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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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A-D, Properties of JY-3-094A, Structure of the 10074-G5 analog JY-3-094. B, Representative EMSA performed with Myc-Max(S) heterodimers [23,28]. C, An identical EMSA performed with Max(L) homodimers. D, Quantification of EMSA results. Triplicate gels from B and C were scanned as previously described [23]. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) (panel B) and Max(L) EMSAs (panel C) are graphed. E-H, Properties of SF-3-103B. E, Structure of SF-3-103B. F, Representative EMSA results performed with Myc-Max(S) heterodimers. G, Representative EMSA performed with Max(L) homodimers. H, Quantification of EMSAs. Triplicate gels were scanned and binding was quantified as described for D. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) and Max(L) EMSAs are graphed. I-L, Properties of SF-4-017. I, Structure of SF-4-017. J, Representative EMSA results performed with Myc-Max(S) heterodimers. K, Representative EMSA performed with Max(L) homodimers. L, Quantification of EMSAs performed as described for D and H.
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Figure 1: A-D, Properties of JY-3-094A, Structure of the 10074-G5 analog JY-3-094. B, Representative EMSA performed with Myc-Max(S) heterodimers [23,28]. C, An identical EMSA performed with Max(L) homodimers. D, Quantification of EMSA results. Triplicate gels from B and C were scanned as previously described [23]. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) (panel B) and Max(L) EMSAs (panel C) are graphed. E-H, Properties of SF-3-103B. E, Structure of SF-3-103B. F, Representative EMSA results performed with Myc-Max(S) heterodimers. G, Representative EMSA performed with Max(L) homodimers. H, Quantification of EMSAs. Triplicate gels were scanned and binding was quantified as described for D. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) and Max(L) EMSAs are graphed. I-L, Properties of SF-4-017. I, Structure of SF-4-017. J, Representative EMSA results performed with Myc-Max(S) heterodimers. K, Representative EMSA performed with Max(L) homodimers. L, Quantification of EMSAs performed as described for D and H.

Mentions: Unlike the case of analogs of the Myc inhibitor 10058-F4 [21], a substantial fraction of which retain the ability to disrupt Myc-Max heterodimers [23], only 2 of 24 tested 10074-G5 analogs, which we refer to as “Group A” compounds, did so (Supplementary Fig. S1 and ref. 28). IC50s for these compounds (JY-3-094 and SF-3-103B), determined in Myc-Max(S) EMSAs were 4.4- and 1.7-fold lower, respectively, than for 10074-G5 (Fig. 1 A–H and Supplementary Fig. S5). The specificity of JY-3-094 and SF-3-103B for Myc-Max(S) heterodimers was also demonstrated by showing that neither compound appreciably affected DNA binding by Max(L) homodimers (IC50s >100 μM) whose free energy of association is actually less than that of Myc-Max heterodimers [38]. Despite these improvements over the parental 10074-G5 compound neither analog showed appreciable activity against HL60 promyelocytic leukemia or Daudi Burkitt lymphoma cells in a standard proliferation assay (IC50s >50 μM, Table 1) [21, 23]. These results indicated that the requirements for 10074-G5 binding to Myc are more stringent than those for 10058-F4 and that further modification was needed to optimize cell-based activities. We reasoned that the ionizable carboxylic acid groups of JY-3-094 and SF-3-103B might be responsible for impeding cell entry.


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)

A-D, Properties of JY-3-094A, Structure of the 10074-G5 analog JY-3-094. B, Representative EMSA performed with Myc-Max(S) heterodimers [23,28]. C, An identical EMSA performed with Max(L) homodimers. D, Quantification of EMSA results. Triplicate gels from B and C were scanned as previously described [23]. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) (panel B) and Max(L) EMSAs (panel C) are graphed. E-H, Properties of SF-3-103B. E, Structure of SF-3-103B. F, Representative EMSA results performed with Myc-Max(S) heterodimers. G, Representative EMSA performed with Max(L) homodimers. H, Quantification of EMSAs. Triplicate gels were scanned and binding was quantified as described for D. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) and Max(L) EMSAs are graphed. I-L, Properties of SF-4-017. I, Structure of SF-4-017. J, Representative EMSA results performed with Myc-Max(S) heterodimers. K, Representative EMSA performed with Max(L) homodimers. L, Quantification of EMSAs performed as described for D and H.
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Related In: Results  -  Collection

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Show All Figures
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Figure 1: A-D, Properties of JY-3-094A, Structure of the 10074-G5 analog JY-3-094. B, Representative EMSA performed with Myc-Max(S) heterodimers [23,28]. C, An identical EMSA performed with Max(L) homodimers. D, Quantification of EMSA results. Triplicate gels from B and C were scanned as previously described [23]. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) (panel B) and Max(L) EMSAs (panel C) are graphed. E-H, Properties of SF-3-103B. E, Structure of SF-3-103B. F, Representative EMSA results performed with Myc-Max(S) heterodimers. G, Representative EMSA performed with Max(L) homodimers. H, Quantification of EMSAs. Triplicate gels were scanned and binding was quantified as described for D. The mean values obtained at each compound concentration ± 1 S.E. for both Myc-Max(S) and Max(L) EMSAs are graphed. I-L, Properties of SF-4-017. I, Structure of SF-4-017. J, Representative EMSA results performed with Myc-Max(S) heterodimers. K, Representative EMSA performed with Max(L) homodimers. L, Quantification of EMSAs performed as described for D and H.
Mentions: Unlike the case of analogs of the Myc inhibitor 10058-F4 [21], a substantial fraction of which retain the ability to disrupt Myc-Max heterodimers [23], only 2 of 24 tested 10074-G5 analogs, which we refer to as “Group A” compounds, did so (Supplementary Fig. S1 and ref. 28). IC50s for these compounds (JY-3-094 and SF-3-103B), determined in Myc-Max(S) EMSAs were 4.4- and 1.7-fold lower, respectively, than for 10074-G5 (Fig. 1 A–H and Supplementary Fig. S5). The specificity of JY-3-094 and SF-3-103B for Myc-Max(S) heterodimers was also demonstrated by showing that neither compound appreciably affected DNA binding by Max(L) homodimers (IC50s >100 μM) whose free energy of association is actually less than that of Myc-Max heterodimers [38]. Despite these improvements over the parental 10074-G5 compound neither analog showed appreciable activity against HL60 promyelocytic leukemia or Daudi Burkitt lymphoma cells in a standard proliferation assay (IC50s >50 μM, Table 1) [21, 23]. These results indicated that the requirements for 10074-G5 binding to Myc are more stringent than those for 10058-F4 and that further modification was needed to optimize cell-based activities. We reasoned that the ionizable carboxylic acid groups of JY-3-094 and SF-3-103B might be responsible for impeding cell entry.

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