Reversible Linkage of Two Distinct Small Molecule Inhibitors of Myc Generates a Dimeric Inhibitor with Improved Potency That Is Active in Myc Over-Expressing Cancer Cell Lines

Abstract

We describe the successful application of a novel approach for generating dimeric Myc inhibitors by modifying and reversibly linking two previously described small molecules. We synthesized two directed libraries of monomers, each comprised of a ligand, a connector, and a bioorthogonal linker element, to identify the optimal dimer configuration required to inhibit Myc. We identified combinations of monomers, termed self-assembling dimeric inhibitors, which displayed synergistic inhibition of Myc-dependent cell growth. We confirmed that these dimeric inhibitors directly bind to Myc blocking its interaction with Max and affect transcription of MYC dependent genes. Control combinations that are unable to form a dimer do not show any synergistic effects in these assays. Collectively, these data validate our new approach to generate more potent and selective inhibitors of Myc by self-assembly from smaller, lower affinity components. This approach provides an opportunity for developing novel therapeutics against Myc and other challenging protein:protein interaction (PPI) target classes.

Target-Directed Self-Assembly of Homodimeric Drugs Against β-Tryptase

“…Each coferon monomer comprises an appropriate “pharmacophore”, with an affinity for a binding site on the target, connected to a small linker moiety capable of a reversible, covalent reaction with a “partner” coferon binding at a proximal site on the target. 4 Thus, the macromolecular biological target serves as a template for the assembly of the higher affinity coferon dimer. The delivery of a larger dimeric drug molecule as two low molecular weight monomeric coferons affords greater flexibility in optimizing drug-like absorption, distribution, metabolism, and excretion (ADME) properties.…”

A general model for predicting the binding affinity of reversibly and irreversibly dimerized ligands

“…Further, dimerizing monomers with relatively short linkers and good dimerization constants permits the transformation of fragment-based screening hits (with K D ≥~10 -4 M) into potential tools for cell based assays with no further optimization. This aspect was well demonstrated using Myc inhibitors, in which the ligands had weak activity in cells, even in combination, but their reversibly linked analogs had low micromolar activities[ 27 ].…”

“…Monomers based off well-established fragments and containing reversible linker moieties can serve as the basis of screening libraries, permitting rapid searches of chemical space for modestly potent and selective inhibitors. This enablement was well illustrated in the search for Myc-specific cell active inhibitors [ 27 ]. While the Myc monomers were distinct and targeted different sites, they were largely inactive in cell based assays.…”

“…Nevertheless, the irreversible formation of dimer dictates that their thermodynamic treatment involves only irreversibly linked bivalents as described above. Reversible bioorthogonal moieties were reviewed in the literature [ 24 , 25 ] and additional ones have been introduced by Barany et al [ 26 , 27 ]. Recent work shows that bivalents employing these reversible moieties in the linkers as part of a reversible linker technology can also penetrate cells and dimerize to yield significant activity gains [ 27 ].…”

“…Reversible bioorthogonal moieties were reviewed in the literature [ 24 , 25 ] and additional ones have been introduced by Barany et al [ 26 , 27 ]. Recent work shows that bivalents employing these reversible moieties in the linkers as part of a reversible linker technology can also penetrate cells and dimerize to yield significant activity gains [ 27 ]. These reversible linkers require a more complex equilibrium description since several of these chemistries are completely reversible under aqueous conditions.…”