A Crystal Structure Based Guide to the Design of Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) Activated ProTides

Kimberly M. Maize, Rachit Shah, Alex Strom, Sidath Kumarapperuma, Andrew Zhou, Carston R. Wagner, Barry C. Finzel

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.

Original languageEnglish (US)
Pages (from-to)3987-3997
Number of pages11
JournalMolecular pharmaceutics
Volume14
Issue number11
DOIs
StatePublished - Nov 6 2017

Bibliographical note

Funding Information:
Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. The authors would also like to acknowledge Jay Nix and the Molecular Biology Consortium of ALS Beamline 4.2.2. This research used resources of the Advanced Light Source, which is a DOE Office of Science User Facility under Contract No. DE-AC02-05CH11231.

Funding Information:
Isothermal titration calorimetry was carried out using an ITC-200 microcalorimeter, funded in part by the NIH Shared Instrumentation Grant S10-OD017982. Support for K.M.M was provided by a University of Minnesota Frieda Martha Kunze Fellowship and an American Foundation for Pharmaceutical Education Pre-Doctoral Fellowship. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with Haupt-man−Woodward Medical Research Institute. This research used resources of the Advanced Photon Source, a U.S.

Publisher Copyright:
© 2017 American Chemical Society.

Keywords

  • ProTide
  • hHint1
  • nucleotide
  • phosphoramidate
  • pronucleotide

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