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Disruption of Myc-Max heterodimerization with improved cell-penetrating analogs of the small molecule 10074-G5.

Wang H, Chauhan J, Hu A, Pendleton K, Yap JL, Sabato PE, Jones JW, Perri M, Yu J, Cione E, Kane MA, Fletcher S, Prochownik EV - Oncotarget (2013)

Bottom Line: Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc.Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association.Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.

View Article: PubMed Central - PubMed

Affiliation: Section of Hematology/Oncology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.

ABSTRACT
The c-Myc (Myc) oncoprotein is a high-value therapeutic target given that it is deregulated in multiple types of cancer. However, potent small molecule inhibitors of Myc have been difficult to identify, particularly those whose mechanism relies on blocking the association between Myc and its obligate heterodimerization partner, Max. We have recently reported a structure-activity relationship study of one such small molecule, 10074-G5, and generated an analog, JY-3-094, with significantly improved ability to prevent or disrupt the association between recombinant Myc and Max proteins. However, JY-3094 penetrates cells poorly. Here, we show that esterification of a critical para-carboxylic acid function of JY-3-094 by various blocking groups significantly improves cellular uptake although it impairs the ability to disrupt Myc-Max association in vitro. These pro-drugs are highly concentrated within cells where JY-3-094 is then generated by the action of esterases. However, the pro-drugs are also variably susceptible to extracellular esterases, which can deplete extracellular reservoirs. Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc. Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association. Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.

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Related in: MedlinePlus

LC-MS/MS quantification of intracellular prodrug and metabolite in HL-60 cellsCells were cultured in the presence of 10 μM prodrug for 72 h. Intracellular prodrug and metabolite (JY-3-094) levels were then quantified by LC-MS/MS. All data are expressed as mean ± standard deviation with N=3 for each prodrug treatment. ND = not detectable.
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Figure 4: LC-MS/MS quantification of intracellular prodrug and metabolite in HL-60 cellsCells were cultured in the presence of 10 μM prodrug for 72 h. Intracellular prodrug and metabolite (JY-3-094) levels were then quantified by LC-MS/MS. All data are expressed as mean ± standard deviation with N=3 for each prodrug treatment. ND = not detectable.

Mentions: Based on the foregoing findings, we asked if we could simultaneously detect intracellular Group B prodrugs and JY-3-094, the common product of esterase-mediated hydrolysis. HL-60 cells were therefore cultured in the presence of 10 μM of each prodrug for 72 h; its intracellular concentrations and that of JY-3-094 were then quantified by LC-MS/MS (Fig. 4). Since our aim was to evaluate the extent of ester hydrolysis of each prodrug to active JY-3-094, we did not search for other metabolites. Given that different prodrugs may be internalized and cleared at different rates, this approach also did not afford the opportunity to correlate total amounts of prodrug with total uptake. Exposure to 10 μM of the acetoxymethyl ester 3JC-91-5 afforded 9.5 μM intracellular concentration of metabolite (JY-3-094) with no detectable 3JC-91-5, suggesting 95% uptake of the prodrug, its complete conversion to JY-3-094 and its long-term intracellular retention in unmodified form. Similarly high rates of conversion were seen with methyl and ethyl esters (3JC-91-1 and 3JC-91-3, respectively), followed closely by phenol ester SF-4-017 and then trifluoroethyl ester 3JC-91-7. Similar degrees of conversion were observed when the above studies were repeated in MDA-MB-231 breast cancer cells (Supplementary Fig. S8).


Disruption of Myc-Max heterodimerization with improved cell-penetrating analogs of the small molecule 10074-G5.

Wang H, Chauhan J, Hu A, Pendleton K, Yap JL, Sabato PE, Jones JW, Perri M, Yu J, Cione E, Kane MA, Fletcher S, Prochownik EV - Oncotarget (2013)

LC-MS/MS quantification of intracellular prodrug and metabolite in HL-60 cellsCells were cultured in the presence of 10 μM prodrug for 72 h. Intracellular prodrug and metabolite (JY-3-094) levels were then quantified by LC-MS/MS. All data are expressed as mean ± standard deviation with N=3 for each prodrug treatment. ND = not detectable.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3757250&req=5

Figure 4: LC-MS/MS quantification of intracellular prodrug and metabolite in HL-60 cellsCells were cultured in the presence of 10 μM prodrug for 72 h. Intracellular prodrug and metabolite (JY-3-094) levels were then quantified by LC-MS/MS. All data are expressed as mean ± standard deviation with N=3 for each prodrug treatment. ND = not detectable.
Mentions: Based on the foregoing findings, we asked if we could simultaneously detect intracellular Group B prodrugs and JY-3-094, the common product of esterase-mediated hydrolysis. HL-60 cells were therefore cultured in the presence of 10 μM of each prodrug for 72 h; its intracellular concentrations and that of JY-3-094 were then quantified by LC-MS/MS (Fig. 4). Since our aim was to evaluate the extent of ester hydrolysis of each prodrug to active JY-3-094, we did not search for other metabolites. Given that different prodrugs may be internalized and cleared at different rates, this approach also did not afford the opportunity to correlate total amounts of prodrug with total uptake. Exposure to 10 μM of the acetoxymethyl ester 3JC-91-5 afforded 9.5 μM intracellular concentration of metabolite (JY-3-094) with no detectable 3JC-91-5, suggesting 95% uptake of the prodrug, its complete conversion to JY-3-094 and its long-term intracellular retention in unmodified form. Similarly high rates of conversion were seen with methyl and ethyl esters (3JC-91-1 and 3JC-91-3, respectively), followed closely by phenol ester SF-4-017 and then trifluoroethyl ester 3JC-91-7. Similar degrees of conversion were observed when the above studies were repeated in MDA-MB-231 breast cancer cells (Supplementary Fig. S8).

Bottom Line: Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc.Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association.Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.

View Article: PubMed Central - PubMed

Affiliation: Section of Hematology/Oncology, Children's Hospital of Pittsburgh of UPMC, Pittsburgh, PA, USA.

ABSTRACT
The c-Myc (Myc) oncoprotein is a high-value therapeutic target given that it is deregulated in multiple types of cancer. However, potent small molecule inhibitors of Myc have been difficult to identify, particularly those whose mechanism relies on blocking the association between Myc and its obligate heterodimerization partner, Max. We have recently reported a structure-activity relationship study of one such small molecule, 10074-G5, and generated an analog, JY-3-094, with significantly improved ability to prevent or disrupt the association between recombinant Myc and Max proteins. However, JY-3094 penetrates cells poorly. Here, we show that esterification of a critical para-carboxylic acid function of JY-3-094 by various blocking groups significantly improves cellular uptake although it impairs the ability to disrupt Myc-Max association in vitro. These pro-drugs are highly concentrated within cells where JY-3-094 is then generated by the action of esterases. However, the pro-drugs are also variably susceptible to extracellular esterases, which can deplete extracellular reservoirs. Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc. Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association. Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.

Show MeSH
Related in: MedlinePlus