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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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Ligand mediated inter-domain motion of hUPP1. While the structural conformations of monomers of hUPP1 vary only subtly between BAU-bound and ligand-free forms, there is substantial movement between the two domains upon inhibitor binding, as illustrated here. In the presence of BAU, phosphate coordination is also promoted within the substrate pocket, despite its absence in purification or crystallization solutions. The presence of both molecules within the ligand pocket causes the enzyme to dramatically close the active site, resulting in backbone carbon motion of 3–5 Å and individual residue repositioning of over twice that magnitude. The open state seen for the ligand-free structure of hUPP1 has not been previously observed in bacterial homologues, possibly because the hexameric assembly of these microbial enzymes restricts their range of inter-domain motion.
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Figure 5: Ligand mediated inter-domain motion of hUPP1. While the structural conformations of monomers of hUPP1 vary only subtly between BAU-bound and ligand-free forms, there is substantial movement between the two domains upon inhibitor binding, as illustrated here. In the presence of BAU, phosphate coordination is also promoted within the substrate pocket, despite its absence in purification or crystallization solutions. The presence of both molecules within the ligand pocket causes the enzyme to dramatically close the active site, resulting in backbone carbon motion of 3–5 Å and individual residue repositioning of over twice that magnitude. The open state seen for the ligand-free structure of hUPP1 has not been previously observed in bacterial homologues, possibly because the hexameric assembly of these microbial enzymes restricts their range of inter-domain motion.

Mentions: Determination of both ligand-free and BAU-bound forms of hUPP1 allows direct analysis of the conformational changes induced in the enzyme upon substrate binding. To this end, there are only subtle differences in the fold and residue orientation between overlaid monomers of hUPP1. However, there is a dramatic inter-domain motion between monomers leading to 3–5 Å changes in the relative positioning of the loops undergoing the greatest movement (Figure 5). This hinge-type conformational change results in a closure of the active site around its substrates and appears to be driven by the formation of interactions between Arg94 and phosphate, His36 and the ribose sugar group, and Tyr35 and the nucleobase. It is noteworthy that this domain motion has not been observed in any of the previously determined microbial structures despite multiple ligand-free structures, suggesting that the human enzyme is more mobile than its bacterial 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)

Ligand mediated inter-domain motion of hUPP1. While the structural conformations of monomers of hUPP1 vary only subtly between BAU-bound and ligand-free forms, there is substantial movement between the two domains upon inhibitor binding, as illustrated here. In the presence of BAU, phosphate coordination is also promoted within the substrate pocket, despite its absence in purification or crystallization solutions. The presence of both molecules within the ligand pocket causes the enzyme to dramatically close the active site, resulting in backbone carbon motion of 3–5 Å and individual residue repositioning of over twice that magnitude. The open state seen for the ligand-free structure of hUPP1 has not been previously observed in bacterial homologues, possibly because the hexameric assembly of these microbial enzymes restricts their range of inter-domain motion.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Ligand mediated inter-domain motion of hUPP1. While the structural conformations of monomers of hUPP1 vary only subtly between BAU-bound and ligand-free forms, there is substantial movement between the two domains upon inhibitor binding, as illustrated here. In the presence of BAU, phosphate coordination is also promoted within the substrate pocket, despite its absence in purification or crystallization solutions. The presence of both molecules within the ligand pocket causes the enzyme to dramatically close the active site, resulting in backbone carbon motion of 3–5 Å and individual residue repositioning of over twice that magnitude. The open state seen for the ligand-free structure of hUPP1 has not been previously observed in bacterial homologues, possibly because the hexameric assembly of these microbial enzymes restricts their range of inter-domain motion.
Mentions: Determination of both ligand-free and BAU-bound forms of hUPP1 allows direct analysis of the conformational changes induced in the enzyme upon substrate binding. To this end, there are only subtle differences in the fold and residue orientation between overlaid monomers of hUPP1. However, there is a dramatic inter-domain motion between monomers leading to 3–5 Å changes in the relative positioning of the loops undergoing the greatest movement (Figure 5). This hinge-type conformational change results in a closure of the active site around its substrates and appears to be driven by the formation of interactions between Arg94 and phosphate, His36 and the ribose sugar group, and Tyr35 and the nucleobase. It is noteworthy that this domain motion has not been observed in any of the previously determined microbial structures despite multiple ligand-free structures, suggesting that the human enzyme is more mobile than its bacterial 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