Bioconjugate Chemistry最新文献

Multiple studies have demonstrated the benefit of engineering hybrid ligands that combine the unique benefits of small molecules and proteins or peptides. However, the molecular complexity of hybrid ligands generates a parameter space so large it cannot be exhaustively explored. We systematically evaluated the impact of one molecular design element, conjugation site, on the discovery of functional protein-small molecule hybrids (PriSMs). We utilized a library of yeast-displayed fibronectin domain variants with amino acid and loop length diversity in the paratope and a single cysteine at one of 18 possible conjugation sites. The protein variants were coupled with maleimide-functionalized acetazolamide and sorted via competitive flow cytometry to discover potent and selective inhibitors of three isoforms of carbonic anhydrase. Deep sequencing of the resultant populations of functional PriSMs revealed an isoform-dependent distribution of conjugation site preferences. The top PriSMs showed potency and selectivity gains up to 23- and 100-fold (in this case, for CA-II vs CA-XII, with a 43-fold selectivity gain for CA-II vs CA-IX) relative to PEG₂-acetazolamide alone. The presented study expands our fundamental understanding of the role of conjugation site in PriSM function and informs future PriSM engineering efforts by highlighting the benefit of conjugation site diversity in PriSM libraries.

Membrane proteins are biologically and pharmaceutically significant, and determining their 3D structures requires a membrane-mimetic system to maintain protein stability. Detergent micelles are widely used as membrane mimetics; however, their dynamic structures often lead to the denaturation and aggregation of encapsulated membrane proteins. To address the limitations of classical detergents in stabilizing membrane proteins, we previously reported a class of glucose-neopentyl glycols (GNGs) and their pendant-bearing versions (P-GNGs), several of which proved more effective than DDM in stabilizing membrane proteins. In this study, we synthesized additional GNG derivatives by varying the lengths of the pendant (P-GNGs), and by introducing hemifluorinated pendants to the GNG scaffold (fluorinated pendant-bearing GNGs or FP-GNGs). The synthetic flexibility of the GNG chemical architecture allowed us to create a diverse range of derivatives, essential for the effective optimization of detergent properties. When tested with two model membrane proteins (a transporter and a G-protein coupled receptor (GPCR)), most of the new (F)P-GNGs demonstrated superior stabilization of these membrane proteins compared to DDM, the original GNG (OGNG)), and a previously developed P-GNG (i.e., GNG-3,14). Notably, several P-GNGs synthesized in this study were as effective as or even better than lauryl maltose neopentyl glycol (LMNG) in stabilizing a human GPCR, beta2 adrenergic receptor (β2AR). Enhanced protein stability was particularly observed for the P-GNGs with a butyl (C4) or pentyl (C5) pendant, indicating that these pendant sizes are optimal for membrane protein stability. The volumes of these pendants appear to minimize the empty spaces in the micelle interiors, thereby enhancing detergent-detergent interactions in micelles complexed with the membrane proteins. Additionally, we identified one FP-GNG that was more efficient at extracting the transporter and more effective at stabilizing the GPCR than DDM. Thus, the current study demonstrates that both chain length and number of fluorine atoms in the pendants of the P-GNGs were crucial determinants for membrane protein stability. We not only developed a few (F)P-GNGs that are significantly more effective than maltoside detergents (LMNG/DDM) for protein extraction and stability but we also provided an effective strategy for detergent design through the utilization of partially fluorinated pendants of varying length.

Chelation approaches that are compatible with a multitude of isotopes are an important area of development. Here, we introduce the design, synthesis, and evaluation of 2,3-dihydroxyterephthalate/catechol chelator conjugates compatible with the positron emission tomography (PET) isotopes ⁶⁸Ga³⁺ and ⁴⁵Ti⁴⁺, targeting the prostate-specific membrane antigen (PSMA). The conjugates are made in a multistep organic synthesis incorporating 2,3-dihydroxyterephthalate, linked to the amino hexanoic acid-extended, urea-dipeptides EuE or KuE (substrates of the PSMA active site). The radiochemical complexes, [⁴⁵Ti][Ti(TREN-CAM-hex-EuE)]^2-, [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2-, and [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3- form readily at room temperature within 15 min with a molar activity of 24-29 mCi/μmol. The corresponding chelates are stable in phosphate-buffered saline (PBS) solution prior to injection. Subsequent in vivo studies in a bilateral tumor xenograft mouse model were conducted, including 90- and 270-min PET, followed by biodistribution and metabolite analysis at 2 or 5 h postinjection. These studies demonstrated selective uptake of the radiochemical complexes in the PSMA-expressing tumor (17.25 ± 4.15, 13.84 ± 3.85, 15.64 ± 6.37% ID/g for [⁴⁵Ti][Ti(TREN-CAM-hex-EuE)]^2-, [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2- and [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3- respectively), with pharmacokinetics dominated by renal clearance. Delayed clearance of the [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2- complex is observed when compared with that of [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3- as indicated by elevated activity retention in the blood, which we attribute to the charge difference and partial complex dissociation. Urine metabolite analysis shows that [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3- is excreted >98% intact, while [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2- exhibited signs of dechelation. Conclusively, our data support further investigation of bifunctional TREN-CAM derivatives as a synthetically accessible bifunctional chelator class for ⁶⁸Ga³⁺ and ⁴⁵Ti⁴⁺ isotopes.

Chelation approaches that are compatible with a multitude of isotopes are an important area of development. Here, we introduce the design, synthesis, and evaluation of 2,3-dihydroxyterephthalate/catechol chelator conjugates compatible with the positron emission tomography (PET) isotopes ⁶⁸Ga³⁺ and ⁴⁵Ti⁴⁺, targeting the prostate-specific membrane antigen (PSMA). The conjugates are made in a multistep organic synthesis incorporating 2,3-dihydroxyterephthalate, linked to the amino hexanoic acid-extended, urea-dipeptides EuE or KuE (substrates of the PSMA active site). The radiochemical complexes, [⁴⁵Ti][Ti(TREN-CAM-hex-EuE)]^2–, [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2–, and [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3– form readily at room temperature within 15 min with a molar activity of 24–29 mCi/μmol. The corresponding chelates are stable in phosphate-buffered saline (PBS) solution prior to injection. Subsequent in vivo studies in a bilateral tumor xenograft mouse model were conducted, including 90- and 270-min PET, followed by biodistribution and metabolite analysis at 2 or 5 h postinjection. These studies demonstrated selective uptake of the radiochemical complexes in the PSMA-expressing tumor (17.25 ± 4.15, 13.84 ± 3.85, 15.64 ± 6.37% ID/g for [⁴⁵Ti][Ti(TREN-CAM-hex-EuE)]^2–, [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2– and [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3– respectively), with pharmacokinetics dominated by renal clearance. Delayed clearance of the [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2– complex is observed when compared with that of [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3– as indicated by elevated activity retention in the blood, which we attribute to the charge difference and partial complex dissociation. Urine metabolite analysis shows that [⁶⁸Ga][Ga(TREN-CAM-hex-KuE)]^3– is excreted >98% intact, while [⁴⁵Ti][Ti(TREN-CAM-hex-KuE)]^2– exhibited signs of dechelation. Conclusively, our data support further investigation of bifunctional TREN-CAM derivatives as a synthetically accessible bifunctional chelator class for ⁶⁸Ga³⁺ and ⁴⁵Ti⁴⁺ isotopes.

Synthetic cells are a rapidly maturing platform with emerging applications in biomedicine and biotechnology. The specific novelty of this work is that we develop synthetic cells that respond to an extracellular stimulus by performing the folding of an encapsulated polypeptide into a functional enzyme. To this end, we developed artificial transmembrane signaling receptors. These contain an extracellular enzyme-responsive group, a self-immolative linker as the mechanism of signal transduction, and a secondary messenger molecule with intracellular activity. The secondary messenger is chosen such that it initiates protein refolding from the denatured polypeptide. Results of this study expand the molecular toolbox for the design of synthetic cells with life-like, responsive behavior.

Synthetic cells are a rapidly maturing platform with emerging applications in biomedicine and biotechnology. The specific novelty of this work is that we develop synthetic cells that respond to an extracellular stimulus by performing the folding of an encapsulated polypeptide into a functional enzyme. To this end, we developed artificial transmembrane signaling receptors. These contain an extracellular enzyme-responsive group, a self-immolative linker as the mechanism of signal transduction, and a secondary messenger molecule with intracellular activity. The secondary messenger is chosen such that it initiates protein refolding from the denatured polypeptide. Results of this study expand the molecular toolbox for the design of synthetic cells with life-like, responsive behavior.

Increasingly, street mixtures of opioids are reported to contain combinations of synthetic opioids, such as fentanyl with fentanyl analogues or counterfeit oxycodone pills containing fentanyl. While antiopioid immunotherapeutics have been investigated as a possible approach to address the opioid epidemic, the efficacy of vaccines and antibodies is limited to specific target opioids, based on the chemical structure of the haptens used in vaccines. Hence, there is a need for rational design of antiopioid conjugate vaccines that simultaneously target multiple opioids. Here, four novel haptens were synthesized, which were designed to elicit antibodies capable of binding to fentanyl other target opioids, including carfentanil, alfentanil, or oxycodone. Haptens were conjugated to CRM carrier protein and formulated with an aluminum salt adjuvant, and vaccines containing bivalent haptens were compared to admixtures of individual conjugate vaccines targeting the two opioids separately. Rats were immunized with monovalent, admixed, or novel bivalent vaccines, and the blockade of opioid effects was assessed against the individual drugs and their mixtures. Opioid-specific antibody titer was measured, and in vivo effects of vaccines were assessed in terms of preventing opioid-induced antinociception and respiratory depression and opioid distribution to the brain. While the bivalent vaccines reduced the effects of some target opioids, the admixed vaccine formulations were more effective against fentanyl/carfentanil and fentanyl/alfentanil mixtures. The bivalent fentanyl/oxycodone vaccine was as effective as the monovalent vaccines against a single drug challenge. These results inform the design of future vaccines against opioids and other drugs, particularly in the context of vaccines against polysubstance use that require optimization of response against multiple drugs of interest.

Increasingly, street mixtures of opioids are reported to contain combinations of synthetic opioids, such as fentanyl with fentanyl analogues or counterfeit oxycodone pills containing fentanyl. While antiopioid immunotherapeutics have been investigated as a possible approach to address the opioid epidemic, the efficacy of vaccines and antibodies is limited to specific target opioids, based on the chemical structure of the haptens used in vaccines. Hence, there is a need for rational design of antiopioid conjugate vaccines that simultaneously target multiple opioids. Here, four novel haptens were synthesized, which were designed to elicit antibodies capable of binding to fentanyl other target opioids, including carfentanil, alfentanil, or oxycodone. Haptens were conjugated to CRM carrier protein and formulated with an aluminum salt adjuvant, and vaccines containing bivalent haptens were compared to admixtures of individual conjugate vaccines targeting the two opioids separately. Rats were immunized with monovalent, admixed, or novel bivalent vaccines, and the blockade of opioid effects was assessed against the individual drugs and their mixtures. Opioid-specific antibody titer was measured, and in vivo effects of vaccines were assessed in terms of preventing opioid-induced antinociception and respiratory depression and opioid distribution to the brain. While the bivalent vaccines reduced the effects of some target opioids, the admixed vaccine formulations were more effective against fentanyl/carfentanil and fentanyl/alfentanil mixtures. The bivalent fentanyl/oxycodone vaccine was as effective as the monovalent vaccines against a single drug challenge. These results inform the design of future vaccines against opioids and other drugs, particularly in the context of vaccines against polysubstance use that require optimization of response against multiple drugs of interest.

Fibroblast growth factor 21 (FGF21) is a crucial regulator of glucose and lipid metabolism, showing significant therapeutic promise for metabolic disorders. However, its clinical application is limited by poor pharmacokinetics. One potential strategy to improve its half-life is to facilitate albumin binding through fatty acid derivation. Despite this promise, achieving site-specific modifications of FGF21 while preserving its biological activity has been challenging. In this study, we applied a rational design approach to create site-specific fatty acid derivatives of FGF21, guided by the structure of the FGF21-receptor complex. This strategy successfully enhances albumin binding without interfering with receptor interactions. The modified FGF21 derivatives exhibited dramatically extended half-lives in mice, increasing from 0.73 h to 11.36 and 13.36 h, respectively. Furthermore, these analogues showed superior biological activity in the presence of albumin, outperforming the C-terminal-derived variant zalfermin. This rational design approach not only improves the pharmacokinetic profile of FGF21 but also provides a framework for enhancing the therapeutic potential of other small proteins.

Fibroblast growth factor 21 (FGF21) is a crucial regulator of glucose and lipid metabolism, showing significant therapeutic promise for metabolic disorders. However, its clinical application is limited by poor pharmacokinetics. One potential strategy to improve its half-life is to facilitate albumin binding through fatty acid derivation. Despite this promise, achieving site-specific modifications of FGF21 while preserving its biological activity has been challenging. In this study, we applied a rational design approach to create site-specific fatty acid derivatives of FGF21, guided by the structure of the FGF21-receptor complex. This strategy successfully enhances albumin binding without interfering with receptor interactions. The modified FGF21 derivatives exhibited dramatically extended half-lives in mice, increasing from 0.73 h to 11.36 and 13.36 h, respectively. Furthermore, these analogues showed superior biological activity in the presence of albumin, outperforming the C-terminal-derived variant zalfermin. This rational design approach not only improves the pharmacokinetic profile of FGF21 but also provides a framework for enhancing the therapeutic potential of other small proteins.