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Identification of a new selective chemical inhibitor of mutant isocitrate dehydrogenase-1.

Kim HJ, Choi BY, Keum YS - J Cancer Prev (2015)

Bottom Line: We have found that IDH1, but not IDH1-R132H, can catalyze the conversion of isocitrate into α-ketoglutarate (α-KG).We have observed an underlying biochemical mechanism explaining how a heterozygous IDH1 mutation contributes to the generation of R-2HG and increases cellular histone H3 trimethylation levels.We have also identified a novel selective IDH1-R132H chemical hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one, which could be used for a future lead development against IDH1-R132H.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Dongguk University, Goyang, Seowon University, Cheongju, Korea.

ABSTRACT

Background: Recent genome-wide sequencing studies have identified unexpected genetic alterations in cancer. In particular, missense mutations in isocitrate dehydrogenase-1 (IDH1) at arginine 132, mostly substituted into histidine (IDH1-R132H) were observed to frequently occur in glioma patients.

Methods: We have purified recombinant IDH1 and IDH1-R132H proteins and monitored their catalytic activities. In parallel experiments, we have attempted to find new selective IDH1-R132H chemical inhibitor(s) from a fragment-based chemical library.

Results: We have found that IDH1, but not IDH1-R132H, can catalyze the conversion of isocitrate into α-ketoglutarate (α-KG). In addition, we have observed that IDH1-R132H was more efficient than IDH1 in converting α-KG into (R)-2-hydroxyglutarate (R-2HG). Moreover, we have identified a new hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one as a new selective IDH1-R132H inhibitor.

Conclusions: We have observed an underlying biochemical mechanism explaining how a heterozygous IDH1 mutation contributes to the generation of R-2HG and increases cellular histone H3 trimethylation levels. We have also identified a novel selective IDH1-R132H chemical hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one, which could be used for a future lead development against IDH1-R132H.

No MeSH data available.


Related in: MedlinePlus

Measurement of the catalytic activities of recombinant isocitrate dehydrogenase-1 (IDH1) and IDH1-R132H enzymes. (A) Purification of recombinant IDH1 and IDH1-R132H proteins. (B) Measurement of IDH1 and IDH1-R132H activities converting isocitrate (ICT) into α-ketoglutarate (α-KG) by measuring the reduction of NADP+ into nicotinamide adenine dinucleotide phosphate (NADPH). The enzyme activity is plotted as an increasing absorbance at the 320 nm wavelength. (C) Measurement of IDH1 and IDH1-R132H activities converting α-KG into a stereoselective (R)-2-hydroxyglutarate (R-2HG) by measuring the oxidation of NADPH into NADP+. The enzyme activity is plotted as a decreasing absorbance at the 320 nm wavelength. (D) The effect of IDH1 and IDH1-R132H on cellular histone H3 trimethylation levels. The 293T cells was transfected with pcDNA3 vector, pcDNA3-HA-IDH1 and pcDNA3-HA-IDH1-R132H plasmids and Western blot was conducted with indicated primary antibodies.
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f1-jcp-20-78: Measurement of the catalytic activities of recombinant isocitrate dehydrogenase-1 (IDH1) and IDH1-R132H enzymes. (A) Purification of recombinant IDH1 and IDH1-R132H proteins. (B) Measurement of IDH1 and IDH1-R132H activities converting isocitrate (ICT) into α-ketoglutarate (α-KG) by measuring the reduction of NADP+ into nicotinamide adenine dinucleotide phosphate (NADPH). The enzyme activity is plotted as an increasing absorbance at the 320 nm wavelength. (C) Measurement of IDH1 and IDH1-R132H activities converting α-KG into a stereoselective (R)-2-hydroxyglutarate (R-2HG) by measuring the oxidation of NADPH into NADP+. The enzyme activity is plotted as a decreasing absorbance at the 320 nm wavelength. (D) The effect of IDH1 and IDH1-R132H on cellular histone H3 trimethylation levels. The 293T cells was transfected with pcDNA3 vector, pcDNA3-HA-IDH1 and pcDNA3-HA-IDH1-R132H plasmids and Western blot was conducted with indicated primary antibodies.

Mentions: We have prepared recombinant IDH1 and IDH1-R132H enzymes via IPTG induction in Escherichia coli, followed by a nickel-based affinity purification and dialysis. Coomassie blue staining illustrates that both IDH1 and IDH1-R132H proteins are soluble (Fig. 1A). Because IDH1 requires NADP+ as a cofactor, the IDH1 activity converting ICT into α-KG can be indirectly quantified by measuring the amount of resulting NADPH at 340 nm by spectrophotometry (Fig. 1B, upper panel). Our results show that IDH1 efficiently converts ICT into α-KG through a coupled reduction of NADP+ into NADPH, while IDH1-R132H fails to do so (Fig. 1B, lower panel). As stated earlier, it is known that IDH1-R132H gains a new enzyme activity, e.g., catalysis of α-KG into R-2HG, which can be indirectly assessed by measuring the formation of NADP+ from NADPH (Fig. 1C, upper panel). However, it is unclear whether this activity also occurs in IDH1. Our result illustrates that both IDH1 and IDH1-R132H can convert α-KG into R-2HG, although the degree of this metabolic conversion by IDH1-R132H was far more efficient, when compared with that by IDH1 (Fig. 1C, lower panel).


Identification of a new selective chemical inhibitor of mutant isocitrate dehydrogenase-1.

Kim HJ, Choi BY, Keum YS - J Cancer Prev (2015)

Measurement of the catalytic activities of recombinant isocitrate dehydrogenase-1 (IDH1) and IDH1-R132H enzymes. (A) Purification of recombinant IDH1 and IDH1-R132H proteins. (B) Measurement of IDH1 and IDH1-R132H activities converting isocitrate (ICT) into α-ketoglutarate (α-KG) by measuring the reduction of NADP+ into nicotinamide adenine dinucleotide phosphate (NADPH). The enzyme activity is plotted as an increasing absorbance at the 320 nm wavelength. (C) Measurement of IDH1 and IDH1-R132H activities converting α-KG into a stereoselective (R)-2-hydroxyglutarate (R-2HG) by measuring the oxidation of NADPH into NADP+. The enzyme activity is plotted as a decreasing absorbance at the 320 nm wavelength. (D) The effect of IDH1 and IDH1-R132H on cellular histone H3 trimethylation levels. The 293T cells was transfected with pcDNA3 vector, pcDNA3-HA-IDH1 and pcDNA3-HA-IDH1-R132H plasmids and Western blot was conducted with indicated primary antibodies.
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f1-jcp-20-78: Measurement of the catalytic activities of recombinant isocitrate dehydrogenase-1 (IDH1) and IDH1-R132H enzymes. (A) Purification of recombinant IDH1 and IDH1-R132H proteins. (B) Measurement of IDH1 and IDH1-R132H activities converting isocitrate (ICT) into α-ketoglutarate (α-KG) by measuring the reduction of NADP+ into nicotinamide adenine dinucleotide phosphate (NADPH). The enzyme activity is plotted as an increasing absorbance at the 320 nm wavelength. (C) Measurement of IDH1 and IDH1-R132H activities converting α-KG into a stereoselective (R)-2-hydroxyglutarate (R-2HG) by measuring the oxidation of NADPH into NADP+. The enzyme activity is plotted as a decreasing absorbance at the 320 nm wavelength. (D) The effect of IDH1 and IDH1-R132H on cellular histone H3 trimethylation levels. The 293T cells was transfected with pcDNA3 vector, pcDNA3-HA-IDH1 and pcDNA3-HA-IDH1-R132H plasmids and Western blot was conducted with indicated primary antibodies.
Mentions: We have prepared recombinant IDH1 and IDH1-R132H enzymes via IPTG induction in Escherichia coli, followed by a nickel-based affinity purification and dialysis. Coomassie blue staining illustrates that both IDH1 and IDH1-R132H proteins are soluble (Fig. 1A). Because IDH1 requires NADP+ as a cofactor, the IDH1 activity converting ICT into α-KG can be indirectly quantified by measuring the amount of resulting NADPH at 340 nm by spectrophotometry (Fig. 1B, upper panel). Our results show that IDH1 efficiently converts ICT into α-KG through a coupled reduction of NADP+ into NADPH, while IDH1-R132H fails to do so (Fig. 1B, lower panel). As stated earlier, it is known that IDH1-R132H gains a new enzyme activity, e.g., catalysis of α-KG into R-2HG, which can be indirectly assessed by measuring the formation of NADP+ from NADPH (Fig. 1C, upper panel). However, it is unclear whether this activity also occurs in IDH1. Our result illustrates that both IDH1 and IDH1-R132H can convert α-KG into R-2HG, although the degree of this metabolic conversion by IDH1-R132H was far more efficient, when compared with that by IDH1 (Fig. 1C, lower panel).

Bottom Line: We have found that IDH1, but not IDH1-R132H, can catalyze the conversion of isocitrate into α-ketoglutarate (α-KG).We have observed an underlying biochemical mechanism explaining how a heterozygous IDH1 mutation contributes to the generation of R-2HG and increases cellular histone H3 trimethylation levels.We have also identified a novel selective IDH1-R132H chemical hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one, which could be used for a future lead development against IDH1-R132H.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Dongguk University, Goyang, Seowon University, Cheongju, Korea.

ABSTRACT

Background: Recent genome-wide sequencing studies have identified unexpected genetic alterations in cancer. In particular, missense mutations in isocitrate dehydrogenase-1 (IDH1) at arginine 132, mostly substituted into histidine (IDH1-R132H) were observed to frequently occur in glioma patients.

Methods: We have purified recombinant IDH1 and IDH1-R132H proteins and monitored their catalytic activities. In parallel experiments, we have attempted to find new selective IDH1-R132H chemical inhibitor(s) from a fragment-based chemical library.

Results: We have found that IDH1, but not IDH1-R132H, can catalyze the conversion of isocitrate into α-ketoglutarate (α-KG). In addition, we have observed that IDH1-R132H was more efficient than IDH1 in converting α-KG into (R)-2-hydroxyglutarate (R-2HG). Moreover, we have identified a new hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one as a new selective IDH1-R132H inhibitor.

Conclusions: We have observed an underlying biochemical mechanism explaining how a heterozygous IDH1 mutation contributes to the generation of R-2HG and increases cellular histone H3 trimethylation levels. We have also identified a novel selective IDH1-R132H chemical hit molecule, e.g., 2-(3-trifluoromethylphenyl)isothioazol-3(2H)-one, which could be used for a future lead development against IDH1-R132H.

No MeSH data available.


Related in: MedlinePlus