Abstract 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.
Abstract 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.
Hydroxypyridyl-substituted 1,2,4,5-tetrazines have recently been developed as advanced scissors for bioorthogonal click-to-release chemistry. In contrast to the otherwise characteristic pink color of 1,2,4,5-tetrazines, these compounds were observed to give yellow solutions in methanol and buffered aqueous media. This study shows that the solvatochromism of hydroxypyridyl-tetrazines has its origin in varying protonation states of the phenolic OH group and reveals the impact of deprotonation on bioorthogonal click reactivity. We demonstrate that the rate of the Diels-Alder cycloaddition is significantly affected by the pH of the aqueous solution, thereby providing critical insights for the development of molecular tools for bioorthogonal bond cleavage.
Ovarian cancer remains a lethal malignancy due to chemoresistance and toxicity, which limits the dose of chemotherapy that can be used, necessitating the development of more targeted therapies such as antibody-drug conjugates (ADCs). However, conventional ADCs suffer from heterogeneity. This study aimed to develop a stable, homogeneous ADC by utilizing a bifunctional dibromomaleimide (DBM) linker to cross-link antibody cysteine residues via disulfide-bridging. A DBM linker was synthesized from 3,4-dibromofuran-2,5-dione and bound to the cytotoxic agent triptolide. This triptolide payload was then conjugated to trastuzumab via site-specific disulfide rebridging to yield a homogeneous trastuzumab-triptolide conjugate to target human epithelial growth factor receptor 2 (HER2) on ovarian cancer cells. The study evaluated the ADC's efficacy against SKOV-3 (high HER2 expression) and OVCAR-8 (low HER2 expression) cell lines. The results showed that the ADC was slightly more potent in SKOV-3 cells, yielding lower IC50 values compared to OVCAR-8. Mechanistic studies of the ADC via flow cytometry revealed that the ADC induced significant apoptosis and cell cycle arrest, characterized by a concentration-dependent increase in Caspase-3/7 expression and distinct alterations in cell population distribution. Furthermore, ADC treatment led to a concentration-dependent decrease in HER2 levels in SKOV-3 cells, confirming successful targeting. The study demonstrates that converting conventional maleimides into bifunctional DBM linkers allows the production of a homogeneous ADC via disulfide-bridging. This approach offers a promising strategy for developing potent anticancer therapeutics with improved selectivity for HER2-overexpressing ovarian cancers.
Abstract 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.
5-Fluorouracil (5-FU) is a chemotherapeutic drug that is widely used to treat gastrointestinal cancers (e.g., hepatocellular carcinoma). Unfortunately, its systemic toxicity limits its clinical utility; therefore, new means for its targeted delivery at the pathological site are highly sought after to enhance therapeutic efficacy. In this work, we describe a 5-FU covalent conjugate with the self-assembling, heterochiral tripeptide DLeu-Phe-Phe (lFF) via a redox-sensitive linker (5-FU-SS-lFF). The conjugate yields supramolecular hydrogels with rheological properties similar to those of lFF hydrogels and enables 5-FU release under reducing conditions, such as those present in the tumor microenvironment. We demonstrate the selectivity of the drug release under such conditions over a period of 24 h, with consequent significant cytotoxicity on cancer cells, as demonstrated on hepatocellular carcinoma spheroids. Overall, this study opens new opportunities for peptide-based supramolecular hydrogels as versatile and smart vehicles for the selective release of anticancer drugs under tumor-like reducing conditions.
Abstract Escherichia coli-derived l-asparaginase (EcA) is a crucial frontline therapy for the treatment of acute lymphoblastic leukemia (ALL), yet its use in clinical settings remains considerably compromised due to its immunogenicity, hypersensitivity reactions, hepatotoxicity, rapid proteolytic degradation, and a markedly short plasma half-life. However, given the clinical benefits of l-asparaginase, an improved variant of it, combined with a biocompatible immobilizing agent, can plausibly circumvent the drawbacks mentioned above. Herein, this study, in order to mitigate these limitations, first, we rationally developed a less immunogenic variant of l-asparaginase, namely, KHY-17-EcA, by a site-directed mutagenesis approach, and then we utilized biocompatible carboxyl-functionalized multiwalled carbon nanotubes (MWCNTs) to covalently immobilize the KHY-17-EcA variant along with the WT-EcA. The resulting nanobiocatalysts (MWCNT-WT-EcA and MWCNT-KHY-17-EcA) exhibited a 96% enzyme-immobilizing capacity. Interestingly, these two immobilized enzymes exhibited excellent thermal and serum stability as well as superior catalytic activity across a wide range of pH and temperature conditions compared to their free forms. Notably, both immobilized enzymes retained almost 90% of their original activity after 4 weeks of storage and exhibited complete resistance to proteolytic degradation by asparaginyl endopeptidase and cathepsin B after 24 h of incubation. Furthermore, the immobilized KHY-17-EcA variant exhibited significantly reduced glutaminase activity, a low Km of 0.312 mM, and high substrate specificity, along with the potent cytotoxic activity against human leukemia cell lines (Jurkat, MOLT-4, K562), while sparing noncancerous human bronchial epithelial cells (HBEC-5i). Apoptosis analysis of treated leukemia cells revealed marked nuclear morphological alterations and a significant increase in the level of apoptotic cell death. These findings collectively demonstrate that the newly synthesized MWCNT-KHY-17-EcA nanobiocatalyst has the potential to be utilized in leukemia treatment. Taken altogether, these results highlight the efficacy of MWCNT-based immobilization as a robust strategy to prolong enzyme shelf life, confer resistance to proteolytic degradation, and potentiate biological functionality, thereby broadening its translational scope in both biotechnology and cancer therapeutics.
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 toxicitya 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.
Elucidating protein-protein interactions plays a crucial part in understanding disease mechanisms and advancing pharmacological research. Photocatalytic proximity labeling using antibody-catalyst conjugates enables the highly target-specific analysis of protein-protein interactions on the cell surface without altering the native cellular state through genetic manipulation. Here, we describe an extension of the deazaflavin-diazirine energy-transfer (DarT) labeling platform through the development and evaluation of trastuzumab-deazaflavin (Tra-dFl) conjugates for mapping the extracellular microenvironment of human epidermal growth factor receptor 2 (HER2). Four Tra-dFl conjugates were synthesized via azide-DBCO click chemistry, varying in PEG linker size and catalyst loading: Tra-PEG0-dFl, Tra-PEG6-dFl, Tra-PEG12-dFl, and Tra-bis-dFl, exhibiting a branched linker for dual attachment. Imaging and proteomic pulldown experiments revealed that linker size influences biotinylation efficiency and proteomic enrichment, resulting in Tra-PEG12-dFl emerging as the most effective construct, enabling the enrichment of cancer-associated cell surface proteins in HER2-positive SK-BR-3 cells. Evaluation of catalyst valency using a branched linker resulted in fewer enriched proteins, suggesting that linker architecture is a more critical parameter for conjugate performance than increased catalyst loading. Together, these findings provide guidelines for antibody-based deazaflavin conjugates and expand the applicability of DarT labeling for target-directed surfaceome mapping.
There is an unmet need to develop vehicles for delivering protein cargo and ribonucleoprotein complexes to cells. Virus capsids, or virus-like particles, have numerous advantages as self-assembling protein platforms, but there remains a need to develop robust methods for the specific internal packaging of cargo. In this work, we use the Hepatitis B Virus (HBV) capsid as our delivery vehicle. To specifically associate protein cargo with the capsid, we covalently attached a derivative of an HBV sulfamoylbenzamide (SBA) antiviral to green fluorescent protein (GFP). We show that SBA-tagged GFP can associate with HBV capsids, but efficient internal packaging requires engineering the assembly of capsids around a cargo. Under standard co-assembly conditions, SBA-tagged GFP predominantly displayed on the capsid exterior, reaching ∼40 GFP cargo proteins per capsid. We tested two strategies to drive encapsidation of protein cargo. First, remodeling the capsid interior was nominally effective. Conversely, His-tag/Ni2+-mediated clustering converts the cargo into a multivalent capsid-binding scaffold that templates assembly, enabling internal packaging of up to ∼30 cargo proteins per capsid. Based on capsid geometry, we suggest the practical limit is approximately 30 cargo proteins per T = 4 HBV capsid, indicating that this strategy approaches maximal loading. Together, these results provide a highly effective method for packaging proteins inside VLPs.
This study investigates how the structural design of secondary PNA probes affects their ability to carry the cytotoxic agent DM1 in a novel affibody-PNA-based pretargeting approach directed at Human Epidermal growth factor Receptor 2 (HER2)-expressing cells. Six different DM1-conjugated secondary 8-mer PNA probes with strategic hydrophilic modifications were designed and evaluated. Surface plasmon resonance analysis confirmed that the modifications preserved hybridization to the HER2-targeting affibody-PNA primary probe, with all secondary probes showing similar binding kinetics (KD = 310-660 pM). Biodistribution studies in nontumor-bearing NMRI mice with lutetium-177 (177Lu)-labeled secondary probes revealed that probe modifications dramatically influenced organ distribution patterns. The secondary probes SP2, SP5, and SPc demonstrated favorable biodistribution profiles with reduced accumulation in critical organs, i.e., liver and kidneys. Receptor-mediated endocytosis was also affected by secondary probe design, showing an increase in cellular internalization for SP5 (20.4% after 24 h) compared to SP2 (12.1%). Our findings highlight how rational design can optimize in vivo pharmacokinetics while preserving binding properties essential for effective pretargeting, providing a foundation for developing novel targeted cytotoxic delivery systems with potentially improved therapeutic indexes.
DNA-encoded library (DEL) technology has emerged as a powerful platform for small-molecule discovery, in which on-DNA reaction development plays a central role in determining accessible chemical space. Early on-DNA chemistry mainly focused on establishing robust DNA-compatible transformations under mild aqueous conditions but often generated structurally limited libraries. Recent advances in photochemistry, electrochemistry, biocatalysis, and complexity-generating reactions have substantially expanded the scope of accessible on-DNA transformations and enabled the incorporation of increasingly diverse and medicinally relevant scaffolds into DELs. In this Viewpoint, we discuss the recent progress and emerging trends in on-DNA reaction development, with particular emphasis on the transition from compatibility-driven chemistry toward function-oriented DEL synthesis. We further highlight current challenges and future opportunities for developing precision on-DNA chemistry to support next-generation ligand discovery.
As a highly symmetrical and endogenous protein-based drug delivery system, ferritin nanocage has garnered tremendous attention in nanomedicine due to its exceptional biocompatibility, dynamic self-assembly behavior, and intrinsic receptor-mediated targeting capabilities. With the rapid evolution of bioconjugation chemistry and protein engineering, the functional manipulation of ferritin has transitioned from passive physical encapsulation to precise, site-specific engineering of ferritin architectures. This Viewpoint systematically highlights the multidimensional bioconjugation strategies of ferritin and its relatives. We specifically focus on the biochemical principles driving genetic fusion engineering, classic covalent chemical conjugation, modular bioorthogonal assembly, and the in situ construction of catalytic centers within the inner cavity. These chemistries are not merely incremental refinements. They unlock entirely new therapeutic and diagnostic capabilities, including single-dose tumor-lethal chemotherapy, high-sensitivity multimodal/nuclear imaging, targeted protein degradation, ultrapure nanovaccines, and inflammation intervention. This Viewpoint aims to provide a chemically grounded and forward-looking roadmap for the next generation of ferritin engineering.
With the increasing cancer incidence and death tolls worldwide, antibody-drug conjugates (ADCs) have emerged as a promising platform to selectively deliver cytotoxic payloads to cancerous tissues. This platform has the potential to spare patients from off-target effects compared to traditional chemotherapy. Despite the clinical success of some ADCs, many in the pipeline did not even reach the clinical stage. We turn our attention to their linker chemistry that connects the antibody and the drug molecules together. Despite their small size, linkers play a key role in governing the payload release kinetics and location. In this Viewpoint, we collect and discuss a plethora of linkers employed in ADC development, specifically focusing on advancements in antibody modification and linker cleavage chemistries. The choice of linker chemistry has evolved over time, going from stochastically attached non-stimuli-responsive to site-specifically modified cleavable linkers. Then we correlate the linker technologies to their pharmacokinetic outcomes in clinics, followed by a discussion of the upcoming "linker-free" technology for more facile and efficient antibody-conjugated targeted delivery.
To improve the theranostic efficacy of PSMA-targeted radiopharmaceuticals on patients with medium-to-low PSMA expression, we radiolabeled a series of novel designed PSMA-targeting dimer agents to improve tumor imaging and treatment outcomes under challenging conditions. All agents were successfully radiolabeled with 68Ga in moderate decay-corrected labeling yield and high radiochemical purity. Among them, [68Ga]Ga-TVS-PSMA-1 exhibited high tumor uptake in both LNCaP and 22RV1 models. [177Lu]Lu-TVS-PSMA-1 demonstrated significantly improved treatment efficacy with a lower dose and good safety profile compared with [177Lu]Lu-PSMA-617. A first-in-human [68Ga]Ga-TVS-PSMA-1 PET/CT scan was performed in a 66-year-old male prostate cancer patient. Two metastatic lesions in the lumbar vertebrae were clearly visualized, and the primary tumor exhibited an SUVmax of 30.3. The [68Ga]Ga-TVS-PSMA-1 showed lower salivary gland uptake compared with previously reported values for [68Ga]Ga-PSMA-11; however, confirmation in larger patient cohorts is required. This study demonstrates that the dimer agent TVS-PSMA-1 holds great potential as a next-generation theranostic agent for prostate cancer.
Influenza A virus is a medically important target. Hemagglutinin (HA), a trimeric glycoprotein on the viral surface, recognizes sialylated glycans through three receptor-binding sites. Glycoligands that interact with HA through multivalent binding can serve as viral inhibitors. To achieve efficient multivalent binding, precise control over glycoligand structures is essential. Here, we synthesized glycooligomers displaying sialyllactose units at defined intramolecular spacings and investigated how ligand structure affects binding to HA. Glycooligomers displaying one, two, or three sialyllactose units at regular intervals exhibited enhanced binding as carbohydrate valency increased. When the spacing between two sialyllactose units was varied, shorter spacing enhanced binding, likely because of a statistical rebinding effect. Interestingly, the presentation of an excessive number of sialyllactose units weakened the interaction with HA, presumably because of steric hindrance. These findings highlight the importance of precise carbohydrate arrangement in the design of effective multivalent ligands.
Polymer conjugation is a widely used strategy to improve the pharmacokinetics of protein therapeutics by extending their half-life and protecting them from degradation. Poly(ethylene glycol) (PEG) is the most commonly used polymer for this purpose. However, its use can trigger unwanted immune responses, leading to the production of anti-PEG antibodies that compromise efficacy. This issue has driven the search for alternative polymers, with poly(sarcosine) (PSAR), derived from an endogenous amino acid, emerging as a promising candidate. Despite this potential, the immunogenicity of PSAR-protein conjugates (PSARylation) has not been systematically compared with that of PEGylated counterparts. In this report, we show that conjugating a model protein antigen to high-molecular-weight PSAR more effectively suppresses immune responses against both the protein and the polymer than PEG. Although polymer conjugation has traditionally been viewed as a means of steric "masking" of a protein, our results demonstrate that the chemical nature of the polymer is a critical, independent factor, as PSAR conjugates elicited lower antibody production than PEG conjugates of identical hydrodynamic size. This study highlights polymer selection as an important design parameter for minimizing immunogenicity in next-generation protein therapeutics. Polymers derived from biological building blocks, such as PSAR, offer a promising route to developing safer, more effective biopharmaceuticals with better tolerance and reduced risk of adverse immune reactions.
The folate receptors (FR) are highly overexpressed in various solid tumors and metastatic cancers, whereas they are low or negligible in healthy cells. This provides a window of opportunity to deliver any payload by specifically targeting these receptors. This study develops a delivery strategy for exatecan (payload), a Top1 inhibitor, via the Fol-SS-Exa (8) conjugate, demonstrating its potency against FR-positive cancer cells in vitro and in preclinical studies. The design of the Fol-SS-Exa (8) conjugate features folate as a ligand targeting FRα and a cleavable disulfide linker that remains stable under physiological conditions. Exatecan (6), a highly potent cytotoxic payload, arrests cell division in cancer cells. Fol-SS-Exa (8) exhibited an IC50 value of 4.88 nM in the FR-positive MDA-MB-231 cell line. The Fol-SS-Exa conjugate (8), administered to tumor-induced C57BL/6 mice, exhibited complete regression after three injections (7.5 mg/kg body weight), highlighting its potent antitumor activity. In addition, the conjugate (8) demonstrated the ability to eradicate the toxicity of exatecan (6) when administered alone.
Although DNA-functionalized hydrogels have been widely explored for sensing, controlled release, and smart materials, the potential for strong, noncovalent recognition between DNA and hydrated polymer networks remains largely unexplored. Here, we report the selection of DNA aptamers that specifically bind agarose hydrogels. Using a structured DNA library and agarose beads as the target, a dominant guanine-rich sequence, Agar-1, emerged after 11 rounds of selection. Quantitative PCR and fluorescence assays confirmed that the enriched sequences bind agarose substantially more strongly than a random DNA library. Truncation yielded a 42-nucleotide aptamer that retained binding activity, whereas further truncation that preserved only the guanine-rich region abolished binding, indicating a strict structural requirement. Notably, binding required Mg2+ and was inhibited by K+, suggesting a non-G-quadruplex recognition mechanism. In contrast to previously reported C/T-rich sequences that bind microplastics, the G-rich agarose aptamers highlight the versatility of DNA-polymer interactions and demonstrate how simple changes in sequence composition can drive recognition of distinct materials. These findings establish the feasibility of evolving aptamers against hydrogels and provide a foundation for engineering programmable DNA-hydrogel interfaces for biosensing, responsive materials, and controlled-release applications.
