IDENTIFICATION OF NEW BIOACTIVE MOLECULES AND THEIR POTENTIAL APPLICATION FOR C-MYC MODULATION IN TUMOR PROCESSES.

DESPITE SIGNIFICANT ADVANCES IN SURGICAL AND MEDICAL THERAPIES THAT HAVE BEEN MADE OVER THE PAST DECADES, MALIGNANT TUMORS REMAIN A LEADING CAUSE OF MORBIDITY AND MORTALITY WORLDWIDE, AND THE PURSUIT OF TARGETED THERAPIES CONTINUES TO DEFINE THE FRONTIER OF MODERN ONCOLOGY. CANCER DEVELOPMENT IS A COMPLEX PATHOLOGICAL PROCESS THAT EXPLOITS A VARIETY OF BIOLOGICAL “ACTORS” AND INTERRELATED MECHANISMS. THUS, THE IDENTIFICATION OF EFFECTIVE THERAPIES AND/OR NOVEL MOLECULAR ENTITIES ABLE TO INTERFERE WITH SPECIFIC TUMOR TARGETS IS URGENTLY NEEDED. AMONG THE MOST PROMINENT ONCOGENIC DRIVERS IS C-MYC, A TRANSCRIPTION FACTOR THAT ORCHESTRATES KEY CELLULAR PROCESSES, INCLUDING PROLIFERATION, METABOLISM, AND APOPTOSIS. HOWEVER, ITS DEREGULATION AND/OR OVEREXPRESSION IS A HALLMARK OF VARIOUS HUMAN MALIGNANCIES CORRELATED WITH POOR CLINICAL OUTCOMES. ALTHOUGH THE CENTRAL ROLE OF THIS PROTEIN IN TUMORIGENESIS, TARGETING C-MYC STILL POSES SIGNIFICANT CHALLENGES: ITS INTRINSICALLY DISORDERED STRUCTURE, LACK OF HYDROPHOBIC POCKETS, NUCLEAR LOCALIZATION, AND DEPENDENCE ON PROTEIN-PROTEIN INTERACTIONS HAVE LONG HINDERED THE DEVELOPMENT OF SELECTIVE AND POTENT MODULATORS. TO ADDRESS THESE LIMITATIONS, SEVERAL ALTERNATIVE STRATEGIES CAN BE EXPLORED, INCLUDING THE DISRUPTION OF PROTEIN–PROTEIN INTERACTIONS AND TARGETED PROTEIN DEGRADATION (TPD). THIS PHD THESIS TACKLES THESE CHALLENGES THROUGH AN INTEGRATED STRATEGY THAT COMBINES THE IDENTIFICATION OF DIRECT C-MYC INHIBITORS WITH DEGRADATION-BASED APPROACHES TO DEVELOP NEW BIOACTIVE MOLECULES MODULATING C-MYC. FIRST, A MULTIDISCIPLINARY WORKFLOW BASED ON VIRTUAL SCREENING, MOLECULAR DOCKING, SURFACE PLASMON RESONANCE (SPR), TARGETED LIMITED PROTEOLYSIS (T-LIP), MOLECULAR DYNAMICS (MD) SIMULATIONS AND CELLULAR ASSAYS LED TO THE IDENTIFICATION OF A 1,3-DIPHENYLUREA-BASED INHIBITOR CAPABLE OF ENGAGING THE C-MYC/MAX INTERFACE AND MODULATING C-MYC ACTIVITY IN CANCER CELL LINES. STRUCTURAL AND MECHANISTIC INSIGHTS FROM THIS CHEMOTYPE ENABLED THE RECOGNITION OF A MINIMAL PHARMACOPHORIC MOTIF AND ITS TRANSLATION INTO A SMALL COLLECTION OF C-MYC PROTACS, FOUR OF WHICH INDUCED C-MYC DEGRADATION IN U937 CELLS. IN PARALLEL, THIS WORKFLOW WAS FURTHER EXPANDED WITH SATURATION TRANSFER DIFFERENCE (STD) NMR EXPERIMENTS ON FULL-LENGTH C-MYC, LEADING TO THE IDENTIFICATION OF A SECOND CLASS OF LIGANDS BASED ON A BENZENESULFONAMIDE SCAFFOLD. MORE THAN FIFTEEN SULFONAMIDE DERIVATIVES SHOWED GOOD BINDING AFFINITY TO C-MYC, AND FOUR COMPOUNDS REDUCED C-MYC LEVELS IN LEUKEMIA CELLS, HIGHLIGHTING THIS SCAFFOLD AS A VERSATILE PLATFORM, BOTH FOR THE DEVELOPMENT OF NEW ATTRACTIVE ANTICANCER CANDIDATES AND FOR THE DESIGN OF FUTURE DEGRADERS. FINALLY, THIS THESIS EXPLORES THE APPLICATION OF BIOORTHOGONAL CHEMISTRY TO GENERATE A NEW CLASS OF DEGRADERS DIRECTLY INSIDE LIVING CELLS USING THE SO CALLED CLIPTAC (CLICK-FORMED PROTAC) TECHNOLOGY. BCN- AND DIBAC-BASED PRECURSORS DERIVED FROM THE KNOWN C-MYC INHIBITOR 10058-F4 WERE SHOWN TO UNDERGO EFFICIENT SPAAC LIGATION WITH AZIDO E3 LIGASE RECRUITERS, YIELDING CLICK-FORMED DEGRADERS WITH MEASURABLE BINDING TO C-MYC AND DETECTABLE EFFECTS ON ITS CELLULAR LEVELS. BUILDING ON THIS PROOF OF CONCEPT, A SECOND GENERATION OF IEDDA-BASED CLIPTACS WAS DEVELOPED, EXPLOITING THE ULTRAFAST KINETICS OF TETRAZINE–DIENOPHILE LIGATIONS TO ENABLE MORE EFFICIENT INTRACELLULAR ASSEMBLY. TAKEN TOGETHER, THE STRATEGIES DEVELOPED DURING MY PHD STUDIES PROVIDE NEW OPPORTUNITIES TO MODULATE C-MYC ACTIVITY IN CANCER CELLS. THEY OFFER AN INTEGRATED PLATFORM FOR THE DISCOVERY AND DEVELOPMENT OF NOVEL DIRECT BINDERS AND TARGETED PROTEIN DEGRADATION TOOLS, SUPPORTING FUTURE EFFORTS TOWARD MORE EFFECTIVE THERAPEUTIC STRATEGIES AGAINST C-MYC AND ITS RELATED ONCOPROTEINS.