Obesity is associated with a wide range of public health and economic problems throughout diverse demographic groups. As there is a high incidence of insulin resistance and type-2 diabetes among the obese, a mechanistic understanding of the relationship between insulin resistance and caloric consumption / obesity is needed. In cases of obesity and insulin resistance, triacylglycerides (TAG) accumulate within skeletal muscle cell; however, the mechanism of this relationship is not well understood. The research program described herein therefore sought small molecule probes that inhibit TAG accumulation. Toward this end, screening of 227,000 compounds resulted in the identification of MLS-0308942, which inhibited TAG accumulation with an IC₅₀ of 1.7 μM. Subsequent medicinal chemistry follow up identified MLS-0472732 (ML377), which had similar potency (IC₅₀ 1.1 μM) with some improved ADME/T properties. Data were confirmed in the H9c2 high content assay (IC₅₀ = 0.84 and 0.79 μM, respectively, with full response) and in a human primary cell assay. Furthermore, the compounds tested negatively for DGAT inhibition and cytotoxicity. Several additional analogs with comparable potency were observed, and the established SAR suggests the synthesis of new analogs in the future, which may address the poor microsomal stability exhibited thus far with the new series of compounds.

Probe Structure & Characteristics

This Center Probe Report describes ML377, a selective inhibitor of triacylglyceride accumulation. The chemical structure and data summary are shown in (Table 1).

Chemical Structure of ML377

1. Recommendations for Scientific Use of the Probe

Across nations, cultures, and demographic groups, we are witnessing a dramatic increase in the prevalence of obesity.1-4 This poses enormous public health problems and economic costs due to associated burdens of chronic disease and disability related largely to the high incidence of insulin resistance and, ultimately type 2 diabetes, among the obese. Rational therapeutic approaches to this pervasive medical problem require a comprehensive understanding of the molecular mechanisms that link insulin resistance to excess caloric consumption and obesity.

In the obese state, complex lipids, chiefly triacylglycerides (TAG), accumulate within cells of tissues that do not typically store fat, including skeletal muscle. An association between muscle lipid accumulation and insulin resistance is widely recognized.5,6 However, the development of effective therapeutics aimed at this linkage has been hampered because the mechanistic underpinnings of the association remain elusive, and experimental evidence to support a clear causal relationship is lacking. Indeed, there are circumstances where intramyocellular TAG accumulation is associated with improved muscle performance and metabolic flexibility. Taken together, these observations have led to dogma and confusion, defining a critical gap in scientific knowledge. We hypothesize that myocellular lipid accumulation can trigger both adaptive and maladaptive (lipotoxic) responses relevant to muscle insulin resistance, and that a dynamic balance between these responses determines the evolution of muscle insulin resistance and diabetes. We believe this problem demands an unbiased approach. To this end, we sought to identify molecular probes of cellular processes that control skeletal myocyte lipid homeostasis, and its cross-talk with insulin-stimulated glucose utilization in our R24 Seed Grant proposal (NIDDK R24-DK084969).

There are no current selective and specific inhibitors of the pathways associated with striated muscle lipotoxicity, other than inhibitors of the TAG synthesis pathway (e.g. A-922500, an inhibitor of diacylglycerol acyltransferase (DGAT-1). We specifically are not interested in TAG synthesis pathway inhibitors, so will eliminated them through countersceening against DGAT-1, and in our literature and patent search terms.

The probe, ML377, identified in this proposal will take us to the next step of mechanistic exploration. The following types of tertiary assays and follow-up studies will be conducted (and are described in greater detail in our full R24 proposal, R24 DK092781).

1. Assessment of the effects of the molecular probe hits on cultured primary skeletal myocyte insulin-stimulated myotube glucose uptake (ISGU) and glycogen synthesis (ISGS) to define whether the probe is linked to adaptive versus maladaptive responses.
2. Given the importance of mitochondrial function in linking skeletal myotube lipid accumulation to insulin sensitivity and glucose utilization, rigorous studies of the effects of the probes on mitochondrial respiratory function and mitochondrial biogenesis in primary skeletal myotubes in culture will be performed.
3. Further pharmacologic analyses and optimization will be conducted in order to assess the suitability of the candidate probes for in vivo studies of insulin sensitivity and glucose tolerance in rodents.
4. In parallel, the probes will be used as a perturbagen in primary skeletal myocytes in culture to identify downstream signaling responses and corresponding target pathways. This will involve exposing primary human skeletal myotubes in culture to a relevant concentration of the probe followed by transcriptional profiling, metabolomic profiling, and measurement of substrate fluxes. This approach has worked well for our independent studies focused on the effects of gene “knockout” or overexpression to identify pathways affected by the perturbation.
5. Based on the results of the studies described above, candidate targets will then be assessed. Standard approaches will be used including evaluation of direct interaction of the molecular probe with putative candidate target proteins using biochemical approaches, including mass spectrometry.

In the long run, based on the results of these next-phase experiments, we hope to establish molecular probes of adaptive/maladaptive responses of the skeletal myocyte to lipid overload. These probes will allow us, as delineated above, to identify new pathways and corresponding mechanisms that link alterations in myotube lipid metabolism to glucose utilization and insulin sensitivity. Such probes will serve as highly valuable experimental tools for the field. In addition, the targets identified in these planned experiments could prove useful as new targets for the development of novel therapeutic approaches aimed at the early stages of insulin resistance in the obese population. Further development of “hit” to “lead” will involve continued collaboration with medicinal chemists and pharmacologists. In the long-term, this work could lead to the development of a new class of drugs aimed at preventing the development of insulin resistance, and reducing the end-organ complications of type 2 diabetes.

Examples of the types of scientists in our broad scientific community that would likely utilize this probe are as follows:

• Mitochondrial biologists interested in changing mitochondrial function and biogenesis in cells in culture.
• Scientists with interests in conducting small animal studies aimed at assessing the effects of changing muscle lipid accumulation relevant to the development of insulin resistance.
• Biomedical engineers and nanoscientists who are developing innovative tissue culture techniques to assess muscle cell function in vitro, including assessing fatigability and tension development as a surrogate for exercise performance.
• iPSC-based studies conducted in human cells to assess the effects of changing lipid metabolism and mitochondrial function on progenitor cell differentiation lineages and specific disease phenotypes (e.g. metabolic diseases, cardiovascular diseases, muscular diseases).
• Studies by chemists interested in the structure-function relationship of molecules that change cellular capacity to handle lipid load and alter insulin responsiveness.
• Molecular biologists interested in the gene regulatory control of fat metabolism, who would conduct combined functional genomics (siRNA or cDNA screens) with the effects of the probe to identify relevant pathways downstream of each. This would be a first step towards novel therapeutic target discovery relevant to the broad area of metabolic and cardiovascular disease.

2. Materials and Methods

Primary assay: “HTS identification of small molecule Triacylglycerol inhibitors in a fluorescence assay (from PubChem website (http://pubchem.ncbi.nlm.nih.gov/). Comprises a small molecule screening campaign to identify molecular probes relevant to muscle lipid accumulation and insulin resistance (lipotoxicity) and of cellular processes that control skeletal myocyte lipid homeostasis, and its crosstalk with insulin-stimulated glucose utilization (see AID 651582 In Table 2)

Table 2

Summary of Assays and AIDs.

2.2. Probe Chemical Characterization

Chemical name of probe compound. The IUPAC name of the probe is N-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-2-(2-ethoxyethyl)-3-oxoiso-indoline-4-carboxamide. The actual batch prepared, tested and submitted to the MLSMR is archived as SID 163875672 corresponding to CID 71677755 (Figure 1). ML377 does not have any stereogenic centers. This probe is not commercially available. A 25 mg sample of ML377 synthesized at SBCCG has been deposited in the MLSMR (Evotec) (see Probe Submission Table 3, that summarizes the deposition of the Probe and 5 analogs).

Figure 1

Structure of ML377.

Table 3

Probe and Analog Submissions to MLSMR (Evotec) for TAG inhibitors.

Solubility and Stability of ML377 in PBS at room temperature. The stability of ML377 was investigated (Figure 2) in aqueous buffers at room temperature by monitoring the amount of starting ML377 apparently remaining after incubation at room temperature in either PBS (pH 7.4) or 1:1 PBS:acetonitrile (v/v). ML377 was stable in PBS: acetonitrile with 91.9% of the parent compound remaining after 48 hrs. The apparently lower stability (45.7%) in neat PBS is a reflection of relatively low solubility. Thus, the stability value is artificially low due to compound precipitation vs. degradation. As noted in the Summary of in vitro ADME/T properties, ML377 has ∼20 μM solubility in pH 5.0, 6.2 and 7.4 pION buffer. In pH 7.4 PBS, the solubility of ML377 was 8.4 μM. The scaffold structure represented by ML377 has no substantial chemical liabilities.7

Figure 2

Stability of ML377 in 1× PBS at RT.

2.3. Probe Preparation

ML377 probe SID 163875672 corresponding to CID 71677755 was synthesized according to Scheme 1.

Scheme 1

Synthesis of ML377.

Experimental

A: A solution of 4-methylisobenzofuran-1,3-dione (3.4 g, 21 mmol) in 100 mL methanol was treated with 1 mL concentrated sulfuric acid and was stirred at reflux overnight. The solvent was removed in vacuo and the resulting oil was partitioned with ethyl acetate and aqueous sodium bicarbonate. Evaporation of the organic phase provided dimethyl 3-methylphthalate (A) as a clear oil (2.0 g. 46%), which was, used without further purification. ¹H NMR (500 MHz, CDCl₃) δ 7.76 (d, J = 7.6 Hz, 1H), 7.34 (d, J = 7.4 Hz, 1H), 7.29 (t, J = 7.7 Hz, 1H), 3.88 (s, 3H), 3.82 (s, 3H), 2.28 (s, 3H). MS (ESI+ve): Calculated for C₁₁H₁₃O₄, [M+H] = 209.08, observed [M+H] = 209.07.

B: A flask was charged with A (2.0 g, 9.6 mmol) followed by carbon tetrachloride (80 mL), N-bromosuccinimide (1.8 g, 10 mmol), and AIBN (0.1 g, 0.6 mmol). The mixture was placed in a preheated bath and was stirred at 80 °C overnight. The solvent was removed in vacuo prior to partitioning the residue with ethyl acetate and aqueous sodium bicarbonate. The organic phase was condensed in vacuo to provide a residual oil which was purified by chromatography on silica gel eluted with 3-25% ethyl acetate in hexanes to afford 1.9 g (69%) of dimethyl 3-(bromomethyl)phthalate (B) as a clear oil. ¹H NMR (500 MHz, CDCl₃) δ 7.95 (d, J = 7.8 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 4.57 (s, 2H), 4.00 (s, 3H), 3.93 (s, 3H). MS (ESI+ve): Calculated for C₁₁H₁₂BrO₄, [M+H] = 286.99, observed [M+H] = 286.99.

C: Intermediate B (82 mg, 0.28 mmol) was dissolved in 3.5 mL of acetonitrile and charged with 0.06 mL (0.43 mmol) of triethylamine. 0.50 mL of acetonitrile containing 28 mg (0.31 mmol) of 2-ethoxyethylamine was added and the mixture was heated at 80 C. LC-MS analysis indicated clean conversion after 1 hour. The mixture was diluted with 15 mL of water and extracted with three 3 mL portions of ethyl acetate. The organics were concentrated to give 62 mg of a clear film which was purified by flash chromatography (25 mL silica gel, eluting with 25% and then 50% ethyl acetate / hexanes followed by neat ethyl acetate) to return 49.2 mg (67%) of methyl 2-(2-ethoxyethyl)-3-oxoisoindoline-4-carboxylate (C) as a clear film ¹H NMR (500 MHz, Chloroform-d) δ 7.55 – 7.45 (m, 3H), 4.49 (s, 2H), 3.93 (s, 3H), 3.71 (t, J = 5.1 Hz, 2H), 3.60 (t, J = 5.1 Hz, 2H), 3.42 (q, J = 7.0 Hz, 2H), 1.12 (t, J = 7.0 Hz, 3H). MS (ESI+ve): Calculated for C₁₄H₁₈NO₄, [M+H] = 264.12, observed [M+H] = 264.11.

D: The ester intermediate C (49 mg, 0.19 mmol) was dissolved in 3 mL of tetrahyrofuran and treated with 1 mL of 0.5 M aqueous lithium hydroxide. After 40 hours, the mixture was diluted with 5 mL water, acidified with 1 mL 1N aqueous hydrochloric acid, and extracted thrice (4 mL, 2 mL, 2 mL) with chloroform. The extracts were filtered through magnesium sulfate and concentrated to give 129 mg of crude white solid product, which was triturated with chloroform. Concentration of the solution phase returned 50.4 mg (ca. 100%) of 2-(2-ethoxyethyl)-3-oxoisoindoline-4-carboxylic acid (D) as a white solid. ¹H NMR (500 MHz, Chloroform-d) δ 15.83 (s, 1H), 8.44 – 8.26 (m, 1H), 7.77 – 7.54 (m, 2H), 4.65 (s, 2H), 3.81 (t, J = 5.1 Hz, 2H), 3.65 (t, J = 5.1 Hz, 2H), 3.45 (q, J = 7.0 Hz, 2H), 1.13 (t, J = 7.0 Hz, 3H). MS (ESI+ve): Calculated for C₁₃H₁₆NO₄, [M+H] = 250.10, observed [M+H] = 250.09.

ML377: The acid D (49 mg, 0.20 mmol) and the solids ethyl dimethylaminopropylcarbodiimide hydrochloride (94 mg, 0.49 mmol) and 1-hydroxybenzotriazole (33 mg, 0.22 mmol) were charged with 1 mL of N,N-dimethylformamide. The mixture was treated with triethylamine (0.16 mL, 1.18 mmol) and stirred at room temperature for 15 minutes before adding solid 3-(1H-benzo[d]imidazol-2-yl)aniline (CAS #7596-74-9, 41 mg, 0.20 mmol). After stirring for 18 hours, the mixture was diluted with 10 mL water. After standing for 4 hours during which a white solid settled, the supernatant was drawn off. The residue was transferred with 33% methanol / chloroform. Concentration gave 85.4 mg of crude solid product, which was purified by flash chromatography (25 mL silica gel, eluting with 50% ethyl acetate / hexanes followed by neat ethyl acetate). The purified material was taken up in 4 mL of 75% acetonitrile / water and lyophilized to yield 64.5 mg of N-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-2-(2-ethoxyethyl)-3-oxoisoindoline-4-carboxamide (ML377) as a white solid. ¹H NMR (500 MHz, DMSO-d6) δ 13.70 (s, 1H), 12.99 (br. s, 1H), 8.65 (br. s, 1H), 8.37 (d, J = 7.5 Hz, 1H), 8.05 – 7.80 (m, 4H), 7.60 (m, 3H), 7.28 – 7.20 (m, 2H), 4.75 (s, 2H), 3.87 (t, J = 5.4 Hz, 2H), 3.72 (t, J = 5.4 Hz, 2H), 3.52 (q, J = 7.0 Hz, 2H), 1.14 (t, J = 7.0 Hz, 3H). ¹³C NMR (126 MHz, DMSO) δ 168.97, 162.74, 151.54, 144.34, 140.31, 132.35, 132.19, 131.40, 131.05, 130.04, 128.79, 127.25, 122.16, 121.73, 118.36, 68.11, 65.96, 51.40, 43.07, 15.57. HRMS (ESI+ve): Calculated for C₂₆H₂₅N₄O₃, [M+H] = 441.1882, observed [M+H] = 441.1928.

¹H NMR Spectrum of ML377 (500 MHz, DMSO-d₆)

¹³C NMR Spectrum of ML377 (126 MHz, DMSO-d₆)

LC/MS for ML377

MS spectrum for M377

3. Results

3.1. Dose Response Curves for Probe

ML377 did not inhibit cell viability of H9c2 cells as determined by ATP levels and DAPI nuclei staining (Figure 3) while it was was efficacious for inhibition of TAG accumulation in both rat H9c2 skeletal in human primary myotube cells.

Figure 3

Potency of ML377 inhibition for triacylglycerol accumulation in both H9c2 cells and differentiated human skeletal myotubes. Error bars are standard deviation of triplicate runs tested on at least two separate days.

3.2. Cellular Activity

The differential dose responsiveness for inhibition of TAG accumulation in H9c2 and human primary cells while not affecting H9c2 viability are consistent with the ability of ML377 to penetrate cell membranes. Additional profiling indicated ML377 was not toxic to human hepatocytes at >50 μM (Table 4).

Table 4

Summary of in vitro ADME Properties of TAG inhibitor probe ML377.

4. Discussion

5. References

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Morino K, Petersen KF, Shulman GI. Molecular mechanisms of insulin resistance in humans and their potential links with mitochondrial dysfunction. Diabetes. 2006;55 Suppl 2:S9–S15. [PMC free article: PMC2995546] [PubMed: 17130651]

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