A quantitative, surface plasmon resonance-based approach to evaluating DNA binding by the c-Myc oncoprotein and its disruption by small molecule inhibitors

Abstract

The use of small molecules to interfere with protein-protein interactions has tremendous therapeutic appeal and is an area of intense interest. Numerous techniques exist to assess these interactions and their disruption. Many, however, require large amounts of protein, do not allow interactions to be followed in real time, are technically demanding or require large capital expenditures and high levels of expertise. Surface plasmon resonance (SPR) represents a convenient alternative to these techniques with virtually none of their disadvantages. We have devised an SPR-based method that allows the heterodimeric association between the c-Myc (Myc) oncoprotein and its obligate partner Max to be quantified in a manner that agrees well with values obtained by other methods. We have adapted it to examine the ability of previously validated small molecules to interfere with Myc-Max heterodimerization and DNA binding. These inhibitors comprised two distinct classes of molecules that inhibit DNA binding by preventing Myc-Max interaction or distorting pre-formed heterodimers and rendering them incapable of DNA binding. Our studies also point out several potential artifacts and pitfalls to be considered when attempting to employ similar SPR-based methods. This technique should be readily adaptable to the study of other protein-protein interactions and their disruption by small molecules.

Direct inhibition of c-Myc-Max heterodimers by celastrol and celastrol-inspired triterpenoids

“…To confirm on-target binding, we employed a surface plasmon resonance (SPR)-based approach [ 59 ] in which an E-box-containing oligonucleotide is tethered to the sensor chip of the SPR instrument and is followed by the addition of Myc and Max(S) in the presence of increasing amounts of inhibitor. Because compounds such as 10058-F4 and 10074-G5 prevent Myc-Max(S) heterodimerization by distorting Myc's bHLH-ZIP domain, they are most effective when added prior to heterodimerization (Figure 2A ) [ 33 , 34 ].…”

“…A. A biotin-tagged E-box-containing double-stranded oligonucleotide was attached to a streptavadin-impregnated biosensor chip so as to achieve a response of 700-800 units as previously described [ 31 , 59 ]. The reading was then re-set to 0.…”

“…C. Selective inhibition of a Myc-responsive promoter. HeLa cells stably expressing a highly labile luciferase under the control of either a Myc or AP-1-responsive promoter [ 59 ] were exposed for 4 hr to the indicated concentrations of celastrol or SBI compounds and then assayed for luciferase activity The results show the mean of triplicate samples +/− 1 S.E. ***: p < 0.05; ****: < 0.005 Immunoblotting for total Myc protein showed no changes compared to untreated control cells (not shown).…”

“…SPR experiments were performed as described previously [ 59 ] using a Biacore™ 3000 instrument and streptavidin-coated biosensor chips (SA-Chip, GE Healthcare, Inc. Piscataway, NJ). The running buffer used was HBS-EP (10mM HEPES, pH 7.4; 500 mM NaCl; 3 mM EDTA; 0.005% v/v Surfactant P20) [ 59 ].…”

OsMADS57 together with OsTB1 coordinates transcription of its target OsWRKY94 and D14 to switch its organogenesis to defense for cold adaptation in rice

“…All experiments were performed using a Biacore 3000 instrument and streptavadin‐coated biosensor chips (SA‐Chip; GE Healthcare) (Henriksson‐Peltola et al ., ; Wang et al ., ). All buffers were freshly prepared, filtered using 0.22‐μm syringe filters and de‐gassed.…”