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Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Bol GM, Vesuna F, Xie M, Zeng J, Aziz K, Gandhi N, Levine A, Irving A, Korz D, Tantravedi S, Heerma van Voss MR, Gabrielson K, Bordt EA, Polster BM, Cope L, van der Groep P, Kondaskar A, Rudek MA, Hosmane RS, van der Wall E, van Diest PJ, Tran PT, Raman V - EMBO Mol Med (2015)

Bottom Line: We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity.Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells.Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus

Specific binding of RK-33 to DDX3 and induction of radiosensitization in lung cancer cell linesA Chemical structure of RK-33.B A predicted molecular model of RK-33 docked into the ATP-binding cleft of DDX3. RK-33 is displayed in pink, the surface of the DEADc domain is in green, and the surface of the HELICc domain is in red.C Hydrogen bond interactions between RK-33 and DDX3. Alpha helices are displayed in green, and β-sheets are shown in maroon.D, E Chemical structures of biotin-linked RK-33 at R3 position with ethylene amine linker (AK-298) and biotin-labeled RK-33 at R2 position with (PEG)2 ethylene amide linker (SK-153). The two structural differences of AK-298 and SK-153 are the length of the biotin linker and attachment position at RK-33.F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.G Schematic representation of helicase assay.H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4–6).I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.Source data are available online for this figure.
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fig02: Specific binding of RK-33 to DDX3 and induction of radiosensitization in lung cancer cell linesA Chemical structure of RK-33.B A predicted molecular model of RK-33 docked into the ATP-binding cleft of DDX3. RK-33 is displayed in pink, the surface of the DEADc domain is in green, and the surface of the HELICc domain is in red.C Hydrogen bond interactions between RK-33 and DDX3. Alpha helices are displayed in green, and β-sheets are shown in maroon.D, E Chemical structures of biotin-linked RK-33 at R3 position with ethylene amine linker (AK-298) and biotin-labeled RK-33 at R2 position with (PEG)2 ethylene amide linker (SK-153). The two structural differences of AK-298 and SK-153 are the length of the biotin linker and attachment position at RK-33.F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.G Schematic representation of helicase assay.H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4–6).I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.Source data are available online for this figure.

Mentions: Based on the role of DDX3 in proliferation and as a potential marker of aggressive cancer, we rationally designed small molecules to bind specifically to the ATP-binding cleft of DDX3 (Kondaskar et al, 2010). We identified a fused diimidazodiazepine molecule (RK-33; Fig2A) that exhibited promising cell death kinetics and has a computed binding affinity of −8 kcal/mol between RK-33 and DDX3 (Fig2B and C). To evaluate binding of RK-33 to DDX3, we synthesized two biotinylated RK-33 molecules (Fig2D and E) and demonstrated that RK-33 binds specifically to DDX3, but not to the closely related proteins DDX5 and DDX17 (Fig2F).


Targeting DDX3 with a small molecule inhibitor for lung cancer therapy.

Bol GM, Vesuna F, Xie M, Zeng J, Aziz K, Gandhi N, Levine A, Irving A, Korz D, Tantravedi S, Heerma van Voss MR, Gabrielson K, Bordt EA, Polster BM, Cope L, van der Groep P, Kondaskar A, Rudek MA, Hosmane RS, van der Wall E, van Diest PJ, Tran PT, Raman V - EMBO Mol Med (2015)

Specific binding of RK-33 to DDX3 and induction of radiosensitization in lung cancer cell linesA Chemical structure of RK-33.B A predicted molecular model of RK-33 docked into the ATP-binding cleft of DDX3. RK-33 is displayed in pink, the surface of the DEADc domain is in green, and the surface of the HELICc domain is in red.C Hydrogen bond interactions between RK-33 and DDX3. Alpha helices are displayed in green, and β-sheets are shown in maroon.D, E Chemical structures of biotin-linked RK-33 at R3 position with ethylene amine linker (AK-298) and biotin-labeled RK-33 at R2 position with (PEG)2 ethylene amide linker (SK-153). The two structural differences of AK-298 and SK-153 are the length of the biotin linker and attachment position at RK-33.F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.G Schematic representation of helicase assay.H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4–6).I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.Source data are available online for this figure.
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Related In: Results  -  Collection

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fig02: Specific binding of RK-33 to DDX3 and induction of radiosensitization in lung cancer cell linesA Chemical structure of RK-33.B A predicted molecular model of RK-33 docked into the ATP-binding cleft of DDX3. RK-33 is displayed in pink, the surface of the DEADc domain is in green, and the surface of the HELICc domain is in red.C Hydrogen bond interactions between RK-33 and DDX3. Alpha helices are displayed in green, and β-sheets are shown in maroon.D, E Chemical structures of biotin-linked RK-33 at R3 position with ethylene amine linker (AK-298) and biotin-labeled RK-33 at R2 position with (PEG)2 ethylene amide linker (SK-153). The two structural differences of AK-298 and SK-153 are the length of the biotin linker and attachment position at RK-33.F Immunoblots of pull-down assay of DDX3 with biotin, AK-298, and SK-153. Lower panels display results using DDX5 and DDX17 antibodies.G Schematic representation of helicase assay.H Immunoblot displaying increasing concentrations of RK-33 (50, 100, 200 nM) resulting in increased inhibition of unwinding of oligomer products (lanes 4–6).I MTS viability assay of various lung cancer cell lines treated with RK-33 for 72 h. Mean from 3 replicates with SD.J, K Colony-forming assay of A549 and H3255 cells treated with RK-33 and with various doses of radiation 4 h later. Curves were fitted with a quadratic polynomial equation. Mean from 2 replicates with SD. P-values were determined by the extra sum of squares F-test.Source data are available online for this figure.
Mentions: Based on the role of DDX3 in proliferation and as a potential marker of aggressive cancer, we rationally designed small molecules to bind specifically to the ATP-binding cleft of DDX3 (Kondaskar et al, 2010). We identified a fused diimidazodiazepine molecule (RK-33; Fig2A) that exhibited promising cell death kinetics and has a computed binding affinity of −8 kcal/mol between RK-33 and DDX3 (Fig2B and C). To evaluate binding of RK-33 to DDX3, we synthesized two biotinylated RK-33 molecules (Fig2D and E) and demonstrated that RK-33 binds specifically to DDX3, but not to the closely related proteins DDX5 and DDX17 (Fig2F).

Bottom Line: We designed a first-in-class small molecule inhibitor, RK-33, which binds to DDX3 and abrogates its activity.Mechanistically, loss of DDX3 function either by shRNA or by RK-33 impaired Wnt signaling through disruption of the DDX3-β-catenin axis and inhibited non-homologous end joining-the major DNA repair pathway in mammalian somatic cells.Overall, inhibition of DDX3 by RK-33 promotes tumor regression, thus providing a compelling argument to develop DDX3 inhibitors for lung cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiology and Radiological Science, Johns Hopkins University School of Medicine, Baltimore, MD, USA Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands.

No MeSH data available.


Related in: MedlinePlus