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Targeted delivery of ubiquitin-conjugated BH3 peptide-based Mcl-1 inhibitors into cancer cells.

Muppidi A, Doi K, Edwardraja S, Pulavarti SV, Szyperski T, Wang HG, Lin Q - Bioconjug. Chem. (2014)

Bottom Line: J., Gulick, A.Chem.Soc. 134 , 14734 ).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, State University of New York at Buffalo , Buffalo, New York 14260-3000, United States.

ABSTRACT
BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 ( Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. ( 2012 ) J. Am. Chem. Soc. 134 , 14734 ). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents.

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(A) Reaction scheme for the Bpy-mediated cross-linkingof the NoxaBH3–ubiquitin conjugate. Bpy is rendered in a ball-and-stickmodel in the Bpy-cross-linked conjugate. (B,C) Deconvoluted massesof NoxaBH3–ubiquitin-Bpy (PU-Bpy) and analogous ubiquitin–NoxaBH3-Bpy(UP-Bpy).
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fig2: (A) Reaction scheme for the Bpy-mediated cross-linkingof the NoxaBH3–ubiquitin conjugate. Bpy is rendered in a ball-and-stickmodel in the Bpy-cross-linked conjugate. (B,C) Deconvoluted massesof NoxaBH3–ubiquitin-Bpy (PU-Bpy) and analogous ubiquitin–NoxaBH3-Bpy(UP-Bpy).

Mentions: In exploiting the ubiquitin/CXCR4 axisfor targeted delivery of the NoxaBH3 peptide-based Mcl-1 inhibitors,we had the following considerations: (1) the fusion of NoxaBH3 peptideto ubiquitin should not interfere with ubiquitin binding to CXCR4;and (2) the conjugation of ubiquitin to NoxaBH3 peptides should notnegatively affect NoxaBH3 binding to Mcl-1. Accordingly, we fusedthe NoxaBH3 peptide at either the N- or the C-terminus of ubiquitinseparated by a long flexible linker (GGGGS)3 (Figure 1A) to ensure that the two domains function properly.Thus, synthetic genes encoding the fusion proteins were inserted intothe pET28a expression vector, and the His-tagged ubiquitin–peptideconjugates, NoxaBH3 peptide–ubiquitin (PU) and ubiquitin–NoxaBH3peptide (UP), were purified to homogeneity with Ni-NTA-based affinitychromatography. SDS–PAGE and mass spectrometry analyses confirmedthe identity and high purity of the conjugates (Figure 1B–C). Since the internalized extracellular ubiquitinmay potentially be susceptible to polyubiquitination and subsequentproteasome-mediated degradation,15 we furthermutated ubiquitin lysine-48 and -63, two sites for ubiquitin chaingrowth,24 to arginines to obtain a mutantconjugate, NoxaBH3–ubiquitin-K48R/K63R (PU-KKmt). Since cross-linkingof the NoxaBH3 peptide with a biaryl derivative has been shown togreatly increase the inhibitory activity against Mcl-1,20 we also performed the cross-linking reactionwith the ubiquitin–peptide conjugates. Figure 2A shows the scheme for the cross-linking of the NoxaBH3–ubiquitinconjugate by Bpy to generate the Bpy-cross-linked NoxaBH3–ubiquitinconjugate (PU-Bpy). On the basis of mass spectrometry analysis, thereaction proceeded cleanly to give rise to PU-Bpy with a yield of87% (Figure 2B). Similarly, the cross-linkingreactions with UP (Figure 2C) and PU-KKmt gavethe corresponding Bpy-cross-linked products in 80% and 85% yield,respectively (Table 1).


Targeted delivery of ubiquitin-conjugated BH3 peptide-based Mcl-1 inhibitors into cancer cells.

Muppidi A, Doi K, Edwardraja S, Pulavarti SV, Szyperski T, Wang HG, Lin Q - Bioconjug. Chem. (2014)

(A) Reaction scheme for the Bpy-mediated cross-linkingof the NoxaBH3–ubiquitin conjugate. Bpy is rendered in a ball-and-stickmodel in the Bpy-cross-linked conjugate. (B,C) Deconvoluted massesof NoxaBH3–ubiquitin-Bpy (PU-Bpy) and analogous ubiquitin–NoxaBH3-Bpy(UP-Bpy).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3974624&req=5

fig2: (A) Reaction scheme for the Bpy-mediated cross-linkingof the NoxaBH3–ubiquitin conjugate. Bpy is rendered in a ball-and-stickmodel in the Bpy-cross-linked conjugate. (B,C) Deconvoluted massesof NoxaBH3–ubiquitin-Bpy (PU-Bpy) and analogous ubiquitin–NoxaBH3-Bpy(UP-Bpy).
Mentions: In exploiting the ubiquitin/CXCR4 axisfor targeted delivery of the NoxaBH3 peptide-based Mcl-1 inhibitors,we had the following considerations: (1) the fusion of NoxaBH3 peptideto ubiquitin should not interfere with ubiquitin binding to CXCR4;and (2) the conjugation of ubiquitin to NoxaBH3 peptides should notnegatively affect NoxaBH3 binding to Mcl-1. Accordingly, we fusedthe NoxaBH3 peptide at either the N- or the C-terminus of ubiquitinseparated by a long flexible linker (GGGGS)3 (Figure 1A) to ensure that the two domains function properly.Thus, synthetic genes encoding the fusion proteins were inserted intothe pET28a expression vector, and the His-tagged ubiquitin–peptideconjugates, NoxaBH3 peptide–ubiquitin (PU) and ubiquitin–NoxaBH3peptide (UP), were purified to homogeneity with Ni-NTA-based affinitychromatography. SDS–PAGE and mass spectrometry analyses confirmedthe identity and high purity of the conjugates (Figure 1B–C). Since the internalized extracellular ubiquitinmay potentially be susceptible to polyubiquitination and subsequentproteasome-mediated degradation,15 we furthermutated ubiquitin lysine-48 and -63, two sites for ubiquitin chaingrowth,24 to arginines to obtain a mutantconjugate, NoxaBH3–ubiquitin-K48R/K63R (PU-KKmt). Since cross-linkingof the NoxaBH3 peptide with a biaryl derivative has been shown togreatly increase the inhibitory activity against Mcl-1,20 we also performed the cross-linking reactionwith the ubiquitin–peptide conjugates. Figure 2A shows the scheme for the cross-linking of the NoxaBH3–ubiquitinconjugate by Bpy to generate the Bpy-cross-linked NoxaBH3–ubiquitinconjugate (PU-Bpy). On the basis of mass spectrometry analysis, thereaction proceeded cleanly to give rise to PU-Bpy with a yield of87% (Figure 2B). Similarly, the cross-linkingreactions with UP (Figure 2C) and PU-KKmt gavethe corresponding Bpy-cross-linked products in 80% and 85% yield,respectively (Table 1).

Bottom Line: J., Gulick, A.Chem.Soc. 134 , 14734 ).

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, State University of New York at Buffalo , Buffalo, New York 14260-3000, United States.

ABSTRACT
BH3 peptides are key mediators of apoptosis and have served as the lead structures for the development of anticancer therapeutics. Previously, we reported the application of a simple cysteine-based side chain cross-linking chemistry to NoxaBH3 peptides that led to the generation of the cross-linked NoxaBH3 peptides with increased cell permeability and higher inhibitory activity against Mcl-1 ( Muppidi, A., Doi, K., Edwardraja, S., Drake, E. J., Gulick, A. M., Wang, H.-G., Lin, Q. ( 2012 ) J. Am. Chem. Soc. 134 , 14734 ). To deliver cross-linked NoxaBH3 peptides selectively into cancer cells for enhanced efficacy and reduced systemic toxicity, here we report the conjugation of the NoxaBH3 peptides with the extracellular ubiquitin, a recently identified endogenous ligand for CXCR4, a chemokine receptor overexpressed in cancer cells. The resulting ubiquitin-NoxaBH3 peptide conjugates showed increased inhibitory activity against Mcl-1 and selective killing of the CXCR4-expressing cancer cells. The successful delivery of the NoxaBH3 peptides by ubiquitin into cancer cells suggests that the ubiquitin/CXCR4 axis may serve as a general route for the targeted delivery of anticancer agents.

Show MeSH
Related in: MedlinePlus