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.

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) and 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, and 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.

Innovative Approach for a Classic Target: Fragment Screening on Trypanothione Reductase Reveals New Opportunities for Drug Design.

Trypanothione reductase (TR) is a key factor in the redox homeostasis of trypanosomatid parasites, critical for survival in the hostile oxidative environment generated by the host to fight infection. TR is considered an attractive target for the development of new trypanocidal agents as it is essential for parasite survival but has no close homolog in humans. However, the high efficiency and turnover of TR challenging targets since only potent inhibitors, with nanomolar IC50, can significantly affect parasite redox state and viability. To aid the design of effective compounds targeting TR, we performed a fragment-based crystal screening at the Diamond Light Source XChem facility using a library optimized for follow-up synthesis steps. The experiment, allowing for testing over 300 compounds, resulted in the identification of 12 new ligands binding five different sites. Interestingly, the screening revealed the existence of an allosteric pocket close to the NADPH binding site, named the "doorstop pocket" since ligands binding at this site interfere with TR activity by hampering the "opening movement" needed to allow cofactor binding. The second remarkable site, known as the Z-site, identified by the screening, is located within the large trypanothione cavity but corresponds to a region not yet exploited for inhibition. The fragments binding to this site are close to each other and have some remarkable features making them ideal for follow-up optimization as a piperazine moiety in three out of five fragments.

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.

Balanced dual acting compounds targeting aromatase and estrogen receptor α as an emerging therapeutic opportunity to counteract estrogen responsive breast cancer.

Breast Cancer (BC) is a leading cause of death in women, currently affecting 13% of female population worldwide. First-line clinical treatments against Estrogen Receptor positive (ER+) BC rely on suppressing estrogen production, by inhibiting the aromatase (AR) enzyme, or on blocking estrogen-dependent pro-oncogenic signaling, by targeting Estrogen Receptor (ER) α with selective Modulators/Degraders (SERMs/SERDs). The development of dual acting molecules targeting AR and ERα represents a tantalizing alternative strategy to fight ER + BC, reducing the incidence of adverse effects and resistance onset that limit the effectiveness of these gold-standard therapies. Here, in silico design, synthesis, biological evaluation and an atomic-level characterization of the binding and inhibition mechanism of twelve structurally related drug-candidates enable the discovery of multiple compounds active on both AR and ERα in the sub-µM range. The best drug-candidate 3a displayed a balanced low-nanomolar IC50 towards the two targets, SERM activity and moderate selectivity towards a BC cell line. Moreover, most of the studied compounds reduced ERα levels, suggesting a potential SERD activity. This study dissects the key structural traits needed to obtain optimal dual acting drug-candidates, showing that multitarget compounds may be a viable therapeutic option to counteract ER + BC.

Multifaceted activity of polyciclic MDR revertant agents in drug-resistant leukemic cells: Role of the spacer.

A small library of derivatives carrying a polycyclic scaffold recently identified by us as a new privileged structure in medicinal chemistry was designed and synthesized, aiming at obtaining potent MDR reverting agents also endowed with antitumor properties. In particular, as a follow-up of our previous studies, attention was focused on the role of the spacer connecting the polycyclic core with a properly selected nitrogen-containing group. A relevant increase in reverting potency was observed, going from the previously employed but-2-ynyl- to a pent-3-ynylamino moiety, as in compounds 3d and 3e, while the introduction of a triazole ring proved to differently impact on the activity of the compounds. The docking results supported the data obtained by biological tests, showing, for the most active compounds, the ability to establish specific bonds with P-glycoprotein. Moreover, a multifaceted anticancer profile and dual in vitro activity was observed for all compounds, showing both revertant and antitumor effects on leukemic cells. In this respect, 3c emerged as a "triple-target" agent, endowed with a relevant reverting potency, a considerable antiproliferative activity and a collateral sensitivity profile.

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.

Targeting Orthosteric and Allosteric Pockets of Aromatase via Dual-Mode Novel Azole Inhibitors.

Breast cancer (BC) is the most diffused cancer type in women and the second leading cause of death among the female population. Effective strategies to fight estrogen responsive (ER+) BC, which represents 70% of all BC cases, rely on estrogen deprivation, via the inhibition of the aromatase enzyme, or the modulation of its cognate estrogen receptor. Current clinical therapies significantly increased patient survival time. Nevertheless, the onset of resistance in metastatic BC patients undergoing prolonged treatments is becoming a current clinical challenge, urgently demanding to devise innovative strategies. In this context, here we designed, synthesized, and performed in vitro inhibitory tests on the aromatase enzyme and distinct ER+/ER- BC cell line types of novel azole bridged xanthones. These compounds are active in the low µM range and behave as dual-mode inhibitors, targeting both the orthosteric and the allosteric sites of the enzyme placed along one access channel. Classical and quantum-classical molecular dynamics simulations of the new compounds, as compared with selected steroidal and nonsteroidal inhibitors, provide a rationale to the observed inhibitory potency and supply the guidelines to boost the activity of inhibitors able to exploit coordination to iron and occupation of the access channel to modulate estrogen production.

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).