Synthetic lethality has proven to be a tactical paradigm to design synergistic anticancer drug combinations. In this context, we leveraged BRCA2 and PARP as a synthetic lethal target pair to consolidate the use of small molecule inhibitors of RAD51-BRCA2 protein-protein interaction as inducers of the BRCAness phenotype that sensitizes BRCA2-functional cancer cells to PARP inhibitors. Starting from compound 1, a phenyl furan-carboxyquinoline, we developed a series of analogues, leading to derivative 19. This compound effectively inhibits RAD51-BRCA2 interaction, impairs homologous recombination, and synergizes with olaparib in BxPC-3 pancreatic cancer cells, inducing synthetic lethality in both 2D and 3D spheroids. Additionally, 19 showed efficacy in human pancreatic cancer cells and no toxicity in normal pancreatic cells, positioning it as an early tool compound and a starting point for further optimization.

Alzheimer's disease is a neurodegenerative disorder characterized by progressive cognitive decline and memory loss. It is associated with the self-assembly of the amyloid-β peptide, a soluble intrinsically disordered protein naturally present in the brain parenchyma in various alloforms. This study presents a computational approach to identify possible modulators of the monomeric aggregation-prone conformations of amyloid-β, a critical intermediate in the fibrillation process. A structure-based virtual screening campaign was designed using a structural ensemble to identify potential binders. The workflow included binding site identification, small molecule-peptide docking, protein-protein docking, and molecular dynamics simulations to evaluate interaction stability and aggregation propensity. From this pipeline, a set of novel molecules was identified as capable of interacting with aggregation-prone forms of amyloid-β, potentially reducing their tendency to form toxic aggregates.

The lack of efficient treatments and reliable biomarkers for neurodegenerative diseases requires the development of a late-stage powerful therapy. To this aim, we focused on Fyn and GSK-3β because both kinases are strictly involved in regulating neurodevelopmental processes, besides orchestrating neurotoxic aggregates' deposition and neuroinflammatory processes development. Based on these premises, we developed dual kinase inhibitors to verify at the cellular level the suitability of Fyn and GSK-3β modulation in pursuing the recovery of neural trophism paired to the activation of a neuroprotective profile. Starting from the mild inhibitory potency of the 3-aminothiazole-7-azaindole scaffold, we identified nanomolar dual and selective inhibitors among the kinases of interest. In-depth biological evaluations were performed with the best compounds of the series to assess the neuroprotective and neuromodulatory properties, like enabling neurogenesis or glial polarization, as well as triggering immunomodulation with different patterns relating to their inhibitory profile, setting the stage for potential development of neuroregenerative treatments.

Aiming at developing a new class of quaternary pyridinium salts, the lead compound 1, characterized by a pyridine-3-yl chalcone framework, was rationally modified by inserting alkyl functions varying from 6 to 18 carbon units. Among the set, some valuable lead compounds were identified. Derivatives 4-6 were primarily active against Staphylococcus aureus and Candida albicans, respectively (MIC = 1.56 and 3.125 μM). In comparison, analogs 4 and 5 showed significant activities against Escherichia coli (MIC = 6.25 μM). Interestingly, the antimicrobial property of compounds 4-6, as well as their antibiofilm activity, occurred at lower concentrations than their cyto- and erythrocyte toxicities, thus ensuring a favorable safety profile. Structure-activity relationship analysis highlighted the critical role of the alkyl tail length in the antimicrobial activity, and optimal results were observed for moieties ranging from 10 to 14 carbon units. Molecular dynamics studies performed on 2 and 5 by modeling them on Gram-positive and Gram-negative membranes showed that the derivatives, upon diffusing across periodic boundary conditions, were able to intercalate into the microbial membranes. The difference in diffusion rates provides useful information to support the diverse antimicrobial potencies of the newly designed quaternary pyridinium salt.

Molecular-targeted therapies for the treatment of cystic fibrosis (CF) rely on small-molecule modulators that rescue the activity of the defective CF transmembrane conductance regulator (CFTR) anion channel. ARN23765 is a small molecule with subnanomolar potency in rescuing the function of mutant CFTR in bronchial epithelial cells from CF patients carrying the F508del-CFTR mutation. Considering the multifaceted interactions of CFTR with the plasma membrane and the complexity of the protein network within the cellular compartments, here we report the investigation of ARN23765's molecular mechanism in live cells. We used the photoaffinity labeling (PAL) approach to demonstrate the interaction of ARN23765-derived probes with CFTR in cells. We showed that ARN23765 contributes to F508del-CFTR rescue by stabilizing the membrane-spanning domain-1 and interacting with CFTR at the same site as other type I CFTR correctors. Our study characterizes ARN23765's mode of action and highlights the potential of studying the interactions between CFTR and its correctors in live cells.

The RAD51-BRCA2 interaction is central to DNA repair through homologous recombination. Emerging evidence indicates RAD51 overexpression and its correlation with chemoresistance in various cancers, suggesting RAD51-BRCA2 inhibition as a compelling avenue for intervention. We previously showed that combining olaparib (a PARP inhibitor (PARPi)) with RS-35d (a BRCA2-RAD51 inhibitor) was efficient in killing pancreatic ductal adenocarcinoma (PDAC) cells. However, RS-35d impaired cell viability even when administered alone, suggesting potential off-target effects. Here, through multiple, integrated orthogonal biological approaches in different 2D and 3D PDAC cultures, we characterised RS-35d enantiomers, in terms of mode of action and single contributions. By differentially inhibiting both RAD51-BRCA2 interaction and sensor kinases ATM, ATR and DNA-PK, RS-35d enantiomers exhibit a ‘within-pathway synthetic lethality’ profile. To the best of our knowledge, this is the first reported proof-of-concept single small molecule capable of demonstrating this built-in synergism. In addition, RS-35d effect on BRCA2-mutated, olaparib-resistant PDAC cells suggests that this compound may be effective as an anticancer agent possibly capable of overcoming PARPi resistance. Our results demonstrate the potential of synthetic lethality, with its diversified applications, to propose new and concrete opportunities to effectively kill cancer cells while limiting side effects and potentially overcoming emerging drug resistance.

Abstract The innovative framework of a fully small molecule-induced synthetic lethality gained increasing attention for its promising application in the selective eradication of cancer cells. Disrupting the interaction between BRCA2 and RAD51, key players in DNA repair by homologous recombination (HR), represents an interesting option within the synthetic lethality paradigm. We previously showed that combining the PARP inhibitor (PARPi) olaparib with the BRCA2-RAD51 disruptor RS-35d, a racemic dihydroquinolone pyrazoline-derivative previously synthetized by us, was efficient in tackling pancreatic ductal adenocarcinoma (PDAC). To deepen our knowledge on this approach, we investigated RS-35d mode of action by characterising the single contribution of the purified two enantiomers, R-35d and S-35d respectively. RS-35d racemate was unveiled to possess a built-in synthetic lethal profile, falling within the paradigm of “within-pathway synthetic lethality”. This opens the way for novel anticancer synthetic lethality strategies towards more robust personalised medicine approaches for unmet medical needs like BRCA-competent and PARP-resistant PDAC.

Understanding the allosteric mechanisms within biomolecules involved in diseases is of paramount importance for drug discovery. Indeed, characterizing communication pathways and critical hotspots in signal transduction can guide a rational approach to leverage allosteric modulation for therapeutic purposes. While the atomistic signatures of allosteric processes are difficult to determine experimentally, computational methods can be a remarkable resource. Network analysis built on Molecular Dynamics simulation data is particularly suited in this respect and is gradually becoming of routine use. Herein, we collect the recent literature in the field, discussing different aspects and available options for network construction and analysis. We further highlight interesting refinements and extensions, eventually providing our perspective on this topic.

In cystic fibrosis (CF), deletion of phenylalanine 508(F508del)in the CF transmembrane conductance regulator (CFTR) is associatedto misfolding and defective gating of the mutant channel. One of themost promising CF drug targets is the ubiquitin ligase RNF5, whichpromotes F508del-CFTR degradation. Recently, the first ever reportedinhibitor of RNF5 was discovered, i.e., the 1,2,4-thiadiazol-5-ylidene inh-2. Here, we designed and synthesized a series of new analoguesto explore the structure-activity relationships (SAR) of thisclass of compounds. SAR efforts ultimately led to compound 16, which showed a greater F508del-CFTR corrector activity than inh-2, good tolerability, and no toxic side effects. Analogue 16 increased the basal level of autophagy similar to whathas been described with RNF5 silencing. Furthermore, co-treatmentwith 16 significantly improved the F508del-CFTR rescueinduced by the triple combination elexacaftor/tezacaftor/ivacaftorin CFBE41o(-) cells. These findings validate the 1,2,4-thiadiazolylidenescaffold for the discovery of novel RNF5 inhibitors and provide evidenceto pursue this unprecedented strategy for the treatment of CF.

In cystic fibrosis (CF), the deletion of phenylalanine 508 (F508del) in the CF transmembrane conductance regulator (CFTR) leads to misfolding and premature degradation of the mutant protein. These defects can be targeted with pharmacological agents named potentiators and correctors. During the past years, several efforts have been devoted to develop and approve new effective molecules. However, their clinical use remains limited, as they fail to fully restore F508del-CFTR biological function. Indeed, the search for CFTR correctors with different and additive mechanisms has recently increased. Among them, drugs that modulate the CFTR proteostasis environment are particularly attractive to enhance therapy effectiveness further. This Perspective focuses on reviewing the recent progress in discovering CFTR proteostasis regulators, mainly describing the design, chemical structure, and structure–activity relationships. The opportunities, challenges, and future directions in this emerging and promising field of research are discussed, as well.

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS patients and transplant recipients, and in congenitally infected newborns. The utility of available drugs is limited by poor bioavailability, toxicity, and emergence of resistant strains. Therefore, it is crucial to identify new targets for therapeutic intervention. Among the latter, viral protein–protein interactions are becoming increasingly attractive. Since dimerization of HCMV DNA polymerase processivity factor ppUL44 plays an essential role in the viral life cycle, being required for oriLyt-dependent DNA replication, it can be considered a potential therapeutic target. We therefore performed an in silico screening and selected 18 small molecules (SMs) potentially interfering with ppUL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the selected SMs led to the identification of four active compounds. The most active one, B3, also efficiently inhibited HCMV AD169 strain in plaque reduction assays and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, B3 specifically reduced viral gene expression starting from 48 h post infection, consistent with the inhibition of viral DNA synthesis measured by qPCR starting from 72 h post infection. Therefore, our data suggest that inhibition of ppUL44 dimerization could represent a new class of HCMV inhibitors, complementary to those targeting the DNA polymerase catalytic subunit or the viral terminase complex.

Commonly used non-antibiotic drugs have been associated with changes in gut microbiome composition, paving the way for the possibility of repurposing FDA-approved molecules as next-generation microbiome therapeutics. Herein, we developed and validated an ex vivo high-throughput screening platform─the mini gut model─to underpin human gut microbiome response to molecular modulators. Ten FDA-approved compounds, selected based on maximum structural diversity of molecular fingerprints, were screened against the gut microbiome of five healthy subjects to characterize the ability of human-targeted drugs to modulate the human gut microbiome network. Three compounds, THIP hydrochloride, methenamine, and mesna, have shown promise as novel gut microbiome therapeutics in light of their capability of promoting health-associated features of the gut microbiome. Our findings provide a resource for future research on drug-microbiome interactions and lay the foundation for a new era of more precise gut microbiome modulation through drug repurposing, aimed at targeting specific dysbiotic events.

Human cytomegalovirus (HCMV) is a leading cause of severe diseases in immunocompromised individuals, including AIDS and transplanted patients, and in congenitally infected newborns. Despite the availability of several antiviral drugs, their utility is limited by poor bioavailability, toxicity, and resistant strains emergence. Therefore, it is crucial to identify new targets of therapeutic intervention. The dimerization of HCMV DNA polymerase processivity factor UL44 plays an essential role in the viral life cycle being required for ori Lyt-dependent DNA replication. We validated the existence of UL44 homodimers both in vitro and in living cells by a variety of approaches, including GST pulldown, thermal shift, FRET and BRET assays. Dimerization occurred with an affinity comparable to that of the UL54/UL44 interaction, and was impaired by amino acid substitutions at the dimerization interface. Subsequently, we performed an in-silico screening to select 18 small molecules (SMs) potentially interfering with UL44 homodimerization. Antiviral assays using recombinant HCMV TB4-UL83-YFP in the presence of the 18 selected SMs led to the identification of four active SMs. The most active one also inhibited AD169 in plaque reduction assays, and impaired replication of an AD169-GFP reporter virus and its ganciclovir-resistant counterpart to a similar extent. As assessed by Western blotting experiments, treatment of infected cells specifically reduced viral gene expression starting from 48 h post infection, consistent with activity on viral DNA synthesis. Therefore, SMs inhibitors of UL44 dimerization could represent a new class of HCMV inhibitors, alternative to those targeting the DNA polymerase catalytic subunit or the viral terminase complex. IMPORTANCE HCMV is a ubiquitous infectious agent causing life-lasting infections in humans. HCMV primary infections and reactivation in non-immunocompetent individuals often result in life-threatening conditions. Antiviral therapy mainly targets the DNA polymerase catalytic subunit UL54 and is often limited by toxicity and selection of drug-resistant viral strains, making the identification of new targets of therapeutic intervention crucial for a successful management of HCMV infections. The significance of our work is in identifying the dimerization of the DNA polymerase processivity factor UL44 as an alternative antiviral target. We could show that full length UL44 dimerizes in a cellular context with high affinity and that such interaction could be targeted by small molecules, thus inhibiting the replication of several HCMV strains, including a drug-resistant mutant. Thus, our work could pave the way to the development of a new class of anti-HCMV compounds that act by targeting UL44 dimerization.

Synthetic lethality is an innovative framework for discovering novel anticancer drug candidates. One example is the use of PARP inhibitors (PARPi) in oncology patients with BRCA mutations. Here, we exploit a new paradigm based on the possibility of triggering synthetic lethality using only small organic molecules (dubbed “fully small-molecule-induced synthetic lethality”). We exploited this paradigm to target pancreatic cancer, one of the major unmet needs in oncology. We discovered a dihydroquinolone pyrazoline-based molecule (35d) that disrupts the RAD51-BRCA2 protein–protein interaction, thus mimicking the effect of BRCA2 mutation. 35d inhibits the homologous recombination in a human pancreatic adenocarcinoma cell line. In addition, it synergizes with olaparib (a PARPi) to trigger synthetic lethality. This strategy aims to widen the use of PARPi in BRCA-competent and olaparib-resistant cancers, making fully small-molecule-induced synthetic lethality an innovative approach toward unmet oncological needs.

Urease is a nickel-containing enzyme that is essential for the survival of several and often deadly pathogenic bacterial strains, including Helicobacter pylori. Notwithstanding several attempts, the development of direct urease inhibitors without side effects for the human host remains, to date, elusive. The recently solved X-ray structure of the HpUreDFG accessory complex involved in the activation of urease opens new perspectives for structure-based drug discovery. In particular, the quaternary assembly and the presence of internal tunnels for nickel translocation offer an intriguing possibility to target the HpUreDFG complex in the search of indirect urease inhibitors. In this work, we adopted a theoretical framework to investigate such a hypothesis. Specifically, we searched for putative binding sites located at the protein–protein interfaces on the HpUreDFG complex, and we challenged their druggability through structure-based virtual screening. We show that, by virtue of the presence of tunnels, some protein–protein interfaces on the HpUreDFG complex are intrinsically well suited for hosting small molecules, and, as such, they possess good potential for future drug design endeavors.

Molecular dynamics (MD) and enhanced sampling MD was performed for 100 ns on the biological assembly of the COVID-19 protease (6LU7), and a template of the COVID-19 S-protein:ACE2 receptor interface (99.88% coverage of 6M0J; model03, swissmodel). Apo-site pharmacophores of the resulting structural clusters were used to mine the FDA database (8700 compounds), and a multi-target library was developed from MD-based hits in high affinity sites across 100 ns. Consensus hits from high throughput docking in crystal structures 5R82, 6LU7 and 6Y2F (protease), and 6VW1 (S-protein:ACE2) were also added, and the resulting libraries were re-docked into MD sites to collect potential COVID-19 re-purposed therapeutics by estimated binding energies.

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell-cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates.

In cystic fibrosis (CF), deletion of phenylalanine 508 (F508del) in the CFTR channel is associated with misfolding and premature degradation of the mutant protein. Among the known proteins associated with F508del-CFTR processing, the ubiquitin ligase RNF5/RMA1 is particularly interesting. We previously demonstrated that genetic suppression of RNF5 in vivo leads to an attenuation of intestinal pathological phenotypes in CF mice, validating the relevance of RNF5 as a drug target for CF. Here, we used a computational approach, based on ligand docking and virtual screening, to discover inh-02, a drug-like small molecule that inhibits RNF5. In in vitro experiments, treatment with inh-02 modulated ATG4B and paxillin, both known RNF5 targets. In immortalized and primary bronchial epithelial cells derived from CF patients homozygous for the F508del mutation, long-term incubation with inh-02 caused significant F508del-CFTR rescue. This work validates RNF5 as a drug target for CF, providing evidence to support its druggability.