(150o) Multi-Site Esterification: A Reversible Approach to Encapsulate Therapeutic Peptides

As natural molecules with low off-site toxicity, peptides have massive therapeutic potential; however, their small size and limited enzymatic stability, among other factors, leads to their rapid removal from circulation and limits their efficacy. Encapsulating therapeutic peptides within carriers can overcome these obstacles by increasing size and stability; however, since encapsulation often relies on hydrophobic and/or electrostatic interactions between the carrier and drug, it can be particularly challenging to encapsulate hydrophilic peptides with low net charge. Therefore, we developed a prodrug scheme in which we esterify the carboxylic acids on a hydrophilic, mixed charge therapeutic peptide to temporarily increase both the hydrophobicity and net charge to promote encapsulation into drug carriers. Subsequent hydrolysis of the installed esters provides both a way to release the peptide from the carrier and restore it to its unmodified, therapeutically active form. Specifically, we installed methyl esters on the aspartic acids (D), glutamic acid (E), and carboxyl terminus of αCT11 (RPRPDDLEI), a connexin-derived therapeutic peptide. We used reverse phase-high performance liquid chromatography (RP-HPLC) to characterize and separate the reaction mixture of peptides with numbers and positions of installed esters, and then determined the number of esters installed on each αCT11 formulation by mass spectrometry. We found the RP-HPLC elution times to increase with the number of installed esters, indicating that esterification indeed increases hydrophobicity. Interestingly, mass spectrometry confirmed three distinct peaks in the product mixture to be composed of αCT11 with 3 methyl esters, suggesting that ester position also impacts hydrophobicity. To further investigate ester position, we identified the residues containing installed esters in each observed RP-HPLC peak through a 2D NMR procedure, combining correlation spectroscopy, total correlation spectroscopy, and heteronuclear multiple bond correlation spectroscopy. Next, we investigated the reversion of the esterified peptides to their therapeutically active form at physiological pH and through accelerated hydrolysis experiments (pH 10), where increasing installed ester number slowed hydrolysis rate, and αCT11 activation. Finally, as a proof of concept that installing methyl esters onto αCT11 would endow greater efficacy relative to the unmodified derivative, we performed an in vitro cell culture scratch wound assay, which showed the esterified derivatives to increase cell migration into wounds relative to cells treated with unmodified αCT11. Together, this work shows the potential of multi-site peptide esterification as a tunable, reversible prodrug scheme that promotes encapsulation, enables release, and augments functionality.