PDI (Kd = 62 nM)

LOC-14 (0.01-100 μM; 24 hours) exhibits the ability to inhibit recombinant (r)PDIA3 at an IC50 of about 5 μM.Treatment with a PDI inhibitor, LOC14 inhibited PDIA3 activity in lung epithelial cells, decreased intramolecular disulfide bonds and subsequent oligomerization (maturation) of HA in both H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68) infected lung epithelial cells[2].

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LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a Kd of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures[1].

LOC-14 (orally administered by gavage; 20 mg/kg; once daily; 12-28 weeks) prolongs survival, attenuates brain atrophy, and significantly improves motor function in N171-82Q HD mice. Male N171-82Q HD mice are the animal model. 20 mg per kilogram. 20 mg/kg, given once daily, orally, for a period of 12 to 28 weeks. Improved motor performance in HD mice as a result. LOC14 was freshly made by first combining it with 1-Methyl-2-pyrrolidinone (NMP) to create an 80 mg/ml stock solution, and then diluting it with 0.5% methyl cellulose to the desired concentration (40× dilution)[3].

Enzymatic Insulin Reduction Assay.[1]
The assay was carried out in a 384-well black, clear bottom plate. Each well contained 80 μL of the reaction mixture in buffer A (10 mM Tris⋅HCl, pH 8, 150 mM NaCl, and 2 mM EDTA) with 5 μM PDIa, 100 μM bovine insulin, 350 μM DTT, and 75 μM test compound. All experiments were done in duplicate. The assay plate was incubated at 25 °C for 1 h, and then the absorbance at 650 nm was read on a Tecan Infinite 200 microplate reader for each sample consecutively at 5-min intervals for 1 h. Increase in absorbance is indicative of insulin’s β-chain aggregation and precipitation out of solution[1].

Cells MTEC Concentration: 0.01 μM; 0.1 μM; 0.5 μM; 1 μM; 5 μM; 10 μM; 100 μM 24-hour incubation period; result: reduced activity of recombinant (r)PDIA3.
Primary MTECs were isolated and cultured from age and sex matched wild type (WT) C57BL/6NJ mice as previously described. Cells were plated at 2 × 106 cells/dish and when greater than 90% confluent, infected with mouse-adapted H1N1 influenza A virus Puerto Rico 8/34 (PR8) or H3N2 A X-31, A/Aichi/68 (X31) at 2.5 Egg infectious units (EIU)/cell in a DMEM/F12 (Gibco) growth factor–free medium. Ultraviolet light (UV)–irradiated virus that was replication-deficient (mock) was used as a control. Following infection the cells were incubated for 1 h at 37 °C, the plates were then washed twice with 2 mL PBS to remove unbound virus, and supplemented with growth factor–free medium. MTECs were pretreated for 2 h with 10 μM LOC14, during viral infection, and 1 h post viral infection, DMSO was used as a control. All treatments were performed in growth factor–free medium[2].

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High-Throughput Screen of LOC Library. LOC mother plates with compounds at 4 mg/mL were thawed and spun down (215 × g, 20 °C, 1 min) before use. Biomek FX robotic liquid dispenser was used to handle all liquid transferring and mixing. Replica daughter plates (D1) were prepared by transferring 2 μL of compound from the mother plate into 384-deepwell clear, round bottom, polypropylene plates containing 98 μL of PC12 medium without selective agent geneticin to obtain compound concentration at 80 μL/mL in 2% (vol/vol) DMSO. Twofold serial dilution was performed across five daughter plates by transferring 50 μL of compounds (at 80 μL/mL) from the D1 plate into 50 μL of PC12 medium in daughter plate D2, mixing, and then repeating the process for the remaining three plates. Daughter plate D1 with compounds at 80 μL/mL, daughter plate D3 with compounds at 20 μL/mL, and daughter plate D5 with compounds at 5 μL/mL were then used for the screen. Assay plates were set up by seeding tebufenozide-induced PC12 mHTTQ103 cells into 384-well black, clear-bottom plates at a density of 7,500 cells per well in 57 μL PC12 medium without geneticin. Three microliters of compound from the daughter plates (D1, D3, and D5) were added to the assay plates for a final compound concentration of 4, 1, and 0.25 μL/mL. Four wells containing uninduced PC12 mHTTQ103 cells and four wells containing medium only were also included on each plate as controls. The assay plates were incubated at 37 °C, 9.5% CO2 for 48 h. Twenty microliters of 40% (vol/vol) Alamar blue (cat. no. DAL1100; Life Technologies) solution in PC12 medium was added to each well (1:10 final dilution), and the plates were incubated for an additional 12–24 h at 37 °C, 9.5% CO2. Alamar blue fluorescence was read on a fluorescence plate reader with a 530-nm excitation filter and 590-nm emission filter. Each compound concentration was tested in triplicate.[1]

Chronic administration of a reversible, brain penetrable small molecule PDI modulator, LOC14 (20 mg/kg/day), significantly improved motor function, attenuated brain atrophy and extended survival in the N171–82Q HD mice. Moreover, LOC14 preserved medium spiny neuronal marker dopamine- and cyclic-AMP-regulated phosphoprotein of molecular weight 32 000 (DARPP32) levels in the striatum of HD mice. Mechanistic study revealed that LOC14 suppressed mHtt-induced ER stress, indicated by repressing the abnormally upregulated ER stress proteins in HD models. These findings suggest that LOC14 is promising to be further optimized for clinical trials of HD, and modulation of signaling pathways coping with ER stress may constitute an attractive approach to reduce mHtt toxicity and identify new therapeutic targets for treatment of HD.[3]

[1]. Small molecule-induced oxidation of protein disulfide isomerase is neuroprotective. Proc Natl Acad Sci U S A. 2015 Apr 28;112(17):E2245-52.

[2]. Lung epithelial protein disulfide isomerase A3 (PDIA3) plays an important role in influenza infection, inflammation, and airway mechanics. Redox Biol. 2019 Apr;22:101129.

[3]. Small molecule modulator of protein disulfide isomerase attenuates mutant huntingtin toxicity and inhibits endoplasmic reticulum stress in a mouse model of Huntington's disease. Hum Mol Genet. 2018 May 1;27(9):1545-1555

Protein disulfide isomerase (PDI) is an endoplasmic reticulum molecular chaperone protein upregulated in mouse models of neurodegenerative diseases involving protein misfolding and in patient brain tissue. However, the role of PDI in these diseases remains unclear. This article reports the discovery of a reversible neuroprotective lead compound (LOC)14, which can act as a regulator of PDI. The screening method for LOC14 involved high-throughput screening of approximately 10,000 lead compounds to identify those that effectively rescued the viability of PC12 cells expressing mutant huntingtin protein; then, the effects of the compounds on PDI reductase activity were assessed through in vitro screening. Isothermal titration calorimetry and fluorescence experiments showed that the binding of LOC14 to PDI is reversible, with a dissociation constant (Kd) of 62 nM, indicating that LOC14 is the most potent PDI inhibitor reported to date. Using two-dimensional heteronuclear single quantum correlation nuclear magnetic resonance experiments, we determined that the binding site of LOC14 is located near the active site and observed that the binding of LOC14 forces PDI to adopt an oxidative conformation. In addition, we found that LOC14-induced PDI oxidation has a neuroprotective effect not only in cell culture but also in corticostriatal brain slice culture. LOC14 exhibits high stability, low intrinsic microbody clearance and low plasma protein binding in mouse liver microsomes and plasma. These results suggest that LOC14 is a promising lead compound that can be used to evaluate the potential therapeutic effects of modulating PDI in animal disease models. [1]

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Protein disulfide isomerases (PDIs) are a class of redox chaperone proteins that catalyze the formation or isomerization of disulfide bonds in proteins. Previous studies have shown that one of the members of the PDIA3 protein family can interact with influenza A virus (IAV) hemagglutinin (HA), and this interaction is necessary for the efficient oxidative folding of HA in vitro. However, it remains unclear whether these host-virus protein interactions occur during viral infection and whether such interactions could serve as potential therapeutic targets for influenza infection. This study demonstrates that PDIA3 is specifically upregulated in IAV-infected mouse or human lung epithelial cells, and that PDIA3 directly interacts with IAV-HA. Treatment with the PDI inhibitor LOC14 inhibited PDIA3 activity in lung epithelial cells and reduced intramolecular disulfide bond formation and subsequent oligomerization (maturation) of HA in H1N1 (A/PR8/34) and H3N2 (X31, A/Aichi/68)-infected lung epithelial cells. The reduction in disulfide bond formation significantly decreased viral load and suppressed the pro-inflammatory response in lung epithelial cells. Specific knockout of the PDIA3 gene in mouse lung epithelial cells significantly reduced viral load and lung inflammatory immune marker levels after influenza virus (IAV) infection and significantly improved airway mechanics. In summary, these results indicate that PDIA3 is essential for the effective pathogenicity of influenza virus in vivo, and pharmacological inhibition of PDIA3 is expected to become a novel anti-influenza treatment strategy for influenza pandemics and severe influenza seasons. [2] Huntington's disease (HD) is caused by the amplification of the cytosine-adenine-guanine (CAG) trinucleotide repeat sequence in the huntingtin protein (HTT) gene, which encodes an extended polyglutamine sequence at the N-terminus of the huntingtin protein (Htt), leading to Htt misfolding, abnormal protein aggregation, and the gradual appearance of disease symptoms. Chronic activation of endoplasmic reticulum (ER) stress caused by mutant Htt (mHtt) leads to cell dysfunction and ultimately cell death. Protein disulfide isomerase (PDI) is a molecular chaperone protein located in the endoplasmic reticulum. Our previous research has shown that mHtt leads to the accumulation of PDI on the mitochondrial-associated endoplasmic reticulum membrane and induces cell death. However, small-molecule modulators of PDI activity can protect cells from mHtt toxicity, as reported in cell and brain slice models of Huntington's disease (HD). In this study, we demonstrated that PDI is highly expressed in brain tissue, cells, and a mouse model of HD patients. Long-term administration of the reversible, blood-brain barrier-penetrating small-molecule PDI modulator LOC14 (20 mg/kg/day) significantly improved motor function, reduced brain atrophy, and prolonged survival in N171-82Q HD mice. Furthermore, LOC14 maintained the level of DARPP32 (a 32,000 molecular weight phosphoprotein regulated by dopamine and cyclic adenosine monophosphate), a marker of medium-sized spinous neurons, in the striatum of HD mice. Mechanistic studies have shown that LOC14 inhibits mHtt-induced endoplasmic reticulum stress by suppressing abnormally upregulated endoplasmic reticulum stress proteins in HD models. These findings suggest that LOC14 may be further optimized for clinical trials in HD, and that modulating signaling pathways in response to endoplasmic reticulum stress may be an effective way to reduce mHtt toxicity and find new therapeutic targets for HD. [3]

C16H19N3O2S

317.405962228775

317.12

C, 60.55; H, 6.03; N, 13.24; O, 10.08; S, 10.10

877963-94-5

877963-94-5

9117962

White to off-white solid powder

1.3

0

4

3

22

460

0

YVBSNHLFRIVWFQ-UHFFFAOYSA-N

InChI=1S/C16H19N3O2S/c20-15(12-5-6-12)18-9-7-17(8-10-18)11-19-16(21)13-3-1-2-4-14(13)22-19/h1-4,12H,5-11H2

2-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,2-benzothiazol-3-one

LOC14; LOC 14; LOC-14; 2-[(4-cyclopropanecarbonylpiperazin-1-yl)methyl]-2,3-dihydro-1,2-benzothiazol-3-one; 2-[[4-(Cyclopropylcarbonyl)-1-piperazinyl]methyl]-1,2-benzisothiazol-3(2H)-one; LOC-14; CHEMBL4637290; 2-((4-(Cyclopropanecarbonyl)piperazin-1-yl)methyl)benzo[d]isothiazol-3(2H)-one; 2-[[4-(cyclopropanecarbonyl)piperazin-1-yl]methyl]-1,2-benzothiazol-3-one;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). This product is not stable in solution, please use freshly prepared working solution for optimal results.

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

DMSO: ~50 mg/mL (~157.5 mM)
Water: ˂1 mg/mL
Ethanol: ~19 mg/mL (~60 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.55 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.55 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.55 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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