Small-Molecule Inhibition of c-MYC:MAX Leucine Zipper Formation Is Revealed by Ion Mobility Mass Spectrometry

Abstract

The leucine zipper interaction between MAX and c-MYC has been studied using mass spectrometry and drift time ion mobility mass spectrometry (DT IM-MS) in addition to circular dichroism spectroscopy. Peptides comprising the leucine zipper sequence with (c-MYC-Zip residues 402-434) and without a postulated small-molecule binding region (c-MYC-ZipΔDT residues 406-434) have been synthesized, along with the corresponding MAX leucine zipper (MAX-Zip residues 74-102). c-MYC-Zip:MAX-Zip complexes are observed both in the absence and in the presence of the reported small-molecule inhibitor 10058-F4 for both forms of c-MYC-Zip. DT IM-MS, in combination with molecular dynamics (MD), shows that the c-MYC-Zip:MAX-Zip complex [M+5H](5+) exists in two conformations, one extended with a collision cross section (CCS) of 1164 ± 9.3 Å(2) and one compact with a CCS of 982 ± 6.6 Å(2); similar values are observed for the two forms of c-MYC-ZipΔDT:MAX-Zip. Candidate geometries for the complexes have been evaluated with MD simulations. The helical leucine zipper structure previously determined from NMR measurements (Lavigne, P.; et al. J. Mol. Biol. 1998, 281, 165), altered to include the DT region and subjected to a gas-phase minimization, yields a CCS of 1247 Å(2), which agrees with the extended conformation we observe experimentally. More extensive MD simulations provide compact complexes which are found to be highly disordered, with CCSs that correspond to the compact form from experiment. In the presence of the ligand, the leucine zipper conformation is completely inhibited and only the more disordered species is observed, providing a novel method to study the effect of interactions of disordered systems and subsequent inhibition of the formation of an ordered helical complex.

Therapeutic Strategies to Inhibit MYC

“…However, many of the active species belong to structural classes annotated as nonselective ligands in pharmaceutical screening libraries, so-called pan-assay interference compounds (PAINS) (Baell and Holloway 2010;Whitty 2011), establishing important first directions for follow-up chemistry. Perhaps consistent with this behavior, incisive and important studies have observed multiple binding events of bioactive compounds along the MYC protein by circular dichroism (Hammoudeh et al 2009), nuclear magnetic resonance (NMR) (Hammoudeh et al 2009), and most recently by mass spectrometry (Harvey et al 2012). The relatively low potency, demonstrable selectivity, and durable biostability of lead compounds have prevented use in MYC model systems in vivo (Guo et al 2009;Clausen et al 2010), but insights are emerging from this work and structural modeling.…”

Druggability of Intrinsically Disordered Proteins

“…Its activity is dependent on heterodimerisation with the disordered protein Max (Blackwood and Eisenman 1991). Both c-Myc and Max are highly disordered in their free forms and undergo mutual coupled binding and folding to form the heterodimer complex via a basic helix-loop-helix leucine zipper domain present in both proteins (Hammoudeh et al 2009;Harvey et al 2012;Michel and Cuchillo 2012). This interaction has been reported to be druggable by small molecules that bind to the monomeric and disordered c-Myc (Hammoudeh et al 2009;Harvey et al 2012;Michel and Cuchillo 2012).…”

“…Both c-Myc and Max are highly disordered in their free forms and undergo mutual coupled binding and folding to form the heterodimer complex via a basic helix-loop-helix leucine zipper domain present in both proteins (Hammoudeh et al 2009;Harvey et al 2012;Michel and Cuchillo 2012). This interaction has been reported to be druggable by small molecules that bind to the monomeric and disordered c-Myc (Hammoudeh et al 2009;Harvey et al 2012;Michel and Cuchillo 2012). NMR spectroscopy revealed that these molecules bind to a disordered site in the c-Myc monomer located at the interface between the helix-loop-helix and leucine zipper in the c-Myc-Max complex (Hammoudeh et al 2009).…”

Novel allosteric mechanism of p53 activation by small molecules for targeted anticancer therapy

“…Modulation of protein conformation by a weak binding ligand has previously been shown by IMMS (Harvey et al, 2012). Interestingly, IMMS can detect the changes in p53 conformation induced by single point mutations in the p53 core domain .…”

“…IMMS is an attractive addition to native mass spectrometry as it allows study of the topology of proteins under various conditions (Jurneczko et al 2013;Harvey et al 2012). We performed IMMS analysis of both wt and The mass spectra for Np53 without RITA, with RITA and the control spectra show no mass .…”