Inhibitors of Ketohexokinase: Discovery of Pyrimidinopyrimidines with Specific Substitution that Complements the ATP-Binding Site

Abstract

Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC 50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC 50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket.

Specific regions of the brain are capable of fructose metabolism

“…The [fructose] of 1 mM is at the high range of physiological concentration in blood immediately after ingestion of fructose (Topping and Mayes, 1971) and at sufficient concentration to get a signal as well as allow competition with 2.5 mM glucose. The specificity of the pyrimindinopyrimidine KHK inhibitor is high; with a panel of 31 kinases the IC 50 values were less than 800-fold higher than for KHK (Maryanoff et al, 2011). In the reactions using fructose alone, these brain regions oxidized fructose at similar rates as the individually dissected brain regions (see Table 2).…”

“…Given there was no significant difference in oxidative capacity of fructose between the regions, and to increase total oxidative signal (see Table 2), these brain regions were pooled to afford as large a difference between uninhibited and inhibited treatments. To answer the question whether the Fru-1-P pathway has an important role, Fru-1-P-positive-brain regions were used for oxidation assays using 14 C-fructose (1 mM) in four ways: 1) using 14 C-fructose (1 mM) alone to measure overall metabolism, 2) including 2.5 mM glucose to measure any change due to completion with HK using the Fru-6-P pathway, 3) including 1 μM of a pyrimindinopyrimidine inhibitor of KHK to measure any change if the Fru-1-P pathway is blocked (this inhibitor concentration is >80-fold excess of the 0.012 μM IC 50 value determined in vitro and 3-fold in excess of its in vivo IC 50 value in HepG2 cells (Maryanoff et al, 2011)), or 4) using both 2.5 mM glucose and 1 μM the KHK inhibitor, which would measure any metabolism of fructose not blocked by these additions (Fig 7). The [fructose] of 1 mM is at the high range of physiological concentration in blood immediately after ingestion of fructose (Topping and Mayes, 1971) and at sufficient concentration to get a signal as well as allow competition with 2.5 mM glucose.…”

Genome-Wide CRISPR Screen for Essential Cell Growth Mediators in Mutant KRAS Colorectal Cancers

“…We also confirmed that the 2 validation sgRNAs targeted exons 3A or 4 which would knockout KHK-A expression and that the exon 3C-targeting sgRNAs had no effect on the growth of HCT116 MUT xenografts (Figure S2E), consistent with the predominant expression of KHK-A in these cells. To determine the effect of KHK inhibition on xenograft growth, mice were administered a commercially available small molecule KHK inhibitor (Figure 2D and S2F (12)), which binds to the conserved ATP-binding domain present in both KHK isoforms (Figure S2G). This compound significantly inhibited the growth of HCT116 MUT xenografts, but not HCT116 WT xenografts (Figures 2G and 2H), confirming the CRISPR knockdown data and establishing the importance of this enzyme for the growth of KRAS mutant tumors.…”

“…After xenografts were palpable (~7 days after injection), animals were injected intraperitoneally with 100 μL of vehicle, thionicotinamide (Spectrum; 100 mg/kg body weight) in 1% DMSO, or KHK inhibitor (Calbiochem or synthesized according to published methods (12); 25 mg/kg) in PBS. Injections were repeated every other day for 14 days (7 doses total).…”

Recent Progress in Pyrimido[5,4-d]pyrimidine Chemistry

“…In this way, anilino derivatives 103 (96GEP4431867) and, after a second substitution, 105 (97WOP32882) are obtained. The introduction of three different amino groups by three consecutive substitution steps (Scheme 24) is exemplified by the synthesis of PPs 106b (10WOP26262) and 107 (11MCL538). Similar syntheses lead to a 107-analog (12BML5326) and compound 108 (95GEP4325900).…”