Protein cages provide useful scaffolds for nanoscale engineering because of their highly ordered structures and in vivo self-assembly. These scaffolds are amenable to late-stage conjugation, enabling an expansion in functionality. However, many conjugation techniques either lack site selectivity, require unnatural amino acid incorporation, or have bulky recognition motifs to facilitate ligation reactions. Here, an asparaginyl endopeptidase (AEP) enzyme with ligase activity is employed for the highly efficient functionalization of virus-like particles (VLPs) from Enterobacteria phage P22. The capacity of this enzyme to conjugate peptides and proteins onto assembled P22 VLPs under mild reaction conditions via a minimal extension to the P22 coat protein C-terminus is demonstrated. We extend the reaction efficiency to facilitate a one-pot dual-functionalization reaction, in which two therapeutically relevant receptor-targeting domains are conjugated to P22 VLPs in a single step. Finally, we demonstrate the potential for AEP-mediated bioconjugation to bestow P22 VLPs with receptor-binding functionality in vitro. This work demonstrates the efficacy of AEP ligases as bioconjugation tools for the site-selective functionalization of large molecular assemblies such as VLPs.

Proteins and nucleic acids represent nature's most versatile biomolecules. By leveraging the functionality of proteins and the programmability of nucleic acids, along with the exquisite recognition properties of both, protein-nucleic acid conjugates can unlock hybrid systems that outperform either component alone. In this Viewpoint, we discuss emerging applications of protein-nucleic acid conjugates in biorecognition and therapeutics. As detection tools, these structures can identify molecules in complex media and single living cells with high selectivity or amplify signals to create assays with ultralow limits of detection. For therapeutics, they can be engineered to regulate genes in a tissue-selective manner, act as immunotherapies, or form the basis of new biomaterials with unique in vitro and in vivo properties. We end by focusing on the outstanding challenges that remain before the full potential of these structures is realized in revolutionizing diagnostics and medicine.

Peptide and peptoid are two types of peptidomimetics can be used as drugs for diseases. One of the main hallmarks in Alzheimer's Disease (AD) is Aβ aggregation. Due to the natural affinity between peptide and Aβ proteins, peptide therapeutics are found out to be suitable for the disease, as they are almost nontoxic, possess good biocompatibility and are structurally changeable. This review mainly focuses on peptide and peptoid as inhibitors targeting Aβ protein, elaborating on their design strategies and some of their inhibitory effects, providing diverse potential compounds for the treatment of AD. The involved peptide inhibitors are classified into three major categories based on their structural features: inhibitors based on the natural Aβ sequence, inhibitors based on non-natural Aβ sequences, and inhibitors conjugated with small molecule compounds. To facilitate understanding of the development process of each type of inhibitor, we have expounded on each major category in terms of chronological order, specialty and importance. It also summarized the challenges and design strategies faced by peptide inhibitors in targeting Aβ aggregation. This review provides a valuable resource for researchers in the field of synthetic peptide and their biomedical applications, offering structural insights into the design of new peptide for better diagnosis and therapeutic applications.

The recent development of Volumetric Imaging via Photochemical Sectioning (VIPS) has enabled nanoscale imaging of whole-mount tissue samples of virtually any size by embedding intact tissue in a photocleavable, superabsorbent hydrogel. However, the efficacy of sample embedding, imaging, and photochemical sectioning is fundamentally governed by the mechanical stiffness, structural stability, and photodegradation performance of the photocleavable hydrogel (PC-gel) polymer network. To elucidate the effect of the photosensitive cross-linker design on these critical properties, we synthesized a set of photocleavable cross-linkers (PCs) with varying polyethylene glycol (PEG) backbone lengths and prepared the corresponding PC-gels under a fixed monomer formulation and polymerization condition. We quantified and compared the viscoelastic properties of the formed PC-gels in their swollen states, and found that the cross-linker length markedly reshaped the PC-gel mechanics. In addition, we evaluated the light-triggered degradation of the PC-gels using both wide-field and spatially controlled illumination. We found that PC-1000, PC-1500, and PC-2000 gels remained comparably photodegradable, all enabling on-demand, spatially confined decross-linking under such illumination. These results provide practical guidelines for modulating the cross-linker architecture of PC-gel polymer networks to achieve optimal physicochemical properties for whole-mount tissue imaging using VIPS.

Nectin-4 is a validated target for theranostic approaches toward urothelial carcinoma, and radiolabeled ligands for PET and SPECT imaging are gaining increasing interest for noninvasive quantification of Nectin-4 prior to a targeted therapy. The goal of this study was to develop bicyclic peptides that allow radiolabeling with three different radionuclides (61Cu, 64Cu, and 68Ga) and to evaluate their suitability for high-contrast PET imaging in urothelial carcinoma (UC) and triple-negative breast cancer (TNBC). For this purpose, NECT-330, NECT-334, and NECT-346 were designed based on our previously developed ligand NECT-224 but harbor additional linker units between the bicyclic scaffold and the radiometal chelator. Binding kinetics to recombinant Nectin-4 and related isoforms were assessed by surface plasmon resonance (SPR) interaction analysis. Nectin-4-specific cell binding of the radiolabeled probes was determined using UC and TNBC cell lines, and PET/CT imaging was performed in respective tumor xenograft models. All novel ligands showed potent and selective binding to Nectin-4, with improved koff values compared to NECT-224. In small-animal PET/CT imaging studies, the most favorable properties were observed for [64Cu]Cu-NECT-334 (tumor-to-muscle of 20.8 and tumor-to-heart of 11.2) and [64Cu]Cu-NECT-346 (tumor-to-muscle of 15.5 and tumor-to-heart of 8.7) 1-2 h postinjection using a HT-1376 tumor model. Although the novel radioligands provided superior imaging properties to the previously developed radioligand [64Cu]Cu-NECT-224, a first-in-human application of the latter radioligand was initiated to assess the general suitability of PET/CT imaging in humans with a 64Cu-labeled bicyclic peptide directed to Nectin-4. Prospectively, the improved tumor-to-background contrasts achieved with the novel radioligands might further improve diagnostic accuracy and facilitate effective theranostic options in Nectin-4-expressing malignancies.

Despite significant research and investment, antibody-drug conjugates (ADCs) primarily rely on a limited number of broadly cytotoxic payloads that contribute significantly to toxicity. More recently, there has been a migration toward mechanistically targeted payloads, pairing improved antibodies with small molecules that act on disease-specific biology, improving precision and tolerability. Designing these next-generation payloads means rethinking some of our design metrics and guidelines derived from traditional medicinal chemistry: properties such as LogD, pKa, permeability, efflux, and metabolic stability must be carefully balanced to ensure efficient intracellular release while limiting systemic exposure. Here we discuss our observations around these properties and suggest design rules and guidelines for the optimization of next-generation ADC payloads.

The copper-catalyzed azide-alkyne cycloaddition reaction was applied to conjugate four human milk oligosaccharides (HMOs) with antimicrobial fluoroquinolones (FQs) (ciprofloxacin and sparfloxacin) to obtain a total of 12 per-O-acetylated and unprotected HMO-FQ conjugates. These compounds underwent antimicrobial testing against Gram-positive (Streptococcus agalactiae, Enterococcus faecalis, and Staphylococcus aureus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria. The HMO-FQ conjugates exhibited the most significant antimicrobial activity against E. coli and S. aureus reaching minimum inhibitory concentration as low as 1.56 μM and 3.13 μM, respectively. A general trend was that per-O-acetylated HMO-FQ conjugates exhibited higher antimicrobial activity than their unprotected counterparts. The antimicrobial activity of the tested HMO-FQ conjugates is expected to arise from the ciprofloxacin and sparfloxacin moieties because per-O-acetylated and unprotected HMOs did not exhibit any antimicrobial activity. Additionally, the HMO-FQ conjugates displayed no cytotoxicity as evident from test on two cell lines (HepG2 and MRC5).

Immunogenicity remains a major challenge in antibody therapeutics. Antibodies can be recognized as foreign by immune cells, leading to the induction of antidrug antibodies that ultimately compromise therapeutic efficacy. This response is primarily driven by proteolytic processing within antigen-presenting cells and the subsequent presentation of peptide fragments to T cells via major histocompatibility complex molecules. As mirror-image proteins are expected to exhibit resistance to proteolytic degradation, we have been developing mirror-image VHH antibodies composed entirely of d-amino acids. Previously, we generated a d-VHH antibody with specific binding activity to vascular endothelial growth factor A (VEGF-A) and demonstrated that it did not induce antidrug IgG antibodies in a mouse model. However, this d-VHH antibody did not inhibit the VEGF-VEGF receptor interaction, and the immunological processes leading to the lack of IgG antibody induction had not been experimentally demonstrated. In this study, we constructed a new phage-displayed library to improve the hit rate and enable the efficient chemical synthesis of d-VHH antibodies. Using this library, mirror-image screening against d-VEGF identified new VHH antibody sequences with specific binding. The synthesized d-VHH antibody inhibited the VEGF-VEGF receptor interaction. Next, we investigated the immunogenicity of d-VHH antibody using a conventional l-VHH antibody as a reference. The d-VHH antibody resisted enzymatic degradation and, in a mouse model, did not induce inflammatory cytokine production, T-cell responses, or the formation of antidrug antibodies against the administered antibody. Collectively, these findings highlight the potential of inhibitory d-VHH antibodies as a promising approach for developing low-immunogenic therapeutic antibodies.

Peptide-functionalized alginate hydrogels are a promising biomaterial candidate for supporting the expansion of adherent human mesenchymal stem cells (hMSCs) toward therapeutic applications. Despite widespread RGD-modified alginates, critical aspects of peptide conjugation chemistry, including verification of the coupling orientation and role of amino acids flanking the core RGD motif, remain underexplored. Through systematic material characterization and peptide sequence design, this work addresses these important research gaps. We first thoroughly characterized the alginate backbone composition via 1H NMR spectroscopy using peak deconvolution, providing more distinct quantitation of alginate backbone composition and conjugation sites. We then coupled 4 peptide sequences systematically designed to probe the effect of the spacer N-terminal to RGD, either glycine-glycine-phenylalanine-threonine (GGFT) or cysteine-glycine-phenylalanine-threonine (CGFT), and the presence or absence of a C-terminal serine-proline (SP) motif. As a spacer, we capitalized on using phenylalanine (F), as opposed to the previously published tyrosine (Y), to mitigate side reactions with the phenolic hydroxyl group while retaining aromatic protons (δ ∼7.2-7.4 ppm) for a quantitative degree of substitution and coupling efficiency characterization via 1H NMR. Further included in our design was polar threonine (T), to enhance the aqueous solubility of the overall peptide spacer. Critically, we provided evidence (previously only assumed) that amide bond formation occurred through the peptide N-terminus as intended (versus the unwanted side reaction of coupling the guanidinium group of arginine (R) to alginate's carboxyl) using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) coupling. The attachment, spreading, and proliferation of hMSCs to hydrogel films of this well-characterized alginate series (cross-linked with calcium chloride) were then examined to understand the role of peptide sequence on performance. Alginates functionalized with GGFTRGD or CGFTRGD failed to support hMSC expansion, whereas inclusion of the C-terminal SP sequence (yielding GGFTRGDSP or CGFTRGDSP) promoted robust cell attachment and a >10-fold increase in the final cell number, demonstrating the essential role of SP immediately C-terminal to RGD, which more closely resembles the sequence of native fibronectin. Together, the characterization methods and sequence/activity relationships established herein provide a foundation for the rational design of well-characterized and reproducible peptide-alginate hydrogels for scalable hMSC expansion and therapeutic applications.

Radiotherapy (RT), a cornerstone of modern oncology, is often hindered by the hypoxic tumor microenvironment (TME), inherent radioresistance, and collateral damage to surrounding healthy tissues. To circumvent these limitations, we developed a biomimetic, interfacially engineered nanoplatform (CMHHND) designed for precision synergistic therapy. The nanoplatform features hafnium-doped hollow Prussian blue (HHPB) nanocatalysts as a functional core, which are loaded with nuclear-targeted doxorubicin and encapsulated within a homologous cancer cell membrane (CM) shell. This macromolecular camouflage grants the system exceptional colloidal stability and superior homotypic targeting capabilities through interfacial molecular recognition. Mechanistically, CMHHND functions as a multimodal agent: it acts as a photothermal agent, a dual-enzyme mimic with catalase (CAT)- and peroxidase (POD)-like activities to modulate the TME, and a radiosensitizer leveraging the high atomic number (Z) of Hf to enhance X-ray deposition. Systematic in vivo experiments confirmed that CMHHND-mediated synergistic therapy (RT/PTT/Chemo) effectively eradicated 4T1 tumors with no apparent systemic toxicity. Our findings highlight that integrating biomimetic interfacial engineering with multifunctional nanocatalysts provides a potent strategy to reverse hypoxia-associated radioresistance, offering a robust paradigm for the design of advanced colloidal nanomedicines in precision cancer therapy.

Lipid A, the hydrophobic anchor of lipopolysaccharide in the outer membrane of Gram-negative bacteria, serves as its principal immunostimulatory center. Variations in acyl chain numbers and lengths, phosphorylation status, and substituent groups directly influence the immunological responses elicited by this molecule. A representative example is monophosphoryl lipid A (MPLA), a dephosphorylated derivative of lipid A. This 1-O-dephosphorylated variant largely preserves the immunostimulatory strength of lipid A while showing substantially lower toxicity─a property that has made it clinically successful as a vaccine adjuvant. The synthesis of lipid A and its derivatives has attracted extensive attention from synthetic chemists. Understanding the structure-activity relationships (SARs) of these molecules has therefore remained a central goal in the field. However, systematic SAR work and the creation of structurally defined variants depend on reliable synthetic access, which continues to pose significant difficulties. In particular, the stereoselective assembly of the diglucosamine core and the precise installation of diverse acyl and phosphate groups present substantial synthetic hurdles. While numerous synthetic routes have emerged for lipid A and its derivatives over the past few decades, a general and effective method for assembling the diglucosamine core remains to be established. This review systematically summarizes these synthetic advances, with a particular emphasis on rational design and structural considerations. We highlight strategies and concepts developed to address key chemoselective, stereoselective, and enantioselective challenges during assembly of the characteristic bisphosphorylated β-(1→6)-linked diglucosamine backbone. Finally, we outline the fundamental relationships between unique molecular conformations of lipid A and their resulting biological activities. This review provides theoretical guidance for designing rational synthetic routes of novel lipid A and its derivatives MPLA, selectively regulating stereoselectivity issues, and the development of targeted, more effective immunotherapies and vaccine adjuvants.

When trihydroxypiperidine (PIP) or DAB-1 pyrrolidine (PYR) iminosugars are multimerized on the prism[5]arene scaffold, they generate highly efficient multivalent inhibitors of GCase. Binding trihydroxypiperidine to the rims of prism[5]arene (PrS[5]PIP) results in a multivalent derivative that is more effective than others reported in the literature with the same bioactive unit. This efficacy is attributed to the role of prism[5]arene, whose barrel-like shape exposes an external π-surface that enables van der Waals contacts and other secondary interactions with the enzyme, thereby enhancing affinity for GCase through dimer stabilization. The high efficiency of PrS[5]PIP is supported by in silico calculations that showed how the crowding of the bioactive units on the rims of the macrocycle promotes a rearrangement allowing the active site to accommodate two trihydroxypiperidine units, which engage in hydrogen bonding with the catalytically active Glu residues. Due to their inhibitory potency, these prismarene derivatives have been tested as pharmacological chaperones in intact cell lines from Gaucher patients carrying different mutations. The results indicated that the PrS[5]PIP is more effective toward the N370S mutation, while the DAB-1 pyrrolidine iminosugar PrS[5]PYR unexpectedly enhances L444P mutant GCase activity. This suggests that the type of iminosugar linked to the prismarene scaffold influences GCase activity rescue in a mutation-dependent manner and opens the way for the future development of increasingly efficient pharmacological chaperones.

The aberrant self-assembly of amyloid β (Aβ) peptides into toxic oligomers is a central event in Alzheimer's disease, motivating the development of molecular systems capable of modulating or degrading these species. Porphyrins and porphyrin-peptide bioconjugates have emerged as promising photoactive platforms due to their known ability to generate the highly oxidant singlet oxygen (1O2) upon light excitation. Still, the mechanisms underlying their oxidative effects on Aβ remain unclear. Here, we investigate the photophysical properties and the 1O2-mediated oxidation capability of two porphyrin-peptide conjugates, Ph-Pep and its Zn-metallated analogue ZnPh-Pep, both incorporating the Aβ recognition motif KLVFF. Steady-state and time-resolved spectroscopic studies reveal the good preservation of the photophysical and photochemical properties of the porphyrin chromophore units after their conjugation with the peptide appendage. Population of the long-lived triplet state of both compounds under visible-light excitation results in an effective generation of 1O2. High-resolution mass spectrometry shows that the 1O2 photogenerated by ZnPh-Pep induces extensive oxidation, yielding peroxidic and hydroperoxidic species, and provides evidence of partial Aβ42 degradation. In contrast, Ph-Pep leads to a rapid loss of detectable Aβ signal, suggesting the formation of higher-order aggregates rather than peptide cleavage. These distinct outcomes highlight the roles of Zn2+ coordination and peptide conjugation in modulating the 1O2-driven pathways. Overall, this study provides a mechanistic insight into designing photoactivatable porphyrin-based constructs capable of degrading or modulating toxic Aβ assemblies, opening intriguing prospects for light-mediated therapeutic strategies for neurodegenerative diseases.

Despite its potent anti-aging efficacy, the practical application of retinol (ROH) remains highly restricted by its poor photostability, low aqueous solubility, and concentration-dependent cytotoxicity. Herein, we report the synthesis of a hyaluronic acid-retinol (HA-ROH) conjugate to overcome these limitations and improve transdermal delivery. The successful conjugation was confirmed via proton nuclear magnetic resonance (1H NMR) spectroscopy, high-performance liquid chromatography (HPLC) and Fourier-transform infrared spectroscopy (FT-IR). Notably, the HA-ROH conjugate exhibited remarkable photostability under UVA irradiation compared to free retinol. HA-ROH presented a sustained, hyaluronidase-responsive ROH release profile, indicating its potential as an enzyme-responsive controlled delivery system. Furthermore, the conjugation significantly mitigated the cytotoxicity of retinol in both HaCaT keratinocytes and human dermal fibroblasts. The conjugate suppressed matrix metalloproteinase-1 (MMP-1) expression more effectively than free ROH. In vitro cellular uptake and ex vivo skin permeation studies demonstrated enhanced transdermal delivery of the HA-ROH conjugate. Taken together, these results suggest that the HA-ROH conjugate is a highly promising, stable, and safe candidate for advanced dermatologic and cosmetic applications.

Glycosylated self-assembling molecules enable the construction of nanostructures whose surfaces are decorated with saccharides under aqueous conditions, reminiscent of biological systems at both the molecular and cellular levels. Such glyco-nanostructures thus hold great promise as soft materials for biological applications. Herein, we report the facile synthesis of glycosylated diarylmethyloxy-triazines, which serve as new building blocks for fabricating self-assembled nanostructures in aqueous media and forming supramolecular hydrogels. The triazine scaffold enables the protecting-group-free synthesis of glycosylated compounds with reasonable β-anomer selectivity. Notably, both the substituents on the aryl groups attached to the triazine core and the geometry of the glycosidic linkages in the disaccharides strongly influence the hydrogelation behavior.