Classic Galactosemia is a rare genetic metabolic disorder (1/60,000 births) that is characterized by decreased production of galactose-1-phosphate uridyltransferase (GALT), an enzyme responsible for the conversion of galactose-1-phosphate (gal-1-p) to glucose-1-phosphate. The resulting elevated intracellular concentrations of gal-1-p are believed to be the major pathogenic mechanism in Classic Galactosemia that leads to a myriad of secondary symptoms and, if untreated, death of the patient. Galactokinase (GALK) is an upstream enzyme in the Leloir pathway that is responsible for conversion of galactose to gal-1-p. Therefore, it was hypothesized that the identification of a small-molecule inhibitor of GALK would act to decrease levels of gal-1-p and allow for a novel entry into therapies for this disorder. In collaboration with Professor Kent Lai of the University of Utah, a quantitative high-throughput screening (qHTS) assay has been performed at the NIH Chemical Genomics Center (NCGC) and has identified a potent and selective inhibitor of GALK bearing a 1,4-dihydropyrimidine core. Several rounds of medicinal chemistry were performed and generated a small molecule capable of inhibiting GALK with an IC₅₀ of 1.0μM. The probe ML152 (CID-664331; SID-87550830) is selective for GALK against CDP-ME, and thus represents an advancement over previously reported inhibitors. The probe was the best analog for inhibition of GALK in a purified enzyme assay. Its mechanism of action, as determined via substrate competition and kinetic assays, are consistent with it being ATP competitive.

Probe Structure & Characteristics

ML152

Recommendations for scientific use of the probe

Classic Galactosemia is a potentially lethal disease caused by deficient galactose-1-phosphate uridyltransferase (GALT) that results in the buildup of galactose-1-phosphate (gal-1-P) in the blood. Galactokinase (GALK) is the enzyme responsible for converting galactose into gal-1-p. A pharmacological inhibitor of GALK is therefore sought for a potential therapy for galactosemia by reducing levels of gal-1-P. The probe developed herein inhibits GALK with a potency of 1.0μM in a luminescence based biochemical assay and should be useful for studying the role of GALK modulation in the progression of Classic Galactosemia.

1. Introduction

Classic Galactosemia is a potentially lethal disorder with a high mortality rate when left untreated. Typically, removal of lactose and galactose from the diet of individuals with deficient GALT function is sufficient for the prevention of lethality. While morbidity rates for those affected with Classic Galactosemia have decreased, the lack of an effective therapy has been shown to cause developmental delay, neurological disorders, and premature ovarian failure1. Although the exact pathogenic mechanism of Classic Galactosemia is not known, elevated galactose-1-phosphate (gal-1-p) levels have been denoted as a probable cause. The high levels of gal-1-p are believed to arise from deficient GALT, which is the second enzyme in the Leloir pathway that converts gal-1-p to uridine diphosphogalactose (UDP-gal) and uridine diphosphoglucose (UDP-glu) to glucose-1-phosphate (glu-1-p). Upstream from this enzyme liess GALK, which converts galactose to gal-1-p. This, and the fact that GALK-deficient patients have much milder and benign phenotypes, position GALK as a key target for reduction of gal-1-p levels and a potential therapeutic target for Classic Galactosemia2–4. The current probe (CID-664331) represents the first small molecule GALK inhibitor with good potency against the purified GALK enzyme and high selectivity against CMK kinase (a related kinase in the GHMP kinase family).

2. Materials and Methods

All air and/or moisture sensitive reactions were performed under positive pressure of nitrogen with oven-dried glassware. Anhydrous solvents such as dichloromethane, N, N-dimethylforamide (DMF), acetonitrile, methanol and triethylamine were obtained by purchasing from Sigma-Aldrich. Preparative purification was performed on a Waters semi-preparative HPLC. The column used was a Phenomenex Luna C18 (5 micron, 30 × 75 mm) at a flow rate of 45 ml/min. The mobile phase consisted of acetonitrile and water (each containing 0.1% trifluoroacetic acid). A gradient of 10% to 50% acetonitrile over 8 minutes was used during the purification. Fraction collection was triggered by UV detection (220 nM). Analytical analysis was performed on an Agilent LC/MS (Agilent Technologies, Santa Clara, CA).

Method 1: A 7 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with an 8 minute run time at a flow rate of 1 mL/min. A Phenomenex Luna C18 column (3 micron, 3 × 75 mm) was used at a temperature of 50°C.

Method 2: A 3 minute gradient of 4% to 100% Acetonitrile (containing 0.025% trifluoroacetic acid) in water (containing 0.05% trifluoroacetic acid) was used with a 4.5 minute run time at a flow rate of 1 mL/min. A Phenomenex Gemini Phenyl column (3 micron, 3 × 100 mm) was used at a temperature of 50 °C.

Purity determination was performed using an Agilent Diode Array Detector on both Method 1 and Method 2. Mass determination was performed using an Agilent 6130 mass spectrometer with electrospray ionization in the positive mode. ¹H NMR spectra were recorded on Varian 400 MHz spectrometers. Chemical Shifts are reported in ppm with tetramethylsilane (TMS) as internal standard (0 ppm) for CDCl₃ solutions or undeuterated solvent (DMSO-h6 at 2.49 ppm) for DMSO-d6 solutions. All of the analogs for assay have purity greater than 95% based on both analytical methods. High resolution mass spectrometry was recorded on an Agilent 6210 Time-of-Flight LC/MS system. Confirmation of molecular formula was accomplished using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02).

2.1. Assays

Table 1Screens Deposited in PubChem

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PubChem AID	Type	Target	Conc. Range	Samples Tested
1868	Primary qHTS	GALK	57.5 μM – 3.7 nM	274,749
2499	Confirmatory	GALK	57.5 μM – 0.3 nM	43
2506	Anti-target	CDP-ME (CMK)	57.5 μM – 0.3 nM	43
2502	Anti-target	Redox activity	57.5 μM – 0.3 nM	43
2547	Anti-target	Cytotoxicity	57.5 μM – 0.3 nM	174
2114	Summary	GALK	NA	NA

Primary qHTS assay for inhibitors of GALK [AID-1868]

Assay details and protocol: The primary assay monitored ATP depletion using Promega’s KinaseGlo™ technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzyme5, 6. ATP was held at 35μM, near its reported K_M value, and the K_M for galactose was determined under the 1536-well assay conditions to be 50–100μM (Figure 1A). As well, we confirmed the IC₅₀ for a commercially available CD45 inhibitor (N-(9,10-dioxo-9,10-dihydrophenanthren-2-yl)pivalamide), previously found by Dr. Lai to inhibit GALK (Figure 1B)1. This was used as the positive control for the assay. The assay used 5 nM GalK and a 1 hr incubation time, which gave sufficient signal:background and stability for the HTS. The percent conversion of ATP under these conditions was estimated to be approximately 50% using an ATP standard curve.

Figure 1

Galactose K_M determination and sensitivity to a control inhibitor in the 1536-well GalK assay. A. Galactose variation in the 1536-well assay using ATP depletion. Rates (from determining the remaining ATP at time seven points starting at 5 min to 55 min; (more...)

Table 2Stepwise Protocol Used for the 1536-Well Assay

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Step	Value	Description
1a	3μl	35μM ATP buffer (2–48)
1b	4μl	Buffer only to column 1
2	23nl	ATP titration (Col 1), CD45 inhibitor, DMSO
3	23nl	40μM to 0.24nM
4a	1μl	Col 4, Buffer (no GalK)
4b	1μl	Col 2&3, 5–48 GalK (5 nM), 100 μM galactose
5	1 hr	Room temperature incubation
6	4μl	KinaseGlo-Plus detection
7	10 min	Room temperature incubation
8	Read	ViewLux
Step Notes
1	White solid Kalypsys plates 1a: (HEPES pH 8.0, 5mM MgCl2, 60mM NaCl, 1 mM DTT, 0.01% BSA, 35μM ATP) 1b: no ATP buffer
2	Pintool addition of compound. Controls (column 2–4); and ATP 35μM titration 1:2 dilution (column 1)
3	Pintool transfer (DMSO; iPA, MeOH, 3-sec dry wash cycle)
4	4a: buffer, 4b: buffer + 5 nM GalK & 100μM galactose (kept on ice)
5	Room temperature incubation
6	Kinase-Glo Plus at room temperature
7	Room temperature incubation
8	ViewLux, 1 sec exposure 2x bin

Confirmatory assay. [AID-2499]

As in the primary screen, the confirmatory assay monitored ATP depletion using Promega’s KinaseGlo™ technology, where ATP levels are measured through luminescence generated from firefly luciferase, a bioluminescent ATP-dependent enzyme5, 6. ATP was held at 35μM, near its reported K_M value, and the K_M for galactose was determined under the 1536-well assay conditions to be 50 – 100μM. In this assay, NCGC00187642 (CID-664331; SID-87550830) was found to be a 1μM inhibitor of GALK (Figure 2).

Figure 2

Dose response curve for NCGC00187642 (CID-664331; SID-87550830) in confirmatory assay.

Secondary assays

The selectivity for these analogs against CDP-ME, another member of the GHMP kinase family, was performed using a bioluminescent Kinase Glo™ assay that detects ATP depletion after kinase reaction (PubChem ID 2506). NCGC00187642 (CID-664331; SID-87550830); all analogs showed no activity in this assay (up to 57μM), highlighting the selectivity of this chemotype. All analogs were also tested for cytotoxicity using Promega CellTiter Glo on HEK293 cells that were treated with compounds and analyzed after 48 hours. All compounds shown in table 5 showed no cytotoxic effect (PubChem ID 2547). In addition, the ability of these compounds to undergo redox recycling, which may lead to false positive results, was also evaluated using an endpoint colorimetric assay; this assay detects the presence of H₂O₂ in the kinase reaction buffer (PubChem ID 2502). The lead compound and analogs are inactive in the redox recycling, further confirming that these compounds have genuine GALK target activity.

2.2. Probe Chemical Characterization

Scheme 1Synthesis of CID-664331

¹H NMR (400 MHz, DMSO-d₆); 10.37 (br. s. 1H), 9.98 (br. S. 1H), 7.40 (m, 2H), 7.16 (m, 2H), 2.52 (m, 2H), 2.39 (m, 2H), 2.26 (m, 2H), 1.83 (m, 6H), 1.62 (m, 2H). Method 1, retention time, 5.671 min; Method 2, retention time 3.704 min; HRMS: m/z (M+H⁺) = 336.1592 (Calculated for C₁₉H₂₀N₄O₂ = 336.1586). Solubility (PBS, pH 7.4, 23°C) = 2.3 μg/ml. Stability profile over 48 hrs (PBS, pH 7.4, 23°C) is shown below in Figure 3.

Figure 3

PBS Buffer Stability of NCGC00187642/CID664331. Stability of NCGC00187642/CID664331 in PBS buffer (pH 7.4, 23°C) plotted as AUC vs. time for a 48 hr period using LCMS Method 1 described in Section 2. No instability was observed, and 100% of compound (more...)

MLS Numbers for Probe and Analogs

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Probe	NCGC00187642	MLS000039538
Analog	NCGC00187643	MLS003178546
Analog	NCGC00188578	MLS003178547
Analog	NCGC00188580	MLS003178548
Analog	NCGC00188579	MLS003178549
Analog	NCGC00188583	MLS003178550

3. Results

Please check subsections for a detailed description of the results.

3.1. Summary of Screening Results

Primary assay summary: The primary qHTS showed excellent performance, and the statistics of the screen are shown below.

Table 3Primary Assay Statistics

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Parameter	qHTS
System	Kalypsys/Viewlux detection
Plates Screened	1192 (4 batches)
Plates Failed QC	30 (4 titration series)
Compound (total # tested)	277,329
Concentration-Response Titrations	277,329
Sample wells	1,561,627
Number of data points	1,561,627
Z′	0.48 ± 0.16
Signal/Background	4.5 ± 1.6
CV	17 ± 9
MSR of CD45 control	2

Identification of lead: A very low active rate was observed, and the results of the qHTS are shown in Table 4. A total of 149 compounds were found to show certain inhibition. These were re-confirmed in the primary assay. Several counter-screens for assay related artifacts were then applied. These included an assay for inhibitors of KinaseGlo5, redox-activity8 and selectivity against the related GHMP kinase, CDP-Me kinase (CMK) from bacteria. The latter used KinaseGlo again for detection. This identified NCGC00187642 (CID-664331; SID-87550830) as a selective inhibitor of GALK.

Table 4

Concentration-Response Curve Class Distribution from Primary Screen.

3.2. Dose Response Curve for Probe

Figure 4Potency and selectivity of NCGC00187642 (CID-664331; SID-87550830) against CMK for the lead compound

The lead compound was observed to be active against GALK with an IC₅₀ of 1μM but inactive against CDP-Me kinase. Results showed no redox or cytotoxic activity of the compound (cytotoxicity was determined after 48 hours). All assays were performed at least in duplicate on at least two separate days.

3.4. SAR Table

Table 5SAR of select analogs in GALK purified enzyme assay

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Entry	Structure	Compound IDa	IC50 (μM)b	%Max Resp.c
1		NCGC00187643-01 (CID-44607593) SID-87357364)	16.8	−74%
2		NCGC00187641-01 (CID-44607596) (SID-87357362)	16.8	−74%
3		NCGC00187642-02 (CID-664331) (SID-87550830)	1.0	−86%
4		NCGC00186052-01 CID-1294862) (SID-85256913)	11.9	−99%
5		NCGC00188034-01 (CID-1286615) (SID-87357368)	6.0	−82%
6		NCGC00188035-01 (CID-44607600) (SID-87357369)	14.9	−36%
7		NCGC00188036-01 (CID-44607594) (SID-87357370)	13.3	−75%
8		NCGC00188578-01 (CID-44623889) (SID-87550843)	6.0	−81%
9		NCGC00188580-01 (CID-44623882) (SID-87550845)	21	−58%
10		NCGC00188577-01 (CID-44623881) (SID-87550842)	37	−38%
11		NCGC00188572-01 (CID-44623883) (SID-87550837)	6.7	−73%
12		NCGC00188579-01 (CID-44623888) (SID-87550844)	7.5	−73%
13		NCGC00188581-01 (CID-44623886) (SID-87550846)	>57	NA
14		NCGC00188583-01 (CID-44623884) (SID-87550848)	>57	NA

a

All compounds were synthesized

b

IC₅₀ values were determined utilizing the luminescen GALK-luminescent ATP-depletion assay.

c

Max Resp. represents the % inhibition at 57 μM compound.

4. Discussion

SAR was seen with potencies ranging from 1μM to > 50μM (Table 5). Entries 1 – 4 show the effect of varying the ketone starting material, giving variations of the spiro group, with the 5-membered ring giving the best results (entry 3). The dimethyl, 4- and 6-membered rings resulted in a ~10-fold loss of potency (compare entries 1, 2 and 4). The dione was also varied, giving a 5-membered compound as well as 5-phenyl and 5,5-dimethyl analogs that showed loss in potency (entries 5 – 7). Adding hydrophobic groups to the benzoxazole guanidine showed moderate to large loss of potency (6- to >50-fold). Changing the benzoxazole to a N-methylbenzimidazole was accompanied by a >50-fold loss in potency as well. From these results, it was determined that NCGC00187642 (CID-664331; SID-87550830) was the best analog for the inhibition of GALK in the purified enzyme assay.

4.2. Mechanism of Action Studies

The mechanism of action of NCGC00187642 (CID-664331; SID-87550830) was determined via substrate competition and kinetic assays. Results are consistent with the lead compound being ATP competitive (Figure 5).

Figure 5

Substrate competition with NCGC00187642 (CID-664331; SID-87550830). A Kinase reaction run at two different ATP concentrations, while keeping the galactose concentration at K_M, showed increase in potency of the compound as exhibited by a left shift of (more...)

Probe properties

5. References

1.

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2.

Gitzelmann R. Galactose-1-phosphate in the pathophysiology of galactosemia. Eur J Pediatr. 1995;154:S45–9. [PubMed: 7671964]

3.

Lai K, Langley SD, Khwaja FW, Schmitt EW, Elsas LJ. GALT deficiency causes UDP-hexose deficit in human galactosemic cells. Glycobiology. 2003;13:285–94. [PubMed: 12626383]

4.

Lai K, Willis AC, Elsas LJ. The biochemical role of glutamine 188 in human galactose-1- phosphate uridyltransferase. J Biol Chem. 1999;274:6559–66. [PubMed: 10037750]

5.

Auld DS, et al. A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution. J Med Chem. 2009;52:1450–1458. [PMC free article: PMC3430137] [PubMed: 19215089]

6.

Inglese J, et al. Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci U S A. 2006;103:11473–11478. [PMC free article: PMC1518803] [PubMed: 16864780]

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Shukla SJ, et al. Identification of pregnane X receptor ligands using time-resolved fluorescence resonance energy transfer and quantitative high-throughput screening. Assay Drug Dev Technol. 2009;7:143–169. [PMC free article: PMC3116688] [PubMed: 19505231]

8.

Soares KM, et al. Profiling the NIH Small Molecule Repository for Compounds That Generate H(2)O(2) by Redox Cycling in Reducing Environments. Assay Drug Dev Technol. 2010;8(2):152–74. [PMC free article: PMC3098569] [PubMed: 20070233]