Type Ia Congenital Disorders of Glycosylation (CDG-Ia) is the most common form of Congenital Disorders of Glycosylation, an autosomal recessive defects in the synthesis of N-linked oligosaccharide chains caused by defects in the PMM2 gene. PMM2 encodes phosphomannomutase 2, which is responsible for the conversion of mannose-6-P [Man-6-P] to Man-1-P. Currently, there is no treatment for CDG-Ia patients. The current project aimed to identify novel non-competitive inhibitors of PMI that can be used as potential therapeutic treatments for these patients. The developed probe ML089 (CID-22416235) inhibits human phosphomannose isomerase (PMI) and may inhibit other PMI orthologs due to the highly conserved nature of the enzyme. The probe is membrane permeable and, as a result ,can also be used to inhibit PMI in living cells. Thus, the probe may serve as a therapeutic treatment for CDG-Ia patients by blocking the catabolism of Man-6-P in cells, thereby redirecting it towards protein glycosylation using this residual PMM2 activity.

Probe Structure & Characteristics

Recommendations for the scientific use of this probe

The probe can be used to inhibit human phosphomannose isomerase and due to the high homology between other eukaryotic orthologs, it will likely inhibit them as well. The probe can also be used to inhibit this enzyme in living cells since it is membrane permeable. One use of the probe would be to block catabolism of mannose-6-P in cells and direct it toward protein glycosylation. It may have therapeutic potential in phosphomannomutase 2-deficient CDG-Ia patients who are given mannose dietary supplements.

1. Scientific Rationale for Project

Congenital Disorders of Glycosylation (CDG) are autosomal recessive defects in the synthesis of N-linked oligosaccharide chains (1). CDG group I (CDG-I) defects are defined as those caused by mutations in genes encoding enzymes used for the synthesis and transfer of lipid linked oligosaccharide (LLO) to newly synthesized proteins in the lumen of the ER. The steps in this pathway and the genes encoding them are very similar from yeast to human. It requires 30–40 single gene products, each dependent on the previous step in the linear sequence to produce and transfer the LLO to protein. Therefore, mutations in any step may cause a type of CDG. There is considerable overlap in the clinical presentations between different types of CDG and a broad diversity within each type. The most common form of CDG, called Type Ia (CDG-Ia), is caused by defects in PMM2 (Man-6-P to Man-1-P), the gene that encodes phosphomannomutase. Mortality is 20% in the first 5 years, but then patients stabilize. Currently, there is no treatment for the CDG-Ia.

CDG-Ib patients, who are deficient in phosphomannose isomerase (PMI) catalyzing conversion of Man-6-P to Fru-6-P, are successfully treated with free mannose (2 – 6). Unfortunately, mannose therapy is not effective for CDG-Ia patients, most likely due to efficient Man-6-P consumption in the PMI reaction (7,8). It is believed that patients with Congenital Disorder of Glycosylation Type Ia (CDG-Ia) will benefit from dietary mannose if there is a simultaneous reduction of phosphomannose isomerase (PMI) activity. This would allow a modest intracellular accumulation of Man-6-P and drive metabolic flux into the glycosylation pathway using the residual PMM2 activity (9). It is assumed that a non-competitive inhibitor would work best in this setting; however, identification of chemical probes with diverse modes of action (MOA) would be advantageous for further characterization of PMI and PMM variants.

We proposed that CDG-Ia patients will benefit from dietary mannose if we simultaneously reduce PMI activity with a non-competitive or un-competitive inhibitor. This would allow a modest intracellular accumulation of Man-6-P and drive metabolic flux into the glycosylation pathway using the residual PMM2 activity.

Novel chemical probes elucidated in this way are invaluable tools to help explain the role of PMI in various biochemical pathways, and will ultimately lead to the first therapy for the growing number of CDG-Ia patients.

2. Project Description

b. Assay implementation and screening

i. PubChem Bioassay Name(s), AID(s), Assay-Type (Primary, DR, Counterscreen, Secondary)

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PubChemBioAssay Name	AIDs	Probe Type	Assay Type	Assay Format	Assay Detection & well format
HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay	1209	Inhibitor	Primary/ Confirmatory	Biochemical	Fluorescence 1536 well
HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate [Confirmatory]	1220	Inhibitor	Primary/ Confirmatory/ Mechanistic	Biochemical	Fluorescence 1536 well
Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay. [Confirmatory]	1535	Inhibitor	SAR	Biochemical	Fluorescence 384 well
Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate. [Confirmatory]	1536	Inhibitor	SAR/ mechanistic	Biochemical	Fluorescence 384 well
uHTS Identification of Diaphorase Inhibitors and Chemcical Oxidizers: Counter Screen for Diaphorase-based Primary Assays [Primary Screening]	1217	Inhibitor	Primary/ Counter screen	Biochemical	Fluorescence 1536 well
Counter Screen for Glucose-6-Phosphate Dehydrogenase-based Primary Assay	1020	Inhibitor	Primary/ Counter Screen	Biochemical	Fluorescence 1536 well
Phosophomannose Mutase 2 (PMM2)	1655	Inhibitor	Confirmatory/ Counter screen	Biochemical	Fluorescence 1536 well
PHOSPHO1	1666	Inhibitor	Secondary DR	Biochemical	Fluorescence 384 well
Screening for Phosphomannose Isomerase inhibitors in cellular based assay using Hela cells.	1553	Inhibitor	Secondary Assay Provider	Cell-based	Radioactive water 3H20 formation
Toxicity Screening of PMI Inhibitors in Hela cells	1620	Inhibitor	Secondary Assay Provider	Cell-based	35S protein incorporation

ii. Assay Rationale & Description

The purpose of this assay was to identify inhibitors of human PMI. This was accomplished by using a G6PD- NADPH-coupled assay. In the assay PMI activity was detected through conversion of its product, fructose-6-phosphate, to glucose-6-phosphate catalyzed by phosphoglucose isomerase (PGI) and subsequent oxidation of glucose-6-phosphate to 6-phosphogluconolactone concomitant with NADP-to-NADPH conversion catalyzed by glucose-6-phosphate dehydrogenase (G6PDH). The NADPH was then detected via a resazurin-diaphorase fluorogenic reaction.

This assay was performed in the presence of near-Km concentrations of the PMI substrate, mannose-6-phosphate, to ensure the identification of inhibitors with diverse Mechanism of Actions (MOAs). Keeping in mind that non- or un-competitive inhibitors might provide a better probe, we also developed and utilized for the full-deck screening the assay performed in the presence of 10xKm of the PMI substrate be better for to help with prioritization of hits for follow-up work. The idea being, that the competitive inhibitors would show much less inhibition in these conditions comparing to the normal screening. Thus, we selected scaffolds that inhibit in both assays with similar potency for further work; the described herein probe originated from this type of scaffold. In addition, we also developed and implemented the following assays for counter screening: G6PDH (primary HTS), diaphorase (primary HTS). Compounds inhibitory in any of these counter-screen assays were excluded from further consideration.

Protocol

PMI assay materials

1. Human PMI protein was provided by Dr. Hudson Freeze (Burnham Institute for Medical Research, San Diego, CA). The target protein, recombinant Phosphomannose Isomerase (PMI), was made in 1 L batches from high expressing baculovirus sytems in SF9 insect cells in Dr Hudson Freeze laboratory. .
2. Substrate working solution: 50 mM HEPES, pH 7.4, 0.4 mM Mannose-6- phosphate, 1.6 U/ml Diaphorase, 0.2 mM Resazurin.
3. Enzyme working solution: 50 mM HEPES, pH 7.4, 0.44 mM NADP+, 9.048 mM MgCl2, 0.01% Tween 20, 4.6 ug/ml phosphoglucose isomerase, 30 ng/ml PMI, 1.8 ug/ml G6PDH.

Table of Reagents and source used in experiments

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Reagents name	Vendors name
PhosphoGlucose Isomerase (PGI)	Roche Diagnostics
Glucose-6 phosphate Dehydrogenase (G6PD)	USB
Mannose-6 Phosphate	Sigma-Aldrich
NADP	ISC/Bioexpress
Hepes Buffer	Sigma-Aldrich
Magnesium Chloride	Sigma-Aldrich

PMI HTS protocol

1. 2 uL of Substrate working solution was added to columns 3–48 of a Costar 1536- well black plate (cat #3724) using a Thermo Multidrop Combi dispenser
2. 2 ul of Substrate working solution without mannose-6-p was added to columns 1 and 2 (positive control) of a Costar 1536-well black plate (cat #3724) using a Thermo Multidrop Combi dispenser
3. 40 nL of 100% DMSO was added to columns 1–4 using a HighRes biosolutions pintool and V&P Scientific pins
4. 40 nL of 2 mM compounds in 100% DMSO were dispensed in columns 5–48 using a HighRes biosolutions pintool and V&P Scientific pins
5. 2 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
6. Plates were incubated at room temperature for 20 min.
7. After 20 minutes the plates were read on a ViewLux plate reader (Perkin Elmer), Ex544, Em590.
8. The screening was performed using a HighRes biosolution fully integrated HTS POD-based system
9. Data analysis was performed using CBIS software (ChemInnovations, Inc).

Compounds showing more than 50% inhibition were requested from MLSMR for dose-response confirmation.

PMI dose-response assay protocol

1. 9 uL of Substrate working solution was added to columns 3–24 of a Greiner 384-well black plates (cat #784076) using a Thermo Multidrop Combi dispenser
2. 9 ul of Substrate working solution without mannose-6-p was added to columns 1 and 2 (positive control) using a Thermo Multidrop Combi dispenser
3. 2 uL of serially diluted compounds in 10% DMSO were added to columns 3–22 using Biomek FX
4. 2 uL of 10% DMSO were added to columns 1–2, 23–24
5. 9 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
6. Plates were incubated at room temperature for 20 min.
7. After 20 minutes the plates were read on an Analyst plate reader (Molecular Devices), Ex544, Em590.
8. Data analysis was performed using CBIS software (ChemInnovations, Inc).

The confirmatory screen

The coupled assay is complex and needs to be validated using a simple system. We elected to confirm the results by adding various inhibitors to purified PMI in the presence of [2–3H]Mannose-6-P. The direct action of PMI on this substrate produces ³H₂O which can be quantified in a simple “blow-off” (evaporation). This method can be modified for use in cells by providing [2-³H-mannose into glycosylation pathway. This assay provides ³H-mannose to monolayer of human hepatoma cells (C3A) then measures release of ³H₂O into growth media by scintillation counting at a fixed time point (AID-1553).

Assay was conducted in 24-well tissue culture plates and Dr. Hudson Freeze laboratory ran the capacity of 6 compounds at 3 concentrations in duplicate (1 plate w/ controls). As additional confirmation, the amount of ³H-mannose incorporated into cellular protein was measured by TCA precipitation. Successful candidates increased the amount of incorporation into protein and decreased the amount of ³H₂O formed.

iii. Summary of Results

194,158 compounds were tested at 20µM and 926 had an activity ≥ 50% in the assay. The average Z′ for the screen was 0.81, signal to background was 3.9, signal to noise was 52.4 and signal window was 19.1. Hits confirmed using DMSO solutions were clustered into 3 series, the benzoisothiazolone series, thiol-triazole series and the dithiazole series. Analog-by-catalog (ABC) SAR studies on the Benzoisothiazolone series are summarized below:

7 confirmed HTS hits along with 50 analogs were purchased from various vendors for SAR studies. Some SAR can be concluded from this round of study (Figure 1) and some representative examples are shown in Table 1.

Figure 1

Table 1

SAR Studies.

c. Probe Optimization

i. SAR & chemistry strategy (including structure and data) that led to the probe

Figure 2SAR strategy for CID-22416235

The SAR around CID-22416235 is illustrated in Figure 1. Three key structural features amenable to chemical derivatization were recognized to be responsible for biological activity: (a) the benzoisothiazolone fused aryl ring substituents, (b) the benzoisothiazolone ring heteroatoms, and (c) the aryl substituents. The overall goals of the SAR optimization efforts were two-fold: (1) produce inhibitors showing increased potency in vitro against the PMI enzyme that show minimal activity against PMM and (2) demonstrate cell-based efficacy and reduced cellular toxicity of these analogues. Cell based efficacy was determined at 3 concentrations, and the compounds which were toxic showed decreasing activity as concentration was increased. The in vitro enzyme inhibition and cell-based efficacy data for selected analogues of CID-22416235 are shown in Table 2. Compound CID-1510389 (MLS-0315771) was the primary lead compound based on the initial in vitro HTS data. While this compound was active in both the in vitro enzyme assay and the cell-based assay, it showed significant cellular toxicity, as can be seen with the dramatic reduction in cellular efficacy as concentration was increased. Thus, a variety of analogues were prepared to both reduce toxicity of these analogues and elucidate the key features of CID-1510389 (MLS-0315771) that were responsible for its activity. As can be seen in Table 1, removal of either nitrogen or sulfur from the benzoisothiazolone core completely abolishes both PMI and cellular activity [CID-4617895 (MLS-0390881) and CID-25067464 (MLS-0315862) respectively]. Also, replacing the sulfur with another heteroatom, e.g. nitrogen as in CID-13947703 (MLS-0315920), also furnishes analogues without detectable activity. This demonstrated the importance of both the nitrogen and sulfur atoms in the core. The initial lead (CID-1510389 (MLS-0315771)) had a fluorine atom in the 4 position. Additional substitution patterns were synthesized with the goal of lessening cellular toxicity. When the fluorine substituent was moved to the 5 position, as seen in CID-25181201 (MLS-0315923), a lessening of the toxicity was seen as indicated by more activity being retained at 50 µM as compared to the 4-F analogue. It was evident from the observation that the 25 µM dose had more activity than the 50 µM dose, there was still appreciable toxicity. However, the 5-fluoro analogues indeed furnish potent compounds with reduced cellular toxicity. Additional compounds with 5-F substitution patterns showed dose dependent increases in activity, indicative of less toxicity, such as CID-25181243 (MLS-0390936). Finally, optimization of the aromatic portion of the molecule was conducted on both the 5-F and unsubstituted benzoisothiazolone. It was found that para and bis-ortho, meta substituted analogues achieved potency requirements in the enzyme assay. However, dramatic differences were seen in cellular toxicity amongst these. After judicious exploration of aryl substituents, two compounds showed promising in vitro inhibition as well as dose-dependent cellular efficacy with minimal indications of toxicity (AID-1620), CID-22416276 (MLS-0390876) and CID-25181243. In fact, these compounds had almost identical activity profiles. However, when tested in the counter assay against the PMM enzyme, CID-22416276 (MLS-0390876) had an IC₅₀ of 22.3 µM while the probe compound CID-25181243 was completely inactive when tested at concentrations up to 100 µM. Thus, CID-22416235 had the most favorable profile of enzyme activity and selectivity, cellular efficacy, and minimum toxicity.

Table 2

In vitro enzyme activity and cellular efficacy data. Cellular data is presented at 3 concentrations. Decreasing cell efficacy with increasing concentration is indicative of cellular toxicity.

3. Probe

b. Probe chemical structure

c. Structural Verification Information of probe SID

SID-57287553.

h. Detailed synthetic pathway for making probe

Scheme 1Synthesis of CID-22416235. ^aphenyliodine(III) bis(trifluoroacetate)

4. Appendices

5. Bibliography

1.

Freeze HH. Genetic defects in the human glycome. Nat Rev Genet. 2006;7:537–551. [PubMed: 16755287]

2.

Alper J. Searching for medicine’s sweet spot. Science. 2001;291:2338–2343. [PubMed: 11269308]

3.

Niehues R, Hasilik M, Alton G, Körner C, Schiebe-Sukumar M, Koch HG, Zimmer KP, Wu R, Harms E, Reiter K, von Figura K, Freeze HH, Harms HK, Marquardt T. Carbohydratedeficient glycoprotein syndrome type Ib. Phosphomannose isomerase deficiency and mannose therapy. J Clin Invest. 1998;101:1414–1420. [PMC free article: PMC508719] [PubMed: 9525984]

4.

Babovic-Vuksanovic D, Patterson MC, Schwenk WF, O’Brien JF, Vockley J, Freeze HH, Mehta DP, Michels VV. Severe hypoglycemia as a presenting symptom of carbohydrate deficient glycoprotein syndrome. J Pediatr. 1999;135:775–781. [PubMed: 10586187]

5.

de Lonlay P, Cuer M, Vuillaumier-Barrot S, Beaune G, Castelnau P, Kretz M, Durand G, Saudubray JM, Seta N. Hyperinsulinemic hypoglycemia as a presenting sign in phosphomannose isomerase deficiency: A new manifestation of carbohydrate-deficient glycoprotein syndrome treatable with mannose. J Pediatr. 1999;135:379–383. [PubMed: 10484808]

6.

Westphal V, Kjaergaard S, Davis JA, Peterson SM, Skovby F, Freeze HH. Genetic and metabolic analysis of the first adult with congenital disorder of glycosylation type ib: long-term outcome and effects of mannose supplementation. Mol Genet Metab. 2001;73:77–85. [PubMed: 11350186]

7.

Rush JS, Panneerselvam K, Waechter CJ, Freeze HH. Mannose supplementation corrects GDP-mannose deficiency in cultured fibroblasts from some patients with Congenital Disorders of Glycosylation (CDG). Glycobiology. 2000;10:829–835. [PubMed: 10929009]

8.

Panneerselvam K, Freeze HH. Mannose corrects altered N-glycosylation in carbohydratedeficient glycoprotein syndrome fibroblasts. J Clin Invest. 1996;97:1478–1487. [PMC free article: PMC507208] [PubMed: 8617881]

9.

Freeze HH. Chapter 6: Monosaccharide Metabolism. In: Varki A, Cummings R, Esko JD, Freeze HH, Hart G, Marth JD, editors. Essentials of Glycobiology. New York: Cold Spring Harbor Laboratory Press; 1999. pp. 69–84.