The pathophysiology of phenylketonuria (PKU) is linked to the toxic effects of chronically elevated levels of phenylalanine (Phe) in the blood and other tissues, particularly the brain. Untreated PKU is associated with progressive cognitive and intellectual impairment. PKU is caused by a mutation in the Phe hydroxylase (PAH) gene and results in inefficient or absence of metabolic conversion of phenylalanine to tyrosine. The only available treatments for PKU patients is a life-long, Phe-restricted diet, which works for all patients, or sapropterin, for a subset of patients carrying mutations that destabilize PAH.<br/>Hoskins et al. reported in 1980 that an encapsulated Phenylalanine Ammonia Lyase (PAL) when orally administered to a single PKU patients reduced blood Phe levels by 25%, presumably by degrading Phe upon its liberation from protein in the duodenum to cinnamic acid (CA). This promising result led to various investigations of potential oral enzyme treatments in the PKU mouse, as well as chemically induced PKU rats. While initial results appeared promising, an oral enzyme therapy was not advanced clinically and a polyethylene glycol modified PAL from <i>Anabaena variabilis</i> (AvPAL) was developed for subcutaneous injection instead.<br/>Using CodeEvolver® directed evolution technologies, we generated variants of AvPAL that were sufficiently stable to the conditions in the gastrointestinal tract to provide a potential oral enzyme therapy for PKU. In addition to the discovery path of this therapeutic candidate, we will discuss the insights that were obtained in PAL structure and heretofore unknown mechanistic steps of PAL catalysis.

Development of a protease-stable Phenylalanine Ammonia Lyase as orally-administered enzyme therapy for potential treatment for PKU

256th National Meeting (2018)

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