Allosteric Small Molecule Inhibitors of LMPTP

Obesity is frequently complicated by a constellation of metabolic and cardiovascular anomalies, called the metabolic syndrome, which significantly increases morbidity and mortality of affected individuals. Insulin resistance is an important component of the metabolic syndrome. Protein tyrosine phosphatases (PTPs) that regulate insulin signaling are in principle excellent therapeutic targets for insulin resistance syndromes. The low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene, represents an attractive target in this family. LMPTP is highly expressed in adipocytes and there is strong in vitro and in vivo evidence that LMPTP is a negative regulator of insulin signaling and a promising drug target for obesity. Genetic association studies in humans support a role for LMPTP in insulin resistance and the metabolic complications of obesity. In vivo, partial knock-down of LMPTP expression by specific antisense oligonucleotides (ASOs) led to improved glycemic and lipid profiles and decreased insulin resistance in diet-induced obese C57BL/6 mice. This probe report describes the first (and first-in-class) selective allosteric LMPTP inhibitor, ML400 (CID 73050863, SID 173019983). ML400 is potent (EC₅₀∼1μM), selective against other phosphatases, and displays good cell-based activity as well as rodent pharmacokinetics. As such it should be a valuable tool to explore the effects of selective LMPTP inhibition in vitro and in vivo.

Probe Structure & Characteristics

This Center Probe Report describes ML400, a selective allosteric inhibitor of LMPTP. ML400 has a central quinazoline core. The chemical structure and data summary are shown in (Table 1)

Chemical Structure of ML400

Table 1

Potency and selectivity characteristics for probe ML400.

1. Recommendations for Scientific Use of the Probe

Background: Obesity is frequently complicated by a constellation of metabolic and cardiovascular anomalies, called the metabolic syndrome, which significantly increases morbidity and mortality of affected individuals1. Insulin resistance is an important component of the metabolic syndrome. Protein tyrosine phosphatases (PTPs) that regulate insulin signaling are in principle excellent therapeutic targets for insulin resistance syndromes2. Indeed, PTP1B, a critical negative regulator of insulin signaling in liver and skeletal muscle, is currently an important drug target in obesity and type2 diabetes. We focus herein on another PTP, the low molecular weight protein tyrosine phosphatase (LMPTP), encoded by the ACP1 gene. LMPTP is highly expressed in adipocytes3. There is strong in vitro and in vivo evidence that LMPTP is a negative regulator of insulin signaling and a promising drug target in obesity. Genetic association studies in humans support a negative role for LMPTP in insulin resistance and the metabolic complications of obesity4. In vivo, partial knock-down of LMPTP expression by specific antisense oligonucleotides (ASOs) led to improved glycemic and lipid profiles and decreased insulin resistance in diet-induced obese C57BL/6 mice5. Interestingly, anti-LMPTP ASOs did not induce any metabolic phenotype in lean mice. Our current working model is that LMPTP plays a critical negative role in adipocyte insulin signaling6, while it is less important in liver and muscle, where it can be at least partially compensated for by PTP1B and/or other prominent PTPs. We hypothesize that a specific small-molecule inhibitor of LMPTP will significantly reduce obesity associated insulin resistance and decrease the severity of the metabolic syndrome in obesity. One important and innovative aspect of our screening effort was that we screened for non-competitive allosteric inhibitors. Searches for active site competitive inhibitors of PTPs by HTS are notoriously plagued by problems with low selectivity and lack of cell-permeability of the identified hits7. Although several prominent experts have hypothesized that non active site allosteric inhibitors could be the solution to the above-mentioned problem, there is still a considerable paucity of publications in this field8. Currently only three allosteric inhibitors of PTPs have been published9-11, and there is no known allosteric inhibitor of LMPTP.

Obesity and the metabolic syndrome. It has been estimated that every year in the U.S. more than 70 billion dollars are spent for the treatment of obesity-related conditions12 and almost 300,000 deaths/year can be attributed to the complications of obesity13. Obese patients often show multiple metabolic and cardiovascular anomalies known as “the metabolic syndrome”, including glucose intolerance, hyperlipidemia (especially high triglycerides with low HDL), and hypertension1. New pharmacologic approaches to correct the metabolic syndrome in obese individuals are urgently needed.

Promise and problems of PTPs as drug targets for the metabolic syndrome. Obesity-induced insulin resistance is believed to be a central pathogenic factor in the metabolic syndrome14. Obese patients are routinely treated with oral hypoglycemic agents, however even combinations of multiple agents are often insufficient to ensure adequate glycemic control, requiring the addition of parenteral insulin to the regimen. Reduced signal transduction at several levels after engagement of the insulin receptor (IR) has been observed in multiple insulin resistance syndromes, including the metabolic syndrome15, 16. The IR is a protein tyrosine kinase, and tyrosine phosphorylation plays an important role in insulin signal transduction. Pharmacological treatments able to increase the activity of the IR and/or tyrosine phosphorylation of IR targets are currently viewed as promising ways to reduce insulin resistance17. Several PTPs are involved in the regulation of insulin signaling18,19. In 1999 the Kennedy group reported compelling evidence that the tyrosine phosphatase PTP1B is an important in vivo inhibitor of insulin signaling. Mice carrying a deletion of PTP1B showed reduced obesity and insulin resistance while lacking major side effects20. This seminal paper triggered a major search for pharmacological inhibitors of PTP1B2,21. In addition to PTP1B, several other PTPs are now known to downregulate insulin signaling in vivo and are considered good pharmacological targets for insulin resistance22. Anti-PTP agents are envisioned as a possible new class or hypoglycemic agents and in principle could be combined with currently available oral antidiabetic medications. Due to their mechanism of action on the IR, they also could in principle act as potent insulin-sparing agents when used in combination with parenteral insulin. Although PTPs are good targets for insulin resistance and other human diseases, drugging this family of enzymes is considered a difficult task7. Searches for active-site competitive inhibitors of PTPs are plagued by problems with low selectivity and lack of cell-permeability of the hits. These problems stem in part from the particular features of the PTPs active site, which is small, well conserved among different members of the family, and highly charged7 as it has evolved to bind and “recognize” the phosphate moiety of phosphorylated proteins. Several experts have hypothesized that targeting secondary allosteric sites could be a solution to these three above-mentioned problems8. The first allosteric inhibitor of PTP1B was published in 2004 by Sunesis, Inc.9. This uncharged compound does not bind to the highly charged active site and is promising, since it shows superior selectivity properties and is active in cell-based assays. However, this is a very new field, and there is still a considerable paucity of publications. One additional allosteric inhibitor of PTP1B has been reported10, and we recently published an allosteric inhibitor series for PTPN2211, and both compounds were active in cell-based assays. There is no known allosteric inhibitor of any other PTP, including LMPTP.

LMPTP: a novel drug target for the metabolic syndrome. This grant proposal focuses on the low molecular weight PTP, a class II PTP23, encoded by the gene ACP1. LMPTP is a small (18 kD) cytosolic enzyme that is expressed ubiquitously but has particularly high expression in adipocytes3,24. As a result of an alternative mRNA splicing mechanism, LMPTP is usually found as two isozymes, called LMPTP-A and –B (the rodent isoforms are called respectively LMPTP-IF1 and -IF2)25,26. In humans the total enzymatic activity of LMPTP is variable and is determined by a common genetic polymorphism27. This unique feature makes it possible to test the involvement of LMPTP in human diseases by genetic association studies (for a review of associations between LMPTP and human diseases see ref. 28). Several studies have shown that LMPTP is an inhibitor of insulin signaling. In cell lines LMPTP is able to inhibit both the metabolic and growth-inducing effects of insulin29. Also in vitro the phosphatase dephosphorylates peptides derived from the phosphorylated IGF-1 receptor and IR30. Increased insulin signaling was observed in the adipose tissue of obese mice treated with anti-LMPTP antisense oligonucleotides (ASO). In the same study LMPTP was also easily co-precipitated with the IR. Multiple lines of evidence suggest that LMPTP plays an important role in the metabolic syndrome. The first line of evidence comes from human genetic studies. The ACP1 gene is located in one of the candidate genome regions for obesity on chromosome 2p25 and is currently included in the obesity gene map31. We and others carried out several genetic studies in obesity and type 2 diabetes. These studies showed that carriers of ACP1 alleles associated with low enzymatic activity tend to have lower non-fasting glucose levels and are protected from obesity-associated lipid anomalies4,32-34. Strong in vivo evidence suggesting that inhibition of LMPTP decreases the insulin resistance associated with obesity has been obtained recently at Isis Pharmaceuticals Inc., by treating mice with anti-LMPTP ASOs5. Leptin-deficient or diet-induced obese mice treated with specific anti-LMPTP ASOs showed a marked improvement of lipid profiles, and of glucose and insulin tolerance, in the absence of significant side effects5. These data strongly suggest that LMPTP is a promising pharmacological target for obesity-associated metabolic syndrome. Active site inhibitors of the LMPTP are under development in several laboratories35. The Ottaná group reported 5-arylidene-2,4-thiazolidinediones as a novel inhibitor class for PTPs, including LMPTP36-38. More recently Forghieri et al. reported the synthesis of flavonoid derivatives, which inhibited LMPTP and PTP1B and showed activity in cell-based assays of insulin signaling39. Although these studies show that there is a high level of interest in LMPTP, so far all the published LMPTP inhibitors have been obtained using conventional approaches, and they have low potential to yield leads with good drug-like properties. Although there are no known allosteric inhibitors of LMPTP, it has been reported that hypoxanthine is an allosteric stimulator of LMPTP-A, strongly suggesting that the enzyme has at least one allosteric site40.

Prior Art: A comprehensive review of all of the published LMPTP compounds is available41. All of these are competitive inhibitors w/ limited selectivity or cell permeability, or non-competitive but of poor potency with no selectivity of cell-activity data. Additionally, a series of benzoic acid derivatives that inhibited by a competitive mechanism both PTP1B and LMPTP-A has been reported.42 Treatment of mouse C2C12 skeletal muscle cells with several of these compounds increased tyrosine phosphorylation of the insulin receptor. Lastly, there is a recent report of a series of inhibitors of LMPTP-B that were first identified in silico.43 The compounds were not tested against LMPTP-A, or any other PTP1B, nor were they tested in cell-based assays. An additional SciFinder search was performed on March 20, 2014 and returned no additional relevant references.

Use of Probes. There are no known allosteric inhibitors of LMPTP, the only member of the class II of tyrosine phosphatases. Orthosteric inhibitors are plagued by lack of selectivity against other phosphatases, especially among enzymes belonging the class I of PTPs, as well as lack of cellular efficacy. Our probe displays selectivity against two PTPs that are representative of the two major types of class I phosphatases, Lymphoid Phosphatase isoform 1(LYP-1, a tyrosine-specific phosphatase) and VH1-related (VHR, a dual-specificity phosphatase). Although LMPTP appears to be a promising therapeutic target for metabolic syndrome, the current probes available that inhibit the enzymatic activity of LMPTP are unsuitable for preclinical experimentation in rodent models of disease. ML400 will be used for exploring the effect of selective in vitro, in cell and in vivo inhibition of LMPTP. In vivo, ML400 will be used to test whether acute inhibition of the activity of LMPTP is efficacious in treating metabolic syndrome in diet-induced obese mice. Additionally, a selective, cell-permeable inhibitor of LMPTP will provide an invaluable resource to the tyrosine phosphatase community for studies of LMPTP biology and in the areas of diet-induced obesity and type 2 diabetes therapies.

Therefore, there still remains an unmet need for novel potent, selective, not competitive, cell-permeable and cell-active LMPTP inhibitors not targeted to the conserved active site of phosphatases.

2. Materials and Methods

2.1. Assays

Table 2 summarizes details for the assays that enabled this probe discovery project. A detailed description of the Primary assay can be found in the PubChem AIDs listed

Table 2

Summary of Assays and AIDs.

2.2. Probe Chemical Characterization

Chemical name of probe compound. The IUPAC name of the probe is 2-(4-methoxyphenyl)-N-(3-(piperidin-1-yl)propyl)quinazolin-4-amine. The actual batch prepared, tested and submitted to the MLSMR is archived as SID 73019983 corresponding to CID 73050863. The probe ML400 does not have any chiral centers (Figure 1)

Figure 1

Structure of ML400.

Solubility and Stability of ML400 in PBS at room temperature. The stability of ML400 was investigated (Figure 2) in aqueous buffers at room temperature by monitoring the amount of starting ML400 apparently remaining after incubation at room temperature in either PBS (pH 7.4) or 1:1 PBS:acetonitrile (v/v). ML400 was stable in both PBS or PBS:acetonitrile with 100% remaining after 48 hrs. (Fitted curves are not plotted to show that data points are virtually superimposable). As noted in the Summary of in vitro ADME/T properties ML400 has excellent solubility (>103 μg/mL, >24.4 μM) at pH 5.0, 6.2 and 7.4 in pION buffer and comparable solubility in PBS at pH 7.4. The scaffold structure represented by ML400 has no substantial chemical liabilities.

Figure 2

Stability of ML400 in 1× PBS and 1:1 PBS:ACN at RT.

Table 3 summarizes the deposition of the Probe and 5 analogs.

Table 3

Probe and Analog Submissions to MLSMR (Evotec) for Small Molecule Inhibitors of LMPTP.

2.3. Probe Preparation

This probe is not commercially available. A 27 mg sample of ML400 synthesized at SBCCG has been deposited in the MLSMR (Evotec) (see Probe Submission Table 4). The probe ML400 was readily prepared in 4 steps from commercially available starting materials. (Compounds are numbered as in Scheme 1).

Table 4

Summary of in vitro ADME Properties of selected LMPTP inhibitors.

Scheme 1

Synthesis of ML400, conditions: a. 4-Methoxybenzoyl chloride, DIPEA, DCM, 0°C to RT, overnight (80%); b. t-BuOK, t-BuOH, 75°C, overnight (84%); c. POCl3, 90°C, overnight (61%); d. 3-(Piperidin-1-yl)propan-1-amine, t-BuOK 10%, Dry (more...)

Preparation of N-(2-acetylphenyl)-4-methoxybenzamide [2]

To a solution of 1-(2-aminophenyl)ethanone 1 (5.0 g, 37 mmol) and DIPEA (13 mL, 74 mmol) in 200 mL of THF in an ice bath was added 4-methoxybenzoyl chloride(7.5 mL, 56 mmol) dropwise. After 30 min at 0°C, the mixture was stirred at room temperature overnight and poured in 50 mL of ice water. The precipitate was collected and washed with water and then methanol. The solid was dried under vacuum to yield 8.0 g of crude product 2 (80% yield). MS (EI) m/z 270 (M+1).

Preparation of 2-(4-methoxyphenyl)quinolin-4-ol [3]

N-(2-acetylphenyl)-4-methoxybenzamide 2 (4.0 g, 15 mmol) was suspended in 100 mL of tert-butyl alcohol. Potassium tert-butoxide (3.3 g, 30 mmol) was added. The mixture was heated at 75°C overnight at nitrogen atmosphere. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled and poured into 50 mL of ice water. 10% aqueous HCl was added until pH=6. The solid was collected and washed several times with water to afford 3.1 g of crude product 3 (84 % yield). MS (EI) m/z 252 (M+1).

Preparation of 4-chloro-2-(4-methoxyphenyl)quinoline [4]

2-(4-methoxyphenyl)quinolin-4-ol 3 (3.1 g, 12.4 mmol) was added to phosphorus oxychloride POCl₃ (50 mL, 540 mmol) to give an dark solution, then several drops of DMF was added if necessary. The reaction was heated at 90°C overnight. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The resulting oil was basified with 1N NaOH solution, extracted with ethyl acetate and dried over magnesium sulfate. The organic layer was concentrated under reduced pressure to the crude product, which was chromatographed on silica gel and eluted with ethyl acetate and dichoromethane (0:100 to 30:70 gradient) to yield 2.0 g of product 4 (61 % yield). ¹H NMR (400 MHz, DMSO-d) δ 3.84 (s, 3H), 7.09 (m, 2H), 7.70 (m, 1H), 7.88 (m, 1H), 8.09 (m, 1H), 8.18 (m, 1H), 8.27 (m, 2H), 8.36 (m, 1H). MS (EI) m/z 270 (M+1).

Preparation of 2-(4-methoxyphenyl)-N-(3-(piperidin-1-yl)propyl)quinolin-4-amine [ML400]

ML400

Potassium tert-butoxide (50 mg, 0.5 mmol)) was added to a solution of 4-chloro-2-(4-methoxy-phenyl)quinoline 4 (1.0 g, 3.7 mmol) and 3-(Piperidin-1-yl)propan-1-amine (1.1 g, 7.7 mmol) in dry DMA (50 ml). The reaction was heated at 135°C overnight at nitrogen atmosphere. When the reaction was determined to be complete by HPLC, the reaction mixture was cooled to room temperature and evaporated under vacuum to give a residue. 20 mL of water was added and extracted with chloroform. The organic layer was dried over magnesium sulfate and concentrated under reduced pressure to give the crude product, which was subjected to be purified by preparative HPLC to afford 0.8 g of ML400 (57% yield). ¹H NMR (400 MHz, DMSO-d) δ 1.44 (m, 2H), 1.60 (m, 4H), 1.97 (m, 2H), 2.72 (m, 6H), 3.45 (m, 2H), 3.82 (s, 3H), 4.94 (s, 2H), 6.91 (s, 1H), 7.04 (m, 2H), 7.39 (m, 2H), 7.61 (m, 1H), 7 .82 (m, 1H), 8.13 (m, 3H), 8.26 (s, 2H). ¹³C NMR (400 MHz, DMSO-d) δ(ppm) 22.8, 23.7, 24.1, 53.0, 55.3, 94.5, 113.8, 117.7, 121.5, 123.6, 128.6, 129.3, 132.0, 147.7, 150.9, 156.0, 160.2, 164.4. MS (EI) m/z 376 (M+1).

Figure 3¹H NMR Spectrum of ML400 (400 MHz, CDCl₃)

Figure 4¹³C NMR Spectrum of ML400 (125 MHz, CDCl₃)

Figure 5Composite LC/MS for ML400

3. Results

3.1. Dose Response Curves for Probe

Figure 6Dose response curves of ML400 in the LMPTP enzyme inhibition assays with OMFP and pNPP as substrates, as well as in the LYP-1 and VHR-1 phosphatase selectivity assays

Selectivity against additional phosphatases

The probe ML400 and initial screening hits were found to be selective for LMPTP versus LYP-1 and VHR (usually IC50 > 80 μM for both of these). Then throughout the SAR development most of the analogs were also not active against LYP-1 and VHR, so selectivity was maintained for this preferred scaffold. Of note two analogs did have weak yet measurable activity against LYP-1 and VHR (7 -15 μM) giving us confidence that the apparent selectivity was not an artifact of the assay set up.

3.2. Cellular Activity

The probe ML400 was also profiled in an insulin-based mouse 3T3-L1 adipogenesis assay. We have previously found that deficiency in LMPTP expression impairs 3T3-L1 adipogenesis (Bottini laboratory unpublished observations). In this assay, 3T3-L1 pre-adipocytes are grown to 2-days post-confluence in DMEM with 10% bovine calf serum, and then induced to differentiate to adipocytes following stimulation for 2 days with an induction cocktail containing 1 μg/ml insulin, 1 μM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine in DMEM containing 10% fetal bovine serum (FBS).44 2 days later, the media is replaced with DMEM with 10% FBS and 1 μg/ml insulin, and after 2 additional days, the media is replaced with DMEM with 10% FBS for 2 additional days, at which point adipogenesis is measured using the AdipoRed Adipogenesis Assay Reagent from Lonza, according to the manufacturer's instructions. In brief, the assay reagent is added to the wells containing cells, where it partitions into the fat droplets of differentiated adipocytes, and emits fluorescence at 572 nm that can be detected with a plate-reader. To test the effect of probe ML400, cells were plated into 48-well plates and allowed to grow to confluence. Cells were then treated with 10 μM ML400 or 0.025% DMSO, and after 2 days induced to differentiate in the presence of 10 μM ML400 or 0.025% DMSO. Fresh compound or DMSO was added during each media replacement. As shown in the Figure 7, we found that treatment with 10 μM ML400 completely abolished 3T3-L1 adipogenesis.

Figure 7

ML400 Prevents Adipogenesis in 3T3-L1 Cells.

4. Discussion

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