Crystal Structure of Pyridoxal Kinase in Complex with Roscovitine and Derivatives

Abstract

Pyridoxal kinase (PDXK) catalyzes the phosphorylation of pyridoxal, pyridoxamine, and pyridoxine in the presence of ATP and Zn 2؉ . This constitutes an essential step in the synthesis of pyridoxal 5-phosphate (PLP), the active form of vitamin B 6 , 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, N 6 -methyl-(R)-roscovitine, and O 6 -(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.

A phase I trial of the selective oral cyclin-dependent kinase inhibitor seliciclib (CYC202; R-Roscovitine), administered twice daily for 7 days every 21 days

“…Preclinical toxicology studies with 28 days treatment in rodents reported anaemia, leucocytosis, bone marrow hyperplasia, polydipsia, polyuria and gastric ulceration. Preclinical data indicated that exposure to seliciclib in the concentration range 7.9 -30.2 mM over a 24-h period could achieve clinically relevant biological effects (De la Motte and Gianella-Borradori, 2004;Whittaker et al, 2004;Tang et al, 2005). In vivo studies also indicated that a single dose of 500 mg kg À1 (2750 mg m À2 of mouse body surface area) achieved levels of Z10 mM for 24 h .…”

N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine

“…Chemistry (R)-roscovitine was synthesized as described previously (12,26) and kindly provided by Dr. Hervé Galons and Nassima Oumata (Université René Descartes).…”

“…Its selectivity has been extensively studied using either panels of purified kinases (z152 kinases tested; refs. 23 -25), affinity chromatography on immobilized roscovitine (25,26), a quantitative competition assay (27), and a yeast three-hybrid screen (28). Altogether, these methods have provided a rather precise view on the targets of (R)-roscovitine.…”

“…Affinity chromatography revealed an unexpected interacting protein, pyridoxal kinase (PDXK; refs. 25,26). The pharmacology of (R)-roscovitine has been extensively studied in mice, rat, and human (reviewed in ref.…”

Recombinant expression, purification and characterization of Bombyx mori (Lepidoptera: Bombycidae) pyridoxal kinase

“…Several mammalian PLKs have been successfully expressed in Escherichia coli and the expressed recombinant proteins exhibit the same biochemical characteristics as those of the native protein purified from various mammalian tissues (Gao et al, 1998;Lee et al, 2000;Di Salvo et al, 2004). Recently, the three-dimensional structures of PLKs from bacteria and mammals, alone and in complex with various ligands were determined, providing a better understanding of the catalytic mechanism of PLK (Li et al, 2002(Li et al, , 2004Safo et al, 2004Safo et al, , 2006Tang et al, 2005;Cao et al, 2006). The active sites are structurally very similar among members of the ribokinase superfamily .…”