Oral enzyme therapy for maple syrup urine disease (MSUD) suppresses plasma leucine levels in intermediate MSUD mice and healthy nonhuman primates.

Maple syrup urine disease (MSUD) is an inborn error of branched-chain amino acid metabolism affecting several thousand individuals worldwide. MSUD patients have elevated levels of plasma leucine and its metabolic product α-ketoisocaproate (KIC), which can lead to severe neurotoxicity, coma, and death. Patients must maintain a strict diet of protein restriction and medical formula, and periods of noncompliance or illness can lead to acute metabolic decompensation or cumulative neurological impairment.

An orally administered enzyme therapeutic for homocystinuria that suppresses homocysteine by metabolizing methionine in the gastrointestinal tract.

Classical homocystinuria (HCU) is a rare inborn error of amino acid metabolism characterized by accumulation of homocysteine, an intermediate product of methionine metabolism, leading to significant systemic toxicities, particularly within the vascular, skeletal, and ocular systems. Most patients require lifelong dietary therapy with severe restriction of natural protein to minimize methionine intake, and many patients still struggle to maintain healthy homocysteine levels. Since eliminating methionine from the diet reduces homocysteine levels, we hypothesized that an enzyme that can degrade methionine within the gastrointestinal (GI) tract could help HCU patients maintain healthy levels while easing natural protein restrictions.

Optimizing human α-galactosidase for treatment of Fabry disease.

Fabry disease is caused by a deficiency of α-galactosidase A (GLA) leading to the lysosomal accumulation of globotriaosylceramide (Gb3) and other glycosphingolipids. Fabry patients experience significant damage to the heart, kidney, and blood vessels that can be fatal. Here we apply directed evolution to generate more stable GLA variants as potential next generation treatments for Fabry disease.

Adapting protein sequences for optimized therapeutic efficacy.

Therapeutic proteins alleviate disease pathology by supplementing missing or defective native proteins, sequestering superfluous proteins, or by acting through designed non-natural mechanisms. Although therapeutic proteins often have the same amino acid sequence as their native counterpart, their maturation paths from expression to the site of physiological activity are inherently different, and optimizing protein sequences for properties that 100s of millions of years of evolution did not need to address presents an opportunity to develop better biological treatments. Because therapeutic proteins are inherently non-natural entities, optimization for their desired function should be considered analogous to that of small molecule drug candidates, which are optimized through expansive combinatorial variation by the medicinal chemist.

Enzyme evolution for industrial biocatalytic cascades.

Multi-step, biocatalytic cascades are poised to lead to further adoption of enzymes by the chemical industry. Over the past twenty years, the promise of in vitro enzyme evolution for the sustainable biocatalytic synthesis of complex chemicals at large scale has materialized. Recently, the field of biocatalysis is seeing further expansion, with biocatalytic processes becoming more complex and involving multiple consecutive enzymatic conversions.

Origins of stereoselectivity in evolved ketoreductases.

Mutants of Lactobacillus kefir short-chain alcohol dehydrogenase, used here as ketoreductases (KREDs), enantioselectively reduce the pharmaceutically relevant substrates 3-thiacyclopentanone and 3-oxacyclopentanone. These substrates differ by only the heteroatom (S or O) in the ring, but the KRED mutants reduce them with different enantioselectivities. Kinetic studies show that these enzymes are more efficient with 3-thiacyclopentanone than with 3-oxacyclopentanone.

The role of distant mutations and allosteric regulation on LovD active site dynamics.

Natural enzymes have evolved to perform their cellular functions under complex selective pressures, which often require their catalytic activities to be regulated by other proteins. We contrasted a natural enzyme, LovD, which acts on a protein-bound (LovF) acyl substrate, with a laboratory-generated variant that was transformed by directed evolution to accept instead a small free acyl thioester and no longer requires the acyl carrier protein. The resulting 29-mutant variant is 1,000-fold more efficient in the synthesis of the drug simvastatin than the wild-type LovD.

On the development of new biocatalytic processes for practical pharmaceutical synthesis.

Biocatalysis has established itself as a scalable and green technology for the production of a broad range of pharmaceutical APIs and intermediates. The number and scope of biocatalysts employed on large scale to deliver cost-advantaged and quality-advantaged processes to important substances continue to expand. This review discusses the recent developments in the field, including examples of processes leveraging hydrolases, reductases, transaminases, oxidases and other biocatalysts, focused on the preparation of important investigational and launched therapeutics.

Efficient, chemoenzymatic process for manufacture of the Boceprevir bicyclic [3.1.0]proline intermediate based on amine oxidase-catalyzed desymmetrization.

The key structural feature in Boceprevir, Merck's new drug treatment for hepatitis C, is the bicyclic [3.1.0]proline moiety "P2".

Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture.

Pharmaceutical synthesis can benefit greatly from the selectivity gains associated with enzymatic catalysis. Here, we report an efficient biocatalytic process to replace a recently implemented rhodium-catalyzed asymmetric enamine hydrogenation for the large-scale manufacture of the antidiabetic compound sitagliptin. Starting from an enzyme that had the catalytic machinery to perform the desired chemistry but lacked any activity toward the prositagliptin ketone, we applied a substrate walking, modeling, and mutation approach to create a transaminase with marginal activity for the synthesis of the chiral amine; this variant was then further engineered via directed evolution for practical application in a manufacturing setting.

Practical chiral alcohol manufacture using ketoreductases.

Over the past two years the application of ketoreductases in the commercial synthesis of chiral alcohols has undergone a revolution. Biocatalysts are now often the preferred catalyst for the synthesis of chiral alcohols via ketone reduction and are displacing reagents and chemocatalysts that only recently were considered break-through process solutions themselves. Tailor-made enzymes can now be generated from advanced, non-natural variants using HTP screening and modern molecular biology techniques.

Enzyme optimization: moving from blind evolution to statistical exploration of sequence-function space.

Directed evolution is a powerful tool for the creation of commercially useful enzymes, particularly those approaches that are based on in vitro recombination methods, such as DNA shuffling. Although these types of search algorithms are extraordinarily efficient compared with purely random methods, they do not explicitly represent or interrogate the genotype-phenotype relationship and are essentially blind in nature. Recently, however, researchers have begun to apply multivariate statistical techniques to model protein sequence-function relationships and guide the evolutionary process by rapidly identifying beneficial diversity for recombination.

Improving catalytic function by ProSAR-driven enzyme evolution.

We describe a directed evolution approach that should find broad application in generating enzymes that meet predefined process-design criteria. It augments recombination-based directed evolution by incorporating a strategy for statistical analysis of protein sequence activity relationships (ProSAR). This combination facilitates mutation-oriented enzyme optimization by permitting the capture of additional information contained in the sequence-activity data.

Directed Evolution and Biocatalysis.

This review describes the current state of biocatalysis in the chemical industry. Although we recognize the advantages of chemical approaches, we suggest that the use of biological catalysis is about to expand dramatically because of the recent developments in the artificial evolution of genes that code for enzymes. For the first time it is possible to consider the rapid development of an enzyme that is designed for a specific chemical reaction.

Towards novel processes for the fine-chemical and pharmaceutical industries.

In response to the need in the pharmaceutical industry for more complex, chiral molecules, fine-chemical companies are embracing new manufacturing technologies to produce compounds of these specifications. In particular, recent developments in biocatalysis combined with novel process engineering are providing improved methods for the production of valuable chemical intermediates.