Development of Novel IP6K Inhibitors for the Treatment of Obesity and Obesity-Induced Metabolic Dysfunctions

Abstract

Obesity and obesity-induced metabolic dysfunctions are significant risk factors for nonalcoholic fatty liver disease and cardiovascular diseases. Thus, obesity is an economic and social burden in developed countries. Blocking the synthesis of inositol pyrophosphates by inositol hexakisphosphate kinase (IP6K) has been identified as a potential therapeutic strategy for obesity and related diseases. We have developed a novel and potent IP6K inhibitor 20 (UNC7467) (IC₅₀ values: IP6K1 8.9 nM; IP6K2 4.9 nM; IP6K3 1320 nM). Inositol phosphate profiling of the HCT116 colon cancer cell line demonstrates that 20 reduced levels of inositol pyrophosphates by 66-81%, without significantly perturbing levels of other inositol phosphates. Furthermore, intraperitoneal injection of 20 in diet-induced obese mice improved glycemic profiles, ameliorated hepatic steatosis, and reduced weight gain without altering food intake. Thus, inhibitor 20 can be used as an in vivo probe for IP6K-related research. Moreover, it may have therapeutic relevance in treating obesity and related diseases.

Figure 1.

Examplars for IP6K inhibitors.

Figure 2.

Application of an enzyme coupled assay to assay the potency of TNP against IP6K1. (A) Chair conformations of InsP₆ and 5-InsP₇ in a graphical representation of the coupled enzyme assay. Released Pi was assayed using Malachite Green (see Experimental Section). (B) Application of this assay to determine the dose/response relationship for inhibition of recombinant IP6K1 by TNP. Data represent mean values and standard errors from three independent experiments. The IC₅₀ value for IP6K1 is 1.0 μM. Similar experiments yielded IC₅₀ data of 2.0 and 14.7 μM for IP6K2 and IP6K3, respectively.

Figure 3.

Effects upon IP6Ks of 10 nM of six key inhibitors. These assays were performed by HPLC analysis of [³H]-InsP₆ phosphorylation as described in the Experimental Section. Data represent mean values and standard errors from three to six independent experiments.

Figure 4.

Interactions of 20 with IP6Ks. Dose/response relationships for inhibition by 20 of (A) IP6K1, (B) IP6K2, and (C) IP6K3 by HPLC analysis of [³H]-InsP₆ phosphorylation. Data represent mean values and standard errors from either three or four independent experiments. (D) Thermogram (“DP” = differential power) for ITC analysis of the interaction of 20 with IP6K2. (E) Corresponding Wiseman plot. The ITC data are from a representative experiment, typical of three.

Figure 5.

Evaluation of specificity and potency of 20. (A) Representative HPLC analysis of InsP₅, InsP₆, InsP₇, and InsP₈ in HCT116 cells after a 3 h treatment with either vehicle (black symbols) or 2.5 μM 20 (red). (B) Mean and standard errors from three independent experiments, performed as in panel A. *, p < 0.05; **, p < 0.02. (C) Assay of InsP₇ kinase activity of PPIP5K2 in the presence of either 2.5 μM 20 (red bar) or vehicle control (black bar). (D, E) Dose/dependent effects of 3 and 18 h treatment, respectively, of 20 upon [³³]P-Pi efflux from HCT116 cells. The broken lines indicate the 30% of total [³³]P-Pi efflux that is not regulated by InsP₈. Data in panels C, D, and E represent the mean values and standard errors from either three or four independent experiments.

Figure 6.

Treatment of DIO mice with 20 reduced body fat and restored insulin sensitivity. (A, B) Total body weight and percent changes in body weight. (C, D) Fat and lean mass before and after treatment. (E) Weight of EWAT and IWAT. (F, G) ITT after 1-week. (H, I) GTT after 3 weeks. (J) Serum insulin levels in vehicle- and 20-treated mice. (K, L) PTT after 4 weeks. (M, N) Liver weight and hepatic steatosis in vehicle- and 20-treated mice.

Scheme 1.. Synthesis of Compound 6

Scheme 2.. Representative Synthetic Routes for Analog Synthesis^a

^a (A) Analogs 22 (general procedure D) and 23 (general procedure E). (B) Analogs 26 (general procedure F) and 27 (general procedure G). (C) Analog 31 (general procedure I).