TNP and its analogs: Modulation of IP6K and CYP3A4 inhibition

Abstract

Inositol hexakisphosphate kinase (IP6K) is an important mammalian enzyme involved in various biological processes such as insulin signalling and blood clotting. Recent analyses on drug metabolism and pharmacokinetic properties on TNP (N²-(m-trifluorobenzyl), N⁶-(p-nitrobenzyl)purine), a pan-IP6K inhibitor, have suggested that it may inhibit cytochrome P450 (CYP450) enzymes and induce unwanted drug-drug interactions in the liver. In this study, we confirmed that TNP inhibits CYP3A4 in type I binding mode more selectively than the other CYP450 isoforms. In an effort to find novel purine-based IP6K inhibitors with minimal CYP3A4 inhibition, we designed and synthesised 15 TNP analogs. Structure-activity relationship and biochemical studies, including ADP-Glo kinase assay and quantification of cell-based IP7 production, showed that compound 9 dramatically reduced CYP3A4 inhibition while retaining IP6K-inhibitory activity. Compound 9 can be a tool molecule for structural optimisation of purine-based IP6K inhibitors.

Keywords: Inositol hexakisphosphate kinase; cytochrome P450 3A4; structure-activity relationship.

Figure 1.

Chemical structure of TNP.

Figure 2.

The selective inhibitory effect of TNP on CYP3A4. (A) Screening the activities of human microsomal CYP450 isoforms. The CYP450 metabolic capacities were measured by LC-MS/MS and isoform-specific substrates (phenacetin for CYP1A2, chlorzoxazone for 2E1, midazolam [M] for 3A4, and testosterone [T] for 3A4). (B) In vitro analysis of recombinant microsomal CYP3A4 against TNP or ketoconazole (KCZ) treatment with designated concentrations on graphs. Values in all graphs are presented as mean ± SEM.

Figure 3.

Interaction of CYP3A4 and TNP. (A) Type I spectral shift induced by TNP interaction with CYP3A4. (B) Determination of TNP dissociation constant.

Scheme 1.

Synthesis of purine-based TNP analogs. Reagents and conditions: (a) appropriate benzylamines (1.1 eq), Et₃N (1.1 eq), DMF, 100 °C, 8 h; (b) appropriate benzylamines (5.0 eq), n-butanol, NaBF₄ (1.5 eq), 180 °C.

Figure 4.

The cellular effects of three compounds (9, 15, and 20) on IP7 synthesis and Akt activation in the HCT116 cell line. (A–C) HPLC analysis of cellular IPs (IP5, IP6, and IP7) and IP7/IP6 ratio from radiolabeled HCT116. The effects of the tested compounds on (A) IP7 quantification and (B) IP7/IP6 ratio of three compounds and TNP. (C) The inhibitory effect of compound 9 on IP7 synthesis. (D) Akt activation (phosphorylation on Thr308) was analysed by Western blotting with HCT116 cell extracts. Representative immunoblot with the relative levels of pAkt/Akt (an arrowhead) using Image J is shown. All experiments are prepared from HCT116 treated with DMSO, TNP (10 μM), and the selected compounds (50 μM) for 4 h. All values are presented as mean ± SEM. Student's t test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

The best-docked pose of compound 9 with an IP6K homology model. The dotted lines represent hydrogen-bonding interactions.