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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.

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Probe Reports from the NIH Molecular Libraries Program [Internet].

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Therapeutic Inhibitors of Phosphomannose Isomerase - Probe 2

, , , , , , , , , , , , , and .

Received: ; Last Update: September 2, 2010.

Type Ia Congenital Disorders of Glycosylation (CDG-Ia) is the most common form of the Congenital Disorders of Glycosylation. This autosomal recessive disorder impairs the synthesis of N-linked oligosaccharide chains and is 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 phosphomannose isomerase, PMI. as potential therapeutic treatments for these patients. The developed probe ML096 (CID-25199533) inhibits human 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.

Assigned Assay Grant #: R03 MH082386-01

Screening Center Name & PI: Conrad Prebys Center for Chemical Genomics (formerly Burnham Center for Chemical Genomics) & Dr. John C. Reed

Chemistry Center Name & PI: Conrad Prebys Center for Chemical Genomics (formerly Burnham Center for Chemical Genomics) & Dr. John C. Reed

Assay Submitter & Institution: Dr. Hudson Freeze, Sanford-Burnham Medical Research Institute (formerly Burnham Institute for Medical Research)

PubChem Summary Bioassay Identifier (AID): AID-1545

Probe Structure & Characteristics

Image ml096fu1
CID/MLTarget NameIC50/EC50 (nM) [SID, AID]Anti-target Name(s)IC50/EC50 (μM) [SID, AID]SelectivitySecondary Assay(s) Name: IC50/EC50 (nM) [SID, AID]
CID-25199533

ML096
Phosphomannose
Isomerase (PMI)
1,070 nM IC50 SID-57309177
AID-1535
PMM2>100 µM IC50 SID-57309177 AID-1655>93 X

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 would 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

a. The original goal for probe characteristics

The original overall goal for this project was to identify novel non-competitive inhibitors of phosphomannose isomerase (PMI) that can be used as a therapeutic for treating patients with Congenital Disorder of Glycosylation Type Ia (CDG-Ia). A non-competitive inhibitor is preferred (yet not required) to avoid competition by the increased Man-6-P level. The main aims undertaken were: 1) Identify small molecule compounds in the MLSMR collection screening 2) Validated the specificity, activity, stability and kinetic behavior of the selected compound(s) 3) Test selected inhibitors of PMI for in cell based assay to determine their effects on glycosylation in intact cells.

b. Assay implementation and screening

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

PubChemBioAssay NameAIDsProbe TypeAssay TypeAssay FormatAssay Detection & well format
HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay1209InhibitorPrimary/ConfirmatoryBiochemicalFluorescence 1536 well
HTS identification of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate [Confirmatory]1220InhibitorPrimary/Confirmatory/MechanisticBiochemicalFluorescence 1536 well
Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay. [Confirmatory]1535InhibitorSARBiochemicalFluorescence 384 well
Confirmation of compounds inhibiting phosphomannose isomerase (PMI) via a fluorescence intensity assay using a high concentration of mannose 6-phosphate. [Confirmatory]1536InhibitorSAR/mechanisticBiochemicalFluorescence 384 well
uHTS Identification of Diaphorase Inhibitors and Chemical Oxidizers: Counter Screen for Diaphorase-based Primary Assays [Primary Screening]1217InhibitorPrimary/Counter screenBiochemicalFluorescence 1536 well
Counter Screen for Glucose-6-Phosphate Dehydrogenase-based Primary Assay1020InhibitorPrimary/Counter ScreenBiochemicalFluorescence 1536 well
Phosophomannose Mutase 2 (PMM2)1655InhibitorConfirmatory/Counter screenBiochemicalFluorescence 1536 well

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 systems 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

Reagents nameVendors name
PhosphoGlucose Isomerase (PGI)Roche Diagnostics
Glucose-6 phosphate Dehydrogenase (G6PD)USB
Mannose-6 PhosphateSigma-Aldrich
NADPISC/Bioexpress
Hepes BufferSigma-Aldrich
Magnesium ChlorideSigma-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 3) 2 uL of 10% DMSO were added to columns 1–2, 23–24
  4. 9 uL of Enzyme working solution was added to the whole plate using a Thermo Multidrop Combi dispenser.
  5. Plates were incubated at room temperature for 20 min.
  6. After 20 minutes the plates were read on an Analyst plate reader (Molecular Devices), Ex544, Em590.
  7. Data analysis was performed using CBIS software (ChemInnovations, Inc).

iii. Summary of Results

A total of 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. Analog-by-catalog (ABC) SAR studies on the dithiazolimine series are summarized in Table 1 below.

Table 1. In vitro enzyme activity & SAR for dithiazolimine PMI inhibitors.

Table 1

In vitro enzyme activity & SAR for dithiazolimine PMI inhibitors.

c. Probe Optimization

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

Development of initial SAR around the dithiazolimine series of PMI inhibitors via purchased and synthesized analogues established 3 structural features critical for the inhibitory activity of CID-25199533 (MLS-0390940) and its derivatives: (a) a dithiazole core ring, (b) a pendant aromatic or heteroaromatic ring, and (c) a chlorine in the 5 position of the dithiazole ring. These required features are exemplified in Figure 1. Systematic replacement of each of these key moieties resulted in derivates having significantly diminished inhibitory activity (examples not shown). Thus, having established these required features, further SAR optimization efforts focused on two main goals: (1) development of inhibitors with increased potency in vitro against PMI and (2) optimization of the selectivity of these potent inhibitors over PMM. The in vitro enzyme inhibition and cell-based efficacy data for selected analogues of CID-25199533 (MLS-0390940) are shown in Table 1. Compound CID-715715 (MLS-0053966) was the primary lead compound based on the initial in vitro HTS data. This compound showed acceptable PMI inhibition (IC50 = 2.91 µM) and selectivity over PMM (IC50 >100 µM), thus analogue synthesis was executed around this hit. A variety of analogues were prepared to elucidate the optimal substitutions of the aryl ring. As can be seen in Table 1, methoxy substituents as in CID-602897 (MLS-0390826) and CID-483894 (MLS-0390822) suggested that the presence of hydrogen-bonding moieties were essential for activity. A key discovery was made when ortho nitro substituents were incorporated, yielding potent analogues such as CID-3919997 (MLS-0390820), which had acceptable potency (PMI IC50 = 3.52 µM). However, this analogue showed slight inhibition of PMM (IC50 = 58.4 µM). A companion series having a heteroaromatic substituent in place of the phenyl ring was showing potent PMI inhibition and PMM selectivity, as exemplified by the thiadiazole-containing analogue CID-6114286 (MLS-0390818; PMI IC50 = 2.3 µM; PMM IC50 > 100 µM). It was envisioned that this replacement represented an opportunity to maintain PMI potency while improving selectivity over PMM. Thus, selected analogues were synthesized incorporating small heteroaryl rings at this position, yielding potent and selective derivatives such as CID-5527821 (MLS-0390816) (PMI IC50 = 4.34 µM; PMM IC50 > 100 µM) and the probe compound CID-25199533 (MLS-0390940), a potent and selective PMI inhibitor with IC50 = 1.07 µM and completely inactive at PMM when tested at concentrations up to 100 µM.

Figure 1. Summary of required features for CID-25199533 and analogues.

Figure 1

Summary of required features for CID-25199533 and analogues.

3. Probe

a. Chemical name

4-chloro-N-(5-methyl-1,3,4-oxadiazol-2-yl)dithiazol-5-imine [ML096]

b. Probe chemical structure

Image ml096fu11

c. Structural Verification Information of probe SID

SID-57309177

Image ml096fu12

d. PubChem CID (corresponding to the SID)

CID-25199533

e. Availability from a vendor

This probe is not commercially available.

f. MLS#'s of probe molecule and five related samples that were submitted to the SMR collection

Probe/AnalogMLS-# (BCCG#)CIDSIDSource (vendor or BCCG syn)Amt (mg)Date ordered/submitted
ProbeMLS-03909402519953357309177BCCG syn155/18/09
Analog 1MLS-03909322518124057287676BCCG syn155/18/09
Analog 2MLS-00539667157154245711Chemical Block155/18/09
Analog 3MLS-039082660289757287569Chemical Block155/18/09
Analog 4MLS-0390818611428657287561BCCG syn155/18/09
Analog 5MLS-039082971569857287572BCCG syn155/18/09

g. Mode of action for biological activity of probe

The mode of action is not yet fully characterized. The dithiazolimine scaffold with all its primary hits was identified as non- or un-competitive since it inhibited irrespective of substrate concentration. As alterations to the structure of the primary hits that were made to arrive at the probe are limited, the probe is very likely to have the same MOA.

h. Detailed synthetic pathway for making probe

Scheme 1. Synthesis of CID-25199533.

Scheme 1Synthesis of CID-25199533

i. Probe properties (solubility, absorbance/fluorescence, reactivity, toxicity, etc.)

CID-25199533 (MLS-0390940) demonstrated potent inhibition and also demonstrated selectivity within the class.

j. Properties Computed from Structure

Molecular Weight234.68652
Molecular FormulaC5H3ClN4OS2
XLogP3-AA2.1
H-Bond Donor0
H-Bond Acceptor5
Rotatable Bond Count1
Exact Mass233.94368
Topological Polar Surface Area114
Heavy Atom Count13
Formal Charge0

4. Appendices

a. Comparative data on (1) probe, (2) similar compound structures (establishing SAR) and (3) prior probes

See list of compounds ordered for SAR by catalog attached separately to end of this probe report.

b. Comparative data showing probe specificity for target

See Table 1 above for selectivity

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.

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