Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications

Pan-Pathway Based Interaction Profiling of FDA-Approved Nucleoside and Nucleobase Analogs with Enzymes of the Human Nucleotide Metabolism

To identify interactions a nucleoside analog library (NAL) consisting of 45 FDA-approved nucleoside analogs was screened against 23 enzymes of the human nucleotide metabolism using a thermal shift assay. The method was validated with deoxycytidine kinase; eight interactions known from the literature were detected and five additional interactions were revealed after the addition of ATP, the second substrate. The NAL screening gave relatively few significant hits, supporting a low rate of “off target effects.” However, unexpected ligands were identified for two catabolic enzymes guanine deaminase (GDA) and uridine phosphorylase 1 (UPP1). An acyclic guanosine prodrug analog, valaciclovir, was shown to stabilize GDA to the same degree as the natural substrate, guanine, with a ΔTagg around 7°C. Aciclovir, penciclovir, ganciclovir, thioguanine and mercaptopurine were also identified as ligands for GDA. The crystal structure of GDA with valaciclovir bound in the active site was determined, revealing the binding of the long unbranched chain of valaciclovir in the active site of the enzyme. Several ligands were identified for UPP1: vidarabine, an antiviral nucleoside analog, as well as trifluridine, idoxuridine, floxuridine, zidovudine, telbivudine, fluorouracil and thioguanine caused concentration-dependent stabilization of UPP1. A kinetic study of UPP1 with vidarabine revealed that vidarabine was a mixed-type competitive inhibitor with the natural substrate uridine. The unexpected ligands identified for UPP1 and GDA imply further metabolic consequences for these nucleoside analogs, which could also serve as a starting point for future drug design

User-centered requirement elicitation for the procurement of medical equipment used by different services and types of end-users

Abstract We propose a method to collect data on user requirements of medical equipment shared by different users and services, relate those requirements with the equipment's technical features, and rank the most important features to be considered when procuring the equipment, leading to more effective procurement decision making. Our method is structured in three phases: (i) elicit the device's technical characteristics, (ii) determine user requirements, and (iii) relate technical characteristics to user requirements. The method is applied to rank the most relevant features of a hospital recliner used by seven services and three types of users. Our results indicate the 10 most relevant factors (requirements) for an “ideal” hospital recliner with potential impact on outputs (importance scores). A final list of 32 items was useful for comparing user requirements and identifying key features that address the most relevant requirements. User‐centered approaches to requirements elicitation in medical equipment procurement promote healthcare benefits, safety, and end‐user satisfaction. Potential use of our approach goes beyond our application case study into many other categories of procurement decisions and into other industry and business applications, wherever multiple stakeholders' requirements should be considered in decisions with multiple value dimensions of value

Emphysematous Cholecystitis: An Unusual Presentation of a Rare Disease

We report a case of radiological diagnosis of emphysematous cholecystitis, a relatively rare form of acute cholecystitis, in a non-diabetic 55-year-old woman. This case is unique given that this pathology is most commonly seen in diabetic male patients. The purpose of this article is to highlight a case of emphysematous cholecystitis in an unexpected population and discuss the clinical features, the diagnostic methods and the treatment.</jats:p