Bioconjugate Chemistry最新文献

We report the chemical conjugation of a recombinant Adeno Associated Virus (rAAV) capsid with various functionalities, including proteins, using a bioorthogonal strategy. rAAVs were azido-coated or dibenzylcyclooctyne (DBCO)-coated by chemically modifying lysine or tyrosine residues. Lysine residues were modified using a phenyl isothiocyanate anchor, and tyrosine residues using either an aryl diazonium salt or a N-methyl luminol derivative. We demonstrate anchor-dependent labeling levels, as observed with biochemical assays and mass spectrometry. Strain-promoted azide–alkyne cycloaddition (SPAAC) was then implemented and evaluated on the rAAV to append functionalities such as fluorescein, biotin, and carbohydrates to the azido-coated capsids. We confirmed the efficiency of the bioorthogonal reaction and observed a stronger reactivity with dibenzylcyclooctyne (DBCO) compared to bicyclononyne (BCN). The optimized SPAAC reaction was finally used to label the viral vectors with two relevant nanobodies targeting specific immune cell receptors (CD62L and CD45). In vitro transduction assays conducted with one rAAV-nanobody conjugate demonstrated the promising targeting properties of these chemically modified vectors. Thus, we anticipate that this strategy will positively impact the field of rAAV capsid engineering and contribute in tissue-specific targeting for the optimization of gene therapy treatments.

Lysosomal enzyme replacement therapy (ERT) holds potential for treating lysosomal storage disorders, but achieving targeted delivery of deficient therapeutic enzymes remains a significant challenge. This study presents a novel approach for the lysosome-specific delivery of the β-glucosidase (B8CYA8) enzyme by covalently conjugating lysosome-targeting mannose-6-phosphate functionalized glycopolypeptides (M6P-GP). We used a protein-glycopolypeptide conjugate developed through advanced protein engineering and bioconjugation techniques. By conjugating β-glucosidase to M6P-GP that has a high affinity for the cation-independent mannose-6-phosphate receptors (CI-MPR) and lysosomal receptors, we enhance the enzyme’s selective intracellular uptake and lysosome-specific localization. To attain maximum activity of the near-native enzyme after delivery, we have designed and synthesized an acetal linkage containing the pH-responsive linker maleimide-acetal-azide (MAA), which will cleave in the lysosomal acidic pH to detach the glycopolypeptide from the protein backbone. We demonstrated the efficient cellular uptake of the protein-glycopolypeptide conjugate and showed targeted lysosome delivery, leading to increased enzymatic activity compared to untreated cells. Our results proved that the approach mainly improves the specificity and efficiency of enzyme delivery, particularly into lysosomes, which may enable new methods for ERT. These findings suggest that protein-glycopolypeptide conjugates could represent a class of bioconjugates to design targeted enzyme therapies, offering a pathway to the effective treatment of Gaucher disease (GD) and potentially other related lysosomal storage disorders.

A reliable method for the efficient chemical synthesis and poly(ethylene glycol) PEG-like modification of fluorogenic RNA aptamers is reported. The 43-mer version of Mango-II RNA (MangoII-v1), which binds tightly and specifically to the green fluorophore TO1-Biotin (TO1-B), was synthesized by automated phosphoramidite chemistry using 2′-O-[(triisopropylsilyl)oxy]methyl] (2′-O-TOM)-protected ribonucleosides. Solid-phase phosphoramidite chemistry was also used as a single tool to prepare MangoII-v1 modified with a PEG-like oligophosphate synthetic segment (MangoII-v1-P). After cleavage from the resin, deprotection, and purification, the capacity to activate the fluorescence of TO1-B and the degradation behavior of the chemically synthesized RNAs MangoII-v1 and MangoII-v1-P, were deeply investigated in comparison with those of the enzymatically synthesized 48 nucleotides long RNA MangoII. Interestingly, the chemically synthesized MangoII-v1 RNA aptamer demonstrated great activity toward its target, compared to the enzymatically synthesized analogue. Moreover, it was found to be highly stable, retaining its structural integrity and bioactivity, even after seven days of incubation in 20% fetal bovine serum. MangoII-v1-P also showed a high affinity for TO1-B and excellent degradation resistance.

Cardiovascular diseases remain the leading cause of mortality worldwide. Specifically, atherosclerosis is a primary cause of acute cardiac events. However, current therapies mainly focus on lipid-lowering versus addressing the underlying inflammatory response that leads to its development and progression. Nanoparticle-mediated drug delivery offers a promising approach for targeting and regulating these inflammatory responses. In atherosclerotic lesions, inflammatory cascades result in increased T helper (Th) 1 and Th17 activity and reduced T regulatory activation. The regulation of T cell responses is critical in preventing the inflammatory imbalance in atherosclerosis, making them a key therapeutic target for nanotherapy to achieve precise atherosclerosis treatment. By functionalizing nanoparticles with targeting modalities, therapeutic agents can be delivered specifically to immune cells in atherosclerotic lesions. In this Review, we outline the role of T cells in atherosclerosis, examine current nanotherapeutic strategies for targeting T cells and modulating their differentiation, and provide perspectives for the development of nanoparticles specifically tailored to target T cells for the treatment of atherosclerosis.

Malignant glioma highly expresses carbonic anhydrase IX (CAIX). This study aimed to develop [¹⁷⁷Lu]Lu-XYIMSR-01, a small-molecule therapeutic agent CAIX, to assess its potential for treating malignant glioma. [¹⁷⁷Lu]Lu-XYIMSR-01 was synthesized by radiolabeling DOTA-XYIMSR-01 with ¹⁷⁷Lu. In vitro assays were conducted to evaluate the affinity for U87MG tumor cells. The probe was injected via the tail vein into subcutaneous and orthotopic U87MG models for micro-SPECT/CT imaging. The survival rates of tumor-bearing mice were assessed after [¹⁷⁷Lu]Lu-XYIMSR-01 injection by intratumoral in orthotopic models, including untreated controls and those treated with Temozolomide or combination therapy. After purification, the radiochemical yield of [¹⁷⁷Lu]Lu-XYIMSR-01 was 86.47 ± 2.42%, with a radiochemical purity (RCP) of 99%. Its cell uptake in U87MG cells was 3.70 ± 0.57 ‰ AD/10⁵ cells, significantly higher than that in HCT116 cells (0.68 ± 0.16 ‰ AD/10⁵ cells, P = 0.001). In the biodistribution study, [¹⁷⁷Lu]Lu-XYIMSR-01 uptake in U87MG tumors was 6.19 ± 1.37%ID/g, with a tumor/muscle ratio of 20.14 ± 3.24. In the orthotopic glioma model, local injection combined with Temozolomide significantly improved survival and inhibited tumor growth. The results indicate that [¹⁷⁷Lu]Lu-XYIMSR-01 is a promising therapeutic molecular probe for targeting CAIX, and its combination with Temozolomide significantly enhances treatment outcomes for malignant glioma.

SNAP-tag, a mutant of the O⁶-alkylguanine-DNA-alkyltransferase, self-labels by reacting with benzylguanine (BG) substrates, thereby forming a thioether bond. SNAP-tag has been genetically fused to a wide range of proteins of interest in order to covalently modify them. In the context of both diagnostic and therapeutic applications, as well as use as a biological recording device, precise control in a spatial and temporal fashion over the covalent bond-forming reaction is desired to direct inputs, readouts, or therapeutic actions to specific locations, at specific time points, in cells and organisms. Here, we introduce a comprehensive suite of six caged BG molecules: one light-triggered and five others that can be activated through various chemical and biochemical stimuli, such as small molecules, transition metal catalysts, reactive oxygen species, and enzymes. These molecules are unable to react with SNAP-tag until the trigger is present, which leads to near complete SNAP-tag conjugation, as illustrated both in biochemical assays and on human cell surfaces. This approach holds promise for targeted therapeutic assembly at disease sites, offering the potential to reduce off-target effects and toxicity through precise trigger titration.

Antibodies have gained clinical success in the last two decades for the targeted delivery of highly toxic small molecule chemotherapeutics. Yet antibody-drug conjugates (ADCs) often fail in the clinic due to the development of resistance. The delivery of two mechanistically distinct small molecule drugs on one antibody is of increasing interest to overcome these challenges with single-drug ADCs. We have developed a modular synthetic strategy for the construction of a library of 19 dual-drug ADCs where drugs are conjugated through unnatural cyclopentadiene-containing amino acids and native cysteine residues on an anti-HER2 trastuzumab scaffold. Importantly, this strategy utilizes the same functional group on the linker-drug construct; this allows for the facile addition of drugs at either conjugation site and enables the evaluation of different drug-to-antibody ratios and combinations of drug pairs. We tested the library on high- and mid-HER2 expressing cell lines and observed increased toxicity in several dual-drug ADCs compared with single-drug constructs. The strategy developed herein provides a method for the facile synthesis, characterization, and evaluation of dual-payload ADCs. Simultaneous delivery of combinations of drugs with distinct mechanisms of action is critical for the next generation of targeted drug delivery.

Hydrophobic payloads incorporated into antibody-drug conjugates (ADCs) typically are superior to hydrophilic ones in tumor penetration and "bystander killing" upon release from ADCs. However, they are prone to aggregation and accelerated plasma clearance, which lead to reduced efficacies and increased toxicities of ADC molecules. Shielding the hydrophobicity of payloads by incorporating polyethylene glycol (PEG) elements or sugar groups into the ADC linkers has emerged as a viable alternative to directly adopting hydrophilic payloads. In this study, ADC linkers incorporating PEG or sugar groups were synthesized by modifying dipeptide linkers, with hydrophobic monomethyl auristatin E (MMAE) serving as an exemplary hydrophobic payload. All drug-linkers (DLs) were conjugated to RS7, a humanized antibody targeting Trop-2, with drug-to-antibody ratio (DAR) values set at 4 or 8. Among these, the ADC molecule RS7-DL 11, featuring a methyl-PEG₂₄ (mPEG₂₄) moiety as a side chain to the Valine-Lysine-PAB (VK) linker, demonstrated maximum hydrophilicity, biophysical stability, and tumor suppression, along with prolonged half-life and enhanced animal tolerability. In conclusion, through PEGylation of the traditional dipeptide linker, we have demonstrated an optimized ADC conjugation technology that can be employed for conjugating ultrahydrophobic payloads, thus enhancing both the therapeutic index and pharmacokinetics profile.

ENPP-1 is a transmembrane enzyme involved in nucleotide metabolism, and its overexpression is associated with various cancers, making it a potential therapeutic target and biomarker for early tumor diagnosis. Current detection methods for ENPP-1 utilize a colorimetric probe, TMP-pNP, which has significant limitations in sensitivity. Here, we present probe CL-ENPP-1, the first nucleic acid-based chemiluminescent probe designed for rapid and highly sensitive detection of ENPP-1 activity. The design of probe CL-ENPP-1 features a phenoxy-adamantyl-1,2-dioxetane luminophore linked to thymidine via a phosphodiesteric bond. Upon cleavage of the enzymatic substrate by ENPP-1, the probe undergoes an efficient chemiexcitation process to emit a green photon. Probe CL-ENPP-1 demonstrates an exceptional signal-to-noise ratio of 15000 and a limit of detection value approximately 4500-fold lower than the widely used colorimetric probe TMP-pNP. A comparison of TMP-pNP activation by ENPP-1 versus alkaline phosphatase (ALP) reveals a complete lack of selectivity. Removal of the self-immolative spacer from probe CL-ENPP-1 resulted in a new chemiluminescent probe, CL-ENPP-2, with an 18.4-fold increase in selectivity for ENPP-1 over ALP. The ability of probe CL-ENPP-2 to detect ENPP-1 activity in mammalian cells was assessed using the human breast cancer cell line MDA-MB-231. This probe demonstrated a 19.5-fold improvement in the signal-to-noise ratio, highlighting its superior ability to detect ENPP-1 activity in a biological sample. As far as we know, to date, CL-ENPP-1 and CL-ENPP-2 are the most sensitive probes for the detection of ENPP-1 catalytic activity. We anticipate that our new chemiluminescent probes will be valuable for various applications requiring ENPP-1 detection, including enzyme inhibitor-based drug discovery assays. The insights gained from our probe design principles could advance the development of more selective probes for ENPP-1 and contribute to future innovations in chemiluminescence research.