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Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase - PubMed

  • ️Wed Jan 01 2014

Benzothiophene carboxylate derivatives as novel allosteric inhibitors of branched-chain α-ketoacid dehydrogenase kinase

Shih-Chia Tso et al. J Biol Chem. 2014.

Abstract

The mitochondrial branched-chain α-ketoacid dehydrogenase complex (BCKDC) is negatively regulated by reversible phosphorylation.BCKDC kinase (BDK) inhibitors that augment BCKDC flux have been shown to reduce branched-chain amino acid (BCAA) concentrations in vivo. In the present study, we employed high-throughput screens to identify compound 3,6- dichlorobenzo[b]thiophene-2-carboxylic acid (BT2) as a novel BDK inhibitor (IC(50) = 3.19 μM). BT2 binds to the same site in BDK as other known allosteric BDK inhibitors, including (S)-α-cholorophenylproprionate ((S)-CPP). BT2 binding to BDK triggers helix movements in the N-terminal domain, resulting in the dissociation of BDK from the BCKDC accompanied by accelerated degradation of the released kinase in vivo. BT2 shows excellent pharmacokinetics (terminal T(1⁄2) = 730 min) and metabolic stability (no degradation in 240 min), which are significantly better than those of (S)-CPP. BT2, its analog 3-chloro-6-fluorobenzo[ b]thiophene-2-carboxylic acid (BT2F), and a prodrug of BT2 (i.e. N-(4-acetamido-1,2,5-oxadiazol-3-yl)-3,6-dichlorobenzo[ b]thiophene-2-carboxamide (BT3)) significantly increase residual BCKDC activity in cultured cells and primary hepatocytes from patients and a mouse model of maple syrup urine disease. Administration of BT2 at 20 mg/kg/day to wild-type mice for 1 week leads to nearly complete dephosphorylation and maximal activation of BCKDC in heart, muscle, kidneys, and liver with reduction in plasma BCAA concentrations. The availability of benzothiophene carboxylate derivatives as stable BDK inhibitors may prove useful for the treatment of metabolic disease caused by elevated BCAA concentrations.

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Figures

FIGURE 1.
FIGURE 1.

Identification of benzothiophene carboxylate derivatives as novel BDK inhibitors. A, candidate BDK inhibitor 476-I16 identified by high-throughput screening of a 2,000-compound chemical library. B, separation of compound 476-I16 into smaller compound fractions BT1 and BT2 by reversed-phase HPLC. C, absence of BDK inhibition by BT1. D, inhibition of BDK as a function of BT2 concentrations. E, activation of BCKDC in MEF cells by BT1, BT2, and a mixture of both from compound 476-I16, compared with the DMSO control. F, chemical structures, degradation schemes, and molecular masses (m/z) of benzothiophene carboxylate derivatives. BT3 is an analog of BT1. G, chemical structures of (S)-CPP, BT2, and BT2F. Error bars, S.D.

FIGURE 2.
FIGURE 2.

Crystal structures of rat BDK in complex with BDK inhibitors BT2 and (S)-CPP. A, bound BT2 and ADP (in the space-filling model) in the N-terminal domain (green) and C-terminal domain (cyan), respectively, of a BDK monomer. B, the Fo − Fc omit density map of bound BT2 in the allosteric site. C and D, hydrogen bonding networks of bound BT2 and (S)-CPP, respectively, in the allosteric site. E and F, surface maps of the allosteric pocket harboring BT2 and (S)-CPP, respectively.

FIGURE 3.
FIGURE 3.

BT2, BT2F, and BT3 significantly increase BCKDC activity in wild-type and MSUD cells. A, wild-type MEF cells were cultured with 40 μ

m

BT2, BT2F, or BT3 for 48 h with DMSO as a control. BDP−/− cells manifesting intermediate MSUD were grown in the presence of 20 μ

m

BT2 or BT3 for 96 h. BCKDC activity was measured by the intact cell assay using α-keto[1-14C] isovalerate as a substrate. B, primary hepatocytes prepared from wild-type MSUD or iMSUD mice were incubated in the Krebs buffer with BT2 or BT3 (40 μ

m

) for 1 h and assayed for BCKDC activity in a reconstituted reaction mixture. C, harvested lymphoblasts from control subjects and intermediate MSUD patients carrying R252H/E1α or R240C/E2 (both mature sequences) were incubated in the Krebs buffer for 1 h with 40 μ

m

BT2 or BT2F or 20 μ

m

BT3. Wild-type and mutant BCKDC activity was measured by the intact cell assay. Error bars, S.D.

FIGURE 4.
FIGURE 4.

Metabolic stability and pharmacokinetics of BT2 and BT3. A, metabolic stability of BT3 in S9 fractions. BT3 (2 μ

m

) was incubated with S9 factions; concentrations of BT3 and BT2 in aliquots taken at different time points were determined by LC-MS/MS. B, metabolic stability of BT2 in S9 factions. The remaining BT2 levels at different time points were determined by LC-MS/MS and expressed as ln(percentage of initial BT2 concentration). C, pharmacokinetics of plasma BT2 depletion. BT2 (10 mg/kg) was administered to CD-1 male mice by intraperitoneal injection. Plasma was collected from different time points (n = 3). BT2 concentrations were determined by LC-MS/MS. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 when compared with the group treated with DMSO. Error bars, S.D.

FIGURE 5.
FIGURE 5.

Robust simulation of BDKDC activity in mouse tissues with decreased plasma BCAA concentrations. A, BCKDC activity in heart, muscle, kidney, and liver from wild-type C57BL/6J mice treated with vehicle (V) or BT2 (T) for 1 week. Shaded bars represent the mean of BCKDC activities in tissues from an individual mouse (n = 4); *, p < 0.05; ****, p < 0.0001. B, relative protein levels of phosphorylated E1 (pE1) and total E1 (E1) in tissues from vehicle- and BT2-treated mice. GAPDH served as a loading control. The amounts of tissue homogenate applied were 15 μg for liver, 30 μg for heart and kidney, and 60 μg for muscle. C, decreased plasma BCAA concentrations in BT2-treated mice compared with vehicle-treated (n = 4). Leu + Ile, sum of the two amino acids that are indistinguishable in LC-MS/MS. Error bars, S.D.

FIGURE 6.
FIGURE 6.

Protein and mRNA levels of BDK in kidneys and heart from vehicle- and BT2-treated wild-type mice. A, Western blots (top) of kidney and heart homogenates from vehicle-treated (V) and BT2-treated (T) mice with GAPDH as loading controls. The quantification of band densities shows that BDK protein levels in BT2-treated kidney and heart are reduced to 39 and 24%, respectively, compared with the vehicle-treated ones (bottom). B, BDK mRNA levels determined by quantitative PCR in kidneys from BT2-treated mice relative to the vehicle-treated ones (set as 1.0). The GAPDH mRNA level was used as an internal control. Error bars, S.D.

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