Ureidoglycolate lyase (UGL, EC 4.3.2.3) catalyzes the breakdown of ureidoglycolate to glyoxylate and urea, which is the final step in the catabolic pathway leading from purines to urea. Although the sequence of enzymatic steps was worked out nearly 40 years ago, the stereochemistry of the uric acid degradation pathway and the catalytic properties of UGL have remained very poorly described. We now report the first direct investigation of the absolute stereochemistry of UGL catalysis. Using chiral chromatographic analyses with substrate enantiomers, we demonstrate that UGL catalysis is stereospecific for substrates with the (S)-hydroxyglycine configuration. The first potent competitive inhibitors for UGL are reported here. These inhibitors are compounds which contain a 2,4-dioxocarboxylate moiety, designed to mimic transient species produced during lyase catalysis. The most potent inhibitor, 2,4-dioxo-4-phenylbutanoic acid, exhibits a K I value of 2.2 nM and is therefore among the most potent competitive inhibitors ever reported for a lyase enzyme. New synthetic alternate substrates for UGL, which are acyl-␣-hydroxyglycine compounds, are described. Based on these alternate substrates, we introduce the first assay method for monitoring UGL activity directly. Finally, we report the first putative primary nucleotide and derived peptide sequence for UGL. This sequence exhibits a high level of similarity to the fumarylacetoacetate hydrolase family of proteins. Close mechanistic similarities can be visualized between the chemistries of ureidoglycolate lyase and fumarylacetoacetate hydrolase catalysis.Ureidoglycolate lyase (UGL, 1 EC 4.3.2.3) and allantoicase (Acase, EC 3.5.3.4) are the enzymes that catalyze the final steps in the catabolic pathway leading from purines to urea. In this pathway ( Fig. 1), allantoic acid is formed from allantoin by allantoinase, Acase then converts allantoic acid to ureidoglycolate and urea, and, finally, UGL catalyzes the breakdown of ureidoglycolate to glyoxylate and urea. Acase was first found independently in frog liver by Krebs and Weil (1) and in the mycelium of Aspergillus niger by Brunel (2). UGL was first described by Valentine et al. (3,4) in Streptococcus allantoicus and in a strain of Pseudomonas. It has since been established that UGL and Acase are both functional in many bacteria, yeast, fish, and rat (5-16), whereas in some species only one of these activities has been detected (2, 17-23).The stereochemistry of UGL catalysis, and indeed of the uric acid degradation pathway as a whole, has remained poorly described for many years. Conflicting results have been reported as to whether UGL reacts preferentially with (Ϫ)-ureidoglycolate (5, 6, 9, 10, 18) or with (ϩ)-ureidoglycolate (17). Moreover, allantoinase reacts with both enantiomers of allantoin, and Acase has been reported to produce ureidoglycolate exhibiting a negative rotation, but, paradoxically, to also convert ureidoglycolate exhibiting a positive rotation to glyoxylate and urea (11). Thus, the stereospecificity of U...
Treatment advances for soft-tissue sarcomas have been slow. This is, in part, due to their rarity (accounting for 0.7% of all cancers) and heterogeneity (over 50 different diseases fall under this category). Moreover, preclinical models are scarce, often exhibiting slow growth kinetics, which limits their study by large genetic and pharmacological libraries. Here, we present an update on our efforts to harness the power of patient-partnered research to create a platform for rare cancer drug target discovery as a broadly available community resource. We developed a patient-partnered tissue donation pipeline to enable patients anywhere in the United States to participate and piloted our approach for CTNNB1-driven desmoid tumors. To overcome challenges in tissue heterogeneity during ex vivo culture, we optimized a multiplexed sequencing protocol to quantitatively track changes in tumor cell fraction across hundreds of media formulations. Following this strategy, we were able to verify and expand three cell lines that preserve the CTNNB1 mutations at high purity. To identify potential therapeutics, we completed a 6,750-drug repurposing screen, at 2.5uM in duplicate, in two verified cell line models. After extensive quality control assessments and data integration steps to leverage the power of other large scale drug screens, we selected 263 compounds for follow-up based on potency, selectivity, and association with molecular features associated with desmoid tumors. Approximately 70% of selected compounds were validated by an 8-point, 2-fold dilution, dose-response format with a top concentration of 10uM. Of the confirmed active compounds, 80 showed a strong pattern of selectivity, 20 are FDA approved drugs and 13 investigational compounds show a statistical association with CTNNB1 hotspot mutation status or transcriptomic features associated with desmoid tumors. To prioritize potential therapeutic targets, we tested an efficient CRISPR/Cas9 all-in-one library design. The reduction of the CRISPR/Cas9 library size was achieved via multiple gene- and guide-level strategies, which enables statistically powered gene essentiality interrogation in slow-growing patient-derived models. We tested several plating and infection parameters and developed an optimized pipeline for the rapid introduction of this library into early patient-derived samples. Established cell lines of mesenchymal and non-mesenchymal origin, which have previously been tested by genome-wide libraries, were used to control for library and lineage effects. We are developing a biologist-friendly web portal that will enable the research community to easily interact with models and data produced by this effort. Our study provides evidence that a systematic patient-powered approach can facilitate discovery of therapeutic hypotheses for these understudied diseases. Citation Format: Mushriq Al-Jazrawe, Kathryn Cebula, Elisabeth A. Abeyta, Haley S. Curtis, Jane K. McIninch, Jaime H. Cheah, James Berstler, Lisa Miller, James Neiswender, Lisa Brenan, Mike Burger, Francisca Vazquez, Jesse S. Boehm. Drug repurposing and genetic screening strategies for effective treatment discovery in soft-tissue sarcomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5324.
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