Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design

Abstract

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

Thymidine Phosphorylase in Cancer; Enemy or Friend?

“…TP is a homodimer, consisting of two identical subunits that are non-covalently associated with a dimeric molecular mass of 51 kD [4,9]. Each subunit is composed of a small α-helical domain that contains the thymidine-binding site and a large α/β domain that contains the phosphate binding site [10]. High levels of TP are expressed in macrophages, stromal cells, glial cells and some epithelia [11].…”

Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule “crystallization chaperone” 5′-O-tritylinosine (KIN59)

“…5 0 -O-Tritylinosine is a rather lipophilic compound possessing a bulky trityl moiety for which no electron density emerged in the diffraction maps. However, the molecule clearly helped stabilize the conformation of this loop in the crystal lattice [23]. We reasoned this might be due to ligand (5 0 -O-tritylinosine) expulsion following protein packing and local hydrophobic collapse.…”

“…Inspection of the Gly-405-Val-419 loop (which was clipped in the structure of Norman et al [22]), in the crystallographic structure of El Omari et al [23], revealed an intricate hydrogenbonding network, the most relevant part of which is schematically represented in Fig. 2: (i) the backbone NH of Arg-408 is hydrogenbonded to a well-defined water molecule (W98 in subunit A, designated WAT1) that is fixed in place by two additional hydrogen bonds: one with the backbone carbonyl of Gly-149 and another with one carboxylate oxygen from Asp-203, a residue that is situated at the terminus of a-helix 9, (ii) the same carboxylate oxygen of Asp-203 forms a good hydrogen bond with the backbone NH of Gly-149, which is next to Leu-148, a residue whose sidechain provides a hydrophobic surface for interaction with the pyrimidine base of both the dThd substrate and competitive inhibitors such as TPI, (iii) the guanidinium group of Arg-408 establishes two strong hydrogen bonds with the carboxylate oxygens of Asp-156 (not shown) and also a water-mediated (W80 in subunit B, designated WAT2) hydrogen bond with the other Asp-203 carboxylate oxygen, (iv) the carbonyl groups of the backbones of Asp-203 and Arg-408 are both accepting a hydrogen bond from a common water molecule (designated WAT3); (v) the guanidinium group of Arg-410 is partly exposed to the solvent but also engaged in an intersubunit hydrogen bond with the carbonyl group of Ser-65 (not shown); and (vi) the backbone CO and NH groups of Ala-411 in one protomer establish two good hydrogen bonds with the side-chain carboxamide of Gln-67 in the neighboring subunit (not shown).…”

“…The structure was solved at 2.7 Å resolution, revealing a single protomer in the asymmetric unit, but the polypeptide stretch encompassing amino acids 407-414 could not be determined. In contrast, no proteolysis was required and well-ordered crystals with diffraction at 2.3 Å resolution were obtained when both thymidine (2 mM) and 5 0 -O-tritylinosine (1 mM) were present under the crystallization conditions [23]. In this latter structure, two dimers appeared in the asymmetric unit, and residues 406-415 were visible as an arginine-rich surface loop that extends across the active site cleft and appears to stabilize the closed conformation of the enzyme and the dimer interface through hydrogen bonds.…”

The Thymidine Phosphorylase Inhibitor 5′-O-Tritylinosine (KIN59) Is an Antiangiogenic Multitarget Fibroblast Growth Factor-2 Antagonist

“…Unlike all previously described thymidine phosphorylase inhibitors, KIN59 does not interact with the thymidine-or phosphate-binding site of thymidine phosphorylase, indicating that the compound binds to an allosteric site in the enzyme. Computational studies on the thymidine phosphorylase/KIN59 complex identified a possible allosteric site involving amino acid residue Asp203 of human thymidine phosphorylase (45). At variance with thymidine phosphorylase, FGF2 is an extracellular growth factor whose biologic activity is mediated by the activation of tyrosine kinase FGFRs on the cell surface (13,46).…”