A novel small molecule approach for the treatment of propionic and methylmalonic acidemias

Abstract

Propionic Acidemia (PA) and Methylmalonic Acidemia (MMA) are inborn errors of metabolism affecting the catabolism of valine, isoleucine, methionine, threonine and odd-chain fatty acids. These are multi-organ disorders caused by the enzymatic deficiency of propionyl-CoA carboxylase (PCC) or methylmalonyl-CoA mutase (MUT), resulting in the accumulation of propionyl-coenzyme A (P-CoA) and methylmalonyl-CoA (M-CoA in MMA only). Primary metabolites of these CoA esters include 2-methylcitric acid (MCA), propionyl-carnitine (C3), and 3-hydroxypropionic acid, which are detectable in both PA and MMA, and methylmalonic acid, which is detectable in MMA patients only (Chapman et al., 2012). We deployed liver cell-based models that utilized PA and MMA patient-derived primary hepatocytes to validate a small molecule therapy for PA and MMA patients. The small molecule, HST5040, resulted in a dose-dependent reduction in the levels of P-CoA, M-CoA (in MMA) and the disease-relevant biomarkers C3, MCA, and methylmalonic acid (in MMA). A putative working model of how HST5040 reduces the P-CoA and its derived metabolites involves the conversion of HST5040 to HST5040-CoA driving the redistribution of free and conjugated CoA pools, resulting in the differential reduction of the aberrantly high P-CoA and M-CoA. The reduction of P-CoA and M-CoA, either by slowing production (due to increased demands on the free CoA (CoASH) pool) or enhancing clearance (to replenish the CoASH pool), results in a net decrease in the CoA-derived metabolites (C3, MCA and MMA (MMA only)). A Phase 2 study in PA and MMA patients will be initiated in the United States.

Keywords: Methylmalonic acidemia; Propionic acidemia.

Figure 1.

PA and MMA Biochemical Pathways and Biomarkers. PA and MMA are caused by enzymatic deficiency of propionyl-CoA carboxylase (PCC) and methylmalonyl-CoA mutase (MUT). PCC converts propionyl-CoA (P-CoA) to methylmalonyl-CoA (M-CoA) and MUT subsequently converts M-CoA to succinyl-CoA, which feeds into the TCA cycle for energy production. The catabolism of the branched-chain amino acids valine and isoleucine, as well as methionine, threonine, odd-chain fatty acids and the side chain of cholesterol funnel into the TCA cycle through PCC and MUT. Primary metabolites of P-CoA include 2-methylcitric acid (MCA), propionyl-carnitine (C3), 3-hydroxypropionic acid, and propionyl-glycine which are detectable in both PA and MMA, and methylmalonic acid and methylmalonyl-carnitine, which are detectable in MMA patients only. Elevated P-CoA has been shown to inhibit N-acetylglutamate synthase (NAGS), which reduces ureagenesis, thereby causing a secondary hyperammonemia and potential encephalopathy. High concentrations of P-CoA can inhibit PDH, which reduces acetyl-CoA levels and mitochondrial energy production. P-CoA acts as an alternative substrate for citrate synthase (CS), resulting in production of MCA. PA and MMA are multi-systemic diseases affecting renal, gastrointestinal, immune, CNS, hepatic, hematologic, and cardiovascular systems, and are associated with morbidity and mortality in infancy and childhood, and for survivors, debilitating end-organ damage and death. PA and MMA affect sequential steps in the same propionate catabolic pathway, leading to similar acute and chronic disease manifestations, although some later stage disease complications appear to be more specific to either PA or MMA.

Figure 2.

Structure of HST5040

Figure 3.. HST5040 reduces disease-relevant biomarkers in PA and MMA pHep disease models.

Representative data from a PA and MMA pHep donor exposed to HST5040 from 0 μM (represented as 0.01 μM in graphs) to 100 μM for 6 days in the bioreactor. Biomarker levels are normalized to pHep intracellular concentration. Each black dot is a sample with N=8 for each experiment. The calculated EC₅₀ is shown for each biomarker. (A) ¹²C-P-CoA, (B) ¹²C-M-CoA, (C) and C3 and C2 were measured by HT-MS/MS. (D) The ratio of C3/C2 was a calculated from measured C3 and C2 levels in the pHeps. (E) MCA was measured by in cell lysates by LC-MS/MS.

Figure 4.. HST5040 reduces disease-relevant biomarkers in low and high propiogenic conditions in PA and MMA disease models.

Representative data from a PA and MMA pHep donor exposed to HST5040 from 0 μM (represented as 0.01 μM in graphs) to 100 μM for 1.5 hours in static cell culture with low or high propiogenic media formulation to mimic a stable or catabolic metabolic disease state, respectively. Biomarker levels are normalized to pHeps intracellular concentration. Each black dot is a sample with N=4 for each experiment. The calculated EC₅₀ is shown for each biomarker. (A) ¹³C-P-CoA and (B) ¹³C-M-CoA were measured by HT-MS/MS. C) Methylmalonic acid was measured by in MMA pHep lysates by LC-MS/MS.

Figure 5.. The relationship between the HST5040 mechanism of action and CoA pools in the bioreactor.

Representative data from a PA and MMA pHep donor exposed to HST5040 from 0 μM (represented as 0.01 μM in graphs) to 100 μM for 6 days in the bioreactor. Analyte levels are normalized to pHep intracellular concentration. Each black dot is a sample with N=8 for each experiment. The calculated EC₅₀ is shown for each biomarker. (A) ¹²C-P-CoA, (B) ¹²C-Acetyl-CoA, (C) CoASH, and (D) HST5040-CoA were measured by HT-MS/MS.

Figure 6.. The relationship between the HST5040 mechanism of action and CoA pools in static cell culture.

Representative data from a PA and MMA pHep donor exposed to HST5040 from 0 μM (represented as 0.01 μM in graphs) to 100 μM for 1.5 hours in static cell culture with low or high propiogenic media formulation to mimic a stable or catabolic metabolic disease state, respectively. Analyte levels are normalized to pHep intracellular concentration. Each black dot is a sample with N=8 for each experiment. The calculated EC₅₀ is shown for each analyte. (A) ¹³C-P-CoA, (B) ¹²C-Acetyl-CoA, (C) CoASH, and (D) HST5040-CoA were measured by HT-MS/MS.