Development of a succinyl CoA:3-ketoacid CoA transferase inhibitor selective for peripheral tissues that improves glycemia in obesity

Abstract

Many individuals with type 2 diabetes (T2D) cannot take current therapies due to their adverse effects. Thus, new glucose-lowering agents targeting unique mechanisms are needed. Studies have demonstrated that decreasing ketone oxidation, secondary to muscle-specific deletion of succinyl-CoA:3-ketoacid-CoA transferase (SCOT), protects mice against obesity-related hyperglycemia. In silico studies identified that the antipsychotic diphenylbutylpiperidines can inhibit SCOT and alleviate obesity-related hyperglycemia. Because ketones are a major brain fuel, whereas the diphenylbutylpiperidines have central nervous system-related adverse effects, we aimed to develop a peripheral selective SCOT inhibitor (PSSI). Using a pharmacophore derived from the diphenylbutylpiperidine-SCOT interaction, we synthesized PSSI-51, which inhibited SCOT activity in peripheral but not brain tissue, while decreasing myocardial ketone oxidation. Importantly, PSSI-51 treatment improved glycemia in obese mice and demonstrated reduced brain accumulation compared to the diphenylbutylpiperidine pimozide. We propose that PSSI-51 can lay the foundation for optimizing a new class of brain-impermeable SCOT inhibitors for treating T2D.

Keywords: Computational molecular modelling; Diabetology; Medical biochemistry; Pharmaceutical compounds formulation; Pharmacology; Physiology.

Graphical abstract

Figure 1

Development of the SCOT inhibitor PSSI-51 (A) Schematic representation of the discovery process from pharmacophore hypothesis through hit generation to the identification of compound PSSI-51. (B) Depiction of PSSI-51 binding within the oxyanion binding pocket of SCOT, highlighting hydrophobic interactions (red dotted lines) and hydrogen bonding (green dotted lines. (C) The binding interaction fraction of the residues in contact with PSSI-51 during the MD simulation. (D) RMSD comparison between unbound SCOT and SCOT bound with PSSI-51. (E) Superimposed RMSF profiles of side chain residues of unbound SCOT versus SCOT in complex with PSSI-51. (F) PSSI-51 positional stability within the SCOT binding pocket. (G) Detailed schematic of the synthetic route of PSSI-51.

Figure 2

PSSI-51 selectively inhibits SCOT without interacting with canonical targets of DPBPs (A) Quantification of recombinant SCOT enzymatic activity and acetoacetyl CoA production rates in the presence of dimethyl sulfoxide (DMSO) (vehicle control) and PSSI-51 (500, 250, and 125 nmol/L) (n = 7–9 biological replicates). (B) Evaluation of C¹⁴βOHB oxidation rates in the isolated working mouse heart in response to vehicle control or PSSI-51 treatment (n = 5–6 animals). (C) Assessment of potential cerebral uptake of PSSI-51 and pimozide using PAMPA. (D) Fluorescence-based assay in U2OS red _cAMPNomad D2R cells to measure inhibition of dopamine-induced cAMP mobilization by PSSI-51, compared with pimozide and the positive control blenonserin (n = 3 biological replicates). (E) Quantitative fluorescence analysis of dopamine agonistic activity (n = 3 biological replicates). (F) Schematic of targeted site-directed mutagenesis within SCOT binding site. (G) SCOT enzymatic activity of the recombinant mutant SCOT (Y115A/I323A) and rate of acetoacetyl CoA production in the presence of DMSO (vehicle control) and the PSSI-51 (500 nmol/L) (n = 3 technical replicates). (H) Circulating βOHB levels following PSSI-51 administration 16 h prior to oral ketone ester intake. Data are presented as mean ± SEM. Statistical significance was determined using Student’s t test or one-way ANOVA, followed by a Bonferroni post hoc correction where appropriate. Statistical thresholds set at ∗p < 0.05 vs. vehicle control, ^$p < 0.05 vs. pimozide, ^#p < 0.05 vs. dopamine. RMSD; root-mean-square deviation, RMSF; root-mean-square fluctuation, HP Std; high permeability standard, LP Std; low permeability standard; P_e: permeability coefficients.

Figure 3

PSSI-51 inhibits ketone oxidation and improves glycemia in obese mice (A) Schematic diagram outlining the experimental approach. (B) Circulating βOHB levels (n = 6–10 animals). (C) Glucose tolerance tests (performed one day following the final treatment) (n = 6–10 animals); (D) corresponding circulating insulin levels (n = 5–6 animals); (E–G) and body weight and adiposity changes following drug treatment (n = 5–6 animals). (H) Metabolic parameters using indirect calorimetry (24 h) to assess total food and water intake, whole-body energy expenditure, locomotor and ambulatory activity and respiratory exchange ratio (n = 4–5 animals). Data presentation includes mean ± SEM. Statistical analyses were conducted using Student’s t test or one-way ANOVA, supplemented by Bonferroni post hoc tests, with significance thresholds set at ∗p < 0.05 versus vehicle control.

Figure 4

Selective Inhibition of peripheral ketone body metabolism by PSSI-51 (A) SCOT activity assay in the brain, kidney and soleus muscle isolated from mouse treated PSSI-51, and pimozide (n = 3–4 animals). (B) Representative PET images of untreated fed and untreated fasted mice 1 h post injection of S-[¹⁸F]FβOHB and time-activity curves for fasted to fed brain and muscle over 60 min (n = 4 animals). (C) Representative PET images of vehicle treated (fasted) and PSSI-51 treated (fasted) mice 1 h post injection of S-[¹⁸F]FβOHB and time time-activity curves for untreated to treated brain and muscle over 60 min (n = 3–5 animals). (D) Parameters of the activity wheels measured after treatment with PSSI-51 or pimozide after to 24 and 48 after treatment (n = 3 animals per group). (E) Mass spectrometric quantification of PSSI-51:pimozide ratio following a single dose of the drug (n = 3 animals). Data are presented as mean ± SEM. Statistical analyses were conducted using Student’s t test or one-way ANOVA, supplemented by Bonferroni post hoc tests, with significance thresholds set at ∗p < 0.05 vs. vehicle control, ^#p < 0.05 vs. pimozide.