New Compounds for Multifactorial Diseases: Molecular Determinants In the Intracellular Effects

Increased life expectancy has expanded the elderly population and the burden of age-related diseases. Among these, neurodegenerative diseases (NDs) and cancer are major contributors to morbidity and mortality and arise from multifactorial perturbations, including dysregulated monoamine metabolism, mitochondrial dysfunction, oxidative stress, aberrant protein phosphorylation and inflammation. This complexity exposes the limits of single-target pharmacology and motivates mechanism-guided strategies such as polypharmacology and targeted protein degradation. Notably, monoamine oxidases (MAO-A/B) are implicated in neurodegenerative pathways and have also been associated with tumor biology, while the microtubule cytoskeleton plays a dual role: it is a proven target in oncology yet essential for neuronal integrity, so microtubule-targeting agents can elicit peripheral neuropathy. Chapter 2 aimed to identify molecular scaffolds capable of engaging disease-relevant pathways in NDs. A rationally designed library of 2-(phenylamino)-7,8-dihydroquinazolinones was profiled against human recombinant MAO-A/B, GSK-3β, and DYRK1A using enzyme kinetics and cell-based assays. Subtle structural changes markedly influenced potency and isoform selectivity, yielding some potent, mainly competitive MAO inhibitors (nanomolar Kᵢ), highly selective for MAO-B. One compound (1d) also inhibited GSK-3β activity. The most active MAO-B inhibitors retained activity in HepG2 cells with minimal cytotoxicity, nominating this scaffold − particularly compound 1d − as a lead for further optimization in neurodegeneration. Extending the MAO enzyme activity to oncology, Chapter 3 evaluated some polyamine (PA) analogues as dual-acting MAO inhibitors and antiproliferative agents, based on reports of MAO overexpression in several tumours. By considering prior observations that reducing methoctramine flexibility enhances MAO-B inhibition, a small in-house PA library with constrained inner linkers was studied by kinetic (including selectivity versus other amine oxidases) and cell-based assays (antiproliferative activity, ROS production, mitochondrial membrane depolarization, cell-cycle analysis, DNA binding, and thiol/glutathione quantification). Conformational constrain markedly increased both MAO inhibition and antiproliferative effects in LN-229 cells. Two compounds, ELP23 and ELP24, were potent, reversible, and competitive MAO inhibitors (Kᵢ < 1 µM) with strong antiproliferative activity (GI₅₀ < 1 µM). ELP23 showed specificity for MAO over other amine oxidases and inhibited MAO activity in cell lysates; both compounds induced apoptosis. ELP24 elicited a dose-dependent ROS increase attenuated by N-acetylcysteine. Mitochondrial depolarization, DNA binding, and cell-cycle arrest were excluded as primary cytotoxic mechanisms, while both compounds modulated thiol and glutathione homeostasis, motivating further target-identification studies. Recognizing the cytoskeleton’s dual role, Chapter 4 explored tubulin-targeting PROTACs as alternatives to conventional microtubule antitumor agents with the goal of improved neuronal tolerance. Four maytansinol-based PROTACs (lenalidomide or VH032 recruiters) designed to degrade αβ-tubulin were tested in PC12 model neurons. A lead candidate reduced total tubulin at micromolar concentrations while preserving acetylated α-tubulin, increased relative microtubule stability, shortened neurites in a dose-dependent manner without soma toxicity (MTT/LDH), slowed tubulin incorporation (FDAP) without altering polymer mass, and preserved tau–microtubule interactions. Altogether, this research work supports some strategies for tackling multifactorial diseases ─ selective enzyme inhibition, polyamine chemotypes, and targeted degradation ─ to achieve safer and more effective therapies.