecules. Internalization into the endosomal pathway is possible by receptor-targeted approaches; however, endosomal escape is inefficient, and its quantification is challenging. To improve our current understanding of cytosolic delivery, we performed comprehensive studies on a receptor-mediated shuttle system based on the chemokine-like receptor 1 (CMKLR1). As a model cargo, PMIγ was used, a known D-amino acid peptide antagonist of the MDM2/p53 interaction, which survives the harsh conditions in the endocytic pathway. Fluorescence correlation spectroscopy (FCS) was used to demonstrate that biologically meaningful cytosolic concentrations (>100 nM) can be reached by receptor-mediated shuttling, even when no endosomal escape enhancing strategies are used. Attachment of the pH-responsive endosomal escape peptide (EEP) hsLMWP further improved cytosolic delivery but also induced cellular toxicity at higher concentrations. Additionally, the EEP activity was likely limited by its fast degradation after internalization. Intracellular biological activity was confirmed using bioluminescence resonance energy transfer (BRET) studies, which demonstrate binding to MDM2 and inhibition of the p53/MDM2 interaction. This study highlights the potential of receptor-mediated shuttling for cytosolic delivery of therapeutic peptides and provides new insights into achievable intracellular concentrations, advancing the field of peptide therapeutics and drug delivery.

A multi-step structure-based virtual screening (SBVS) campaign was conducted on a 160,000-compound library to identify novel inhibitors targeting an outer surface region of D-amino acid oxidase (DAO), in the absence of known inhibitor binding geometries. The SBVS workflow incorporated druggability-based filtration and employed three docking engines: sievgene, DOCK 6, and AutoDock Vina. After the final screening stage and selection based on consensus among top-ranked compounds, five candidates were evaluated in vitro. Among them, one compound (N-{[(3S,3aS,6aS)-5-benzylhexahydro-2H-furo[2,3-c]pyrrol-3-yl]methyl}pyridine-3-carboximidic acid) exhibited significant inhibitory activity against DAO. To further refine the docking results and identify the most stable binding pose of the compound, we addressed the post-docking challenge using a combination of molecular dynamics (MD) simulations and machine learning-based hierarchical clustering. Poses generated from the three docking engines were classified using four clustering algorithms, and representative poses for each cluster were subjected to MD and binding free energy calculations via the molecular mechanics/generalized Born surface area (MM/GBSA) method. Notably, three poses converged to a common stable state characterized by the lowest binding free energies. The identified inhibitor in this stable state occupied a region spatially distinct from the substrate-binding site, consistent with our design strategy. Furthermore, we evaluated clustering performance based on the consistency between pose classification and calculated binding free energies. Among the tested methods, nearest neighbor and group average clustering exhibited the highest consistency. These findings demonstrate the utility of an integrated virtual screening strategy, including post-docking analysis to refine binding poses, for the robust identification of surface-binding inhibitors.

Chronic wound infection is a major global public health issue, with Enterococcus faecalis among the most commonly isolated pathogens from such wounds. Neutrophils are short-lived immune cells critical for host defense, yet E. faecalis-neutrophil interactions are poorly understood. Here, we show that instead of eliminating E. faecalis, neutrophils provide a niche for intracellular persistence and replication, potentially prolonging infection and inflammation at the wound site. In murine wound beds and ex vivo wound cells, intracellular E. faecalis was detected in recruited neutrophils at 24 h post-infection (h p.i). Unexpectedly, extended infection did not induce neutrophil death. Rather, E. faecalis infection significantly prolonged the life spans of both murine and human neutrophils in vitro compared to uninfected controls. Quantification of intracellular CFU revealed that E. faecalis were phagocytosed regardless of opsonization and persisted intracellularly up to 24 h p.i. This finding was confirmed via transmission electron microscopy and confocal microscopy. Blinded quantification and fluorescent D-amino acid staining, which marks newly synthesized bacterial peptidoglycan, revealed active replication within murine neutrophils between 6 and 18 h p.i., followed by a predominately persistent phase between 18 and 24 h p.i. Infected murine neutrophils remained immunologically active, secreting pro-inflammatory and chemoattractant cytokines. These findings highlight an underappreciated intracellular lifestyle for E. faecalis that may contribute to its ability to persist in chronic wounds and contribute to biofilm-associated infections.

Array-based biosensors hold substantial promise for rapid bacterial identification. However, conventional approaches face two key limitations: their reliance on nonspecific interactions with bacterial surfaces hinders biochemical interpretation, and their detection of bulk populations results in concentration-dependent responses that obscure distinctive bacterial fingerprints. Here, we present a novel, concentration-independent bacteria identification (CIBI) sensor array strategy that profiles the intrinsic biochemical characteristics of bacteria at the single-cell level to generate distinct fingerprints. The array consists of three sensing modules: two unnatural d-amino acid probes targeting different enzymatic pathways in peptidoglycan synthesis, and a phenylboronic acid probe recognizing surface polysaccharides. By measuring per-cell fluorescence from ∼10,000 individually interrogated bacteria rather than population responses, the system achieves concentration-independent profiling. Combined with machine learning, the CIBI strategy accurately identifies nine bacterial strains (105 CFU/mL) with 92.2% accuracy within 100 min. Remarkably, it also predicts the identity of previously unseen strains. In clinical applications, the array identified pathogen-spiked urinary tract infection samples with 95.2% accuracy, improving to 97.6% using a random forest algorithm. Overall, this strategy offers a robust platform for rapid and reliable clinical diagnostics.

Plant resistance to heat stress can be modelled by variation attributable to the genotype, environment, the rhizosphere microbiome, and their interactions. Using this Genotype × Environment × Rhizosphere Microbiome (GERMs) model, we studied three cereal genotypes: two inbred maize lines with contrasting heat sensitivity, and a sorghum inbred that displayed moderate heat tolerance. Plants were grown under optimal and heat stressed conditions across two soil treatments. We developed a systems-level metatranscriptomics approach to examine both plant and microbial transcriptomic profiles and integrated them with microbiome compositional data and plant phenotypes. We compared our strategy to amplicon profiling and found that our metatranscriptomic strategy offers greater functional and taxonomic resolution, allowing us to characterize active microbial pathways and analyze them jointly with plant gene expression profiles within a single system. We show that the microbiome functional profile is driven by host genotype and environmental factors and can enhance plant resilience. Our analyses identified plant genes and microbial pathways consistently associated with heat tolerance and key host–microbe interactions. Specifically, we identified D-amino acid metabolism as a plausible mechanism underlying a synergistic response to heat stress. These results demonstrate that the rhizosphere microbiome is not a passive component but an active participant in plant responses to abiotic stress. This work offers a new perspective on cereal adaptation to high temperatures and underscores the utility of the GERMs framework for dissecting functional relationships among plant genotype, environment, and the rhizosphere microbiome.

Hepatitis E virus (HEV) lacks approved virus-specific antiviral therapies, and off-label treatments with ribavirin and pegylated interferon are limited by toxicity and emerging resistance mutants. This study identifies reactive oxygen species (ROS) promotion mediated by the FDA-approved drug auranofin and D-amino acid oxidase as an effective antiviral strategy against multiple genotypes of HEV, including two globally relevant human-associated genotypes and a ribavirin treatment failure-associated HEV mutant. The observed synergistic anti-HEV activity in vitro for combined treatment with both auranofin and ribavirin suggests a potential clinically effective combinational therapeutic approach. ROS promotion through auranofin or other means represents an underexplored antiviral strategy with potential for broad-spectrum activity against a range of viral diseases.

An augmented strategy for constructing intelligent soft robots includes the transfer of biogenic features from nature to man-made artificial systems serving a range of life-like functions. Inspired by living technology, we have customized macroscale hydrogel boats by encoding them with an enzyme-powered engine that can convert chemical information into a mechanical response to create motion at the air-water interface. The engine's non-homogeneous enzyme distribution causes erratic motion along straight lines, random turns, and turns with high or low curvature-like trajectories. Nevertheless, the structural remodeling of the boat as well as the working system's configuration can permit directed, controlled, turning, bi-directional, rotation and run-and-tumble-like motion. Intriguingly, this boat is capable of sensing the precise chirality of amino acids (D-amino acid vs.L-amino acid) from individual isomer samples by translating the chiral information into variations in the boat's speed. Therefore, such miniaturized enzyme-powered boats are anticipated to be an advantage for the upcoming next-generation materials with a broader spectrum of functionalities.

BackgroudSecond-generation antipsychotics (SGAs) are widely used in the treatment of schizophrenia, however, growing concerns have emerged regarding their adverse effects on glucose and lipid metabolism. This study aimed to investigate the potential mechanisms underlying SGA-induced disturbances in glucose and lipid metabolism by integrating gut microbiota profiling with metabolomic analysis, thereby providing a scientific basis for future clinical interventions.MethodsA self-controlled before-after study was conducted on subjects who were initially medication-free (pre-medication group) and subsequently initiated on second-generation antipsychotics for 3 months (post-medication group). Anthropometric measurements—including waist circumference, hip circumference, body weight—as well as fasting blood samples (for assessment of glucose, insulin, C peptide, blood lipid) and stool samples were collected at baseline and after three months of treatment. Gut microbiota composition and fecal metabolomic profiles were analyzed using high-throughput sequencing and mass spectrometry–based approaches, respectively.ResultsFirstly, Gut microbial diversity differed significantly between groups. At genus level, the abundances of Escherichia and Bifidobacterium were increased significantly in the post-medication patients, while the abundances of Faecalibacterium and Blautia were decreased. Metabolomic analysis revealed decreased levels of oleamide and stearamide in the post-medication group, which exhibited a negative correlation with the abundance of Faecalibacterium. Additionally, the arginine and proline metabolic pathway, D-amino acid metabolic pathway, and arginine biosynthesis pathway were also altered in this group. Ornithine was identified as a key player in these three differential metabolic pathways.ConclusionIn summary, first-time exposure to second-generation antipsychotics in patients with schizophrenia is associated with disturbances in glucose and lipid metabolism, which appear to be closely linked to SGA-induced perturbations in gut microbiota composition and their associated metabolic profiles.

d-serine administration prevents kidney damage in murine models of acute kidney injury, and risperidone inhibits the activity of d-amino acid oxidase, which regulate plasma d-amino acid levels. This pilot randomized controlled trial investigated the effects of risperidone on glucose, amino acid metabolism, and kidney function in healthy adults. Healthy adults with a homeostasis model assessment of insulin resistance (HOMA-IR) of ≥ 1.6 and estimated glomerular filtration rate (eGFR) of ≥ 60 mL/min/1.73m2 were randomly assigned to the risperidone and control groups. The risperidone group received 0.5 mg/day risperidone for 4 days. The primary outcome was mean change in HOMA-IR on day 5, and the secondary outcomes were changes in d-amino acid levels, eGFR, and urinary albumin. Seven participants were randomized to the risperidone and control groups. The changes in HOMA-IR, eGFR, and urinary albumin on day 5 were not significantly different between the two groups (all p > 0.05). Mean changes in plasma d-serine level and urinary d-serine/creatinine ratio were significantly higher in the risperidone group than in the control group (0.2 vs. −0.3 nmol/mL, p = 0.03 and 38.2 vs. −25.8 nmol/mL, p = 0.01, respectively). Short-term risperidone affects d-serine metabolism without instigating acute adverse effects on kidney or glucose homeostasis in healthy individuals.Clinical Trial Registry number: This study was registered with the Japan Registry for Clinical Trials (jRCTs041210165).

Kinetic investigation on d-amino acid containing peptides and carboxypeptidase Y.

Site-specifically immobilized D-amino acid dehydrogenase for the synthesis of D-phenylalanine.

Development of a highly functional thermostable D-amino acid oxidase for efficient L-phosphinothricin biosynthesis using ProteinMPNN-guided engineering.

Back to basic: Using ammonium hydroxide to improve peptide epimer/isomer liquid chromatography separations.