The results presented in this study suggest that phenylbutyrate is a potential adjunctive treatment for selected classes of MSUD patients. The ability of phenylbutyrate to enhance residual flux through the BCKDC pathway by altering the phosphorylation status of the E1α subunit as well as to directly increase E1 enzyme activity is a new and novel finding. In three patients with clinically, late-onset forms of MSUD, phenylbutyrate treatment reduced the blood concentrations of BCAA and their corresponding BCKA. Recent reports suggest that the BCKA, particularly the α-keto acid of leucine, are the toxic metabolites in MSUD (17
). Until now, no pharmacological treatment for MSUD was available and acute decompensation due to leucine intoxication could only be treated with supportive measures and/or hemodialysis (1
The two patients with E2 mutations (patients 4 and 5) responded to phenylbutyrate treatment with significant reductions in plasma leucine and all three BCKA. It is likely that in both of these patients, activation of E1 through the inhibition of BDK activity by phenylbutyrate increased BCKDC flux which enhanced the clearance of BCKA. On the other hand, the patient carrying the homozygous mutation p.V412M (patient 3) responded to phenylbutyrate, whereas the patient found to be a compound heterozygous for the c.887_894del and p.Y438N mutations (patient 2) did not respond. E1 is a heterotetramer of two E1α and two E1β subunits which assemble in the active enzyme. Mutation or deletion of functionally important residues might abort the tetrameric assembly. According to protein structural modeling, the V412 is at the surface accessible area of E1α to form heterotetrameric assembly with E1β. Mutations affecting this residue hamper the assembly of the complex. Addition of phenylbutyrate might stabilize assembly formation and ultimately enhance the oxidation of BCKA. A naturally occurring osmolyte trimethylamine N-oxide has been shown to correct tetrameric assembly defects caused by the Y438N mutation, leading to a partial restoration of E1 activity (19
). The amino acids G290, Y438, and the residues encoded by c.887–895 are localized in the surface accessible area of E1α to form an α2
tetrameric assembly with the E1β subunit. Mutations of the G290 by Arg and Y438 by Asn (20
) and deletion of c.887–895 diminish the assembly formation of E1 and ultimately the total function of E1 catalyzed decarboxylation.
Both patients with E2 mutations and some residual activity responded to phenylbutyrate. The E2 reaction is considered rate-limiting for the overall BCKDC activity (21
). However, the present result suggests that increasing E1 activity can increase BCKDC activity and/or activation of free E1 increases decarboxylation of BCKA sufficiently to reduce BCKA levels. Protein structural analysis shows that the amino acids R301 and S366, detected in patients 4 and 5, respectively, are among the residues involved in CoA binding (22
) and core formation in 24-meric assembly. Mutations of these residues do not show significant differences in the model structure, and may be involved in overall transacylation reaction of E2.
The response to phenylbutyrate is complex and may not be simply correlated with residual BCKDC activity measurements in fibroblasts or with the genotype. The enzymatic activity of BCKDC in patient fibroblasts is known to poorly correlate with the clinical severity (13
). Moreover, the estimates of enzyme activity ex vivo
using cultured patients’ cell lines can be considerably different from estimates of enzyme activity in vivo
). Still, in-depth structural analysis and modeling of phenylbutyrate interaction to this enzyme complex may eventually better help predict genotype–response correlations. Until more patients with a wider range of mutations have been examined, in vivo
loading test may be required to predict phenylbutyrate responsiveness in MSUD patients.
The availability of a novel therapeutic approach to reduce the blood levels of the BCAA and their BCKA may allow for less stringent dietary restrictions as well as a potential treatment during acute metabolic decompensations. The catabolism of BCAA is tightly regulated by the kinase and phosphatase action on the E1α subunit of the E1 decarboxylase of BCKDC.
Finally, we have described a novel mechanism of phenylbutyrate action in vivo
, which is mediated by direct BDK inhibition. A wide range of biological activities have been attributed to phenylbutyrate. In its only FDA approved use in urea cycle disorders, phenylbutyrate acts as a pro-drug leading to the generation of phenylacetate. Here, phenylacetate conjugates glutamine and serves as an alternative route of nitrogen disposal. In addition to this application, phenylbutyrate has been studied for cancer, cystic fibrosis, thalassemia, spinal muscular atrophy, amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease and type 2 diabetes mellitus (14
). Biochemical activities that have been attributed in these scenarios include action as chaperone, histone deacetylase inhibitor, growth inhibition and relief of endoplasmic reticulum stress. However, the mechanistic basis for these activities remains poorly defined. Our data suggest a novel direct effect on BCKDC via action on protein phosphorylation. As a major regulatory mechanism of almost of all biological processes, a potential approach for targeting protein phosphorylation may offer new treatment avenues in disease processes where phosphorylation is central to pathogenesis (24