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Overcoming the Hydrolytic Lability of a Reaction Intermediate in Production of Protein/Drug Conjugates: Conjugation of an Acyclic Nucleoside Phosphonate to a Model Carrier Protein.

Overcoming the Hydrolytic Lability of a Reaction Intermediate in Production of Protein/Drug Conjugates: Conjugation of an Acyclic Nucleoside Phosphonate to a Model Carrier Protein.
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Xu S, Kaltashov IA,


Xu S, Kaltashov IA, (click to view)

Xu S, Kaltashov IA,

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Molecular pharmaceutics 2017 07 28() doi 10.1021/acs.molpharmaceut.7b00410

Abstract

Acquired immunodeficiency syndrome (AIDS) remains one of the most serious public health challenges and a significant cause of mortality for certain populations. Despite the large number of antiretrovirals developed in the past two decades, the inability of small molecule therapeutics to target HIV reservoirs directly creates a significant obstacle to their effective utilization. Indeed, achieving the desired therapeutic outcome in the absence of an effective means of targeted delivery must rely on dosage escalation, which frequently causes severe toxicity. This problem may be solved by conjugation of antiretroviral agents to endogenous proteins that are specifically recognized by HIV reservoirs (such as macrophages) for internalization and catabolism. However, conjugation of a large class of antiretroviral agents (acyclic nucleoside phosphonates, such as adefovir, tenofovir, and cidofovir) to a protein is challenging due to rapid degradation of the activated form of the drug (e.g., adefovir phosphonoimidazolide) in an aqueous environment. A novel synthetic strategy introduced in this work overcomes the instability of the activated form of adefovir by emulating the first step of its metabolic pathway (phosphorylation), making it highly reactive toward primary amine groups of proteins and, at the same time, less prone to hydrolysis by water. Efficient conjugation of the phosphorylated form of adefovir to a protein following activation with EDC (1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride) and imidazole was demonstrated using a model protein. Mass spectrometry (MS) was used to identify conditions that favor formation of conjugates with minimal side products, and online ion exchange chromatography/MS analysis of the products revealed the presence of multiple positional isomers within the 1:1 protein/drug conjugates. Both liquid chromatography/MS data and the analysis of ions produced upon top-down fragmentation of the 1:1 conjugates suggest that conjugation of phosphorylated adefovir to the protein occurs not only at primary amines but also at hydroxyl groups. The new conjugation protocol can be used to attach adefovir and other acyclic nucleoside phosphonates to proteins recognized by the cell surface receptors specific to macrophages (such as the haptoglobin/hemoglobin complex), enabling targeted drug delivery directly to HIV reservoirs.

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