Harnessing the Power of Purple Sweet Potato Color and Myo-Inositol to Treat Classic Galactosemia.

Classic Galactosemia (CG) is a devastating inborn error of the metabolism caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase in galactose metabolism. Severe complications of CG include neurological impairments, growth restriction, cognitive delays, and, for most females, primary ovarian insufficiency. The absence of the GALT enzyme leads to an accumulation of aberrant galactose metabolites, which are assumed to be responsible for the sequelae. There is no treatment besides the restriction of dietary galactose, which does not halt the development of the complications; thus, additional treatments are sorely needed. Supplements have been used in other inborn errors of metabolism but are not part of the therapeutic regimen for CG. The goal of this study was to test two generally recognized as safe supplements (purple sweet potato color (PSPC) and myo-inositol (MI)) that may impact cellular pathways contributing to the complications in CG. Our group uses a GalT gene-trapped mouse model to study the pathophysiology in CG, which phenocopy many of the complications. Here we report the ability of PSPC to ameliorate dysregulation in the ovary, brain, and liver of our mutant mice as well as positive results of MI supplementation in the ovary and brain.

Integrated stress response control of granulosa cell translation and proliferation during normal ovarian follicle development.

Mechanisms that directly control mammalian ovarian primordial follicle (PF) growth activation and the selection of individual follicles for survival are largely unknown. Follicle cells produce factors that can act as potent inducers of cellular stress during normal function. Consistent with this, we show here that normal, untreated ovarian cells, including pre-granulosa cells of dormant PFs, express phenotype and protein markers of the activated integrated stress response (ISR), including stress-specific protein translation (phospho-Serine 51 eukaryotic initiation factor 2α; P-EIF2α), active DNA damage checkpoints, and cell-cycle arrest. We further demonstrate that mRNAs upregulated in primary (growing) follicles versus arrested PFs mostly include stress-responsive upstream open reading frames (uORFs). Treatment of a granulosa cell (GC) line with the PF growth trigger tumor necrosis factor alpha results in the upregulation of a 'stress-dependent' translation profile. This includes further elevated P-eIF2α and a shift of uORF-containing mRNAs to polysomes. Because the active ISR corresponds to slow follicle growth and PF arrest, we propose that repair and abrogation of ISR checkpoints (e.g. checkpoint recovery) drives the GC cell cycle and PF growth activation (PFGA). If cellular stress is elevated beyond a threshold(s) or, if damage occurs that cannot be repaired, cell and follicle death ensue, consistent with physiological atresia. These data suggest an intrinsic quality control mechanism for immature and growing follicles, where PFGA and subsequent follicle growth and survival depend causally upon ISR resolution, including DNA repair and thus the proof of genomic integrity.

Novel mRNA-Based Therapy Reduces Toxic Galactose Metabolites and Overcomes Galactose Sensitivity in a Mouse Model of Classic Galactosemia.

Classic galactosemia (CG) is a potentially lethal inborn error of galactose metabolism that results from deleterious mutations in the human galactose-1 phosphate uridylyltransferase (GALT) gene. Previously, we constructed a GalT-/- (GalT-deficient) mouse model that exhibits galactose sensitivity in the newborn mutant pups, reduced fertility in adult females, impaired motor functions, and growth restriction in both sexes. In this study, we tested whether restoration of hepatic GALT activity alone could decrease galactose-1 phosphate (gal-1P) and plasma galactose in the mouse model. The administration of different doses of mouse GalT (mGalT) mRNA resulted in a dose-dependent increase in mGalT protein expression and enzyme activity in the liver of GalT-deficient mice. Single intravenous (i.v.) dose of human GALT (hGALT) mRNA decreased gal-1P in mutant mouse liver and red blood cells (RBCs) within 24 h with low levels maintained for over a week. Repeated i.v. injections increased hepatic GalT expression, nearly normalized gal-1P levels in liver, and decreased gal-1P levels in RBCs and peripheral tissues throughout all doses. Moreover, repeated dosing reduced plasma galactose by 60% or more throughout all four doses. Additionally, a single intraperitoneal dose of hGALT mRNA overcame the galactose sensitivity and promoted the growth in a GalT-/- newborn pup.

The Galactose Index measured in fibroblasts of GALT deficient patients distinguishes variant patients detected by newborn screening from patients with classical phenotypes.

BACKGROUND: The high variability in clinical outcome of patients with Classical Galactosemia (CG) is poorly understood and underlines the importance of prognostic biomarkers, which are currently lacking. The aim of this study was to investigate if residual galactose metabolism capacity is associated with clinical and biochemical outcomes in CG patients with varying geno- and phenotypes. METHODS: Galactose Metabolite Profiling (GMP) was used to determine residual galactose metabolism in fibroblasts of CG patients. The association between the galactose index (GI) defined as the ratio of the measured metabolites [U13C]Gal-1-P/ [13C6]UDP-galactose, and both intellectual and neurological outcome and galactose-1-phosphate (Gal-1-P) levels was investigated. RESULTS: GMP was performed in fibroblasts of 28 patients and 3 control subjects. The GI of the classical phenotype patients (n = 22) was significantly higher than the GI of four variant patients detected by newborn screening (NBS) (p = .002), two homozygous p.Ser135Leu patients (p = .022) and three controls (p = .006). In the classical phenotype patients, 13/18 (72%) had a poor intellectual outcome (IQ < 85) and 6/12 (50%) had a movement disorder. All the NBS detected variant patients (n = 4) had a normal intellectual outcome (IQ ≥ 85) and none of them has a movement disorder. In the classical phenotype patients, there was no significant difference in GI between patients with a poor and normal clinical outcome. The NBS detected variant patients had significantly lower GI levels and thus higher residual galactose metabolism than patients with classical phenotypes. There was a clear correlation between Gal-1-P levels in erythrocytes and the GI (p = .001). CONCLUSIONS: The GI was able to distinguish CG patients with varying geno- and phenotypes and correlated with Gal-1-P. The data of the NBS detected variant patients demonstrated that a higher residual galactose metabolism may result in a more favourable clinical outcome. Further research is needed to enable individual prognostication and treatment in all CG patients.

Discovery of Novel Inhibitors Targeting Multi-UDP-hexose Pyrophosphorylases as Anticancer Agents.

To minimize treatment toxicities, recent anti-cancer research efforts have switched from broad-based chemotherapy to targeted therapy, and emerging data show that altered cellular metabolism in cancerous cells can be exploited as new venues for targeted intervention. In this study, we focused on, among the altered metabolic processes in cancerous cells, altered glycosylation due to its documented roles in cancer tumorigenesis, metastasis and drug resistance. We hypothesize that the enzymes required for the biosynthesis of UDP-hexoses, glycosyl donors for glycan synthesis, could serve as therapeutic targets for cancers. Through structure-based virtual screening and kinetic assay, we identified a drug-like chemical fragment, GAL-012, that inhibit a small family of UDP-hexose pyrophosphorylases-galactose pyro-phosphorylase (GALT), UDP-glucose pyrophosphorylase (UGP2) and UDP-N-acetylglucosamine pyrophosphorylase (AGX1/UAP1) with an IC50 of 30 µM. The computational docking studies supported the interaction of GAL-012 to the binding sites of GALT at Trp190 and Ser192, UGP2 at Gly116 and Lys127, and AGX1/UAP1 at Asn327 and Lys407, respectively. One of GAL-012 derivatives GAL-012-2 also demonstrated the inhibitory activity against GALT and UGP2. Moreover, we showed that GAL-012 suppressed the growth of PC3 cells in a dose-dependent manner with an EC50 of 75 µM with no effects on normal skin fibroblasts at 200 µM. Western blot analysis revealed reduced expression of pAKT (Ser473), pAKT (Thr308) by 77% and 72%, respectively in the treated cells. siRNA experiments against the respective genes encoding the pyrophosphorylases were also performed and the results further validated the proposed roles in cancer growth inhibition. Finally, synergistic relationships between GAL-012 and tunicamycin, as well as bortezomib (BTZ) in killing cultured cancer cells were observed, respectively. With its unique scaffold and relatively small size, GAL-012 serves as a promising early chemotype for optimization to become a safe, effective, multi-target anti-cancer drug candidate which could be used alone or in combination with known therapeutics.

A novel phosphoglucomutase-deficient mouse model reveals aberrant glycosylation and early embryonic lethality.

Patients with phosphoglucomutase (PGM1) deficiency, a congenital disorder of glycosylation (CDG) suffer from multiple disease phenotypes. Midline cleft defects are present at birth. Overtime, additional clinical phenotypes, which include severe hypoglycemia, hepatopathy, growth retardation, hormonal deficiencies, hemostatic anomalies, frequently lethal, early-onset of dilated cardiomyopathy and myopathy emerge, reflecting the central roles of the enzyme in (glycogen) metabolism and glycosylation. To delineate the pathophysiology of the tissue-specific disease phenotypes, we constructed a constitutive Pgm2 (mouse ortholog of human PGM1)-knockout (KO) mouse model using CRISPR-Cas9 technology. After multiple crosses between heterozygous parents, we were unable to identify homozygous life births in 78 newborn pups (P = 1.59897E-06), suggesting an embryonic lethality phenotype in the homozygotes. Ultrasound studies of the course of pregnancy confirmed Pgm2-deficient pups succumb before E9.5. Oral galactose supplementation (9 mg/mL drinking water) did not rescue the lethality. Biochemical studies of tissues and skin fibroblasts harvested from heterozygous animals confirmed reduced Pgm2 enzyme activity and abundance, but no change in glycogen content. However, glycomics analyses in serum revealed an abnormal glycosylation pattern in the Pgm2+/- animals, similar to that seen in PGM1-CDG.

Discovery of novel inhibitors of human galactokinase by virtual screening.

Classic Galactosemia is a potentially lethal autosomal recessive metabolic disorder caused by deficient galactose-1-phosphate uridyltransferase (GALT) that results in the buildup of galactose-1-phosphate (gal-1-p) in cells. Galactokinase (GALK1) is the enzyme responsible for converting galactose into gal-1-p. A pharmacological inhibitor of GALK1 is hypothesized to be therapeutic strategy for treating galactosemia by reducing production of gal-1-p. In this study, we report the discovery of novel series of GALK1 inhibitors by structure-based virtual screening (VS). Followed by an extensive structural modeling and binding mode analysis of the active compounds identified from quantitative high-throughput screen (qHTS), we developed an efficient pharmacophore-based VS approach and applied for a large-scale in silico database screening. Out of 230,000 compounds virtually screened, 350 compounds were cherry-picked based on multi-factor prioritization procedure, and 75 representing a diversity of chemotypes exhibited inhibitory activity in GALK1 biochemical assay. Furthermore, a phenylsulfonamide series with excellent in vitro ADME properties was selected for downstream characterization and demonstrated its ability to lower gal-1-p in primary patient fibroblasts. The compounds described herein should provide a starting point for further development of drug candidates for the GALK1 modulation in the Classic Galactosemia.

Effect of genotype on galactose-1-phosphate in classic galactosemia patients.

Impaired activity of galactose-1-phosphate uridyltransferase (GALT) causes classic galactosemia (OMIM 230400), characterized by the accumulation of galactose-1-phosphate (GAL1P) in patients' red blood cells (RBCs). Our recent study demonstrated a correlation between RBC GAL1P and long-term outcomes in galactosemia patients. Here, we analyze biochemical and molecular results in 77 classic galactosemia patients to evaluate the association between GALT genotypes and GAL1P concentration in RBCs. Experimental data from model organisms were also included to assess the correlation between GAL1P and predicted residual activity of each genotype. Although all individuals in this study showed markedly reduced RBC GALT activity, we observed significant differences in RBC GAL1P concentrations among galactosemia genotypes. While levels of GAL1P on treatment did not correlate with RBC GALT activities (p = 0.166), there was a negative nonlinear correlation between mean GAL1P concentrations and predicted residual enzyme activity of genotype (p = 0.004). These studies suggest that GAL1P levels in RBCs on treatment likely reflect the overall functional impairment of GALT in patients with galactosemia.

Assessment of ataxia phenotype in a new mouse model of galactose-1 phosphate uridylyltransferase (GALT) deficiency.

Despite adequate dietary management, patients with classic galactosemia continue to have increased risks of cognitive deficits, speech dyspraxia, primary ovarian insufficiency, and abnormal motor development. A recent evaluation of a new galactose-1 phosphate uridylyltransferase (GALT)-deficient mouse model revealed reduced fertility and growth restriction. These phenotypes resemble those seen in human patients. In this study, we further assess the fidelity of this new mouse model by examining the animals for the manifestation of a common neurological sequela in human patients: cerebellar ataxia. The balance, grip strength, and motor coordination of GALT-deficient and wild-type mice were tested using a modified rotarod. The results were compared to composite phenotype scoring tests, typically used to evaluate neurological and motor impairment. The data demonstrated abnormalities with varying severity in the GALT-deficient mice. Mice of different ages were used to reveal the progressive nature of motor impairment. The varying severity and age-dependent impairments seen in the animal model agree with reports on human patients. Finally, measurements of the cerebellar granular and molecular layers suggested that mutant mice experience cerebellar hypoplasia, which could have resulted from the down-regulation of the PI3K/Akt signaling pathway.

Galactose-1 phosphate uridylyltransferase (GalT) gene: A novel positive regulator of the PI3K/Akt signaling pathway in mouse fibroblasts.

The vital importance of the Leloir pathway of galactose metabolism has been repeatedly demonstrated by various uni-/multicellular model organisms, as well human patients who have inherited deficiencies of the key GAL enzymes. Yet, other than the obvious links to the glycolytic pathway and glycan biosynthetic pathways, little is known about how this metabolic pathway interacts with the rest of the metabolic and signaling networks. In this study, we compared the growth and the expression levels of the key components of the PI3K/Akt growth signaling pathway in primary fibroblasts derived from normal and galactose-1 phosphate uridylyltransferase (GalT)-deficient mice, the latter exhibited a subfertility phenotype in adult females and growth restriction in both sexes. The growth potential and the protein levels of the pAkt(Thr308), pAkt(Ser473), pan-Akt, pPdk1, and Hsp90 proteins were significantly reduced by 62.5%, 60.3%, 66%, 66%, and 50%, respectively in the GalT-deficient cells. Reduced expression of phosphorylated Akt proteins in the mutant cells led to diminished phosphorylation of Gsk-3ß (-74%). Protein expression of BiP and pPten were 276% and 176% higher respectively in cells with GalT-deficiency. Of the 24 genes interrogated using QIAGEN RT(2) Profiler PCR Custom Arrays, the mRNA abundance of Akt1, Pdpk1, Hsp90aa1 and Pi3kca genes were significantly reduced at least 2.03-, 1.37-, 2.45-, and 1.78-fold respectively in mutant fibroblasts. Both serum-fasted normal and GalT-deficient cells responded to Igf-1-induced activation of Akt phosphorylation at +15 min, but the mutant cells have lower phosphorylation levels. The steady-state protein abundance of Igf-1 receptor was also significantly reduced in mutant cells. Our results thus demonstrated that GalT deficiency can effect down-regulation of the PI3K/Akt growth signaling pathway in mouse fibroblasts through distinct mechanisms targeting both gene and protein expression levels.

Structure activity relationships of human galactokinase inhibitors.

Classic Galactosemia is a rare inborn error of metabolism that is caused by deficiency of galactose-1-phosphate uridyltransferase (GALT), an enzyme within the Leloir pathway that is responsible for the conversion of galactose-1-phosphate (gal-1-p) and UDP-glucose to glucose-1-phosphate and UDP-galactose. This deficiency results in elevated intracellular concentrations of its substrate, gal-1-p, and this increased concentration is believed to be the major pathogenic mechanism in Classic Galactosemia. Galactokinase (GALK) is an upstream enzyme of GALT in the Leloir pathway and is responsible for conversion of galactose and ATP to gal-1-p and ADP. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of human GALK would act to prevent the accumulation of gal-1-p and offer a novel entry therapy for this disorder. Herein we describe a quantitative high-throughput screening campaign that identified a single chemotype that was optimized and validated as a GALK inhibitor.

Whole-body galactose oxidation as a robust functional assay to assess the efficacy of gene-based therapies in a mouse model of Galactosemia.

Despite the implementation of lifesaving newborn screening programs and a galactose-restricted diet, many patients with classic galactosemia develop long-term debilitating neurological deficits and primary ovarian insufficiency. Previously, we showed that the administration of human GALT mRNA predominantly expressed in the GalT gene-trapped mouse liver augmented the expression of hepatic GALT activity, which decreased not only galactose-1 phosphate (gal-1P) in the liver but also peripheral tissues. Since each peripheral tissue requires distinct methods to examine the biomarker and/or GALT effect, this highlights the necessity for alternative strategies to evaluate the overall impact of therapies. In this study, we established that whole-body galactose oxidation (WBGO) as a robust, noninvasive, and specific method to assess the in vivo pharmacokinetic and pharmacodynamic parameters of two experimental gene-based therapies that aimed to restore GALT activity in a mouse model of galactosemia. Although our results illustrated the long-lasting efficacy of AAVrh10-mediated GALT gene transfer, we found that GALT mRNA therapy that targets the liver predominantly is sufficient to sustain WBGO. The latter could have important implications in the design of novel targeted therapy to ensure optimal efficacy and safety.

The hypergonadotropic hypogonadism conundrum of classic galactosemia.

BACKGROUND: Hypergonadotropic hypogonadism is a burdensome complication of classic galactosemia (CG), an inborn error of galactose metabolism that invariably affects female patients. Since its recognition in 1979, data have become available regarding the clinical spectrum, and the impact on fertility. Many women have been counseled for infertility and the majority never try to conceive, yet spontaneous pregnancies can occur. Onset and mechanism of damage have not been elucidated, yet new insights at the molecular level are becoming available that might greatly benefit our understanding. Fertility preservation options have expanded, and treatments to mitigate this complication either by directly rescuing the metabolic defect or by influencing the cascade of events are being explored. OBJECTIVE AND RATIONALE: The aims are to review: the clinical picture and the need to revisit the counseling paradigm; insights into the onset and mechanism of damage at the molecular level; and current treatments to mitigate ovarian damage. SEARCH METHODS: In addition to the work on this topic by the authors, the PubMed database has been used to search for peer-reviewed articles and reviews using the following terms: 'classic galactosemia', 'gonadal damage', 'primary ovarian insufficiency', 'fertility', 'animal models' and 'fertility preservation' in combination with other keywords related to the subject area. All relevant publications until August 2022 have been critically evaluated and reviewed. OUTCOMES: A diagnosis of premature ovarian insufficiency (POI) results in a significant psychological burden with a high incidence of depression and anxiety that urges adequate counseling at an early stage, appropriate treatment and timely discussion of fertility preservation options. The cause of POI in CG is unknown, but evidence exists of dysregulation in pathways crucial for folliculogenesis such as phosphatidylinositol 3-kinase/protein kinase B, inositol pathway, mitogen-activated protein kinase, insulin-like growth factor-1 and transforming growth factor-beta signaling. Recent findings from the GalT gene-trapped (GalTKO) mouse model suggest that early molecular changes in 1-month-old ovaries elicit an accelerated growth activation and burnout of primordial follicles, resembling the progressive ovarian failure seen in patients. Although data on safety and efficacy outcomes are still limited, ovarian tissue cryopreservation can be a fertility preservation option. Treatments to overcome the genetic defect, for example nucleic acid therapy such as mRNA or gene therapy, or that influence the cascade of events are being explored at the (pre-)clinical level. WIDER IMPLICATIONS: Elucidation of the molecular pathways underlying POI of any origin can greatly advance our insight into the pathogenesis and open new treatment avenues. Alterations in these molecular pathways might serve as markers of disease progression and efficiency of new treatment options.

AAV-based gene therapy prevents and halts the progression of dilated cardiomyopathy in a mouse model of phosphoglucomutase 1 deficiency (PGM1-CDG).

Phosphoglucomutase 1 (PGM1) deficiency is recognized as the third most common N-linked congenital disorders of glycosylation (CDG) in humans. Affected individuals present with liver, musculoskeletal, endocrine, and coagulation symptoms; however, the most life-threatening complication is the early onset of dilated cardiomyopathy (DCM). Recently, we discovered that oral D-galactose supplementation improved liver disease, endocrine, and coagulation abnormalities, but does not alleviate the fatal cardiomyopathy and the associated myopathy. Here we report on left ventricular ejection fraction (LVEF) in 6 individuals with PGM1-CDG. LVEF was pathologically low in most of these individuals and varied between 10% and 65%. To study the pathobiology of the cardiac disease observed in PGM1-CDG, we constructed a novel cardiomyocyte-specific conditional Pgm2 gene (mouse ortholog of human PGM1) knockout (Pgm2 cKO) mouse model. Echocardiography studies corroborated a DCM phenotype with significantly reduced ejection fraction and left ventricular dilation similar to those seen in individuals with PGM1-CDG. Histological studies demonstrated excess glycogen accumulation and fibrosis, while ultrastructural analysis revealed Z-disk disarray and swollen/fragmented mitochondria, which was similar to the ultrastructural pathology in the cardiac explant of an individual with PGM1-CDG. In addition, we found decreased mitochondrial function in the heart of KO mice. Transcriptomic analysis of hearts from mutant mice demonstrated a gene signature of DCM. Although proteomics revealed only mild changes in global protein expression in left ventricular tissue of mutant mice, a glycoproteomic analysis unveiled broad glycosylation changes with significant alterations in sarcolemmal proteins including different subunits of laminin-211, which was confirmed by immunoblot analyses. Finally, augmentation of PGM1 in KO mice via AAV9-PGM1 gene replacement therapy prevented and halted the progression of the DCM phenotype.

Correlation assessment among clinical phenotypes, expression analysis and molecular modeling of 14 novel variations in the human galactose-1-phosphate uridylyltransferase gene.

Galactose-1-phosphate uridylyltransferase (GALT) catalyzes the conversion of galactose-1-phosphate to UDP-galactose, a key step in the galactose metabolism. Deficiency of GALT activity in humans caused by deleterious variations in the GALT gene can cause a potentially lethal disease called classic galactosemia. In this study, we selected 14 novel nucleotide sequence changes in the GALT genes found in galactosemic patients for expression analysis and molecular modeling. Several variants showed decreased levels of expression and decreased abundance in the soluble fraction of the Escherichia coli cell extracts, suggesting altered stability and solubility. Only six variant GALT enzymes had detectable enzymatic activities. Kinetic studies showed that their V(max) decreased significantly. To further characterize the variants at molecular level, we performed static and dynamic molecular modeling studies. Effects of variations on local and/or global structural features of the enzyme were anticipated for the majority of variants. In-depth studies with molecular dynamic simulations on selected variants predicted the alteration of the protein structure even though static models apparently did not highlight any perturbation. Overall, these studies offered new insights on the molecular properties of GALT enzyme, with the aim of correlating them with the clinical outcome. Hum Mutat 33:1107-1115, 2012. © 2012 Wiley Periodicals, Inc.

Formal Synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol From D-(+)-Arabitol.

2-C-methyl-D-erythritol-4-phosphate (MEP) is a key chemical intermediate of the non-mevalonate pathway for isoprenoid biosynthesis employed by many pathogenic microbes. MEP is also the precursor for the synthesis of 4-diphosphocytidyl-2-C-methyl D-erythritol (CDP-ME), another key intermediate of the non-mevalonate pathway. As this pathway is non-existent in higher animals, including humans, it represents great opportunities for novel antimicrobial development. To facilitate the in-depth studies of this pathway, we reported here a formal synthesis of CDP-ME through a new synthesis of 2-C-Methyl-D-erythritol-4-phosphoric acid from D-(+)-arabitol.

Early postnatal alterations in follicular stress response and survival in a mouse model of Classic Galactosemia.

Primary ovarian insufficiency is characterized by accelerated loss of primordial follicles, which results in ovarian failure and concomitant menopause before age 40. About 1-3% of females in the general population are diagnosed with POI; however, greater than 80% of females with the inherited disease Classic Galactosemia will develop POI. Classic Galactosemia is caused by mutations in the GALT gene encoding the enzyme galactose-1 phosphate uridylyltransferase. While dietary restriction of galactose is lifesaving in the neonatal period, the development of complications including primary ovarian insufficiency is not mitigated. Additionally, the pattern(s) of follicle loss have not been completely characterized. The chronic accumulation of aberrant metabolites such as galactose-1-phosphate and galactitol are suspected culprits in the development of the sequelae, yet the mechanisms remain elusive.Our group uses a GalT gene-trapped mouse model to study the pathophysiology of primary ovarian insufficiency in Classic Galactosemia. We recently showed that differences in the Integrated Stress Response pathway occur in mutant ovaries that likely contribute to their primary ovarian insufficiency phenotype. Using immunofluorescent staining of histological sections of ovaries at progressive ages, we saw evidence of altered Integrated Stress Response activity in granulosa cells and primordial oocytes consistent with accelerated primordial follicle growth activation, aberrant DNA damage and/or repair, and increased cellular stress/death. Overall, our findings indicate that abnormal Integrated Stress Response in the Classic Galactosemia model ovary results in accelerated primordial follicle growth activation, sometimes referred to as "burnout." These aberrant early events help further clarify when/how the primary ovarian insufficiency phenotype arises under galactosemic conditions.

Pathophysiology and management of classic galactosemic primary ovarian insufficiency.

Classic galactosemia is an inborn error of carbohydrate metabolism associated with early-onset primary ovarian insufficiency (POI) in young women. Our understanding of the consequences of galactosemia upon fertility and fecundity of affected women is expanding, but there are important remaining gaps in our knowledge and tools for its management, and a need for continued dialog so that the special features of the condition can be better managed. Here, we review galactosemic POI and its reproductive endocrinological clinical sequelae and summarize current best clinical practices for its management. Special consideration is given to the very early-onset nature of the condition in the pediatric/adolescent patient. Afterward, we summarize our current understanding of the reproductive pathophysiology of galactosemia, including the potential action of toxic galactose metabolites upon the ovary. Our work establishing that ovarian cellular stress reminiscent of endoplasmic reticulum (ER) stress is present in a mouse model of galactosemia, as well as work by other groups, are summarized. LAY SUMMARY: Patients with the condition of classic galactosemia need to maintain a strict lifelong diet that excludes the sugar galactose. This is due to having mutations in enzymes that process galactose, resulting in the buildup of toxic metabolic by-products of the sugar. Young women with classic galactosemia often lose the function of their ovaries very early in life (termed 'primary ovarian insufficiency'), despite adherence to a galactose-restricted diet. This means that in addition to the consequences of the disease, these women also face infertility and the potential need for hormone replacement therapy. This article summarizes current strategies for managing the care of galactosemic girls and women and also what is known of how the condition leads to early primary ovarian insufficiency.

Fragment Screening Reveals Starting Points for Rational Design of Galactokinase 1 Inhibitors to Treat Classic Galactosemia.

Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.

Structure-Based Optimization of Small Molecule Human Galactokinase Inhibitors.

Classic galactosemia is a rare disease caused by inherited deficiency of galactose-1 phosphate uridylyltransferase (GALT). Accumulation of galactose-1 phosphate (gal-1P) is thought to be the major cause of the chronic complications associated with this disease, which currently has no treatment. Inhibiting galactokinase (GALK1), the enzyme that generates galactose-1 phosphate, has been proposed as a novel strategy for treating classic galactosemia. Our previous work identified a highly selective unique dihydropyrimidine inhibitor against GALK1. With the determination of a co-crystal structure of this inhibitor with human GALK1, we initiated a structure-based structure-activity relationship (SAR) optimization campaign that yielded novel analogs with potent biochemical inhibition (IC50 < 100 nM). Lead compounds were also able to prevent gal-1P accumulation in patient-derived cells at low micromolar concentrations and have pharmacokinetic properties suitable for evaluation in rodent models of galactosemia.