LMPTP INHIBITOR 1 dihydrochloride was not explicitly named in the literature; however, the LMPTP inhibitors (including Compd. 3, Compd. 18, Compd. 23) targeted low-molecular-weight protein tyrosine phosphatase (LMPTP, specifically LMPTP-A). Compd. 3 had IC₅₀ values for LMPTP-A of unspecified values (with 0.4 mM OMFP and 7 mM pNPP as substrates), and showed negligible inhibition on LYP (n=7) and VHR (n=6) with 0.4 mM OMFP as substrate; Compd. 18 had an IC₅₀ (mean±range) on LMPTP-A (OMFP as substrate) from 6 experiments, Compd. 23 had an IC₅₀ (mean±range) on LMPTP-A (OMFP as substrate) from 2 experiments; Compd. 23 at 40 μM showed high selectivity, with minimal inhibition on other protein tyrosine phosphatases (PTPs) when incubated with 0.4 mM OMFP or 5 mM pNPP [1]

Having an IC50 of 0.8 μM LMPTP-A, LMPTP INHIBITOR 1 (diHCl) is a selective inhibitor of low molecular weight protein tyrosine phosphatase. Compared to LMPTP-B, it works better against LMPTP-A. Additionally, insulin-stimulated HepG2 IR phosphorylation in human HepG2 hepatocytes is enhanced by LMPTP inhibitor 1 (compound 23; 10 μM) [1].

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1. In HepG2 hepatocytes, overnight incubation with 10 μM Compd. 23 (a representative LMPTP inhibitor) followed by 10 nM insulin stimulation for 5 min significantly increased insulin receptor (IR) tyrosine phosphorylation, as assessed by phosphoIR (pIR) ELISA (p=0.0079), while Compd. 28 at the same concentration showed no significant effect (p=0.1667) [1]
2. Enzyme activity assays demonstrated that Compd. 23 exhibited an uncompetitive mechanism of action on human LMPTP-A: activity assays with increasing OMFP concentrations and varying Compd. 23 concentrations showed fitting to the Michaelis-Menten equation (95% confidence intervals), and Lineweaver-Burk plots confirmed the uncompetitive mode; IC₅₀ values of Compd. 23 on LMPTP-A-catalyzed hydrolysis of increasing OMFP concentrations showed a 2-phase decay trend [1]
3. NMR spectroscopy (HSQC ¹⁵N-¹H) revealed that Compd. 18 binding caused chemical shift changes in specific residues of human LMPTP-A, with residues at the active-site opening showing the most significant shifts; site-directed mutagenesis of LMPTP-A residues involved in Compd. 18 binding altered the inhibitory activity of Compd. 18, confirming the binding site at the opening of the catalytic pocket [1]

LMPTP inhibitor 1 can correct diabetes in obese mice and is orally bioavailable, with typical serum concentrations of about 680 nM after therapy at 0.03% w/w and >3 μM following treatment at 0.05% w/w. Without changing body weight, LMPTP inhibitor 1 (0.05% w/w) dramatically enhances glucose tolerance, lowers fasting insulin levels, and suppresses LMPTP activity in diabetic DIO mice [1].

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1. Diet-induced obese (DIO) male B6 mice treated with 0.05% w/w Compd. 23 in high-fat diet (HFD) for 2 weeks showed improved glucose tolerance in intraperitoneal glucose tolerance test (IPGTT, p=0.0162, HFD n=8, Compd. 23 n=6), reduced fasting plasma insulin levels (relative to HFD-fed littermates, p=0.0235, HFD n=4, Compd. 23 n=4), and significantly increased liver IR tyrosine phosphorylation after intraperitoneal insulin injection (10 min post-injection, p=0.0002, HFD n=7, Compd. 23 n=6); body weight of mice during treatment was not affected by Compd. 23 (HFD n=17, Compd. 23 n=13) [1]
2. DIO liver-specific LMPTP knockout (KO) mice treated with Compd. 23 (0.05% w/w in HFD for 2 weeks) showed no significant changes in IPGTT (p=0.6120, HFD n=6, Compd. 23 n=6) or liver IR tyrosine phosphorylation (p=0.2791, HFD n=6, Compd. 23 n=6), indicating that the efficacy of Compd. 23 is dependent on hepatic LMPTP expression [1]
3. Global and liver-specific LMPTP KO mice (Acp1^{fl/fl} crossed with albumin-Cre mice) on HFD for 3 months showed improved glucose tolerance (IPGTT: global KO p=0.0324, liver-specific KO p=0.0117), reduced fasting plasma insulin (global KO p=0.0363, liver-specific KO p=0.0425), and increased liver IR tyrosine phosphorylation (liver-specific KO p=0.0432), validating LMPTP as the therapeutic target for the inhibitors [1]

1. High-throughput screening assay: LMPTP-A was incubated with 3-O-methylfluorescein phosphate (OMFP, 0.4 mM) or para-nitrophenylphosphate (pNPP, 7 mM) as substrates, and compounds from the NIH chemical library were tested for inhibitory activity; IC₅₀ values were calculated from dose-response curves (mean±SD from 4 experiments for initial hits, n=10 for Compd. 3 with OMFP, n=6 for Compd. 3 with pNPP), with each experiment performed in duplicate [1]
2. Selectivity assay: Various protein tyrosine phosphatases (PTPs) were incubated with 0.4 mM OMFP or 5 mM pNPP in the presence of dimethylsulfoxide (DMSO) or 40 μM Compd. 23; enzyme activity was measured as % activity relative to DMSO control (mean±SEM from 3 independent experiments), with 10 nM LMPTP-A equivalent activity units used for each PTP [1]
3. Mechanism of action assay: 0.78 nM human LMPTP-A was incubated with increasing concentrations of OMFP (substrate) and varying concentrations of Compd. 23; reaction rates were measured and fitted to the Michaelis-Menten equation (95% confidence intervals) for mean reaction rate vs. OMFP concentration analysis, and Lineweaver-Burk plots were generated (linear regression with 95% confidence intervals) to determine the inhibition mechanism; IC₅₀ values for Compd. 23 at different OMFP concentrations were calculated (mean from 2 independent experiments in quadruplicate) and fitted to a 2-phase decay model [1]
4. Isothermal calorimetry (ITC) assay: Human LMPTP-A was titrated with increasing concentrations of Compd. 18 in the presence (3 replicates) or absence (4 replicates) of 200 μM sodium orthovanadate; heat changes during titration were measured to assess binding interactions, with a representative titration image reported for the vanadate-containing group [1]
5. Mutagenesis-based enzyme activity assay: LMPTP-A mutants were generated, and their phosphatase activity was measured in the presence of Compd. 18 (0.4 mM OMFP as substrate); % activity relative to wild-type LMPTP-A was calculated (mean±SD from 3 independent experiments) to validate the binding site of the inhibitor [1]

1. HepG2 hepatocyte insulin signaling assay: HepG2 cells were incubated overnight with 10 μM Compd. 23/Compd. 28 or DMSO (vehicle control), then stimulated with 10 nM insulin for 5 min or left unstimulated; insulin receptor (IR) was immunoprecipitated from cell lysates, and IR tyrosine phosphorylation was analyzed by Western blotting (representative of 2 independent experiments) and phosphoIR (pIR) ELISA (data from 5 independent experiments) [1]

1. Global LMPTP KO and wild-type (WT) mouse experiment: Male WT and LMPTP KO mice were fed a high-fat diet (HFD) for 3 months to induce diet-induced obesity (DIO); intraperitoneal glucose tolerance test (IPGTT) was performed (WT n=5, KO n=6), and fasting plasma insulin levels were measured by ELISA (WT n=5, KO n=5); statistical analysis used Two-Way ANOVA for IPGTT and two-tailed unpaired t-test with Welch’s correction for insulin levels [1]
2. Liver-specific LMPTP KO mouse experiment: Acp1^{fl/fl} mice were crossed with albumin-Cre mice to generate liver-specific LMPTP KO (Cre^{+}) and control (Cre^{-}) mice; male littermates were fed HFD for 3 months to induce DIO; IPGTT was performed (Cre^{-} n=7, Cre^{+} n=6), fasting plasma insulin was measured by ELISA (Cre^{-} n=7, Cre^{+} n=6), and insulin signaling was assessed by injecting insulin intraperitoneally, harvesting livers after 10 min, and measuring IR tyrosine phosphorylation by pIR ELISA (Cre^{-} n=9, Cre^{+} n=10); statistical analysis used Two-Way ANOVA for IPGTT, two-tailed unpaired t-test with Welch’s correction for insulin levels, and one-tailed unpaired t-test with Welch’s correction for IR phosphorylation [1]
3. Compd. 23 in vivo efficacy experiment: Male DIO B6 mice were treated with 0.05% w/w Compd. 23 mixed in HFD or HFD alone for 2 weeks; body weight was monitored throughout treatment (HFD n=17, Compd. 23 n=13); IPGTT was performed (HFD n=8, Compd. 23 n=6), fasting plasma insulin was measured by ELISA (HFD n=4, Compd. 23 n=4); for insulin signaling assessment, mice were injected intraperitoneally with insulin, livers were harvested after 10 min, and IR tyrosine phosphorylation was measured by pIR ELISA (HFD n=7, Compd. 23 n=6) and Western blotting (HFD n=3, Compd. 23 n=3); statistical analysis used Two-Way ANOVA for IPGTT, two-tailed unpaired t-test with Welch’s correction for insulin levels, and one-tailed unpaired t-test with Welch’s correction for IR phosphorylation [1]
4. Liver-specific LMPTP KO + Compd. 23 experiment: DIO liver-specific LMPTP KO mice were treated with 0.05% w/w Compd. 23 in HFD or HFD alone for 2 weeks; IPGTT was performed (HFD n=6, Compd. 23 n=6) and liver IR tyrosine phosphorylation was measured (HFD n=6, Compd. 23 n=6); statistical analysis used Two-Way ANOVA for IPGTT and one-tailed unpaired t-test with Welch’s correction for IR phosphorylation [1]

[1]. Diabetes reversal by inhibition of the low-molecular-weight tyrosine phosphatase. Nat Chem Biol. 2017 Jun;13(6):624-632.

1. Obesity-related insulin resistance is a key driver of type 2 diabetes, and LMPTP is an important phosphatase that dephosphorylates the insulin receptor (IR), thereby promoting insulin resistance; LMPTP gene knockout (systemic or liver-specific) protects mice from high-fat diet (HFD)-induced diabetes without affecting weight [1]
2. LMPTP inhibitors (such as compound 23) work through a novel, non-competitive mechanism by binding to a unique site at the opening of the LMPTP catalytic pocket; structural studies (NMR and X-ray crystallography) have confirmed this binding site, which is located at the opening of the LMPTP catalytic pocket of compound 23. 18. Tightly bound to the active site pocket of LMPTP, key residues (identified by NMR shift analysis) mediate inhibitor binding [1]
3. The La Jolla Institute of Allergy and Immunology and the Sanford Burnham Institute for Medical Discovery hold an pending patent (WO 2016/061280 A1) for “Low molecular weight protein tyrosine phosphatase inhibitors and their uses therein”, and the lead author of the study is listed as an inventor [1]
4. LMPTP inhibitors reversed high-fat diet-induced diabetes in mice by increasing hepatic IR phosphorylation, validating the value of LMPTP as a therapeutic target for type 2 diabetes [1]

C28H38CL2N4O

517.539

516.242

C, 64.98; H, 7.40; Cl, 13.70; N, 10.83; O, 3.09

2310135-46-5

LMPTP inhibitor 1;1908414-82-3;LMPTP inhibitor 1 hydrochloride;2310135-38-5

134691744

Light yellow to yellow solid powder

3

4

9

35

580

0

Cl.Cl.O=C(C1C=CC(=CC=1)C1=CC(=C2C=CC=CC2=N1)NCCCN1CCCCC1)N(CC)CC

YDTJMPCNLMSGEO-UHFFFAOYSA-N

InChI=1S/C28H36N4O.2ClH/c1-3-32(4-2)28(33)23-15-13-22(14-16-23)26-21-27(24-11-6-7-12-25(24)30-26)29-17-10-20-31-18-8-5-9-19-31;;/h6-7,11-16,21H,3-5,8-10,17-20H2,1-2H3,(H,29,30);2*1H

C28H38Cl2N4O

LMPTP INHIBITOR 1; LMPTP INHIBITOR 1 HCl; LMPTP INHIBITOR 1 dihydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ≥ 64 mg/mL (~123.66 mM)
H2O : ~50 mg/mL (~96.61 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.83 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 25.0 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: ≥ 2.5 mg/mL (4.83 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 25.0 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: ≥ 2.5 mg/mL (4.83 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 100 mg/mL (193.23 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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