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Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors
[Image: see text] Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. Trypanosoma cruzi, the etiological agent of CD, is a purine au...
Autores principales: | , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Chemical Society
2023
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10357585/ https://www.ncbi.nlm.nih.gov/pubmed/37418678 http://dx.doi.org/10.1021/acs.biochem.3c00116 |
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author | Glockzin, Kayla Meneely, Kathleen M. Hughes, Ryan Maatouk, Sean W. Piña, Grace E. Suthagar, Kajitha Clinch, Keith Buckler, Joshua N. Lamb, Audrey L. Tyler, Peter C. Meek, Thomas D. Katzfuss, Ardala |
author_facet | Glockzin, Kayla Meneely, Kathleen M. Hughes, Ryan Maatouk, Sean W. Piña, Grace E. Suthagar, Kajitha Clinch, Keith Buckler, Joshua N. Lamb, Audrey L. Tyler, Peter C. Meek, Thomas D. Katzfuss, Ardala |
author_sort | Glockzin, Kayla |
collection | PubMed |
description | [Image: see text] Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. Trypanosoma cruzi, the etiological agent of CD, is a purine auxotroph that relies on phosphoribosyltransferases to salvage purine bases from their hosts for the formation of purine nucleoside monophosphates. Hypoxanthine–guanine–xanthine phosphoribosyltransferases (HGXPRTs) catalyze the salvage of 6-oxopurines and are promising targets for the treatment of CD. HGXPRTs catalyze the formation of inosine, guanosine, and xanthosine monophosphates from 5-phospho-d-ribose 1-pyrophosphate and the nucleobases hypoxanthine, guanine, and xanthine, respectively. T. cruzi possesses four HG(X)PRT isoforms. We previously reported the kinetic characterization and inhibition of two isoforms, TcHGPRTs, demonstrating their catalytic equivalence. Here, we characterize the two remaining isoforms, revealing nearly identical HGXPRT activities in vitro and identifying for the first time T. cruzi enzymes with XPRT activity, clarifying their previous annotation. TcHGXPRT follows an ordered kinetic mechanism with a postchemistry event as the rate-limiting step(s) of catalysis. Its crystallographic structures reveal implications for catalysis and substrate specificity. A set of transition-state analogue inhibitors (TSAIs) initially developed to target the malarial orthologue were re-evaluated, with the most potent compound binding to TcHGXPRT with nanomolar affinity, validating the repurposing of TSAIs to expedite the discovery of lead compounds against orthologous enzymes. We identified mechanistic and structural features that can be exploited in the optimization of inhibitors effective against TcHGPRT and TcHGXPRT concomitantly, which is an important feature when targeting essential enzymes with overlapping activities. |
format | Online Article Text |
id | pubmed-10357585 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | American Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-103575852023-07-21 Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors Glockzin, Kayla Meneely, Kathleen M. Hughes, Ryan Maatouk, Sean W. Piña, Grace E. Suthagar, Kajitha Clinch, Keith Buckler, Joshua N. Lamb, Audrey L. Tyler, Peter C. Meek, Thomas D. Katzfuss, Ardala Biochemistry [Image: see text] Over 70 million people are currently at risk of developing Chagas Disease (CD) infection, with more than 8 million people already infected worldwide. Current treatments are limited and innovative therapies are required. Trypanosoma cruzi, the etiological agent of CD, is a purine auxotroph that relies on phosphoribosyltransferases to salvage purine bases from their hosts for the formation of purine nucleoside monophosphates. Hypoxanthine–guanine–xanthine phosphoribosyltransferases (HGXPRTs) catalyze the salvage of 6-oxopurines and are promising targets for the treatment of CD. HGXPRTs catalyze the formation of inosine, guanosine, and xanthosine monophosphates from 5-phospho-d-ribose 1-pyrophosphate and the nucleobases hypoxanthine, guanine, and xanthine, respectively. T. cruzi possesses four HG(X)PRT isoforms. We previously reported the kinetic characterization and inhibition of two isoforms, TcHGPRTs, demonstrating their catalytic equivalence. Here, we characterize the two remaining isoforms, revealing nearly identical HGXPRT activities in vitro and identifying for the first time T. cruzi enzymes with XPRT activity, clarifying their previous annotation. TcHGXPRT follows an ordered kinetic mechanism with a postchemistry event as the rate-limiting step(s) of catalysis. Its crystallographic structures reveal implications for catalysis and substrate specificity. A set of transition-state analogue inhibitors (TSAIs) initially developed to target the malarial orthologue were re-evaluated, with the most potent compound binding to TcHGXPRT with nanomolar affinity, validating the repurposing of TSAIs to expedite the discovery of lead compounds against orthologous enzymes. We identified mechanistic and structural features that can be exploited in the optimization of inhibitors effective against TcHGPRT and TcHGXPRT concomitantly, which is an important feature when targeting essential enzymes with overlapping activities. American Chemical Society 2023-07-07 /pmc/articles/PMC10357585/ /pubmed/37418678 http://dx.doi.org/10.1021/acs.biochem.3c00116 Text en © 2023 The Authors. Published by American Chemical Society https://creativecommons.org/licenses/by-nc-nd/4.0/Permits non-commercial access and re-use, provided that author attribution and integrity are maintained; but does not permit creation of adaptations or other derivative works (https://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Glockzin, Kayla Meneely, Kathleen M. Hughes, Ryan Maatouk, Sean W. Piña, Grace E. Suthagar, Kajitha Clinch, Keith Buckler, Joshua N. Lamb, Audrey L. Tyler, Peter C. Meek, Thomas D. Katzfuss, Ardala Kinetic and Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine Phosphoribosyltransferases and Repurposing of Transition-State Analogue Inhibitors |
title | Kinetic and
Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine
Phosphoribosyltransferases and Repurposing of Transition-State Analogue
Inhibitors |
title_full | Kinetic and
Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine
Phosphoribosyltransferases and Repurposing of Transition-State Analogue
Inhibitors |
title_fullStr | Kinetic and
Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine
Phosphoribosyltransferases and Repurposing of Transition-State Analogue
Inhibitors |
title_full_unstemmed | Kinetic and
Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine
Phosphoribosyltransferases and Repurposing of Transition-State Analogue
Inhibitors |
title_short | Kinetic and
Structural Characterization of Trypanosoma cruzi Hypoxanthine–Guanine–Xanthine
Phosphoribosyltransferases and Repurposing of Transition-State Analogue
Inhibitors |
title_sort | kinetic and
structural characterization of trypanosoma cruzi hypoxanthine–guanine–xanthine
phosphoribosyltransferases and repurposing of transition-state analogue
inhibitors |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10357585/ https://www.ncbi.nlm.nih.gov/pubmed/37418678 http://dx.doi.org/10.1021/acs.biochem.3c00116 |
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