An Albumin-Holliday Junction Biomolecular Modular Design for Programmable Multifunctionality and Prolonged Circulation.

Combinatorial properties such as long-circulation and site- and cell-specific engagement need to be built into the design of advanced drug delivery systems to maximize drug payload efficacy. This work introduces a four-stranded oligonucleotide Holliday Junction (HJ) motif bearing functional moieties covalently conjugated to recombinant human albumin (rHA) to give a "plug-and-play" rHA-HJ multifunctional biomolecular assembly with extended circulation. Electrophoretic gel-shift assays show successful functionalization and purity of the individual high-performance liquid chromatography-purified modules as well as efficient assembly of the rHA-HJ construct. Inclusion of an epidermal growth factor receptor (EGFR)-targeting nanobody module facilitates specific binding to EGFR-expressing cells resulting in approximately 150-fold increased fluorescence intensity determined by flow cytometric analysis compared to assemblies absent of nanobody inclusion. A cellular recycling assay demonstrated retained albumin-neonatal Fc receptor (FcRn) binding affinity and accompanying FcRn-driven cellular recycling. This translated to a 4-fold circulatory half-life extension (2.2 and 0.55 h, for the rHA-HJ and HJ, respectively) in a double transgenic humanized FcRn/albumin mouse. This work introduces a novel biomolecular albumin-nucleic acid construct with extended circulatory half-life and programmable multifunctionality due to its modular design.

Aromatic oligoesters as novel helix mimetic scaffolds.

The design, synthesis, and conformational analysis of a novel aromatic oligoester helix mimetic scaffold is reported. A range of amino acid-type side-chain functionality can be readily incorporated into monomer building blocks over three facile synthetic steps. Analysis of representative dimers revealed a stable conformer capable of effective mimicry of a canonical α-helix and the scaffold was found to be surprisingly stable to degradation in aqueous solutions at acidic and neutral pH.

Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly.

Albumin-nucleic acid biomolecular drug designs offer modular multifunctionalization and extended circulatory half-life. However, stability issues associated with conventional DNA nucleotides and maleimide bioconjugation chemistries limit the clinical potential. This work aims to improve the stability of this thiol conjugation and nucleic acid assembly by employing a fast-hydrolyzing monobromomaleimide (MBM) linker and nuclease-resistant nucleotide analogues, respectively. The biomolecular constructs were formed by site-selective conjugation of a 12-mer oligonucleotide to cysteine 34 (Cys34) of recombinant human albumin (rHA), followed by annealing of functionalized complementary strands bearing either a fluorophore or the cytotoxic drug monomethyl auristatin E (MMAE). Formation of conjugates and assemblies was confirmed by gel shift analysis and mass spectrometry, followed by investigation of serum stability, neonatal Fc receptor (FcRn)-mediated cellular recycling, and cancer cell killing. The MBM linker afforded rapid conjugation to rHA and remained stable during hydrolysis. The albumin-nucleic acid biomolecular assembly composed of stabilized oligonucleotides exhibited high serum stability and retained FcRn engagement mediating FcRn-mediated cellular recycling. The MMAE-containing assembly exhibited cytotoxicity in the human MIA PaCa-2 pancreatic cancer cell line with an IC50 of 342 nM, triggered by drug release from breakdown of an acid-labile linker. In summary, this work presents rHA-nucleic acid module-based assemblies with improved stability and retained module functionality that further promotes the drug delivery potential of this biomolecular platform.

A Plug-and-Play Platform for the Formation of Trifunctional Cysteine Bioconjugates that also Offers Control over Thiol Cleavability.

Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy in vivo. Cysteine residues have been exploited as a route to site-selectively modify proteins, and many successfully approved therapeutics make use of cysteine directed conjugation reagents. However, commonly used linkers, including maleimide-thiol conjugates, are not stable to the low concentrations of thiol present in blood. Furthermore, only a few cysteine-targeting reagents enable the site-selective attachment of multiple functionalities: a useful tool in the fields of theranostics and therapeutic blood half-life extension. Herein, we demonstrate the application of the pyridazinedione motif to enable site-selective attachment of three functionalities to a protein bearing a single cysteine residue. Extending upon previously documented dual modification work, here we demonstrate that by exploiting a bromide leaving group as an additional reactive point on the pyridazinedione scaffold, a thiol or aniline derivative can be added to a protein, post-conjugation. Thiol cleavability appraisal of the resultant C-S and C-N linked thio-bioconjugates demonstrated C-S functionalized linkers to be cleavable and C-N functionalized linkers to be noncleavable when incubated in an excess of glutathione. The plug-and-play trifunctional platform was exemplified by attaching clinically relevant motifs: biotin, fluorescein, a polyethylene glycol chain, and a model peptide. This platform provides a rare opportunity to combine up to three functionalities on a protein in a site-selective fashion. Furthermore, by selecting the use of a thiol or an amine for functionalization, we provide unique control over linker cleavability toward thiols, allowing this novel linker to be applied in a range of physiological environments.

Optimised approach to albumin-drug conjugates using monobromomaleimide-C-2 linkers.

Conjugation of therapeutics to human serum albumin (HSA) using bromomaleimides represents a promising platform for half-life extension. We show here that the Cys-34 crevice substantially reduces the rate of serum stabilising maleimide hydrolysis in these conjugates, necessitating reagent optimisation. This improved reagent design is applied to the construction of an HSA-paclitaxel conjugate, preventing drug loss during maleimide hydrolysis.

One-pot thiol-amine bioconjugation to maleimides: simultaneous stabilisation and dual functionalisation.

Maleimide chemistry is widely used in the site-selective modification of proteins. However, hydrolysis of the resultant thiosuccinimides is required to provide robust stability to the bioconjugates. Herein, we present an alternative approach that affords simultaneous stabilisation and dual functionalisation in a one pot fashion. By consecutive conjugation of a thiol and an amine to dibromomaleimides, we show that aminothiomaleimides can be generated extremely efficiently. Furthermore, the amine serves to deactivate the electrophilicity of the maleimide, precluding further reactivity and hence generating stable conjugates. We have applied this conjugation strategy to peptides and proteins to generate stabilised trifunctional conjugates. We propose that this stabilisation-dual modification strategy could have widespread use in the generation of diverse conjugates.