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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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BAU binding to hUPP1. (A) BAU and phosphate coordination in hUPP1 is consistent with that previously observed in bacterial homologues with the notable exception that R64 (equivalent to R30 in E. coli) does not participate in phosphate binding, possessing instead a conformation that distances its guanidinium group from the negatively-charged oxygen atoms of the anion. (B) Shown is an alignment of three conformations of the E. coli enzyme with the two hUPP1 structures, focusing on the loop toward the back of the active site (human residues 278–284; boxed in Figure 2C). It has been hypothesized that in the bacterial enzyme, this loop undergoes an induced-fit conformational change upon ligand binding. In hUPP1, these residues are more ordered and conformationally restrained. They do not vary significantly in position between the BAU-bound and ligand-free structures, possibly due to the insertion of two additional residues within this region. These differences become increasingly important when considering the impacts of chemical modifications to the initial benzyl moiety of BAU, such as the characterized UPP inhibitor, 5-m-benzyloxybenzylacyclouridine (BBAU) shown (shaded).
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Figure 4: BAU binding to hUPP1. (A) BAU and phosphate coordination in hUPP1 is consistent with that previously observed in bacterial homologues with the notable exception that R64 (equivalent to R30 in E. coli) does not participate in phosphate binding, possessing instead a conformation that distances its guanidinium group from the negatively-charged oxygen atoms of the anion. (B) Shown is an alignment of three conformations of the E. coli enzyme with the two hUPP1 structures, focusing on the loop toward the back of the active site (human residues 278–284; boxed in Figure 2C). It has been hypothesized that in the bacterial enzyme, this loop undergoes an induced-fit conformational change upon ligand binding. In hUPP1, these residues are more ordered and conformationally restrained. They do not vary significantly in position between the BAU-bound and ligand-free structures, possibly due to the insertion of two additional residues within this region. These differences become increasingly important when considering the impacts of chemical modifications to the initial benzyl moiety of BAU, such as the characterized UPP inhibitor, 5-m-benzyloxybenzylacyclouridine (BBAU) shown (shaded).

Mentions: Analysis of the catalytic active site reveals that BAU binds hUPP1 in a manner consistent with that previously characterized using EcUPP [7]. In actuality, all residues interacting with the natural uridine and phosphate ligands are strictly conserved between the two enzymes (Figure 2C). This fact was unclear prior to this study, as regions of these proteins between β2 and β3 lack sufficient sequence similarity for accurate alignment in the absence of additional structural data. Despite strict identity retention in ligand-binding residues, the coordination of the phosphate ion deviates from that observed in previous microbial structures (Figure 4A). Specifically, one of the three pocket arginine residues (Arg64 in hUPP1) is bent away from the anion, leaving only two coordinating guanidinium groups, one from each subunit of the dimer, to bind the substrate. The reason for the unexpected conformation of this arginine is not immediately apparent, as the backbone Cα of this residue is in an equivalent location with respect to the phosphate molecule as in other structurally analyzed UPPs. However, in the ligand-free structure of hUPP1 this residue is found to coordinate a sulfate ion from the crystallization liquor at a position 4.4 Å removed from the catalytic phosphate binding pocket. This finding may imply that in the human enzyme this residue has been adapted to ligand recruitment from the cytoplasm rather than ligand binding in the active site.


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)

BAU binding to hUPP1. (A) BAU and phosphate coordination in hUPP1 is consistent with that previously observed in bacterial homologues with the notable exception that R64 (equivalent to R30 in E. coli) does not participate in phosphate binding, possessing instead a conformation that distances its guanidinium group from the negatively-charged oxygen atoms of the anion. (B) Shown is an alignment of three conformations of the E. coli enzyme with the two hUPP1 structures, focusing on the loop toward the back of the active site (human residues 278–284; boxed in Figure 2C). It has been hypothesized that in the bacterial enzyme, this loop undergoes an induced-fit conformational change upon ligand binding. In hUPP1, these residues are more ordered and conformationally restrained. They do not vary significantly in position between the BAU-bound and ligand-free structures, possibly due to the insertion of two additional residues within this region. These differences become increasingly important when considering the impacts of chemical modifications to the initial benzyl moiety of BAU, such as the characterized UPP inhibitor, 5-m-benzyloxybenzylacyclouridine (BBAU) shown (shaded).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: BAU binding to hUPP1. (A) BAU and phosphate coordination in hUPP1 is consistent with that previously observed in bacterial homologues with the notable exception that R64 (equivalent to R30 in E. coli) does not participate in phosphate binding, possessing instead a conformation that distances its guanidinium group from the negatively-charged oxygen atoms of the anion. (B) Shown is an alignment of three conformations of the E. coli enzyme with the two hUPP1 structures, focusing on the loop toward the back of the active site (human residues 278–284; boxed in Figure 2C). It has been hypothesized that in the bacterial enzyme, this loop undergoes an induced-fit conformational change upon ligand binding. In hUPP1, these residues are more ordered and conformationally restrained. They do not vary significantly in position between the BAU-bound and ligand-free structures, possibly due to the insertion of two additional residues within this region. These differences become increasingly important when considering the impacts of chemical modifications to the initial benzyl moiety of BAU, such as the characterized UPP inhibitor, 5-m-benzyloxybenzylacyclouridine (BBAU) shown (shaded).
Mentions: Analysis of the catalytic active site reveals that BAU binds hUPP1 in a manner consistent with that previously characterized using EcUPP [7]. In actuality, all residues interacting with the natural uridine and phosphate ligands are strictly conserved between the two enzymes (Figure 2C). This fact was unclear prior to this study, as regions of these proteins between β2 and β3 lack sufficient sequence similarity for accurate alignment in the absence of additional structural data. Despite strict identity retention in ligand-binding residues, the coordination of the phosphate ion deviates from that observed in previous microbial structures (Figure 4A). Specifically, one of the three pocket arginine residues (Arg64 in hUPP1) is bent away from the anion, leaving only two coordinating guanidinium groups, one from each subunit of the dimer, to bind the substrate. The reason for the unexpected conformation of this arginine is not immediately apparent, as the backbone Cα of this residue is in an equivalent location with respect to the phosphate molecule as in other structurally analyzed UPPs. However, in the ligand-free structure of hUPP1 this residue is found to coordinate a sulfate ion from the crystallization liquor at a position 4.4 Å removed from the catalytic phosphate binding pocket. This finding may imply that in the human enzyme this residue has been adapted to ligand recruitment from the cytoplasm rather than ligand binding in the active site.

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