Glucuronidation is often the source of unfavorable pharmacokinetics or pharmacodynamics that lead to the failure of drugs during clinical trials, and as such, there is a critical need in drug development for a tool to control glucuronidation. Our long-term goal is to develop drugs that will control glucuronidation. As a first step toward that goal, we will determine the allosteric mechanism that controls human UDP-Glucose Dehydrogenase (hUGDH), the enzyme that produces the essential substrate for glucuronidation. Specifically, hUGDH contains a 30-residue intrinsically disordered C-terminus (the ID-tail) that is important for feedback inhibition by UDP-Xyl, a downstream metabolite. We hypothesize that the ID-tail and an allosteric 'hotspot' interact within a network of dynamic features to favor a conformation of hUGDH with a higher affinity for the allosteric inhibitor. Our hypothesis is based on the observation that: (i) the ID-tail and the hotspot control the rate at which hUGDH is allosterically activated or inhibited; (ii) the ID-tail and the hotspot increase the affinity for the allosteric inhibitor through a long-range mechanism; and (iii) the ID-tail changes the conformational ensemble of hUGDH. The rationale underlying the proposed project is that the knowledge gained by understanding how the inactive state of hUGDH is controlled has the potential to translate into novel strategies for sensitization of drug resistant tumors to existing cancer drugs. This hypothesis will be tested by pursuing three specific aims: 1) determine how the slow isomerization between the inactive and active states is linked to allostery; 2) how the E250/T253 allosteric hotspot and the intrinsically disordered C-terminal tail of hUGDH contributes to feedback inhibition; and 3) determine how the ID-tail stabilizes the inactive conformation of hUGDH. The research proposed in this application is innovative because it focuses on the allosteric inhibition of hUGDH as a global mechanism for controlling glucuronidation, and uses an inhibitory peptide that we discovered. Since the role of intrinsic disorder and the identity of the allosteric switches that control hUGDH were only recently discovered by our lab, this research is distinct from previous attempts that tried to control glucuronidation. The expected outcomes of this work are significant. A detailed description of the inactive state of hUGDH will act as a template for the design of a class of allosteric inhibitos that will act as global regulators of glucuronidation. Because of the persistence of long (>30 residues) intrinsically disordered segments in the proteome, (33% of all proteins), what we learn about the effect of disordered segments on protein structure-function relationships is likely to have a broad impact.
Uncovering the mechanisms of the allosteric regulation of human UDP-glucose dehydrogenase is important to public health because it will facilitate the development of novel strategies in drug discovery. Inhibiting human UDP-glucose dehydrogenase activity will also sensitize some drug resistant tumors to existing chemotherapeutics. This project furthers the mission of NIH by developing and applying fundamental knowledge to enhance health, lengthen life, and reduce the burden of illness.
|Beattie, Nathaniel R; Keul, Nicholas D; Sidlo, Andrew M et al. (2017) Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase. Biochemistry 56:202-211|