Future

Review Special Focus Issue: Rare Diseases

Medicinal Chemistry

For reprint orders, please contact [email protected]

GALK inhibitors for classic galactosemia

Classic galactosemia is an inherited metabolic disease for which, at present, no therapy is available apart from galactose-restricted diet. However, the efficacy of the diet is questionable, since it is not able to prevent the insurgence of chronic complications later in life. In addition, it is possible that dietary restriction itself could induce negative side effects. Therefore, there is a need for an alternative therapeutic approach that can avert the manifestation of chronic complications in the patients. In this review, the authors describe the development of a novel class of pharmaceutical agents that target the production of a toxic metabolite, galactose-1-phosphate, considered as the main culprit for the cause of the complications, in the patients.

Inborn errors of metabolism (IEM; also referred to as inherited metabolic disorders) are a large class of genetic diseases, caused mainly by defects in genes coding for enzymes involved in the metabolism of various substances, leading to accumulation or deficiency of a specific metabolite [1] . Many mutations can impair the structure, function and stability of the enzyme, leading to clinical symptoms that can involve every system of the body, and can be acute or chronic [2] . Typically, the main adverse effects of IEM occur at the gastrointestinal or neurological level. Classical gastrointestinal symptoms are vomiting, failure to thrive, jaundice, hepatomegaly and liver failure, whereas classical neurological symptoms are developmental delay, ataxia and neuropsychiatric manifestations [3] . Other less general symptoms are linked to the specific type of disorder. The onset of symptoms may occur during pregnancy, at birth or later in life, and the severity and type of adverse effects varies with age and with the type of disorder [2] . The management strategy of IEM ranges from intensive care to support therapies, but the main intervention is to remove the toxic metabolites that may have precipitated the illness as soon as they are identified, or to supplement the patients with deficient

10.4155/FMC.14.43 © 2014 Future Science Ltd

metabolites, if known. Indeed, the severity of adverse effects of IEM is often related to the concentration and duration of tissue exposure to toxic metabolites. The specific therapy must be adopted as soon as the diagnosis is confirmed, and generally it must be continued lifelong [4] . Classic galactosemia (OMIM: 230400) is an IEM caused by the impairment of the enzyme GALT (EC 2.7.7.12), which catalyzes the second step in the Leloir pathway of galactose metabolism (Figure 1) [5] . The symptoms of this disease appear in newborns soon after the initiation of breastfeeding or feeding with other milk-based formulas [6] , and some of them (jaundice, vomiting, hepatomegaly and failure to thrive) are among those typical of IEM listed above. In addition, another peculiar symptom is cataracts that develop a few days after galactose exposure [7] . A total of 10% of galactosemic newborns manifest sepsis, mainly caused by Escherichia coli, and this is the main cause of early mortality associated with this disorder [8] . The immediate removal of dietary galactose (e.g., replacing human milk or milk-based formulas with soy-based formulas) usually allows the quick resolution of acute clinical issues [9] . A galactose-free diet, based upon exclusion of milk and of dairy products from the diet, is recommended

Future Med. Chem. (2014) 6(9), 1003–1015

Kent Lai1, Matthew B Boxer2 & Anna Marabotti*,3 Division of Medical Genetics, Department of Pediatrics, University of Utah, 50 N Medical Drive, Salt Lake City, UT 84132, USA 2 National Center for Advancing Translational Sciences, NIH, 9800 Medical Center Drive, Rockville, MD 20850, USA 3 Department of Chemistry & Biology, University of Salerno, Via Giovanni Paolo II, 132, 84084 Fisciano (SA), Italy *Author for correspondence: Tel.: +39 089 969583 Fax: +39 089 969603 [email protected] 1

part of

ISSN 1756-8919

1003

Review  Lai, Boxer & Marabotti

Key Terms Classic galactosemia: Inborn error of metabolism linked to the impairment of the GALT enzyme caused by genetic mutations. Its prevalence is approximately 1:40,00060,000 newborns, with peculiar distribution of common mutations among different geographical areas and ethnic groups. GALT: Key enzyme of galactose metabolism, which catalyzes the reaction between galactose-1-phosphate and UDP-glucose to form glucose-1-phosphate and UDP-galactose in two steps with a ping-pong kinetic mechanism. This enzyme is a member of the branch III of histidine triad superfamily, characterized by a specific nucleoside monophosphate transferase activity. It is a homodimer with two active sites located at the dimer interface and formed by residues belonging to both subunits. Galactose-1-phosphate: Metabolite of galactose, formed by the ATP-dependent phosphorylation of the 1-hydroxyl group of α-D-galactose catalyzed by the enzyme GALK.

lifelong [10] . In addition, vitamin D and calcium supplements should be given to galactosemic patients, in order to ensure that the appropriate calcium intake is provided [11,12] . Despite dietary restriction, during adolescence and adulthood many patients develop long-term negative outcomes, especially affecting neurodevelopment, ovarian function, growth and cataracts [13–16] . Many galactosemic patients manifest intellectual deficits, speech problems, such as dyspraxia [17–19] , and neurologic issues, such as tremors, ataxia or dysarthria [20] . Premature ovarian insufficiency is also frequent in most females and often leads to infertility [21] ; on the contrary, gonads in galactosemic males seem less affected than in females since men with galactosemia show a higher than expected prevalence of cryptorchidism and low semen volumes; however, this does not cause reproductive impairment [22] . Cataracts are very frequent, involving approximately 30% of ­galactosemic patients [14,16] . Galactosemia (type III)

GALK

Galactose

UDP-galactose

GALT Gal-1-P

Searching for a culprit The well-known and typical effect of GALT impairment in galactosemic patients at the biochemical level is the increase in the concentration of galactose1-phosphate (gal-1-P), particularly in red blood cells (RBCs), as well as in other tissues [24] . Typically, this intermediate of the Leloir pathway is converted by GALT into glucose-1-phosphate (glu-1-P) (Figure 1) , and thus the direct consequence of GALT deficiency is the accumulation of gal-1-P in the body (erythrocyte concentrations comprised between 20 and 50 mg/l despite therapy [9]). The importance attributed to the monitoring of the levels of this metabolite in galactosemic patients is attested by the development of many methods to assay its concentration in erythrocytes [25–34] . Actually, gal-1-P concentration in RBCs is the most sensitive index of compliance and effectiveness of dietary therapy. However, high levels of gal-1-P are not only due to the intake of dietary galactose. In galactosemic newborns, gal1-P levels in RBCs generally range above 20 mg/l, and during a galactose-free diet, the levels of gal-1-P oscillate between 10 and 50 mg/l [31] , although a high intra- and inter-individual biological variability is registered [35] . Therefore, the endogenous production of galactose and gal-1-P in these patients seems to sub-

GALE

UDP-glucose

Galactosemia (type II)

One of the most challenging issues about the treatment of galactosemia is that, in addition to diet-independent late-onset complications, many complications in galactosemic patients seem to be related to diet restrictions. For example, many galactosemic patients suffer from poor growth, short stature, reduced bone mineral density and osteoporosis; several physicians are concerned with the fact that diet restriction may have transformed galactosemia into a progressive disease [23] . Therefore, it is crucial to find new treatments to address all these clinical issues for galactosemic patients.

Classic galactosemia (type I) Glu-1-P

(Carbohydrate metabolism)

Figure 1. Leloir pathway of galactose metabolism. The three enzymes participating in the three steps of metabolism (indicated with round arrows) are written in bold italics. The type of galactosemia involving each enzyme is written near the enzyme itself. The final destination of Glu-1-P is the involvement in different steps of carbohydrate metabolism (indicated with the dashed arrow). Gal-1-P: Galactose-1-phosphate; Glu-1-P: Glucose-1-phosphate.

1004

Future Med. Chem. (2014) 6(9)

future science group

GALK inhibitors for classic galactosemia 

stantially contribute to the increased c­ oncentration of this metabolite in the body. Increased levels of gal-1-P are typical to classic galactosemia, while increased levels of galactose are common among all types of galactosemia. Therefore, this metabolite can be considered the culprit – that is, the responsible metabolite for the onset of symptoms ­specific for GALT deficiency [36] . There are several examples in literature that back up this claim. Many studies support the toxicity of gal1-P in model systems, such as yeast [36] . In addition, some studies found a correlation between gal-1-P levels and several clinical signs in galactosemic patients [9,17,34,37–38] . In addition, mild forms of galactosemia such as the Duarte form [39] or the one associated to the GALT p.S135L mutation, typical of patients of African ancestry [40] show near-to-normal levels of RBCs gal-1-P concentrations [41,42] . Moreover, the dietary intervention in order to drastically decrease the high levels of galactose metabolites in the blood does solve the acute clinical issue in newborns. Further indirect evidence for the pathogenic role of gal1-P in the onset of severe symptoms in GALT-deficient people is the typical clinical picture of those patients suffering from impairment of GALK (EC 2.7.1.6). This enzyme is a member of the GHMP family of smallmolecule kinases [43,44] and is devoted to the phosphorylation of galactose, forming gal-1-P in the first step in the Leloir pathway (Figure 1) [45] . The impairment of this enzyme owing to mutations is associated with another IEM, namely type II galactosemia (OMIM: 230200) [1] , and results in the inability to metabolize galactose via the classic pathway, leading to galactose accumulation within the body. However, GALK deficiency is clinically milder than classic galactosemia, resulting mainly in cataracts [46–49] , probably owing to the accumulation of galactitol formed from an alternative metabolism of galactose within the lenses. A few severe clinical features, such as pseudotumor cerebri were reported in literature [50] , but these instances are rare and a causal relationship between these symptoms and GALK deficiency is not certain [49] . In patients suffering from GALK deficiency, gal1-P is not produced, and therefore cannot accumulate. This evidence corroborates the suspicion that gal-1-P is the cause for long-term complications in GALT deficiency. These considerations have opened the way for a new approach in the treatment of classic galactosemia. In fact, it is conceivable that inhibition of GALK activity in GALT-deficient patients would induce in these people a condition similar to the mild type II galactosemia. By preventing the conversion of galactose into gal-1-P, perhaps the onset of long-term, severe complications typical of GALT deficiency could

future science group

Review

be avoided [51] . In addition, it has been proven that a double-knockout (GALK- GALT- ) strain of yeast grows well in the presence of galactose [52,53] . This has been the rationale for developing inhibitors of the GALK enzyme for potential use in classic galactosemic patients in order to improve their clinical outcome. Ways to identify small-molecule human GALK inhibitors In vitro biochemical screens & their results

To date, two high-throughput in vitro biochemical screens have been performed to identify small ­molecules acting as inhibitors for human GALK [51,54] . First pilot screen

For the first screen, the investigators, in collaboration with the scientists at the Broad Institute at Harvard University and Massachusetts Institute of Technology (MA, USA), developed a miniaturized (30 μl total volume), two-step in vitro bio­chemical ­high-throughput screening (HTS) assay for recombinant human GALK [51] . This assay measures the activity of human GALK enzyme in an indirect way, by determining the amount of ATP remaining after completion of the GALK-mediated reaction (Figure 2) . The authors used the ATP analog adenosine 5´-O-(3-thio)-triphosphate (ATP-γ-S) as a proof of principle to demonstrate increasing inhibition of GALK when increasing ATP-γ-S from 0 to 13.3 μM, keeping the concentration of ATP constant at 5 μM [51] . The S/N was calculated by comparing the readouts from reactions without GALK (i.e., 100% inhibition) to reactions with GALK. In this case, the ratio was greater than tenfold. Moreover, since this assay involves luciferase, the authors were aware of the fact that false negatives could be obtained from compounds competing with ATP for luciferase that would not be identified. Despite this apparent ‘undesired outcome’, this assay may, in the long run, reduce the number of nonselective hits for GALK. Indeed, the compounds that were missed were likely to interKey Terms GALK: Enzyme preceding GALT in the galactose metabolism pathway, which converts α-D-galactose into galactose-1-phosphate, in an ATP-dependent ordered reaction. It belongs to a unique class of ATP-dependent enzymes known as the GHMP superfamily and its structure is characterized by two domains with the active site wedged between them. High-throughput screening: An approach in drug discovery in which thousands or even millions of chemicals are extensively screened against one or more targets, in order to find those compounds able to bind to those targets and exert a biological/pharmacological activity.

www.future-science.com

1005

Review  Lai, Boxer & Marabotti

Reaction 1: Galactose + ATP

GALK

Gal-1-P + ADP

Kinase-Glo® recombinant luciferase Oxyluciferin + Reaction 2: ATP + luminescence luciferin Figure 2. High-throughput screening assay. ATP consumed in the first reaction is also the substrate for the coupled reaction. When GALK is active, the quantity of ATP remaining after the reaction is low and the luminescence produced in the coupled reaction will be low. When an inhibitor blocks GALK activity, more ATP is available to the coupled reaction, and therefore the luminescence is higher. Gal-1-P: Galactose-1-phosphate.

act with the ATP binding site of enzymes other than GALK, and are therefore deemed to be less selective inhibitors. Equipped with the newly developed HTS assay, the authors screened 50,000 compounds in duplicate from various libraries of small molecules with diverse structural scaffolds for inhibition of in vitro GALK activity. A Z´ factor assay was included each day as part of the quality control process and was determined using the formula Z´ factor = 1 - (3 × [σp + σn]/|μp - μn|) [55] . It was found to be 0.91 or higher under the optimized experimental conditions. Raw data were submitted to the HTS facility at the Broad Institute to be analyzed by their data analysis team. Duplicate luminescence measurements were corrected for background measurements using the method published in 2007 by Seiler and colleagues [56] . Out of 50,000 compounds, the investigators identified approximately 150 compounds that, at an average concentration of 33.3 μM, inhibited GALK activity in vitro more than 86.5%. The cut off of 86.5% was chosen as it gave a reasonable number of hits to work with in the subsequent confirmatory screens. During a confirmatory screen under identical experimental conditions, these compounds had a reproducibility score of at least 0.7. Following completion of the confirmatory screen of the 150 hits, 34 compounds were selected for further characterization. The other 116 compounds were excluded either because inhibitory properties on GALK activity were not confirmed in the confirmatory screen, or because of known toxicity [57] . A battery of assays was used in the initial ­characterization of these 34 compounds. Determination of IC50

The preferred approach to determine IC50 with high accuracy is to determine reaction rates at initial velocity – that is, zero substrate conversion, as the quantity of substrate conversion can have a negative influence

1006

Future Med. Chem. (2014) 6(9)

on this parameter [58] . Initially, when the investigators determined the IC50 with a luminescence-based assay, they found relatively high percentages of substrate conversion (∼50%). Therefore, they analyzed the dose–responses of the selected compounds using a different assay based on pyruvate kinase/lactate dehydrogenase [59] . In this assay, the galactose present in the reaction was in excess, while ATP consumed by the GALK reaction is recycled by the pyruvate kinase reaction. As a result, the calculated rates of reaction are close to initial velocity [58] . IC50 values were inferred by arbitrarily assigning the reaction rates of the control reactions (with no inhibitors) as 100%. These data were fitted with a standard dose–response inhibition model and, interestingly, the investigators found no significant difference between the results of the two assays, and the IC50 of the compounds ranged from 0.7 μM to 33 μM [51] . Selectivity against other sugar & GHMP kinases

To further prioritize the compounds found with this HTS approach, a small kinase panel made of five kinases from different organisms (glucokinase from Bacillus stearothermophilus, hexokinase from Saccharomyces cerevisiae, human mevalonate kinase, homoserine kinase from Methanococcus jannaschii and 4-diphosphocytidyl2C-methyl-D-erythritol [CDP-ME] kinase from E. coli strain DH5α) was set up in order to test the selectivity of these compounds towards GALK [57] . These kinases were selected because all of them catalyze a similar reaction to GALK by transferring the γ-phosphoryl group of ATP to a six-carbon sugar or their respective substrates. It is worth mentioning that although GALK phosphorylates galactose, it does not belong to the same sugar kinase family as hexokinase and glucokinase. In fact, as told previously, GALK and the other three kinases belong to the GHMP kinase superfamily [43] . Except for one compound, which inhibited glucokinase weakly (IC50 : >60 μM), but not hexokinase, the rest of the selected inhibitors did not recognize hexokinase or glucokinase. When the authors tested the GALK inhibitors against the other three GHMP kinases, they found that a significant number of them also inhibited human mevalonate kinase with IC50 similar to that of GALK. Four compounds weakly inhibited homoserine kinase (IC50 : >60 μM) and eight inhibited bacterial CDP-ME kinase. The crossreactivity of these GALK inhibitors and CDP-ME kinase later led to the identification of some CDP-ME kinase inhibitors, which could p­otentially be developed into novel antimicrobials [60] . However, six compounds showed high selectivity for human GALK, having no detectable inhibition against any of the other tested GHMP kinases at a concentration up to 60 μM. Based on both potency and selectivity, the

future science group

GALK inhibitors for classic galactosemia 

investigators chose three compounds (compounds 1, 4 and 24) for further kinetic and cell-based studies [57] . Kinetic studies

Human GALK catalyzes the reaction in an ordered mechanism in which first ATP and then galactose are bound to the active site of the enzyme [61] . Extensive kinetic analysis of selected inhibitors revealed parabolic competitive, as well as mixed mode, ­inhibition [57] . Toxicity against human primary cell culture

The toxicity of these compounds was assessed by incubating 10–100 μM of each compound with human diploid fibroblasts for 3–6 days and determining their effect on cell viability. The majority of selected compounds had mild to severe toxicity, and only two compounds were not toxic at all up to 100 μM. These results, unfortunately, raised serious concerns about the suitability of some of these compounds for ­therapeutic uses. Lowering gal-1-P in primary & transformed fibroblasts

The inherent toxicity of tested compounds described above hampered earlier attempts to lower intracellular gal-1-P concentrations in cultured diploid primary GALT-deficient fibroblasts challenged with galactose. This motivated the investigators to select another GALT-deficient fibroblast cell line, which was more robust in culture and tolerant to the inhibitors [62] . These cells remain viable in the presence of a 30 μM concentration of these compounds. More importantly, these GALT-deficient cells accumulated gal-1-P upon galactose challenge, and, when treated with selected compounds, there was a dose-dependent effect of this inhibitor on gal-1-P accumulation. For instance, compound 1 at a concentration of 6 μM lowered the intracellular gal-1-P concentration to a level that approached that of the nonchallenged control. Similarly, treatment of compound 24 at a concentration of 20 μM decreased the accumulation of gal-1-P by 70% compared with the positive control [57] . This validated that small molecule inhibitors of GALK can lower gal-1-P in galactose-challenged GALT-deficient cells. Unfortunately, long-term exposure of these first-generation GALK inhibitors was too toxic for diploid cells. The knowledge of kinetic and structural interactions of this first group of selective GALK inhibitors with the enzyme, however, enabled further d­evelopment of nontoxic and more specific GALK inhibitors. Second HTS

Faced with the toxic and nonselective nature of some of the compounds identified from the first pilot screen,

future science group

Review

the investigators proceeded, in collaboration with the scientists at the NIH Chemical Genomics Center (presently at the National Center for Advancing Translational Sciences, MD, USA) to conduct a second HTS of 280,000 compounds [54] . To allow the screening of such large number of compounds, the investigators further miniaturized the established HTS assay to a total assay volume of 10 μl. In addition, the new HTS was performed using multiple concentrations of the compounds in a quantitative HTS format. A total of 149 hits were identified and they were grouped into 25 clusters according to their structural similarity. Among these clusters, they identified a set of three active compounds bearing a dihydropyrimidine core (Figure 3) , which inhibited human GALK at lower micromolar range with no inhibitory activity detected against over 200 other targets in previous screens at National Center for Advancing Translational Sciences. Akin to the primary screen, the confirmatory assay monitored ATP depletion using Promega’s KinaseGlo® technology (WI, USA). Initial ATP concentration was held at 35 μM, near its reported K M value [61] , and the K M for galactose was determined under the assay conditions to be 50–100 μM, which was significantly lower than the concentration used in the first pilot assay. Several rounds of medicinal chemistry were performed on the chemotype looking at the effect of the gem-dimethyl groups, as well as the size of the spiroring. A few substituted benzoxazoles were also made as well as a benzimidazole derivative. Through this preliminary structure–activity relationship analysis, it was determined that benzoxazole substituents decreased activity and the benzimidazole derivative was totally inactive [54] . Removal of the gem-dimethyl groups and incorporation of a cyclopentane ring was optimal and generated a small molecule capable of inhibiting GALK with an IC50 of 1.0 μM (ML152; CID_664331; SID_87550830) (Figure 3) . The characterization of this selected probe focused on parameters similar to those in the first pilot screen. For example, the selectivity for these analogs against CDP-ME kinase was performed using a bioluminescent Kinase-Glo assay that detects ATP depletion after the respective kinase reaction. The probe, ML152, and all analogs showed no activity in this assay (up to 57 μM), highlighting the divergence from previously reported inhibitors. Its mechanism of action, as determined via substrate competition and kinetic assays, is consistent with it being ATP competitive. All analogs were also tested for cytotoxicity using Promega CellTiter Glo® on a human embryonic kidney cell line that was treated with compounds and analyzed after 48 h. All compounds tested showed no cytotoxic effect. In addition, the liability of

www.future-science.com

1007

Review  Lai, Boxer & Marabotti

O

O NH Me Me

N

N H

Preliminary SAR

N O

qHTS hit

NH N

N H

N O

ML152 GALK IC50: 1 µM

Figure 3. Dihydropyrimidine scaffold from quantitative high-throughput screening leading to the GALK inhibitor probe, ML152. qHTS: Quantitative high-throughput screening; SAR: Structure–activity relationship analysis.

these compounds to undergo redox recycling, which may lead to false-positive results, was also evaluated using an end point colorimetric assay that detects the presence of hydrogen peroxide in the kinase reaction buffer; all analogs in the ML152 structural series were inactive in the redox recycling, further confirming that they have genuine GALK target activity. While ML152 provides a useful tool for inhibiting GALK, further work on this series (or identification of new series) to improve potency into the nanomolar range in both the biochemical and cell based assays is required to provide potential leads for galactosemia therapy. Computational approaches & their results

Computational approaches have become an essential part of the process of identifying lead compounds for drug design. In particular, virtual screening strategies enable researchers to screen millions of molecules from very large libraries of chemical compounds in a limited amount of time. The continuous development of supercomputers and infrastructures for innovative Key Terms Virtual screening: Automatic evaluation of very large libraries of small chemical compounds using simplified docking approaches in order to identify those structures which are more likely to bind to a drug target. Docking: Computational technique to predict the preferred binding mode of a small molecule to a large macromolecule. It can also be used to predict the binding affinity of the molecules. It is based on the evaluation of the interactions between the two molecules starting from their 3D structures, using different approaches such as analysis of geometric complementarity, analysis of surface molecular properties and inclusion of flexibility of molecules. A scoring function evaluates the strength of interaction between each possible solution of the docking simulation (called ‘pose’). Molecular dynamics simulations: Computational technique to simulate and investigate the motions of molecules and their temporal evolution. The trajectories are determined generally by solving the Newton’s equation of motion, defining the potential energies using force fields developed by applying classic mechanics to molecular systems.

1008

Future Med. Chem. (2014) 6(9)

strategies, such as distributed computing, has allowed researchers to achieve an impressive deployment of these strategies that have been applied to solve many biological problems [63,64] . Some computational approaches have been applied to the development of inhibitors of GALK enzyme. These strategies allowed a detailed insight on the molecular interaction between the protein and these small m­olecules. Docking of compounds to GALK

During the first HTS, the investigators used a molecular docking program to generate and analyze possible binding models for selected compounds [57] . Among the software programs cited in the literature, Glide (Schrödinger, LLC, NY, USA) [65–67] was regarded as one of the best at the time of the study [68] and was applied to predict the binding modes of the inhibitors. The protein structure used was that of human GALK protein cocrystallized with galactose and the ATP analog phosphoaminophosphonic acid–adenilate ester (deposited in the Protein Data Bank under the code 1WUU) (Figure 4) [69] . The resulting docking poses revealed the binding modes of the compounds and set the stage for further structure–activity relationship analysis and lead optimization [70] . To validate the docking poses, the investigators performed sitedirected mutagenesis experiments and confirmed the important roles of Ser140, Arg228, Glu174, Tyr236, Ser142 and Arg105 in the binding of the selected ­compounds [57] . Virtual screening approach

A virtual screening strategy was also applied to search for inhibitors of the GALK enzyme. Starting from ZINC, a public library of over 21 million commercially available chemicals [71] , 160,000 compounds were randomly selected and docked into the 3D structure of GALK enzyme. Four compounds selected among the top 200 hits were experimentally tested and found to be able to bind and inhibit GALK activity with an IC50 between 70 and 400 μM [70] .

future science group

GALK inhibitors for classic galactosemia 

Docking coupled to molecular dynamics simulations

A further computational approach was applied in order to study in more detail the recognition and the interaction of previously characterized compounds (compounds 1, 4, 24 and ML152) within the active site of the GALK enzyme. In this case, in order to overcome one of the most problematic issues in docking – that is, how to take into account receptor flexibility [72] , an approach was applied in which the selected compounds, once docked into the active site of the enzyme, were then submitted to molecular dynamics (MD) simulations [73] . This combined approach allowed to study the evolution of the interaction of these compounds during the time. In this way, several new highlights on the binding mode of compounds to the enzyme were provided. First, in addition to the known binding site, a secondary site was found. This cavity is placed alongside of the canonical binding site and can host galactose and most of the compounds tested (Figure 5) . The interaction energies of the compounds in this cavity were generally higher with respect to the canonical binding site, indicating that this binding mode is less preferred. However, since all MD simulations starting from this secondary cavity show that the ligand does not move from this cavity to the principal one, this additional cavity could be taken into account to design ligands capable of binding to both sites, thus increasing ligand specificity and/or affinity for the enzyme. In addition, MD simulations showed that the conformations assumed by the ligand during the simulations are not always overlapped with those identified by docking, suggesting that docking provides only one of the possible complex conformations (Figure 6) [73] . In addition, the higher variability in the position of ligands during the MD simulations with respect to galactose suggests that these inhibitors do not bind tightly to the GALK enzyme. This is in agreement with their IC50 values in the micromolar range [57] . Furthermore, the interactions of these ligands with GALK were found to be quite different compared with galactose. Indeed, the sugar interacts with the enzyme mainly forming H-bonds, with a low contribution of hydrophobic contacts, whereas the opposite is true in the case of the analyzed ligands. Finally, from the analysis of MD data, the residues capable of establishing the most stable H-bonds with galactose in the canonical binding site were identified. The information obtained, once implemented into a new virtual screening protocol, can be useful to increase the chance of finding new i­nhibitors of the GALK active site. An alternative approach was the creation of a structure-based pharmacophore model of GALK to identify

future science group

Review

the most important features involved in inhibitor binding [73] . The residues identified in this way belong both to the canonical and to the secondary binding site and are in agreement with those identified with the other approaches. The development of selective and efficient GALK inhibitors would be improved also when the catalytic mechanism of this enzyme will be totally understood. Recent works pointed out the role of Asp186 as the catalytic base and of Arg37 as a residue involved in substrate binding [74,75] , although very recently, performing an analysis of the mechanism of reaction of GALK enzyme using a hybrid quantum mechanics/molecular mechanics approach, researchers identified Arg228 as a residue participating in the mechanism of reaction, stabilizing the negative charge developed during the cleavage of ATP, whereas the role of Asp186 has been questioned [76] . It would be important to clarify definitely this point, since this would open the way to plan the development of GALK inhibitors that could form specific interactions with the enzyme, thus increasing their selectivity.

Figure 4. X-ray structure of GALK enzyme cocrystallized with galactose and ATP analog. Chain A of GALK enzyme extracted from file 1wuu.pdb is represented in the picture. The secondary structures of the enzyme are represented as springs (helices) and arrows (strands). Galactose and the ATP analog bound to enzyme are represented in stick mode and colored with carbon cyan and pink, respectively. Oxygen and nitrogen atoms of both ligands are represented in red and blue, respectively, and phosphorus atoms in ATP analog are represented in orange (hydrogen atoms are not visualized for sake of clarity). Mg2+ ion is represented as a pink sphere. For color images please see online at www.futurescience.com/doi/full/10.2217/fmc.14.43

www.future-science.com

1009

Review  Lai, Boxer & Marabotti

Figure 5. Close up of the active site of the GALK enzyme with galactose in the two possible binding cavities. Galactose and ATP are shown in sticks and colored by atom type; carbons of galactose best docking result in the canonical binding cavity are represented in green, carbons of galactose in the secondary binding cavity are in light blue. Residues establishing H-bonds are in sticks; in yellow residues of the canonical binding site and in magenta residues of the secondary cavity. For color images please see online at www.future-science.com/doi/full/10.2217/fmc.14.43 Reproduced with permission [73] ; © 2013 Elsevier Masson SAS. All rights reserved.

Open questions on the treatment of classic galactosemia Although the search for GALK inhibitors appears to be a promising approach to treat the severe outcomes of classic galactosemia, during the past years other hypotheses have been proposed for the origin of long-term complications associated to this disease. In fact, the role of gal1-P as the main cause has been questioned. For example, some clinical reports show that in adult galactosemic patients, there is not a clear-cut relationship between levels of RBC gal-1-P and severity of symptoms [14–15,77–79] . In some cases, it has been hypothesized that alternate metabolic pathways can reduce or avoid gal-1-P accumulation in these people [80] . Another puzzling case is the first GALT knockout mouse model developed [81] , in which, despite increased concentrations of gal-1-P, galactitol and galactonate in RBCs, no other symptoms typical of classic galactosemia were present [82] . In this case, it has been postulated that pathogenic target(s) of gal-1-P in humans are not present in rodents, thus explaining the fact that the clinical phenotype in these knockout mice is completely different from that of galactosemic patients [83] . However, very recently a new mouse model has been developed [84] , and, in addition to total absence of GALT activity in the homozygotes, it presents several

1010

Future Med. Chem. (2014) 6(9)

of the complications of classic galactosemia. Therefore, this model could be useful to investigate pathogenesis and new therapies for this disease. Recently, another animal model for classic galactosemia has been developed, based on GALT-null fruit fly [85] . This insect was chosen since some evidence highlighted that fruit flies are sensitive to oxidative stress induced by galactose, resulting in shortening of their lifespan [86] . Analogous to galactosemic patients, and in contrast with the first GALT knockout mice [81] , GALTdeficient fruit flies not only accumulate elevated levels of gal-1-P, but show locomotor impairment despite a galactose-free diet initiated early in their life, thereby mimicking someway long-term human complications [87] . This model was used in order to test the hypothesis that oxidative stress, a phenomenon also commonly found in other IEM [88] , can contribute, in part, to the insurgence of clinical symptoms in classic galactosemia. This hypothesis has been suggested by some anecdotal studies on patients with a poor compliance of galactose-free diet, showing that high levels of gal-1-P in blood were significantly associated with low levels of total antioxidant status, whereas the galactose-free diet restored nearto-normal levels [89] . Furthermore, low total antioxidant status and high gal-1-P levels are implicated with high

future science group

GALK inhibitors for classic galactosemia 

blood levels of 8-hydroxy-2-deoxyguanosine (a marker of oxidized DNA damage) in galactosemic patients [90] . Very recently, a study carried out on the GALT-deficient fruit fly supported the hypothesis that oxidative stress can contribute to the acute galactose sensitivity and, by extension, suggested that reactive oxygen species may also contribute to the acute pathophysiology in classic galactosemia [91] . In that study, the activity of two pro-oxidant (paraquat and dimethylsulfoxide) and two anti-oxidant molecules (vitamin C and α-mangostin) administered as food supplements to GALT-null insects, was tested and it was found that the pro-oxidants had a negative impact on the survival of knockout insects exposed to galactose, whereas the antioxidants showed a positive effect on acute galactose sensitivity. Afterwards, two manganese-based superoxide dismutase mimics were tested for their ability to rescue the damages caused by

Review

galactose in the GALT-null insects, and a benefit in terms both of survival and of reduction of long-term movement defects was registered for one of the two molecules tested, when it was administered starting from the larval state [92] . These positive effects appear to be independent of changes in the levels of gal-1-P; however, this does not imply that gal-1-P or other galactose metabolites are not involved in the pathophysiology of the human patients, since antioxidants could act downstream of gal-1-P toxicity pathway or independently from it [91] . Indeed, it has been suggested that two enzymes (G6PD and 6PGDH) catalyzing the production of NADPH are inhibited by gal-1-P [93] . NADPH is a cofactor for glutathione reductase, the major enzyme responsible for the regeneration of the reduced form of glutathione. Thus, the high RBCs levels of gal-1-P in the galactosemic patients could be implicated in an increase of free radicals production by

Figure 6. Positions of GALK enzyme inhibitors after molecular dynamics simulations starting with the ligand into the canonical binding site. Complex with (A) compound 1, (B) compound 4, (C) compound 24 and (D) ML152 are shown. Ligands are shown in stick mode and colored by atom type, carbons are colored based on docking results: conformations from simulations in the presence of ATP are in light green; conformation from simulations in the absence of ATP are in dark green. ATP volume is shown as dots. For color images please see online at www.future-science.com/doi/full/10.2217/fmc.14.43 Reproduced with permission with [73] ; © 2013 Elsevier Masson SAS. All rights reserved.

future science group

www.future-science.com

1011

Review  Lai, Boxer & Marabotti impairing the production of radical scavengers [89] , and the use of antioxidant molecules could reduce this last negative effect without resolving the root cause that g­enerated it. Conclusion This review is focused on the literature on both experimental and theoretical approaches designed for the identification, development and testing of small-molecule inhibitors of the human GALK enzyme, which, in turn, could be used as an improved therapy for patients with classic galactosemia. The information presented here not only summarizes the different approaches to address the issue, but also serves to stimulate further discussion from the scientific community who are interested in this ­subject. Future perspective A galactose-restricted diet is difficult to be kept by galactosemic patients, especially those from western countries, in which there is traditionally a high consumption of dairy products. In addition, given the fact that diet alone is not able to avoid the insurgence of long-term complications, it is likely that in the next 5–10 years more attention will be paid in the identification of a pharmacological therapy for classic galactosemia. At present, inhibitors of GALK enzyme are the most interesting candidates, as they have activity on the direct players of galactose metabolism. A deeper understanding of the

mechanisms of action of current GALK inhibitors may allow the development of more potent and selective molecules. In addition, molecules targeting side processes, such as oxidative stress, could also aid in the treatment of the disease. Other promising approaches such as gene therapy, approaches targeting RNA and enzyme replacement appear at an earlier stage of development. Furthermore, the development of new animal models (hopefully, mammals) to better reproduce the clinical picture of galactosemic patients will allow increased comprehension of biological mechanisms underlying this enigmatic pathology, and will permit testing of potential therapies in a more reliable model. Finally, better knowledge of the interplay between galactose, its metabolites and the overarching metabolic pathways will allow clarification of many aspects of this puzzling problem. Financial & competing interests disclosure This work was supported by research grant 1R01HD074844-01 (NIH/NICHD), another from the Galactosemia Foundation (K Lai), as well as one from ‘Fondi di Ateneo per la Ricerca di Base’ (FARB) 2013, ORSA130225 (A Marabotti). MB Boxer has a patent issued surrounding ML152. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary Background • Galactosemia is a rare metabolic disease in which many questions are still open concerning the causes of its long-term complications. Diet alone is unable to solve all issues related to this disease

Searching for a culprit • Galactose-1-phosphate is considered a responsible for the negative effects in galactosemic patients

Ways to identify small molecule human GALK inhibitors • The development of inhibitors of GALK, the enzyme responsible for galactose-1-phosphate biosynthesis, could be a means for the discovery of a pharmacological treatment for galactosemic people

Open questions on the treatment of classic galactosemia • Other emerging approaches can support the search for a cure for this genetic disease

References

4

Leonard JV, Morris AAM. Diagnosis and early management of inborn errors of metabolism presenting around the time of birth. Acta Paediatr. 95(1), 6–14 (2006).

Papers of special note have been highlighted as: • of interest; •• of considerable interest

1012

1

The Online Metabolic & Molecular Bases of Inherited Disease. Valle D, Beaudet A, Vogelstein B, Kinzler K, Antonarakis S, Ballabio A (Eds). McGraw Hill, NY, USA (2008).

5

Leloir LF. The enzymatic transformation of uridine diphosphate glucose into a galactose derivative. Arch. Biochem. Biophys. 33(2), 186–190 (1951).

2

Hoffmann GF, Nyhan WL, Zschocke J, Kahler SG, Mayatepek E. Inherited Metabolic Diseases. Lippincott Williams & Wilkins, PA, USA (2002).

6

3

Burton BK. Inborn errors of metabolism in infancy: a guide to diagnosis. Pediatrics 102(6), E69 (1998).

Berry GT, Walter JH. Disorders of galactose metabolism. In: Inborn Metabolic Diseases – Diagnosis and Treatment (5th Edition). Fernandes J, Saudubray M, van den Berghe G, Walter JH (Eds). Springer-Verlag, Heidelberg, Germany, 143–150 2012).

Future Med. Chem. (2014) 6(9)

future science group

GALK inhibitors for classic galactosemia 

7

Harley JD, Irvine S, Mutton P, Gupta JD. Maternal enzymes of galactose metabolism and the ‘inexplicable’ infantile cataract. Lancet 2(7875), 259–261 (1974).

25

Kirkman HN, Maxwell ES. Enzymatic estimation of erythrocytic galactose-1-phosphate. J. Lab. Clin. Med. 56, 161–166 (1960).

8

Levy HL, Sepe SJ, Shih VE, Vawter GF, Klein JO. Sepsis due to Escherichia coli in neonates with galactosemia. N. Engl. J. Med. 297(15), 823–825 (1977).

26

Gitzelmann R. Estimation of galactose-1-phosphate in erythrocytes: a rapid and simple enzymatic method. Clin. Chim. Acta 26(2), 313–316 (1969).

9

Classic galactosemia and clinical variant galactosemia. www.ncbi.nlm.nih.gov/books/NBK1518 

27

10

Bosch AM. Classic galactosemia: dietary dilemmas. J. Inherit. Metab. Dis. 34(2), 257–260 (2011).

Dahlqvist A. A fluorometric method for the assay of galactose-1-phosphate in red blood cells. J. Lab. Clin. Med. 78(6), 931–938 (1971).

28

11

Rutherford PJ, Davidson DC, Matthai SM. Dietary calcium in galactosaemia. J. Hum. Nutr. Diet. 15(1), 39–42 (2002).

12

Panis B, van Kroonenburgh MJ, Rubio-Gozalbo ME. Proposal for the prevention of osteoporosis in paediatric patients with classical galactosaemia. J. Inherit. Metab. Dis. 30(6), 982 (2007).

Bozkowa K, Duchnowska A, Chojnacki T, Mankowski T. The use of a radioisotopic method for estimation of galactose-1-phosphate in galactosemia. Biochem. Med. 17(1), 24–30 (1977).

29

13

Komrower CM, Lee DH. Long-term follow-up of galactosemia. Arch. Dis. Child. 45(241), 367–373 (1970).

Pesce MA, Bodourian SH, Nicholson JF. A new microfluorometric method for the measurement of galactose-1-phosphate in erythrocytes. Clin. Chim. Acta 118(2–3), 177–189 (1982).

30

14

Waggoner DD, Buist NR, Donnell GN. Long-term prognosis in galactosaemia: results of a survey of 350 cases. J. Inherit. Metab. Dis. 13(6), 802–818 (1990).

Ning C, Segal S. Plasma galactose and galactitol concentration in patients with galactose-1-phosphate uridyltransferase deficiency galactosemia: determination by gas chromatography/mass spectrometry. Metabolism 49(11), 1460–1466 (2000).

15

Schweitzer S, Shin Y, Jakobs C, Brodehl J. Long-term outcome in 134 patients with galactosaemia. Eur. J. Pediat. 152(1), 36–43 (1993).

31

16

Waisbren SE, Potter NL, Gordon CM et al. The adult galactosemic phenotype. J. Inherit. Metab. Dis. 35(2), 279–286 (2012).

Chen J, Yager C, Reynolds R, Palmieri M, Segal S. Erythrocyte galactose-1-phosphate quantified by isotopedilution gas chromatography-mass spectrometry. Clin. Chem. 48(4), 604–612 (2002).

32

Schadewaldt P, Kamalanathan L, Hammen HW, Wendel U. Stable-isotope dilution analysis of galactose metabolites in human erythrocytes. Rapid Commun. Mass Spectrom. 17(24), 2833–2838 (2003).

33

Jeong JS, Kwon HJ, Yoon HR, Lee YM, Choi TY, Hong SP. A pulsed amperometric detection method of galactose 1-phosphate for galactosemia diagnosis. Anal. Biochem. 376(2), 200–205 (2008).

34

Cangemi G, Barco S, Barbagallo L et al. Erythrocyte galactose-1-phosphate measurement by GC-MS in the monitoring of classical galactosemia. Scand. J. Clin. Lab. Invest. 72(1), 29–33 (2012).

35

Hutchesson AC, Murdoch-Davis C, Green A et al. Biochemical monitoring of treatment for galactosaemia: biological variability in metabolite concentrations. J. Inherit. Metab. Dis. 22(2), 139–148 (1999).

36

Lai K, Elsas LJ, Wierenga KJ. Galactose toxicity in animals. IUBMB Life 61(11), 1063–1074 (2009).

37

Guerrero NV, Singh RH, Manatunga A, Berry GT, Steiner RD, Elsas LJ 2nd. Risk factors for premature ovarian failure in females with galactosemia. J. Pediatr. 137(6), 833–841 (2000).

38

Robertson A, Singh RH, Guerrero NV, Hundley M, Elsas LJ. Outcomes analysis of verbal dyspraxia in classic galactosemia. Genet. Med. 2(2), 142–148 (2000).

39

Langley SD, Lai K, Dembure PP, Hjelm LN, Elsas LJ. Molecular basis for Duarte and Los Angeles variant galactosemia. Am. J. Hum. Genet. 60(2), 366–372 (1997).

40

Lai K, Langley SD, Singh RH, Dembure PP, Hjelm LN, Elsas LJ 2nd. A prevalent mutation for galactosemia among black Americans. J. Pediatr. 128(1), 89–95 (1996).

17

Webb AL, Singh RH, Kennedy MJ, Elsas LJ. Verbal dyspraxia and galactosemia. Pediatr. Res. 53(3), 396–402 (2003).

18

Hoffmann B, Wendel U, Schweitzer-Krantz S. Cross-sectional analysis of speech and cognitive performance in 32 patients with classic galactosemia. J. Inherit. Metab. Dis. 34(2), 421–427 (2011).

19

Potter NL, Nievergelt Y, Shriberg LD. Motor and speech disorders in classic galactosemia. JIMD Rep. 11, 31–41 (2013).

20

Ridel KR, Leslie ND, Gilbert DL. An updated review of the long-term neurological effects of galactosemia. Pediatr. Neurol. 33(3), 153–161 (2005).

21

Fridovich-Keil JL, Gubbels CS, Spencer JB, Sanders RD, Land JA, Rubio-Gozalbo E. Ovarian function in girls and women with GALT-deficiency galactosemia. J. Inherit. Metab. Dis. 34(2), 357–366 (2011).

22

Gubbels CS, Welt CK, Dumoulin JC et al. The male reproductive system in classic galactosemia: cryptorchidism and low semen volume. J. Inherit. Metab. Dis. 36(5), 779–786 (2013).

23

Berry GT, Elsas LJ. Introduction to the Maastricht workshop: lessons from the past and new directions in galactosemia. J. Inherit. Metab. Dis. 34(2), 249–255 (2011).

•

Interesting overview of recent literature on galactosemia, focused on present unsolved problems and future perspectives.

24

Donnell GN, Bergren WR, Perry G, Koch R. Galactose-1phosphate in galactosemia. Pediatrics 31, 802–810 (1963).

future science group

www.future-science.com

Review

1013

Review  Lai, Boxer & Marabotti 41

42

43

56

Seiler KP, George GA, Happ MP et al. ChemBank: a smallmolecule screening and cheminformatics resource database. Nucleic Acids Res. 36(Database issue), D351–D359 (2007).

57

Tang M, Wierenga K, Elsas LJ, Lai K. Molecular and biochemical characterization of human galactokinase and its small molecule inhibitors. Chem. Biol. Interact. 188(3), 376–385 (2010).

••

First characterization of the biological activity of the selected inhibitors of the GALK enzyme.

58

Wu G, Yuan Y, Hodge CN. Determining appropriate substrate conversion for enzymatic assays in high-throughput screening. J. Biomol. Screen. 8(6), 694–700 (2003).

59

Heinrich MR, Howard SM. Galactokinase. Methods Enzymol. 9, 407–412 (1966).

60

Tang M, Odejinmi SI, Allette YM, Vankayalapati H, Lai K. Identification of novel small molecule inhibitors of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase of Gram-negative bacteria. Bioorg. Med. Chem. 19(19), 5886–5895 (2011).

61

Timson DJ, Reece RJ. Functional analysis of disease-causing mutations in human galactokinase. Eur. J. Biochem. 270(8), 1767–1774 (2003).

62

Slepak TI, Tang M, Slepak VZ, Lai K. Involvement of endoplasmic reticulum stress in a novel classic galactosemia model. Mol. Genet. Metab. 92(1–2), 78–87 (2007).

63

Sousa SF, Cerqueira NM, Fernandes PA, Ramos MJ. Virtual screening in drug design and development. Comb. Chem. High Throughput Screen. 13(5), 442–453 (2010).

64

Lill M. Virtual screening in drug design. Methods Mol. Biol. 993, 1–12 (2013).

65

Friesner RA, Banks JL, Murphy RB et al. Glide: a new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. J. Med. Chem. 47(7), 1739–1749 (2004).

66

Halgren TA, Murphy RB, Friesner RA et al. Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J. Med. Chem. 47(7), 1750–1759 (2004).

67

Friesner RA, Murphy RB, Repasky MP et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein–ligand complexes. J. Med. Chem. 49(21), 6177–6196 (2006).

68

Michino M, Abola E, GPCR Dock 2008 participants et al. Community-wide assessment of GPCR structure modelling and ligand docking: GPCR Dock 2008. Nat. Rev. Drug Discov. 8(6), 455–463 (2009).

69

Toward improved therapy for classic galactosemia. www.ncbi.nlm.nih.gov/books/NBK56237  

Thoden JB, Timson DJ, Reece RJ, Holden HM. Molecular structure of human galactokinase: implications for type II galactosemia. J. Biol. Chem. 280(10), 9662–9670 (2005).

•

Follow-up work in the search and optimization of GALK inhibitors.

Presents and discusses the crystallographic structure of the GALK enzyme.

70

Tang M, Odejinmi SI, Vankayalapati H, Wierenga KJ, Lai K. Innovative therapy for classic galactosemia – tale of two HTS. Mol. Genet. Metab. 105(1), 44–55 (2012). 

•

Review on the high-throughput screenings made to find inhibitors of GALK enzyme.

Ficicioglu C, Hussa C, Gallagher PR, Thomas N, Yager C. Monitoring of biochemical status in children with Duarte galactosemia: utility of galactose, galactitol, galactonate, and galactose 1-phosphate. Clin. Chem. 56(7), 1177–1182 (2010). Bork P, Sander C, Valencia A. Convergent evolution of similar enzymatic function on different protein folds: the hexokinase, ribokinase, and galactokinase families of sugar kinases. Protein Sci. 2(1), 31–40 (1993).

44

Timson DJ. GHMP kinases – structures, mechanisms and potential for therapeutically relevant inhibition. Curr. Enz. Inhib. 3(1), 77–94 (2007).

45

Holden HM, Thoden JB, Timson DJ, Reece RJ. Galactokinase: structure, function and role in type II galactosemia. Cell. Mol. Life Sci. 61(19–20), 2471–2484 (2004).

46

Gitzelmann R. Hereditary galactokinase deficiency, a newly recognized cause of juvenile cataracts. Pediatr. Res. 1, 14–23 (1967).

47

Levy NS, Krill AE, Beutler E. Galactokinase deficiency and cataracts. Am. J. Ophthalmol. 74(1), 41–48 (1972).

48

Beutler E, Matsumoto F, Kuhl W et al. Galactokinase deficiency as a cause of cataracts. N. Engl. J. Med. 288(23), 1203–1206 (1973).

49

Bosch AM, Bakker HD, van Gennip AH, van Kempen JV, Wanders RJA, Wijburg FA. Clinical features of galactokinase deficiency: a review of the literature. J. Inherit. Metab. Dis. 25(8), 629–634 (2002).

•

Interesting review on the effects of galactokinase deficiency.

50

Litman N, Kanter AI, Finberg L. Galactokinase deficiency presenting as pseudotumor cerebri. J. Pediatr. 86(3), 410–412 (1975).

51

Wierenga KJ, Lai K, Buchwald P, Tang M. High-throughput screening for human galactokinase inhibitors. J. Biomol. Screen. 13(5), 415–423 (2008).

••

First work looking at GALK inhibitors for classic galactosemia.

52

Douglas HC, Hawthorne DC. Enzymatic expression and genetic linkage of genes controlling galactose utilization in saccharomyces. Genetics 49, 837–844 (1964).

53

Douglas HC, Hawthorne DC Regulation of genes controlling synthesis of the galactose pathway enzymes in yeast. Genetics 54(3), 911–916 (1966).

54

•• 55

1014

Landt M, Ritter D, Lai K, Benke PJ, Elsas LJ, Steiner RD. Black children deficient in galactose 1-phosphate uridyltransferase: correlation of activity and immunoreactive protein in erythrocytes and leukocytes. J. Pediatr. 130(6), 972–980 (1997).

Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4(2), 67–73 (1999).

Future Med. Chem. (2014) 6(9)

future science group

GALK inhibitors for classic galactosemia 

71

Irwin JJ, Sterling T, Mysinger MM, Bolstad ES, Coleman RG. ZINC: a free tool to discover chemistry for biology. J. Chem. Inf. Model. 52(7), 1757–1768 (2012).

83

Lai K, Tang M, Yin X, Klapper H, Wierenga K, Elsas LJ. ARHI: a new target of galactose toxicity in classic galactosemia. Biosci. Hypotheses 1(5), 263–271 (2008).

72

Yuriev E, Ramsland PA. Latest developments in molecular docking: 2010–2011 in review. J. Mol. Recognit. 26(5), 215–239 (2013).

84

73

Chiappori F, Merelli I, Milanesi L, Marabotti A. Static and dynamic interactions between GALK enzyme and known inhibitors: guidelines to design new drugs for galactosemic patients. Eur. J. Med. Chem. 63, 423–434 (2013).

Tang M, Siddiqi A, Witt B et al. Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) – deficient mouse model. Eur. J. Hum. Genet. doi:10.1038/ejhg.2014.12 (2014) (Epub ahead of print).

85

Kushner RF, Ryan EL, Sefton JM et al. A Drosophila melanogaster model of classic galactosemia. Dis. Model. Mech. 3(9–10), 618–627 (2010).

86

Cui X, Wang L, Zuo P et al. D-galactose-caused life shortening in Drosophila melanogaster and Musca domestica is associated with oxidative stress. Biogerontology 5(5), 317–325 (2004).

87

Ryan EL, DuBoff B, Feany MB, Fridovich-Keil JL. Mediators of a long-term movement abnormality in a Drosophila melanogaster model of classic galactosemia. Dis. Model. Mech. 5(6), 796–803 (2012).

88

Mc Guire PJ, Parikh A, Diaz GA. Profiling of oxidative stress in patients with inborn errors of metabolism. Mol. Genet. Metab. 98(1–2), 173–180 (2009).

89

Schulpis KH, Michelakakis H, Tsakiris T, Tsakiris S. The effect of diet on total antioxidant status, erythrocyte membrane Na+,K+ -ATPase and Mg 2+ -ATPase activities in patients with classical galactosaemia. Clin. Nutr. 24(1), 151–157 (2005).

90

Schulpis KH, Papassotiriou I, Tsakiris S. 8-hydroxy-2desoxyguanosine serum concentrations as a marker of DNA damage in patients with classical galactosaemia. Acta Paediatr. 95(2), 164–169 (2006).

91

Jumbo-Lucioni PP, Hopson ML, Hang D, Liang Y, Jones DP, Fridovich-Keil JL. Oxidative stress contributes to outcome severity in a Drosophila melanogaster model of classic galactosemia. Dis. Model. Mech. 6(1), 84–94 (2013).

92

Jumbo-Lucioni PP, Ryan EL, Hopson ML et al. Manganesebased superoxide dismutase mimics modify both acute and long-term outcome severity in a Drosophila melanogaster model of classic galactosemia. Antioxid. Redox Signal. 20(15), 2361–2371  (2014).

93

Gitzelmann R. Galactose-1-phosphate in the pathophysiology of galactosemia. Eur. J. Pediatr. 154(7 Suppl. 2), S45–S49 (1995).

••

Fully computational work on the molecular interactions of GALK inhibitors with the enzyme.

74

Megarity CF, Huang M, Warnock C, Timson DJ. The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases. Bioorg. Chem. 39(3), 120–126 (2011).

75

76

Reinhardt LA, Thoden JB, Peters GS, Holden HM, Cleland WW. pH-rate profiles support a general base mechanism for galactokinase (Lactococcus lactis). FEBS Lett. 587(17), 2876–2881 (2013). Huang M, Li X, Zou JW, Timson DJ. Role of Arg228 in the phosphorylation of galactokinase: the mechanism of GHMP kinases by quantum mechanics/molecular mechanics studies. Biochemistry 52(28), 4858–4868 (2013).

77

Segal S. Of mice and men: galactosemia. Mol. Genet. Metab. 89(4), 401–402 (2006).

78

Hughes J, Ryan S, Lambert D et al. Outcomes of siblings with classical galactosemia. J. Pediatr. 154(5), 721–726 (2009).

79

Widger J, O’Toole J, Geoghegan O, O’Keefe M, Manning R. Diet and visually significant cataracts in galactosaemia: is regular follow up necessary? J. Inherit. Metab. Dis. 33(2), 129–132 (2010).

80

Segal S. Another aspect of the galactosemia enigma. Mol. Genet. Metab. 81(3), 253–254 (2004).

81

Leslie ND, Yager KL, McNamara PD, Segal S. A mouse model of galactose-1-phosphate uridyl transferase deficiency. Biochem. Mol. Med. 59(1), 7–12 (1996).

82

Ning C, Reynolds R, Chen J et al. Galactose metabolism by the mouse with galactose-1-phosphate uridyltransferase deficiency. Pediatr. Res. 48(2), 211–217 (2000).

future science group

www.future-science.com

Review

1015