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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

Model for Group B compound activityEsterification of JY-3-094 to yield Group B compounds greatly promotes cellular uptake but eliminates Myc inhibitory activity. Group B compounds are preferentially concentrated in cells but are susceptible to esterases (grey arrows) at rates that are determined by the identity of the ester group and the amounts and specificities of the esterases. Group B compounds are also susceptible to extracellular serum esterases, with highly esterase-susceptible compounds such as 3JC-91-5 being particularly prone to conversion to JY-3-094 (Fig. 3 and Suppl. Fig. S7). Upon entering cells, Group B compounds may either remain free within the cytoplasm (f) or become localized in a speckled pattern (Fig. 3) that presumably reflects their binding to proteins (b). The rates at which ester hydrolysis of free and bound compounds occur need not be identical although they are depicted as being so here. The hydrolyzed product, JY-3-094, remains bound and unable to interact with Myc (thin arrows). It is not currently known how freely interchangeable the free and bound states are (?). That virtually all of a compound such as 91-5 is converted intracellularly to JY-3-094 yet is a relatively weaker inhibitor of proliferation (Table 1) supports the idea that the bound form of JY-3-094 remains unavailable for Myc interaction despite its prolonged intracellular half-life. In contrast, the more efficient inhibition of Myc-Max heterodimers and proliferation by compounds such as 3JC-91-1, 3JC-91-2 and 3JC-91-3, despite the lower total levels of JY-3-094 suggests that higher levels of the free form of the latter compound are maintained by virtue of the persistence of an extracellular reservoir of the former compounds. (*) indicates that the compound is fluorescent under aqueous conditions.
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Figure 5: Model for Group B compound activityEsterification of JY-3-094 to yield Group B compounds greatly promotes cellular uptake but eliminates Myc inhibitory activity. Group B compounds are preferentially concentrated in cells but are susceptible to esterases (grey arrows) at rates that are determined by the identity of the ester group and the amounts and specificities of the esterases. Group B compounds are also susceptible to extracellular serum esterases, with highly esterase-susceptible compounds such as 3JC-91-5 being particularly prone to conversion to JY-3-094 (Fig. 3 and Suppl. Fig. S7). Upon entering cells, Group B compounds may either remain free within the cytoplasm (f) or become localized in a speckled pattern (Fig. 3) that presumably reflects their binding to proteins (b). The rates at which ester hydrolysis of free and bound compounds occur need not be identical although they are depicted as being so here. The hydrolyzed product, JY-3-094, remains bound and unable to interact with Myc (thin arrows). It is not currently known how freely interchangeable the free and bound states are (?). That virtually all of a compound such as 91-5 is converted intracellularly to JY-3-094 yet is a relatively weaker inhibitor of proliferation (Table 1) supports the idea that the bound form of JY-3-094 remains unavailable for Myc interaction despite its prolonged intracellular half-life. In contrast, the more efficient inhibition of Myc-Max heterodimers and proliferation by compounds such as 3JC-91-1, 3JC-91-2 and 3JC-91-3, despite the lower total levels of JY-3-094 suggests that higher levels of the free form of the latter compound are maintained by virtue of the persistence of an extracellular reservoir of the former compounds. (*) indicates that the compound is fluorescent under aqueous conditions.

Mentions: The results presented here are most consistent with the model depicted in Fig. 5 in which Group B compounds are taken up and concentrated within cells or hydrolyzed extracellularly to JY-3-094. Whereas the former process occurs at similar initial rates in all cases (Fig. 3), hydrolytic rates differ and are dictated by the identity of the ester linkage. When hydrolytic rates are low, sufficient extracellular Group B compound persists so as to establish a longer-term equilibrium between freely interchangeable intra- and extracellular pools. Alternatively, when ester hydrolysis is rapid, as in the case of 3JC-91-5, the extracellular pool is rapidly depleted and reduced to a form (JY-3-094) that is both poorly taken up and rendered incapable of being visualized in its aqueous environment. Group B compounds are similarly susceptible to intracellular esterases, thus further contributing to the loss of signal. Group B compounds and JY-3-094 appear to partition between a diffuse or “free” cytoplasmic state and a more concentrated or “speckled” state, with Group B compounds appearing equally prone to hydrolysis in either state (Fig. 3 and Supplementary video 1). It seems reasonable to speculate that the diffusely distributed JY-3-094 represents the form that is the most available to inhibit Myc. The degree to which this is achieved likely reflects a complex combination of factors that includes the extent of Group B compound uptake, its level and length of persistence in the extracellular compartment, its rate of both extra- and intra-cellular hydrolysis to JY-3-094, its subcellular distribution and the extent to which JY-3-094 can effectively interact with Myc. Another factor, not examined here, includes the rate at which Group B compounds are differentially metabolized within cells to compounds other than JY-3-094.


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)

Model for Group B compound activityEsterification of JY-3-094 to yield Group B compounds greatly promotes cellular uptake but eliminates Myc inhibitory activity. Group B compounds are preferentially concentrated in cells but are susceptible to esterases (grey arrows) at rates that are determined by the identity of the ester group and the amounts and specificities of the esterases. Group B compounds are also susceptible to extracellular serum esterases, with highly esterase-susceptible compounds such as 3JC-91-5 being particularly prone to conversion to JY-3-094 (Fig. 3 and Suppl. Fig. S7). Upon entering cells, Group B compounds may either remain free within the cytoplasm (f) or become localized in a speckled pattern (Fig. 3) that presumably reflects their binding to proteins (b). The rates at which ester hydrolysis of free and bound compounds occur need not be identical although they are depicted as being so here. The hydrolyzed product, JY-3-094, remains bound and unable to interact with Myc (thin arrows). It is not currently known how freely interchangeable the free and bound states are (?). That virtually all of a compound such as 91-5 is converted intracellularly to JY-3-094 yet is a relatively weaker inhibitor of proliferation (Table 1) supports the idea that the bound form of JY-3-094 remains unavailable for Myc interaction despite its prolonged intracellular half-life. In contrast, the more efficient inhibition of Myc-Max heterodimers and proliferation by compounds such as 3JC-91-1, 3JC-91-2 and 3JC-91-3, despite the lower total levels of JY-3-094 suggests that higher levels of the free form of the latter compound are maintained by virtue of the persistence of an extracellular reservoir of the former compounds. (*) indicates that the compound is fluorescent under aqueous conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Model for Group B compound activityEsterification of JY-3-094 to yield Group B compounds greatly promotes cellular uptake but eliminates Myc inhibitory activity. Group B compounds are preferentially concentrated in cells but are susceptible to esterases (grey arrows) at rates that are determined by the identity of the ester group and the amounts and specificities of the esterases. Group B compounds are also susceptible to extracellular serum esterases, with highly esterase-susceptible compounds such as 3JC-91-5 being particularly prone to conversion to JY-3-094 (Fig. 3 and Suppl. Fig. S7). Upon entering cells, Group B compounds may either remain free within the cytoplasm (f) or become localized in a speckled pattern (Fig. 3) that presumably reflects their binding to proteins (b). The rates at which ester hydrolysis of free and bound compounds occur need not be identical although they are depicted as being so here. The hydrolyzed product, JY-3-094, remains bound and unable to interact with Myc (thin arrows). It is not currently known how freely interchangeable the free and bound states are (?). That virtually all of a compound such as 91-5 is converted intracellularly to JY-3-094 yet is a relatively weaker inhibitor of proliferation (Table 1) supports the idea that the bound form of JY-3-094 remains unavailable for Myc interaction despite its prolonged intracellular half-life. In contrast, the more efficient inhibition of Myc-Max heterodimers and proliferation by compounds such as 3JC-91-1, 3JC-91-2 and 3JC-91-3, despite the lower total levels of JY-3-094 suggests that higher levels of the free form of the latter compound are maintained by virtue of the persistence of an extracellular reservoir of the former compounds. (*) indicates that the compound is fluorescent under aqueous conditions.
Mentions: The results presented here are most consistent with the model depicted in Fig. 5 in which Group B compounds are taken up and concentrated within cells or hydrolyzed extracellularly to JY-3-094. Whereas the former process occurs at similar initial rates in all cases (Fig. 3), hydrolytic rates differ and are dictated by the identity of the ester linkage. When hydrolytic rates are low, sufficient extracellular Group B compound persists so as to establish a longer-term equilibrium between freely interchangeable intra- and extracellular pools. Alternatively, when ester hydrolysis is rapid, as in the case of 3JC-91-5, the extracellular pool is rapidly depleted and reduced to a form (JY-3-094) that is both poorly taken up and rendered incapable of being visualized in its aqueous environment. Group B compounds are similarly susceptible to intracellular esterases, thus further contributing to the loss of signal. Group B compounds and JY-3-094 appear to partition between a diffuse or “free” cytoplasmic state and a more concentrated or “speckled” state, with Group B compounds appearing equally prone to hydrolysis in either state (Fig. 3 and Supplementary video 1). It seems reasonable to speculate that the diffusely distributed JY-3-094 represents the form that is the most available to inhibit Myc. The degree to which this is achieved likely reflects a complex combination of factors that includes the extent of Group B compound uptake, its level and length of persistence in the extracellular compartment, its rate of both extra- and intra-cellular hydrolysis to JY-3-094, its subcellular distribution and the extent to which JY-3-094 can effectively interact with Myc. Another factor, not examined here, includes the rate at which Group B compounds are differentially metabolized within cells to compounds other than JY-3-094.

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