Cycloserine enantiomers are reversible inhibitors of human alanine:glyoxylate aminotransferase: implications for Primary Hyperoxaluria type 1

Abstract

Peroxisomal alanine:glyoxylate aminotransferase (AGT) is responsible for glyoxylate detoxification in human liver and utilizes pyridoxal 5′-phosphate (PLP) as coenzyme. The deficit of AGT leads to Primary Hyperoxaluria Type I (PH1), a rare disease characterized by calcium oxalate stones deposition in the urinary tract as a consequence of glyoxylate accumulation. Most missense mutations cause AGT misfolding, as in the case of the G41R, which induces aggregation and proteolytic degradation. We have investigated the interaction of wild-type AGT and the pathogenic G41R variant with d-cycloserine (DCS, commercialized as Seromycin), a natural product used as a second-line treatment of multidrug-resistant tuberculosis, and its synthetic enantiomer l-cycloserine (LCS). In contrast with evidences previously reported on other PLP-enzymes, both ligands are AGT reversible inhibitors showing inhibition constants in the micromolar range. While LCS undergoes half-transamination generating a ketimine intermediate and behaves as a classical competitive inhibitor, DCS displays a time-dependent binding mainly generating an oxime intermediate. Using a mammalian cellular model, we found that DCS, but not LCS, is able to promote the correct folding of the G41R variant, as revealed by its increased specific activity and expression as a soluble protein. This effect also translates into an increased glyoxylate detoxification ability of cells expressing the variant upon treatment with DCS. Overall, our findings establish that DCS could play a role as pharmacological chaperone, thus suggesting a new line of intervention against PH1 based on a drug repositioning approach. To a widest extent, this strategy could be applied to other disease-causing mutations leading to AGT misfolding.

Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase

“…While several crystal structures of AGT‐Ma were solved, 24–30 no structural information on AGT‐Mi was available. Therefore, we first solved the structure of AGT‐Mi by x‐ray crystallography at 2.2 Å resolution (Figure 1, Table S1).…”

“…The crystal structure of AGT-Mi unveils three unexpected frustrated regions and allows to decipher AGT1 hidden plasticity While several crystal structures of AGT-Ma were solved, [24][25][26][27][28][29][30] no structural information on AGT-Mi was available. Therefore, we first solved the structure of AGT-Mi by x-ray crystallography at 2.2 Å resolution (Figure 1, Table S1).…”

Identification of Human Alanine–Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1

“…Molecular docking has been performed using the MOE Dock Tool available on MOE 2018.0101(Chemical Computing Group ULC, Montreal, QC, Canada) as previously reported. 53 Briefly, the crystal structure of human AGT (PDB ID:1H0C) 1 and the ligand (compound 1) have been prepared and protonated using the Protonated 3D Tool and the docking site selected by using the MOE Site Finder Tool. Then, a collection of ligand conformations using the bond rotation method has been generated, placed in the selected site by the Triangle Matcher method, and ranked using the London dG scoring function, which estimates the free energy of binding of the ligand from a given pose.…”

Role of misfolding in rare enzymatic deficits and use of pharmacological chaperones as therapeutic approach

“…Interestingly, only D-cycloserine displays a PC activity on a mutant form of AGT prone to misfolding. The latter data have suggested that a new line of intervention against PH1 could be also based on a drug repositioning approach [174].…”