Crystal Structure of Bovine
Purine Nucleoside Phosphorylase in Complex with AT2172 and Sulphate.
Pereira, H.M1; Berdini, V2; Cleasby, A2; Garratt, R.C1
1- Instituto de Física de
São Carlos, USP; 2- Astex Therapeutics, Cambridge, UK.
Purine nucleoside
phosphorylase (PNP, E.C. 2.4.2.1) catalyses the cleavage of the glycosidic bond
of ribo- and deoxyribonucleosides, in the presence of inorganic phosphate (Pi),
to generate the purine base and ribose (deoxyribose)-1-phosphate. Human PNP is
considered an important target for chemotherapeutic intervention since the
discovery that PNP-deficiency is an inherited disease of purine metabolism,
leading to T-cell immune deficiency. Consequently, potent inhibitors of human
PNP are promising selective immunosuppressive agents for suppression of the
host versus graft response in organ transplantation, for treatment of T-cell
leukemias, and also for some T-cell mediated autoimmune diseases. The known
structure of bovine PNP (bovPNP) was used for Virtual Screening (VS) in order
to discover new potential ligands for PNP.
Approximately 30,000 compounds with MW below 280Da were tested. As a
result, 20 compounds were selected for enzymatic assays. One of these compounds
(AT2172) possesses an IC50 of 5 micromolar against bovPNP. In order
to obtain the complex between AT2172 and the enzyme, the crystallization
procedure of Mao et al., (1998) was followed. The ligand (at 5mM) was dissolved
in mother liquor and introduced into the structure by soaking.
BovPNP-AT2172 data was
collected from a single crystal on the 14.2 Station (SRS, Daresbury-UK) up to
2.1Å using a wavelength of 0.98Å. Data processing was performed with Mosflm and
Scala (CCP4) yielding an overall completeness of 97.4% with an Rsym
of 7,8% to 2.1Å, for a total of 14937 unique reflections. Molecular replacement
using the MOLREP program and the uncomplexed bovPNP, resulted in a single
solution (Rfactor 36.1%). Electron density maps were visualized
using the Quanta program. One peak corresponding to the ligand AT2172 was found
in the active site. The ligand was automatically placed using the X-ligand
module of the Quanta program and refinement carried out using both CNX and
REFMAC. Quanta and Coot were used for
model building and map visualization. The current values of R and Rfree
are 16.6 and 21.1% respectively. This complex structure could help in the
development of new inhibitors for human PNP.
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