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Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy.

Roosild TP, Castronovo S, Fabbiani M, Pizzorno G - BMC Struct. Biol. (2009)

Bottom Line: The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization.The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme.These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Drug Development, Nevada Cancer Institute, Las Vegas, Nevada, USA. troosild@nvcancer.org

ABSTRACT

Background: Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents.

Results: In this report we present the high resolution structures of human uridine phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization.

Conclusion: The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.

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hUPP1 is a dimeric enzyme. In contrast to biophysically analyzed bacterial forms of uridine phosphorylase, human UPP1 is dimeric in solution as analyzed by both calibrated size-exclusion chromatography (blue; x-axis) and multi-angle light scattering analysis (red; y-axis).
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Figure 1: hUPP1 is a dimeric enzyme. In contrast to biophysically analyzed bacterial forms of uridine phosphorylase, human UPP1 is dimeric in solution as analyzed by both calibrated size-exclusion chromatography (blue; x-axis) and multi-angle light scattering analysis (red; y-axis).

Mentions: Recombinantly expressed hUPP1 was noted to possess a smaller than expected quaternary assembly during purification by gel filtration. Analysis by both calibrated size-exclusion chromatography and multi-angle static light scattering (Figure 1) confirmed that the enzyme was dimeric in solution, in contrast to the hexameric assembly predicted from sequence analysis and protein family relationships [7]. Consistent with this observation, both solved crystal forms also yielded dimeric enzymes, verifying that the human form of this enzyme has lost the higher order secondary trimerization of dimers seen in microbial homologues.


Implications of the structure of human uridine phosphorylase 1 on the development of novel inhibitors for improving the therapeutic window of fluoropyrimidine chemotherapy.

Roosild TP, Castronovo S, Fabbiani M, Pizzorno G - BMC Struct. Biol. (2009)

hUPP1 is a dimeric enzyme. In contrast to biophysically analyzed bacterial forms of uridine phosphorylase, human UPP1 is dimeric in solution as analyzed by both calibrated size-exclusion chromatography (blue; x-axis) and multi-angle light scattering analysis (red; y-axis).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664818&req=5

Figure 1: hUPP1 is a dimeric enzyme. In contrast to biophysically analyzed bacterial forms of uridine phosphorylase, human UPP1 is dimeric in solution as analyzed by both calibrated size-exclusion chromatography (blue; x-axis) and multi-angle light scattering analysis (red; y-axis).
Mentions: Recombinantly expressed hUPP1 was noted to possess a smaller than expected quaternary assembly during purification by gel filtration. Analysis by both calibrated size-exclusion chromatography and multi-angle static light scattering (Figure 1) confirmed that the enzyme was dimeric in solution, in contrast to the hexameric assembly predicted from sequence analysis and protein family relationships [7]. Consistent with this observation, both solved crystal forms also yielded dimeric enzymes, verifying that the human form of this enzyme has lost the higher order secondary trimerization of dimers seen in microbial homologues.

Bottom Line: The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization.The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme.These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Drug Development, Nevada Cancer Institute, Las Vegas, Nevada, USA. troosild@nvcancer.org

ABSTRACT

Background: Uridine phosphorylase (UPP) is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. It is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil (5-FU) and its prodrug capecitabine. Additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. However, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. In response to this limitation, specific inhibitors of UPP, such as 5-benzylacyclouridine (BAU), have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-FU and its derivatives, so as to increase the therapeutic index of these agents.

Results: In this report we present the high resolution structures of human uridine phosphorylase 1 (hUPP1) in ligand-free and BAU-inhibited conformations. The structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial UPPs. They also reveal in detail the mechanism by which BAU engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. The observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous UPP structures and may result from the simpler oligomeric organization.

Conclusion: The structural details underlying hUPP1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of BAU and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. Notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hUPP1 than in previously studied microbial homologues. These distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower UPP activity.

Show MeSH
Related in: MedlinePlus