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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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.</p> <p>Conclusion</p> <p>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.</p

Characterization of the family of Mistic homologues

BACKGROUND: Mistic is a unique Bacillus subtilis protein with virtually no detectable homologues in GenBank, which appears to integrate into the bacterial membrane despite an overall hydrophilic composition. These unusual properties have been shown to be useful for high-yield recombinant expression of other membrane proteins through fusion to the C-terminus of Mistic. To better understand the structure and function of Mistic, we systematically searched for and characterized homologous proteins among closely related bacteria. RESULTS: Three homologues of Mistic were found with 62% to 93% residue identity, all only 84 residues in length, corresponding to the C-terminal residues of B. subtilis Mistic. In every case, the Mistic gene was found partially overlapping a downstream gene for a K(+ )channel protein. Residue variation amongst these sequences is restricted to loop regions of the protein's structure, suggesting that secondary structure elements and overall fold have been conserved. Additionally, all three homologues retain the functional ability to chaperone fusion partners to the membrane. CONCLUSION: The functional core of Mistic consists of 84 moderately conserved residues that are sufficient for membrane targeting and integration. Understanding the minimal structural and chemical complexity of Mistic will lead to insights into the mechanistic underpinnings of Mistic-chaperoned membrane integration, as well as how to optimize its use for the recombinant heterologous expression of other integral membrane proteins of interest

Active Site Conformational Dynamics in Human Uridine Phosphorylase 1

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 Å resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an “induced-fit” association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications

5-Fluorouracil binding to hUPP1.

<p>(<b>A</b>) 5-FU is coordinated by residues restricted to the individual monomers of the hUPP1 dimer, in contrast to the binding of BAU that traverses the dimer interface. As expected, Gln217 and Arg219, the key uridine-discriminating residues, form multiple hydrogen bonds with one face of the uracil base. This face also includes a well-coordinated, buried water molecule that associates with 5-FU and creates stabilizing bonds with both Gln217 and Arg275. Additional favourable interactions may be formed by both the backbone carbonyl and side chain hydroxyl groups of Thr141, although the geometry observed in the crystal structure is not consistent with hydrogen bonding. The fluorine moiety resides in a hydrophobic pocket created by Leu272, Leu273 and Ile281, and forms a hydrogen bond with Ser142. Electron density from a 2F_o-F_c map contoured at 1.5σ is shown for the ligand (blue wire). (<b>B</b>) Surface representation from the same perspective emphasizes the depth and fit of the active site for the pyrimidine substrate. The position of Phe213, which was omitted from (A) for clarity, is also illustrated. This residue caps the active site and forms hydrophobic, herringbone stacking interactions with the uracil ring. (<b>C</b>) Schematic map of the contacts between hUPP1 and 5-FU as analyzed by LigPlot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012741#pone.0012741-Wallace1" target="_blank">[37]</a>.</p

Inter-domain flexibility of hUPP1.

<p>Illustration highlights conformational changes at the dimer interface proximate to the active site, overlaying the 5-FU-bound structure (gold), the BAU-bound structure (orange), and ligand-free structure (red). Despite a lack of molecular contacts between residues from the partnering subunit and the 5-FU ligand, the critical residues for binding natural substrates adopt conformations close to those seen in the BAU-bound structure, where they are stabilized by the formation of favourable molecular interactions, and not the conformations revealed in the ligand-free structure. The location of the phosphate ion from the BAU-bound structure is shown for orientation, but not found to be occupied in the 5-FU-bound structure.</p

Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins