Abstract
Pyridoxal kinase (PDXK) catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine in the presence of ATP and Zn2+. This constitutes an essential step in the synthesis of pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, a cofactor for over 140 enzymes. (R)-Roscovitine (CYC202, Seliciclib) is a relatively selective inhibitor of cyclin-dependent kinases (CDKs), currently evaluated for the treatment of cancers, neurodegenerative disorders, renal diseases, and several viral infections. Affinity chromatography investigations have shown that (R)-roscovitine also interacts with PDXK. To understand this interaction, we determined the crystal structure of PDXK in complex with (R)-roscovitine, N6-methyl-(R)-roscovitine, and O6-(R)-roscovitine, the two latter derivatives being designed to bind to PDXK but not to CDKs. Structural analysis revealed that these three roscovitines bind similarly in the pyridoxal-binding site of PDXK rather than in the anticipated ATP-binding site. The pyridoxal pocket has thus an unexpected ability to accommodate molecules different from and larger than pyridoxal. This work provides detailed structural information on the interactions between PDXK and roscovitine and analogs. It could also aid in the design of roscovitine derivatives displaying strict selectivity for either PDXK or CDKs.
Highlights
Pyridoxal kinase (PDXK) catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine in the presence of ATP and Zn2؉
Roscovitine exists as two stereoisomers, (R)-roscovitine and (S)-roscovitine, the (R)-roscovitine isomer being slightly more active on cyclin-dependent kinases (CDKs) [21,22,23]. (R)-Roscovitine has been selected, under the name CYC202, for preclinical and clinical evaluations [24, 25]. (R)-Roscovitine has been crystallized with two protein kinases, CDK2 [21] and CDK5 [26]
Similar to the PDXK/AMP-PCP/pyridoxamine crystal, the PDXK1⁄7(R)-roscovitine complex structure belongs to the P3121 space group, with the complex having one monomer in the asymmetric unit
Summary
NMR spectra were recorded on a Bruker 400 MHz spectrometer. Structural assignments were achieved by one- and two-dimensional methods. The solvent was removed under vacuum, and the residue was purified by HPLC (C18 column, eluted with CH3CN/H2O with 0.05% trifluoroacetic acid) to afford benzyl-(2-chloro-9-isopropyl-9H-purin-6-yl)-methyl-amine [1] as a white solid (64 mg, 94%). The solvent was removed under vacuum, and the residue was purified by column chromatography (silica gel, eluted with ethyl acetate with 10% methanol) to afford benzyl-(2-chloro-9-isopropyl-9H-purin-6-yl)-methylamine [14] as a white solid (0.66 mg, 94%); m/z [Mϩ ϩ 1] 360.1. To a solution of linker-attached N6-methyl-(R)-roscovitine azide [16] (70 mg, 0.10 mmol) in methanol (5 ml) was added Pd/C (10%, 50 mg) It was stirred at room temperature under hydrogen atmosphere for 10 min, and the catalyst was removed by filtration. The spectra was 1H NMR (Me2SO-d6), ␦ 0.84 – 0.89 (m, 3H), 1.45–1.50 (m, 7H), 1.59 –1.62 (m, 1H), 2.95–3.00 (m, 2H), 3.40 –3.60 (m, 29H), 3.78 –3.85 (m, 1H), 4.55– 4.61 (m, 1H), 7.35 (d, 2H, J ϭ 8.0 Hz), 7.73 (b, 3H), 7.79 (d, 2H, J ϭ 8.0 Hz), 7.91 (s, 1H), 8.45 (s, 1H); m/z [Mϩ ϩ 1] 675.1
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
