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Inhibition of tumor growth by a newly-identified activator for epidermal fatty acid binding protein.

Rao E, Singh P, Zhai X, Li Y, Zhu G, Zhang Y, Hao J, Chi YI, Brown RE, Cleary MP, Li B - Oncotarget (2015)

Bottom Line: Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation.Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages.Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
Our previous studies have demonstrated that expression of epidermal fatty acid binding protein (E-FABP) in tumor associated macrophages (TAMs) promotes macrophage anti-tumor activity by enhancing IFNβ responses in tumor models. Thus, E-FABP represents a new protective factor in enhancing tumor immune surveillance against tumor development. Herein, we report the compound 5-(benzylamino)-2-(3-methylphenyl)-1,3-oxazole-4-carbonitrile (designated EI-05) as a novel E-FABP activator for inhibition of mammary tumor growth. EI-05 was selected from the ZINC compound library using molecular docking analysis based on the crystal structure of E-FABP. Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation. Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages. Importantly, administering EI-05 in vivo significantly inhibits mammary tumor growth in a syngeneic mouse model. Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

No MeSH data available.


Related in: MedlinePlus

In-silico screening of EI-05(A) Chemical structure of EI-05 (ZINC00467342) (B) The predicted model of EI-05 binding to the lipid-binding pocket of E-FABP. (C) Normalized melting curves depicting enhanced thermal stability of E-FABP by BMS309413 (blue dashed line), but not by EI-05 (orange and purple solid lines). (D) Excitation and emission spectra of EI-05 solved in methanol. (E) Tyr/Trp emission spectra of E-FABP (0.5 μM) in the 300-500 nm range were measured by step-wise addition of indicated concentrations of EI-05. The Try/Trp emission signal between 300-347 nm (dashed box) is enlarged in the right panel. (F) The emission spectra of indicated concentrations of EI-05 in the absence of E-FABP. (G) Tyr/Trp emission of E-FABP (0.5 μM) in the 300-500 nm range was measured by addition of indicated concentrations of BMS309413. Excitation at 270 nm was used for experiments shown in panels E, F and G.
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Figure 1: In-silico screening of EI-05(A) Chemical structure of EI-05 (ZINC00467342) (B) The predicted model of EI-05 binding to the lipid-binding pocket of E-FABP. (C) Normalized melting curves depicting enhanced thermal stability of E-FABP by BMS309413 (blue dashed line), but not by EI-05 (orange and purple solid lines). (D) Excitation and emission spectra of EI-05 solved in methanol. (E) Tyr/Trp emission spectra of E-FABP (0.5 μM) in the 300-500 nm range were measured by step-wise addition of indicated concentrations of EI-05. The Try/Trp emission signal between 300-347 nm (dashed box) is enlarged in the right panel. (F) The emission spectra of indicated concentrations of EI-05 in the absence of E-FABP. (G) Tyr/Trp emission of E-FABP (0.5 μM) in the 300-500 nm range was measured by addition of indicated concentrations of BMS309413. Excitation at 270 nm was used for experiments shown in panels E, F and G.

Mentions: As identification of small molecule inhibitors of FABP has been proven to be effective in treating atherosclerosis and type II diabetes in mouse models [14], we initially intended to screen small molecules that might specifically bind to the lipid-binding pocket of E-FABP using computational docking analysis [15]. Based on the crystal structure of E-FABP (PDB ID 1B56), EI-05 (Figure 1A) was selected as a potential E-FABP partner due to the predicted binding of E-FABP/EI-05 complex shown by molecular docking modeling (Figure 1B). However, when we purified recombinant E-FABP proteins and conducted in vitro binding assays, we found that E-FABP did not bind to EI-05, despite good binding to the known inhibitor BMS309403 [16], in thermal shift assays (Figure 1C). As EI-05 exhibited a relatively low excitation signal at 270 nm (Figure 1D), which enabled this wavelength to be used to excite Tyr and Trp in E-FABP, we evaluated the binding of E-FABP/EI-05 by Förster resonance energy transfer based on the spectral overlap of the E-FABP Tyr/Trp emission and EI-05 excitation signals. Step-wise addition of EI-05 to E-FABP did not affect the emission of E-FABP Tyr/Trp (Figure 1E). The strong stepwise increases in EI-05 emission signal at 394 nm, shown in Figure 1E, were nearly the same in the absence of E-FABP (Figure 1F), consistent with no energy transfer. In contrast, positive controls performed with BMS309403 showed the expected dose-dependent increase of E-FABP Tyr/Trp emission (Figure 1G). Thus, although predicted to bind E-FABP by computational modeling, our in vitro binding assays clearly indicate that EI-05 has no direct binding to E-FABP.


Inhibition of tumor growth by a newly-identified activator for epidermal fatty acid binding protein.

Rao E, Singh P, Zhai X, Li Y, Zhu G, Zhang Y, Hao J, Chi YI, Brown RE, Cleary MP, Li B - Oncotarget (2015)

In-silico screening of EI-05(A) Chemical structure of EI-05 (ZINC00467342) (B) The predicted model of EI-05 binding to the lipid-binding pocket of E-FABP. (C) Normalized melting curves depicting enhanced thermal stability of E-FABP by BMS309413 (blue dashed line), but not by EI-05 (orange and purple solid lines). (D) Excitation and emission spectra of EI-05 solved in methanol. (E) Tyr/Trp emission spectra of E-FABP (0.5 μM) in the 300-500 nm range were measured by step-wise addition of indicated concentrations of EI-05. The Try/Trp emission signal between 300-347 nm (dashed box) is enlarged in the right panel. (F) The emission spectra of indicated concentrations of EI-05 in the absence of E-FABP. (G) Tyr/Trp emission of E-FABP (0.5 μM) in the 300-500 nm range was measured by addition of indicated concentrations of BMS309413. Excitation at 270 nm was used for experiments shown in panels E, F and G.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480718&req=5

Figure 1: In-silico screening of EI-05(A) Chemical structure of EI-05 (ZINC00467342) (B) The predicted model of EI-05 binding to the lipid-binding pocket of E-FABP. (C) Normalized melting curves depicting enhanced thermal stability of E-FABP by BMS309413 (blue dashed line), but not by EI-05 (orange and purple solid lines). (D) Excitation and emission spectra of EI-05 solved in methanol. (E) Tyr/Trp emission spectra of E-FABP (0.5 μM) in the 300-500 nm range were measured by step-wise addition of indicated concentrations of EI-05. The Try/Trp emission signal between 300-347 nm (dashed box) is enlarged in the right panel. (F) The emission spectra of indicated concentrations of EI-05 in the absence of E-FABP. (G) Tyr/Trp emission of E-FABP (0.5 μM) in the 300-500 nm range was measured by addition of indicated concentrations of BMS309413. Excitation at 270 nm was used for experiments shown in panels E, F and G.
Mentions: As identification of small molecule inhibitors of FABP has been proven to be effective in treating atherosclerosis and type II diabetes in mouse models [14], we initially intended to screen small molecules that might specifically bind to the lipid-binding pocket of E-FABP using computational docking analysis [15]. Based on the crystal structure of E-FABP (PDB ID 1B56), EI-05 (Figure 1A) was selected as a potential E-FABP partner due to the predicted binding of E-FABP/EI-05 complex shown by molecular docking modeling (Figure 1B). However, when we purified recombinant E-FABP proteins and conducted in vitro binding assays, we found that E-FABP did not bind to EI-05, despite good binding to the known inhibitor BMS309403 [16], in thermal shift assays (Figure 1C). As EI-05 exhibited a relatively low excitation signal at 270 nm (Figure 1D), which enabled this wavelength to be used to excite Tyr and Trp in E-FABP, we evaluated the binding of E-FABP/EI-05 by Förster resonance energy transfer based on the spectral overlap of the E-FABP Tyr/Trp emission and EI-05 excitation signals. Step-wise addition of EI-05 to E-FABP did not affect the emission of E-FABP Tyr/Trp (Figure 1E). The strong stepwise increases in EI-05 emission signal at 394 nm, shown in Figure 1E, were nearly the same in the absence of E-FABP (Figure 1F), consistent with no energy transfer. In contrast, positive controls performed with BMS309403 showed the expected dose-dependent increase of E-FABP Tyr/Trp emission (Figure 1G). Thus, although predicted to bind E-FABP by computational modeling, our in vitro binding assays clearly indicate that EI-05 has no direct binding to E-FABP.

Bottom Line: Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation.Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages.Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

View Article: PubMed Central - PubMed

Affiliation: The Hormel Institute, University of Minnesota, Austin, MN, USA.

ABSTRACT
Our previous studies have demonstrated that expression of epidermal fatty acid binding protein (E-FABP) in tumor associated macrophages (TAMs) promotes macrophage anti-tumor activity by enhancing IFNβ responses in tumor models. Thus, E-FABP represents a new protective factor in enhancing tumor immune surveillance against tumor development. Herein, we report the compound 5-(benzylamino)-2-(3-methylphenyl)-1,3-oxazole-4-carbonitrile (designated EI-05) as a novel E-FABP activator for inhibition of mammary tumor growth. EI-05 was selected from the ZINC compound library using molecular docking analysis based on the crystal structure of E-FABP. Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation. Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNβ production, which further promotes the anti-tumor activity of macrophages. Importantly, administering EI-05 in vivo significantly inhibits mammary tumor growth in a syngeneic mouse model. Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNβ responses in macrophages.

No MeSH data available.


Related in: MedlinePlus