Innovative therapy for Classic Galactosemia - tale of two HTS

Abstract

Classic Galactosemia is an autosomal recessive disorder caused by the deficiency of galactose-1-phosphate uridylyltransferase (GALT), one of the key enzymes in the Leloir pathway of galactose metabolism. While the neonatal morbidity and mortality of the disease are now mostly prevented by newborn screening and galactose restriction, long-term outcome for older children and adults with this disorder remains unsatisfactory. The pathophysiology of Classic Galactosemia is complex, but there is convincing evidence that galactose-1-phosphate (gal-1P) accumulation is a major, if not the sole pathogenic factor. Galactokinase (GALK) inhibition will eliminate the accumulation of gal-1P from both dietary sources and endogenous production, and efforts toward identification of therapeutic small molecule GALK inhibitors are reviewed in detail. Experimental and computational high-throughput screenings of compound libraries to identify GALK inhibitors have been conducted, and subsequent studies aimed to characterize, prioritize, as well as to optimize the identified positives have been implemented to improve the potency of promising compounds. Although none of the identified GALK inhibitors inhibits glucokinase and hexokinase, some of them cross-inhibit other related enzymes in the GHMP small molecule kinase superfamily. While this finding may render the on-going hit-to-lead process more challenging, there is growing evidence that such cross-inhibition could also lead to advances in antimicrobial and anti-cancer therapies.

Fig. 1. The metabolic pathway of galactose in humans [57]

Fig. 2. Proposed effects of blockage in galactose metabolic pathway on the myo-inositol metabolic pathway and related signal transduction pathways

Gal-1P (an intermediate) and galactitol (a by-product) of the blocked galactose metabolic pathway interfere inositol turnover and inositol entry by inhibiting IMPase and SMIT, respectively. This could potentially decrease cellular PIP₂ content and adversely affect normal AKT function. Blockage of galactose metabolism has also been shown to up-regulate ARHI gene expression [132], which negatively regulate AKT activity in the cells. (Abbreviations: PIP₂: phosphatidylinositol-4,5-biphosphate; IP₃: inositol-1,4,5-triphosphate; IP₂: inositol bisphosphate; IP₁: inositol monophosphate; PIP₃: phosphatidylinositol-3,4,5-triphosphate; SMIT1: Na⁺/myo-inositol transporter; IMPase: inositol monophosphatase; P13K: phosphatidylinositol 3-kinase; GPCR: G-protein coupled receptor. L: ligand for GPCR; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; PTEN: phosphatase and tensin homolog; ARHI: aplysia ras homolog I.)

Fig. 3. Overview of current therapeutic treatment approaches for inborn error of metabolism at different levels of interaction [58, 59]

Fig. 4. Two high-throughput screening strategies employed to identify GALK inhibitors

Experimental and computational approaches were used in the high-throughput screenings of human GALK inhibitors. The identified inhibitors will be characterized by a series of tertiary assays to determine the efficacy, toxicity, selectivity. Lead compounds with the optimal profile will be selected for further optimization to improve their drug-like properties.

Fig. 5. Molecular structure of ligands binding pocket of human GALK (1wuu)[102]

(a) Amino acid residues of human GALK located within 3.5Å of the ligands were represented here. Molecules in green carbon atoms are galactose and AMPPNP. Amino acid residues are depicted in gray. MI: motif I; MII: motif II; MIII: motif III. (b) Docking pose of compound 4 to GALK crystal structure. Using high-precision docking software GLIDE, compound 4 was docked to the GALK structure. Molecule in green and ball-and-stick conformation is compound 4. Side chain of Ser¹⁴⁰ of GALK is in dark blue, and the side-chains of other amino acid residues selected for the site-directed mutagenesis studies are colored in brown. Side-chains of other amino acid residues surrounding the compound 4 are colored in gray. Yellow dotted lines are hydrogen-bonds formed between compound 4 and the side-chain of Ser¹⁴⁰.

Fig. 5. Molecular structure of ligands binding pocket of human GALK (1wuu)[102]

(a) Amino acid residues of human GALK located within 3.5Å of the ligands were represented here. Molecules in green carbon atoms are galactose and AMPPNP. Amino acid residues are depicted in gray. MI: motif I; MII: motif II; MIII: motif III. (b) Docking pose of compound 4 to GALK crystal structure. Using high-precision docking software GLIDE, compound 4 was docked to the GALK structure. Molecule in green and ball-and-stick conformation is compound 4. Side chain of Ser¹⁴⁰ of GALK is in dark blue, and the side-chains of other amino acid residues selected for the site-directed mutagenesis studies are colored in brown. Side-chains of other amino acid residues surrounding the compound 4 are colored in gray. Yellow dotted lines are hydrogen-bonds formed between compound 4 and the side-chain of Ser¹⁴⁰.

Fig. 6. GALK inhibitor prevents gal-1P accumulation in GALT-deficient fibroblasts [101]

Gal-1P level was measured after SV-40 transformed GALT-deficient fibroblasts were treated with compound BI-01 prior to 0.05% galactose challenge.

Fig. 7. Compounds from commercial sources selected for SAR studies [108]

Nine compounds (41-49) were selected from commercial sources based on their structural similarity to compound 5. These compounds were tested for their in vitro GALK inhibitory properties.

Fig. 8. Synthesized compounds selected for SAR studies [108]

Seven compounds (50-56) were synthesized to test the importance of substitutions at the A-ring in compound 41.

Fig. 9. GALK inhibitors modified with morpholine moiety [108]

Morpholino substitutions at the C-ring were synthesized and tested for potential improvement in binding to the adenine binding pocket.