Crystal Structures of Human Choline Kinase Isoforms in Complex with Hemicholinium-3

Abstract

Human choline kinase (ChoK) catalyzes the first reaction in phosphatidylcholine biosynthesis and exists as ChoKα (α1 and α2) and ChoKβ isoforms. Recent studies suggest that ChoK is implicated in tumorigenesis and emerging as an attractive target for anticancer chemotherapy. To extend our understanding of the molecular mechanism of ChoK inhibition, we have determined the high resolution x-ray structures of the ChoKα1 and ChoKβ isoforms in complex with hemicholinium-3 (HC-3), a known inhibitor of ChoK. In both structures, HC-3 bound at the conserved hydrophobic groove on the C-terminal lobe. One of the HC-3 oxazinium rings complexed with ChoKα1 occupied the choline-binding pocket, providing a structural explanation for its inhibitory action. Interestingly, the HC-3 molecule co-crystallized with ChoKβ was phosphorylated in the choline binding site. This phosphorylation, albeit occurring at a very slow rate, was confirmed experimentally by mass spectroscopy and radioactive assays. Detailed kinetic studies revealed that HC-3 is a much more potent inhibitor for ChoKα isoforms (α1 and α2) compared with ChoKβ. Mutational studies based on the structures of both inhibitor-bound ChoK complexes demonstrated that Leu-401 of ChoKα2 (equivalent to Leu-419 of ChoKα1), or the corresponding residue Phe-352 of ChoKβ, which is one of the hydrophobic residues neighboring the active site, influences the plasticity of the HC-3-binding groove, thereby playing a key role in HC-3 sensitivity and phosphorylation.

New non-symmetrical choline kinase inhibitors

“…16 In this crystal structure, ChoKα1 is in complex with both HC-3 and an ADP fragment. During the crystallization of ChoKβ, HC-3 suffered a phosphorylation on the morpholinium moiety inserted into the choline binding site, and the obtained crystal structures of the complex is constituted by ChoKβ, ADP, and phosphohemicolinium-3 (PHC-3).…”

“…During the crystallization of ChoKβ, HC-3 suffered a phosphorylation on the morpholinium moiety inserted into the choline binding site, and the obtained crystal structures of the complex is constituted by ChoKβ, ADP, and phosphohemicolinium-3 (PHC-3). 16 Human choline kinase ChoKα1 (PDB id: 3G15) was chosen for the docking studies due to the following reasons: i) The above described biological data and other new publication 17 indicate that ChoKα, and not ChoKβ, is the appropriate molecular target for the design of new anticancer drugs; and, ii) In this crystal structure, ChoKα1 is co-crystalized with both ADP and HC-3, being more appropriated for docking studies in both binding sites.…”

Choline kinase alpha—Putting the ChoK-hold on tumor metabolism

“…4) [50]. Reports generally agree on K m values for choline between 100 and 180 µM, K m values for ATP between 410 and 760 µM, and k cat values for the enzyme of 69–83 s −1 [49–51]. …”

“…However, when half-inhibitor fragments were found to be poor inhibitors and highly toxic, it was proposed that the dimeric form of ChoKα must allow each di-cationic inhibitor head-group to interact with a unique catalytic site [93]. With the crystal structures of inhibitors docked at the ChoKα active site having since been elucidated, the distance between catalytic units is now known to be too great for these inhibitors to span between bound ChoKα dimers, although the possibility that the unbound cationic head-group can bind to the choline-binding site of a free ChoKα monomer or to the ATP-binding site on the same protein was never excluded [51]. …”

Novel 4‐Amino Bis‐pyridinium and Bis‐quinolinium Derivatives as Choline Kinase Inhibitors with Antiproliferative Activity against the Human Breast Cancer SKBR‐3 Cell Line

“…Although it is not approved for clinical use, HC-3 has served as the lead compound for the development of ChoK inhibitors with improved potency and selectivity. [11] The number and distribution of the positive charges present in these molecules may affect their intracellular location and influence their activity or toxicity, as has been reported for porphyrins. Each of the oxazinium rings has a positively charged quaternary ammonium and a hydroxy group.…”