human HexA (IC50 = 6.0 µM); human HexB (IC50 = 3.1 µM); OfHex2 (Ki = 2.5 μM)

M-31850 exhibits some activity against Streptomyces plicatus (SpHex) and Jack Bean Hex (JBHex) (IC50 values for SpHex and JBHex are >500 μM and 280 μM, respectively) [1]. M-31850 causes deficient-treated neonatal Sandhoff disease (ISD) cells to exhibit an increase in Mug-treated (Hex S levels) [1]. In comparison to the presence of DMSO, M-31850 increases the half-life of mutant Hex A in Tay-Sachs (ATSD) cells by more than threefold at 44°C. M-31850 is the traditional competitive multiple of Hex (Km rises, M-31850 increases but does not impact Vmax); Ki 0.8 μM [1].

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To conclusively demonstrate that the inhibitor treated cells have increased levels of Hex A in the lysosomes, a lysosome enriched fraction was prepared from DMSO and M-31850-treated fibroblasts (Fig 4c). This method has been previously used to isolate an ER-depleted lysosomal fraction by magnetic chromatography following labeling of fibroblasts by iron-dextran colloid. There was an approximately ten-fold increase in Hex (MUGS) and acid phosphatase (MUP) specific activity in the lysosomal fraction. More importantly MUGS activity was increased three-fold both in the post nuclear supernatant and lysosome enriched fractions, consistent with the hypothesis that increased levels of Hex A in treated cells are found in the lysosome. The activity of lysosomal acid phosphatase was not significantly effected in either fractions from M-31850-treated cells. Fibroblast do not synthesis significant levels of the higher gangliosides, e.g. GM1 ganglioside. Thus, the ATSD and ASD cells used in this study do not store appreciable amounts of GM2 ganglioside. However, our data strongly imply that patient cells that do synthesis higher gangliosides, i.e. neurons, would benefit from M-31850 treatment. [1]

These results validate the approach for identifying novel PC by first performing a HTS for Hex inhibitory compounds. The strongest inhibitors identified, M-22971, M-31850 and M-45373, are novel and structurally distinct from the known human Hex inhibitors, the majority of which are azasugars, e.g., and iminocyclitol derivatives. These HTS derived inhibitors can serve as new, more drug-like, frameworks that can be further optimized using high throughput combinatorial chemistry. This approach could be applied to the naphthalimide derivatives as they can be readily synthesized via a straightforward single step scheme.

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Examination of the mechanism by which compound M-31850 acts as a PC for Hex A [1]
Bisnaphthalimide compound M-31850 was examined in greater detail in terms of its mechanism of binding, because; 1) it was active in cells at the lowest concentration of the HTS compounds examined, 2) several naphthalimide derivatives, Elinafide (LU79533) and alrestatin had been evaluated or approved for use in humans, and 3) and other naphthalimide congeners can be easily synthesized. To confirm that M31850 functions as a PC in a manner similar to NGT, i.e. it binds at (or near) the active site and is capable of increasing the stability of the enzyme, the effects of the compound on enzyme kinetics and thermal denaturation were analyzed. M-31850 increased the half-life of the mutant Hex A from ATSD cells more than two-fold at 44° C, relative to the enzyme heated in the presence of DMSO (Fig. 4a). It acts as a classic competitive inhibitor of Hex (Km increases and Vmax is unaffected by increasing amounts of M-31850), with a Ki of 0.8 ± 0.1 μM (Fig. 4b).

All of the mono-naphthalimide derivatives, M-31860, M-31862 and M-31867 (Table 1 and Table 3) from the secondary screen had IC50 values that were at least 100fold higher than the bis-naphthalimide, M-31850 (Table 3). All of these derivatives contained a small hydrophobic group N-linked to the underlying naphthalimide by a short alkyl chain that lacked a secondary amine. Similarly, the decreased inhibitory activity of the mono-naphthalimides, BTB12933 and 5141402, further underline the importance of the second naphthalimide moiety.

The critical role of the secondary amine group in the linker bridging the two naphthalimide moieties was underscored by the loss of all inhibitory activity when it was replaced by an ether linkage in the analogous position in compound 7916963. The behavior of this compound suggests that the secondary amine in M-31850 may be involved in the formation of a hydrogen bond with an unidentified residue in Hex A. Although reduced more than ten-fold, the inhibitory activity of compound 5141402 suggests that an analogously positioned hydroxyl group could provide the necessary hydrogen atom. The importance of the N-alkylamine linker for inhibitory activity of both the mono- and bis-naphthalimide derivatives, 5141402 and M-31850, is reminiscent of recently described iminocyclitol based Hex inhibitors bearing an N-alkylamine moiety.

The attenuated inhibitory activity of compound 5141402, lacking the second naphthalimide, suggests that it may provide additional hydrophobic contacts important for high affinity binding. It is surprising that there is an approximately 8-fold decrease in the inhibitory activity of LU79553, as it bears two naphthalimide groups and the position of the amine in the linker relative to the naphthalimide group is identical to that in M-31850. This suggests that the two naphthalimide groups must be spaced appropriately in order to effectively bind Hex A. The second active site in Hex A is not a candidate for the site that binds the second naphthamide group in M-31850, as the measured distance between the two active sites is more that 2.5 times the size of the alkylamine linker in M-31850. However the proposed hydrophobic aglycon pockets just outside either active site of Hex A remain possible candidates.

On the basis of the inhibitory activity of the naphthalimide derivatives in Table 2, and the fact that M-31850 is a competitive inhibitor, the following binding model is proposed. One of the naphthalimide groups binds directly in the substrate binding pocket of Hex A, while the second naphthalimide moiety binds to a secondary hydrophobic patch, e.g. an aglycon binding site. An appropriately sized linker between the two naphthalimide groups is needed to span the two sites and must also be able to form a stabilizing hydrogen bond with an acidic acceptor residue on the surface of Hex A.

Compound M-31850 was evaluated in greater detail in part due to its close similarity to LU79553 (Elinafide) which previously had been evaluated in phase I clinical trials as a solid tumor chemotherapeutic due its DNA intercalating ability and inhibitory activity against topoisomerase I. Although, Elinafide did not proceed further in clinical trials due to its toxicity at therapeutic doses, these results demonstrate the utility of using the inhibitory compounds as seeds to identify in-trial or FDA approved drugs. Since the goals of the HTS screen was to identify more drug-like PCs, compounds such as Elinafide would be expected to expedite the development of therapeutics for the treatment of ATSD.

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Mechanism of symmetrical dyads of naphthalimide M-31850 binding to OfHex2 [2]
Inhibitory activity testing suggested M-31850 is a competitive inhibitor against OfHex2 with a Ki value of 2.5 μM. However, the low LE value (0.21) indicated the binding between M-31850 and OfHex2 was not efficient enough. To study the mechanism of M-31850 interacting with OfHex2, molecular docking was performed using the homology modeled structure of OfHex2 using HsHex as template. The molecular docking study revealed that one of the naphthalimide groups of M-31850 localized in the active pocket and the secondary amine in the linker formed a hydrogen bond with the side chain of the residue E345. And the second naphthalimide group stacked poorly in the narrow out-pocket site. As shown in Fig. 2, only part of the imide ring and one aromatic ring of naphthaline of the second naphthalimide group stacked with the imidazole group of the residue H285 at a dihedral of about 35° in the out-pocket site, while the other aromatic ring of naphthaline group was away from the out-pocket site. The inefficiency binding in the out-pocket site might be caused by the oversize of naphthalimide group or the lack of flexibility of the linker between secondary amine and the second naphthalimide group. Thus, the second naphthalimide group is better to be replaced by a smaller group and to be conjugated by an appropriately-lengthed N-alkylamine linker.

For primary and secondary screening of the compounds, enzyme activity was monitored continuously on the Analyst HT fluorometer as describe above. Steady-state kinetic parameters were established using a range of MUG substrate (1.6 mM – 0.003 mM) and enzyme concentrations (1 – 0.01 mg/ml). The inhibitory activity of compounds which were fluorescent (M-31850 and derivatives) or quenched (M-22971) near the emission maxima of MU were also confirmed using the colorimetric substrate pNP-GlcNAc and conditions described for the MUG endpoint assay, except that absorbance was measured at 405 nm.[1]

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Purification of Iron-dextran-labeled Lysosomes [1]
Lysosomal fractions were prepared from ATSD fibroblasts treated with DMSO or M-31850 (1 mM) for five days, followed by labelling with Iron-dextran colloid and subsequent purification by magnetic chromatography as previously described. Lysosomal, acid phosphosphase and Hex A was monitored fluorometrically using the substrate MUP and MUGS, respectively, as described.

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Inhibitory activity and ligand efficiency analysis [2]
The insect GH20 enzyme OfHex2 was prepared as described by Liu et al. the bacterial hexosaminidase from Serratia marcescens and another insect hexosaminidase OfHex1 was prepared as describled by Liu et al. GH20 hexosaminidases from Trichoderma viride, Canavalia ensiformis and Homo sapiens were purchased from Sigma–Aldrich. All of the synthesized compounds were evaluated for their inhibitory activities against hexosaminidases by using the artificial substrate, pNP-GlcNAc. The assay components were incubated in a final volume of 60 μl at 25 °C for 30 min in the presence of Britton-Robinson buffer(OfHex2, pH 6.0; OfHex1, TvHex, and SmHex, pH 7.0; HsHex and CeHex, pH 4.5), ethanol at the final concentration of 20%, enzyme, compounds and pNP-GlcNAc. Then enzyme reaction was stopped by the addition of 60 μl 0.5 M Na2CO3 solution. The inhibition constant (Ki [μM]) was obtained by Dixon plots by changing the concentration of the compound at a constant concentration (0.2 and 0.5 mM) of the substrate. Ligand efficiency values were calculated as follows: LE = −1.35 log(Ki)/N, where N is the number of heavy atoms.

[1]. High-throughput screening for human lysosomal beta-N-Acetyl hexosaminidase inhibitors acting as pharmacological chaperones. Chem Biol. 2007 Feb;14(2):153-64.
[2]. Exploring unsymmetrical dyads as efficient inhibitors against the insect β-N-acetyl-D-hexosaminidase OfHex2. Biochimie. 2014 Feb;97:152-62.

The adult forms of Tay-Sachs and Sandhoff diseases result when the activity of beta-hexosaminidase A (Hex) falls below approximately 10% of normal due to decreased transport of the destabilized mutant enzyme to the lysosome. Carbohydrate-based competitive inhibitors of Hex act as pharmacological chaperones (PC) in patient cells, facilitating exit of the enzyme from the endoplasmic reticulum, thereby increasing the mutant Hex protein and activity levels in the lysosome 3- to 6-fold. To identify drug-like PC candidates, we developed a fluorescence-based real-time enzyme assay and screened the Maybridge library of 50,000 compounds for inhibitors of purified Hex. Three structurally distinct micromolar competitive inhibitors, a bisnaphthalimide, nitro-indan-1-one, and pyrrolo[3,4-d]pyridazin-1-one were identified that specifically increased lysosomal Hex protein and activity levels in patient fibroblasts. These results validate screening for inhibitory compounds as an approach to identifying PCs. [1]

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The GH20 β-N-acetyl-d-hexosaminidase OfHex2 from the insect Ostrinia furnacalis (Guenée) is a target potential for eco-friendly pesticide development. Although carbohydrate-based inhibitors against β-N-acetyl-D-hexosaminidases are widely studied, highly efficient, non-carbohydrate inhibitors are more attractive due to low cost and readily synthetic manner. Based on molecular modeling analysis of the catalytic domain of OfHex2, a series of novel naphthalimide-scaffold conjugated with a small aromatic moiety by an alkylamine spacer linker were designed and evaluated as efficiently competitive inhibitors against OfHex2. The most potent one containing naphthalimide and phenyl groups spanning by an N-alkylamine linker has a Ki value of 0.37 μM, which is 6 fold lower than that of M-31850, the most potent non-carbohydrate inhibitor ever reported. The straightforward synthetic manners as well as the presumed binding model in this paper could be advantageous for further structural optimization for developing inhibitors against GH20 β-N-acetyl-D-hexosaminidases. [2]

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The fact that residues forming the active pocket of HsHex are conserved in OfHex2 inspired us to deduce that the naphthalimide derivatives might be exploited as inhibitors against OfHex2. Binding affinity analysis of M-31850 against OfHex2 confirmed M-31850 was a competitive inhibitor of OfHex2. Ligand efficiency (LE) value, combining the binding free energy to the number of heavy atoms, is a very helpful metric guiding the optimization of hits. The low LE value of M-31850 indicated there was space to increase its efficiency. Molecular docking study suggested this low efficiency of M-31850 was caused by the oversize of the naphthalimide group binding to the small hydrophobic cavity outside of the pocket (named out-pocket site). Thus, the improvement of the binding efficiency in the out-pocket site could be a way to increase inhibitors binding affinity.
To improve the ligand efficiency, a series of novel derivatives were synthesized by replacing one of the naphthalimide in M-31850 with small aromatic rings to afford new unsymmetrical dyads. Optimization on the size of aromatic rings and the length of linker was performed to improve the binding affinity of moieties that bind outside of the active pocket.
The enzymatic testing and efficiency analysis suggested these new unsymmetrical dyads were competitive inhibitors of OfHex2 and had higher ligand efficiency and better inhibitory activity when compared to M-31850. The structure–activity relationship analysis as well as molecular docking studies provided a binding model between these inhibitors and OfHex2. And tryptophan fluorescent assay indicated that the competitive inhibition benefited from the stacking interactions between naphthalimides and tryptophan residues in the active pocket.[2]

C28H21N3O4

463.493

463.153

C, 72.56; H, 4.57; N, 9.07; O, 13.81

281224-40-6

281224-40-6

342139

Typically exists as solid at room temperature

1.4±0.1 g/cm3

715.0±45.0 °C at 760 mmHg

386.2±28.7 °C

0.0±2.3 mmHg at 25°C

1.729

2.12

1

5

6

35

759

0

O=C1C2=C([H])C([H])=C([H])C3C([H])=C([H])C([H])=C(C(N1C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])N1C(C4=C([H])C([H])=C([H])C5C([H])=C([H])C([H])=C(C1=O)C4=5)=O)=O)C2=3

UPLUIYHKSADQOZ-UHFFFAOYSA-N

InChI=1S/C28H21N3O4/c32-25-19-9-1-5-17-6-2-10-20(23(17)19)26(33)30(25)15-13-29-14-16-31-27(34)21-11-3-7-18-8-4-12-22(24(18)21)28(31)35/h1-12,29H,13-16H2

2,2'-(azanediylbis(ethane-2,1-diyl))bis(1H-benzo[de]isoquinoline-1,3(2H)-dione)

M 31850; M31850; 281224-40-6; M-31850; 2,2'-(Azanediylbis(ethane-2,1-diyl))bis(1H-benzo[de]isoquinoline-1,3(2H)-dione); NSC377607; 2-(2-{[2-(1,3-dioxo-2,3-dihydro-1H-benzo[de]isoquinolin-2-yl)ethyl]amino}ethyl)-2,3-dihydro-1H-benzo[de]isoquinoline-1,3-dione; 2-[2-[2-(1,3-dioxobenzo[de]isoquinolin-2-yl)ethylamino]ethyl]benzo[de]isoquinoline-1,3-dione; CHEMBL2333316; SCHEMBL4408773; M-31850

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~5 mg/mL (~10.79 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)