Synthesis of Fyn inhibitors as potential anticancer agents

The Fyn protein, which belongs to the Src family of kinases (SFKs), is involved in many biological activities such as : growth factor and cytokine receptor signalling, integrin-mediated signalling, cell-cell adhesion, platelet activation, ion channel function, differentiation of oligodendrocytes and keratinocytes, T-cell and B-cell receptor signalling. At first, this protein was taken into account for its involvement in some diseases of the central nervous system such as Alzheimer’s and other tauopathies. Fyn, indeed, phosphorylates the Tau protein at its amino-terminus on Tyr 18; phosphorylated Tau is one of the main components of neurofibrillary tangles that characterize Alzheimer’s disease. Then, the involvement of Fyn was highlighted in the development and progression of cancer. The overexpression of this protein leads to morphological changes at the cellular level that underlie the development of cancer cells. The inhibition of Fyn, for example, is associated with a decrease in cell growth; this protein, indeed, promotes the antiapoptotic activity of Akt. Moreover, Fyn participates in other processes involved in the development of malignancies, such as cell migration, which determines the metastatic disease, cell adhesion and EMT (epithelial-mesenchymal transition). Because of all these functions , this protein became a very interesting pharmaceutical target for neurodegenerative pathologies and tumours. During this thesis work, I synthesized some new molecules with potential inhibitory activity against Fyn. The design of these compounds started with virtual screening studies performed on commercially available libraries of compounds to identify new Fyn inhibitors. Some of the resulting compounds proved to efficiently inhibit Fyn. Therefore we planned to synthesize some derivatives bearing the same central scaffold. The new compounds share the same 1,2,4-triazin-5(2H)-one scaffold, differing only for the presence of substituents on the two aromatic rings

Enriching Proteolysis Targeting Chimeras with a Second Modality: When Two Are Better Than One

Proteolysis targeting chimera (PROTAC)-mediated protein degradation has prompted a radical rethink and is at a crucial stage in driving a drug discovery transition. To fully harness the potential of this technology, a growing paradigm toward enriching PROTACs with other therapeutic modalities has been proposed. Could researchers successfully combine two modalities to yield multifunctional PROTACs with an expanded profile? In this Perspective, we try to answer this question. We discuss how this possibility encompasses different approaches, leading to multitarget PROTACs, light-controllable PROTACs, PROTAC conjugates, and macrocycle-and oligonucleotide-based PROTACs. This possibility promises to further enhance PROTAC efficacy and selectivity, minimize side effects, and hit undruggable targets. While PROTACs have reached the clinical investigation stage, additional steps must be taken toward the translational development of multifunctional PROTACs. A deeper and detailed understanding of the most critical challenges is required to fully exploit these opportunities and decisively enrich the PROTAC toolbox

Small molecules exploiting emerging therapeutic opportunities for breast cancer treatment

Among the different types of breast cancer (BC), the estrogen receptor positive (ER+) subtype, which requires estrogens for its growth and proliferation, is the most common, while triple negative BC, characterized by the absence of ER, progesterone receptor and human epidermal growth factor receptor 2, often leads to poor prognosis. First-line therapies for the treatment of ER+ BC act either by suppressing estrogen production, through the inhibition of aromatase (AR) enzyme, or by blocking estrogen prooncogenic activity, via the modulation/degradation of ERs. The serious side effects and the intrinsic or acquired resistance phenomena that arise with prolonged use of these drugs limit their therapeutic application, stimulating the search for new strategies to face this disease. In this context, the development of dual acting aromatase inhibitors, able to target both the orthosteric and the recently identified allosteric pockets of AR could be an opportunity to fight ER+ BC. Another promising strategy could be the development of multitarget compounds, targeting both AR and ERs. In this scenario, here we designed and synthesized two series of new xanthones or more flexible benzophenones as potential dual acting aromatase inhibitors. Moreover, inspired from tamoxifen metabolites and a literature compound endowed with activity on both AR and ER, different structurally related series of potential multitarget compounds were developed. The biological results showed that some of the new molecules were promising candidates for further development. It was recently observed that the lately discovered histamine H4 receptor is expressed in human breast tissue, displaying a key role in biological processes mediated by histamine such as cell proliferation, senescence, and apoptosis in malignant cells, representing a potential target in triple negative BC. Thus, a broad series of methyl quinazoline sulfonamides, carrying different functional groups on the sulfonamide moiety, were designed and synthesized as potential H4 receptor ligands

Reconsidering Aromatase for Breast Cancer Treatment: New Roles for an Old Target

The current therapeutic approach for the treatment of hormone dependent breast cancer includes interference with estrogen receptors via either selective modulators or estrogens deprivation, by preventing their biosynthesis with aromatase inhibitors. Severe side effects and acquired resistance are drawbacks of both drug classes, and the efforts to overcome these issues still allow for research in this field to be animated. This review reports on recent findings that have opened new avenues for reconsidering the role of aromatase enzymes (and estrogen receptors) leading to the possibility of looking at well-known targets in a new perspective

Single-digit nanomolar inhibitors lock the aromatase active site via a dualsteric targeting strategy

The most frequently diagnosed breast cancer (BC) type in women expresses estrogen receptor (ER) , depends on estrogens for its growth, being classified as ER positive (ER+). The gold standard therapy for the treatment of this tumor relies on the inhibition of the aromatase enzyme, which catalyzes estrogen biosynthesis. Despite the clinical success of current aromatase inhibitors (AIs), after prolonged therapeutic regimens, BC ER + patients experience acquired resistance and disease relapse. This points up the urgent need for a newer generation of AIs able to overcome resistance issues, while mitigating toxicity and side effects of current therapies. Here we performed the synthesis, biological evaluation , extensive structural characterization by advanced molecular simulation methods of a new generation of dualsteric non-steroidal AIs, which simultaneously target the enzyme's active and allosteric sites. Notably, 3d, the most active AI of the series, exhibits a single-digit nM potency (IC50 2 nM). A detailed inspection of its binding mode reveals that the ancillary alkoxy chain predatorily takes advantage of the small hydrophobic cavities lining the allosteric site, triggering a remodeling of its residues and completely sealing the active site access-channel. As a result, the inhibitor is effectively locked in. This study sets a conceptual basis to develop a new generation of AIs exploiting a dualsteric targeting strategy

Fragment Merging, Growing, and Linking Identify New Trypanothione Reductase Inhibitors for Leishmaniasis

: Trypanothione reductase (TR) is a suitable target for drug discovery approaches against leishmaniasis, although the identification of potent inhibitors is still challenging. Herein, we harnessed a fragment-based drug discovery (FBDD) strategy to develop new TR inhibitors. Previous crystallographic screening identified fragments 1-3, which provided ideal starting points for a medicinal chemistry campaign. In silico investigations revealed critical hotspots in the TR binding site, guiding our structure- and ligand-based structure-actvity relationship (SAR) exploration that yielded fragment-derived compounds 4-14. A trend of improvement in Leishmania infantum TR inhibition was detected along the optimization and confirmed by the crystal structures of 9, 10, and 14 in complex with Trypanosoma brucei TR. Compound 10 showed the best TR inhibitory profile (Ki = 0.2 μM), whereas 9 was the best one in terms of in vitro and ex vivo activity. Although further fine-tuning is needed to improve selectivity, we demonstrated the potentiality of FBDD on a classic but difficult target for leishmaniasis

Switching from Aromatase Inhibitors to Dual Targeting Flavonoid-Based Compounds for Breast Cancer Treatment

Despite the significant outcomes attained by scientific research, breast cancer (BC) still represents the second leading cause of death in women. Estrogen receptor-positive (ER+) BC accounts for the majority of diagnosed BCs, highlighting the disruption of estrogenic signalling as target for first-line treatment. This goal is presently pursued by inhibiting aromatase (AR) enzyme or by modulating Estrogen Receptor (ER) α. An appealing strategy for fighting BC and reducing side effects and resistance issues may lie in the design of multifunctional compounds able to simultaneously target AR and ER. In this paper, previously reported flavonoid-related potent AR inhibitors were suitably modified with the aim of also targeting ERα. As a result, homoisoflavone derivatives 3b and 4a emerged as well-balanced submicromolar dual acting compounds. An extensive computational study was then performed to gain insights into the interactions the best compounds established with the two targets. This study highlighted the feasibility of switching from single-target compounds to balanced dual-acting agents, confirming that a multi-target approach may represent a valid therapeutic option to counteract ER+ BC. The homoisoflavone core emerged as a valuable natural-inspired scaffold for the design of multifunctional compounds

Binding investigation and preliminary optimisation of the 3-amino-1,2,4-triazin-5(2H)-one core for the development of new Fyn inhibitors

Fyn tyrosine kinase inhibitors are considered potential therapeutic agents for a variety of human cancers. Furthermore, the involvement of Fyn kinase in signalling pathways that lead to severe pathologies, such as Alzheimer’s and Parkinson’s diseases, has also been demonstrated. In this study, starting from 3-(benzo[d][1,3]dioxol-5-ylamino)-6-methyl-1,2,4-triazin-5(2H)-one (VS6), a hit compound that showed a micromolar inhibition of Fyn (IC50= 4.8 μM), we computationally investigated the binding interactions of the 3-amino-1,2,4-triazin-5(2H)-one scaffold and started a preliminary hit to lead optimisation. This analysis led us to confirm the hypothesised binding mode of VS6 and to identify a new derivative that is about 6-fold more active than VS6 (compound 3, IC50= 0.76 μM)