Structural characterization of new deoxycytidine kinase inhibitors rationalizes the affinity-determining moieties of the molecules

Abstract

Deoxycytidine kinase (dCK) is a key enzyme in the nucleoside salvage pathway that is also required for the activation of several anticancer and antiviral nucleoside analog prodrugs. Additionally, dCK has been implicated in immune disorders and has been found to be overexpressed in several cancers. To allow the probing and modulation of dCK activity, a new class of small-molecule inhibitors of the enzyme were developed. Here, the structural characterization of four of these inhibitors in complex with human dCK is presented. The structures reveal that the compounds occupy the nucleoside-binding site and bind to the open form of dCK. Surprisingly, a slight variation in the nature of the substituent at the 5-position of the thiazole ring governs whether the active site of the enzyme is occupied by one or two inhibitor molecules. Moreover, this substituent plays a critical role in determining the affinity, improving it from >700 to 1.5 nM in the best binder. These structures lay the groundwork for future modifications that would result in even tighter binding and the correct placement of moieties that confer favorable pharmacodynamics and pharmacokinetic properties.

Human Biodistribution and Radiation Dosimetry of 18F-Clofarabine, a PET Probe Targeting the Deoxyribonucleoside Salvage Pathway

“…Moreover, dCK provides an escape mechanism when DNA de novo synthesis is inhibited by ribonucleotide reductase inhibitors (6). This resistance can be overcome using small-molecule inhibitors of dCK (6)(7)(8)(9). dCK is also required to phosphorylate and activate inactive prodrugs such as cytarabine, gemcitabine, decitabine, and cladribine (10).…”

Dual protein kinase and nucleoside kinase modulators for rationally designed polypharmacology

“…Additionally, for each ligand-bound structure, a supplementary electron density was present at the top of the cavity (Supplementary Fig. 11 ), likely corresponding to a second molecule of compound interacting weakly with the first one in a similar binding mode as the ‘F-series’ inhibitors reported previously 38 . Moreover, it should be noted that masitinib and imatinib were able to open dCK in situ, since we were able to obtain these 3D structures in the ‘open’ conformation by soaking the protein kinase inhibitors in a ‘closed’ dCK crystal.…”

Structure-Guided Development of Deoxycytidine Kinase Inhibitors with Nanomolar Affinity and Improved Metabolic Stability

“…In contrast, the tighter binding propyl-containing molecules were observed to bind with a single inhibitor molecule, at position 1, per dCK active site. 9 This revealed that binding of two molecules is not required for high affinity. In our previous report, we analyzed the implication of single versus double binding of inhibitor molecules to dCK and concluded that inhibition of dCK is primarily caused by the binding of the inhibitor at position 1, whereas the molecule bound at position 2 does not appreciably enhance the inhibition.…”

“…In previous work we demonstrated that the nature of the substituent at the thiazole ring 5-position (part C of the molecule, Figure 1 A) plays a crucial role in binding affinity. 9 In short, we compared having no substituent at that position to having a methyl, ethyl, or propyl. We found that propyl dramatically improved the binding affinity, and as a result, compounds with a propyl at the 5-position became our lead compounds (i.e., compounds 1 and 2 , Figure 1 ).…”

“…Protein expression and purification were performed exactly as described by us. 9 In brief, we used the S74E-C4S-dCK variant, which is the human dCK protein where four solvent-exposed cysteines are mutated into serines (C4S). We showed that the C4S mutant generates better quality crystals without altering the three-dimensional conformation of the enzyme or its enzymatic activity.…”