Gluten Degradation, Pharmacokinetics, Safety, and Tolerability of TAK-062, an Engineered Enzyme to Treat Celiac Disease.

BACKGROUND AND AIMS: Celiac disease (CeD) is an immune-mediated disorder triggered by the ingestion of gluten. Despite adhering to a gluten-free diet (the only management option available to patients with CeD), many patients continue to experience symptoms and intestinal injury. Degradation of immunogenic fractions of gluten peptides in the stomach has been proposed as an approach to reduce toxicity of ingested gluten; however, no enzymes evaluated to date have demonstrated sufficient gluten degradation in complex meals. TAK-062 is a novel, computationally designed endopeptidase under development for the treatment of patients with CeD. METHODS: Pharmacokinetics, safety, and tolerability of TAK-062 100-900 mg were evaluated in a phase I dose escalation study in healthy participants and patients with CeD. Gluten degradation by TAK-062 was evaluated under simulated gastric conditions in vitro and in healthy participants in the phase I study, with and without pretreatment with a proton pump inhibitor. Residual gluten (collected through gastric aspiration in the phase I study) was quantified using R5 and G12 monoclonal antibody enzyme-linked immunosorbent assays. RESULTS: In vitro, TAK-062 degraded more than 99% of gluten (3 g and 9 g) within 10 minutes. In the phase I study, administration of TAK-062 was well tolerated and resulted in a median gluten degradation ranging from 97% to more than 99% in complex meals containing 1-6 g gluten at 20-65 minutes postdose. CONCLUSIONS: TAK-062 is well tolerated and rapidly and effectively degrades large amounts of gluten, supporting the development of this novel enzyme as an oral therapeutic for patients with CeD. (ClinicalTrials.gov: NCT03701555, https://clinicaltrials.gov/ct2/show/NCT03701555.).

Expanding the product profile of a microbial alkane biosynthetic pathway.

Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.

The response threshold of Salmonella PilZ domain proteins is determined by their binding affinities for c-di-GMP.

c-di-GMP is a bacterial second messenger that is enzymatically synthesized and degraded in response to environmental signals. Cellular processes are affected when c-di-GMP binds to receptors which include proteins that contain the PilZ domain. Although each c-di-GMP synthesis or degradation enzyme metabolizes the same molecule, many of these enzymes can be linked to specific downstream processes. Here we present evidence that c-di-GMP signalling specificity is achieved through differences in affinities of receptor macromolecules. We show that the PilZ domain proteins of Salmonella Typhimurium, YcgR and BcsA, demonstrate a 43-fold difference in their affinity for c-di-GMP. Modulation of the affinities of these proteins altered their activities in a predictable manner in vivo. Inactivation of yhjH, which encodes a predicted c-di-GMP degrading enzyme, increased the fraction of the cellular population that demonstrated c-di-GMP levels high enough to bind to the higher-affinity YcgR protein and inhibit motility, but not high enough to bind to the lower-affinity BcsA protein and stimulate cellulose production. Finally, PilZ domain proteins of Pseudomonas aeruginosa demonstrated a 145-fold difference in binding affinities, suggesting that regulation by binding affinity may be a conserved mechanism that allows organisms with many c-di-GMP binding macromolecules to rapidly integrate multiple environmental signals into one output.

Computational design of an α-gliadin peptidase.

The ability to rationally modify enzymes to perform novel chemical transformations is essential for the rapid production of next-generation protein therapeutics. Here we describe the use of chemical principles to identify a naturally occurring acid-active peptidase, and the subsequent use of computational protein design tools to reengineer its specificity toward immunogenic elements found in gluten that are the proposed cause of celiac disease. The engineered enzyme exhibits a k(cat)/K(M) of 568 M(-1) s(-1), representing a 116-fold greater proteolytic activity for a model gluten tetrapeptide than the native template enzyme, as well as an over 800-fold switch in substrate specificity toward immunogenic portions of gluten peptides. The computationally engineered enzyme is resistant to proteolysis by digestive proteases and degrades over 95% of an immunogenic peptide implicated in celiac disease in under an hour. Thus, through identification of a natural enzyme with the pre-existing qualities relevant to an ultimate goal and redefinition of its substrate specificity using computational modeling, we were able to generate an enzyme with potential as a therapeutic for celiac disease.