Domenico Raimondo

NADPH oxidases (NOXs) are attractive but challenging drug targets. Starting from M41 and its analog MC4876, we synthesized quinoline-based derivatives. SAR studies revealed that modifications at the quinoline C2 position and the amine-moiety influenced potency and isoform preference. Boc-protected analogs preferred NOX5, (compound 1a: IC50 = 1.6 μM), whereas deprotected derivatives favored NOX2. Replacing piperidine with tetrahydropyran afforded 1e, a selective NOX1 inhibitor (IC50 = 3.4 μM). The bisquinoline derivative 2a emerged as the most potent, selective NOX2 inhibitor (IC50 = 1.4 μM; cellular EC50 = 0.89 μM). Computational studies support binding at the NADPH site and attribute 2a′s enhanced potency to additional interactions. MC4876 displayed antiproliferative activity, reducing glioblastoma cell viability, glucose uptake, HK2 expression, and mesenchymal markers, while lowering ROS and inducing mitochondrial dysfunction and DNA damage in U937 cells. These findings identify quinoline as tunable scaffolds for isoform-selective NOX inhibition, with 1e and 2a as promising leads.

Ischemic stroke remains a leading cause of death and long-term disability, and current reperfusion therapies do not prevent progressive neuronal loss and neurological decline. Thus, therapeutic strategies targeting intracellular pathways underlying post-ischemic neurodegeneration remain a critical unmet clinical need. The neuron-enriched kinase c-Jun N-terminal kinase 3 (JNK3) is a key mediator of ischemia-induced neuronal death. Still, efforts to therapeutically inhibit this pathway have been limited by poor isoform selectivity and toxicity. Here, we tested whether specific modulation of pathological JNK3 signaling could provide sustained neuroprotection after stroke. Starting from structural information on the interaction between JNK3 and its selective scaffold protein β-arrestin2 (BA2), we designed a BA2-derived miniprotein to attenuate ischemia-induced JNK3 signaling. This strategy led to the development of SIMBA2, a brain-penetrant miniprotein that selectively modulates JNK3 signaling and reduces phosphorylation of the pro-apoptotic effector c-Jun. In a mouse model of transient middle cerebral artery occlusion, SIMBA2 administered 3 h post-ischemia reduced infarct volume, cerebral edema, and cytotoxic damage, while improving functional recovery and preserving neuronal and synaptic integrity. These effects persisted for at least 7 days, consistent with sustained neuroprotection. Circulating JNK3 blood levels increased after ischemia but were reduced in SIMBA2-treated mice, consistent with reduced neuronal injury. Notably, this reduction of plasma JNK3 correlated with neuroprotection. Together, these findings identify SIMBA2 as a selective JNK3-targeting miniprotein that confers sustained neuroprotection after ischemic stroke, highlighting its potential as a neuroprotective therapy that may complement reperfusion treatments. Moreover, plasma JNK3 levels may represent a peripheral biomarker associated with ischemic neuronal injury.

The convergence of oxidative stress and inflammation drives neurodegeneration and cancer, positioning NADPH oxidases (NOXs) as critical therapeutic targets. Starting from the polyfunctional thiadiazolopyrimidine hit 1, we systematically truncated its peripheral arms to map minimum pharmacophoric requirements. While extensive clipping compromised activity, strategic optimization yielded the streamlined analogue 5. In silico, 5 acted as a competitive NADPH mimic; in cell-free assays, it maintained multi-isoform potency, inhibiting NOX1 and NOX5 at low-micromolar concentrations. In rat brain subcellular fractions, 1 and 5 demonstrated concentration-dependent neuroprotective and antioxidant efficacy (1–10 μM) by suppressing lipid peroxidation and preserving mitochondrial and synaptosomal viability. Notably, chemical profiling proved that 5 successfully stripped away the pro-oxidant liabilities and radical-scavenging artifacts inherent to 1. In cancer, 1 displayed some antiproliferative activity, mainly in hematological malignancies. Compound 5, although aqueous solubility issues limited its cellular antiproliferative performance, represents a specific, artifact-free architectural starting point for future selective NOX inhibitor development.

Fibrous dysplasia (FD) of bone is a fibro-osseous disorder caused by GNAS mutations with defective osteogenic differentiation and increased bone remodeling activity. Inhibition of RANKL leads to the replacement of FD lesions with bone. However, the mechanism and pattern of deposition of the newly formed bone remain unclear. Here, we perform morphological and molecular analyses on EF1α-Gsα ^R201C (FD) mice receiving an anti-mouse RANKL antibody. We show that, although the treatment reduces the expression of osteogenic genes, osteoblastic cells continue to produce bone matrix within FD lesions. However, bone formation does not occur in a diffuse or stochastic manner but follows an ordered spatial pattern that is restricted to the surfaces of the lesional bone. These results suggest that during RANKL inhibition the amount of intra-lesional bone trabecular surfaces is critical to the process of cell differentiation and to the skeletal improvement that FD patients may achieve during the treatment.

Nanopores are key components in several emerging technologies. Nanopore sensors allow the observation of single molecules from the signature they leave in the ionic current flowing through the pore, while nanoporous membranes provide a potentially customizable platform to achieve unprecedented ion selectivity performance. In parallel to the improvements in the fabrication techniques, computational methods have flourished in the last few years. However, despite these advancements, bioinformatic and computational biophysics tools are still not systematically employed in nanopore research when compared to other engineering fields that integrated computer-assisted design (CAD) in their development pipeline decades ago. This review aims to provide a wide-ranging overview of the main bioinformatic tools useful for the engineering of biological nanopores including: analysing the effect of mutations on pore properties, determining the protonation state at different pH and studying the electrostatic environment via adaptive Poisson-Boltzmann solver (APBS). A final section presents recent progress in de novo design using AI-based methods. To favour the widespread adoption of these approaches, the Supplementary Information contains some scripts and protocols that may aid the readers to integrate these tools in their design approaches. Graphical Abstract

Chemokine-like receptor CCRL2 is a nonsignaling atypical GPCR that presents chemerin to its cognate receptor CMKLR1 (ChemerinR1), a process essential for the recruitment of inflammatory cells. Despite their biological importance, the structural determinants of CCRL2-chemerin recognition remain poorly defined. Here, we present a comprehensive multiscale computational study that integrates coarse-grained and all-atom molecular dynamics simulations with structural modeling to investigate CCRL2-chemerin interaction. Our results reveal a flexible yet stable binding interface primarily mediated by chemerin's β1 strand and CCRL2's extracellular loop 2, while the C-terminal region of chemerin remains accessible for CMKLR1 engagement. Electrostatic interactions between CCRL2 N-terminus and chemerin's loop 3 further stabilize the complex without triggering intracellular signaling. A modeled ternary CCRL2-chemerin-CMKLR1 complex provides a putative mechanistic framework in which CCRL2 aligns chemerin to promote efficient CMKLR1 activation. Mapping of naturally occurring missense variants onto this interface suggests that sequence variation at specific residues may influence receptor-ligand stability and function. Together, these findings suggest a structural basis of CCRL2-mediated chemerin presentation and may help improve our understanding of its role in immune signaling.

In this study, we explored the design of linear D‐tripeptides tailored to bind specific cavities of Gadd45β, chosen as a model protein target. To identify peptides that selectively interact with predicted binding sites, we combined computational modeling with biophysical experiments. Gadd45β was selected since it has emerged as a promising therapeutic target involved in multiple disease pathways, including cancer and inflammation. Computational analysis was first employed to characterize the structural features and potential binding sites of Gadd45β. Guided by these insights, linear D‐tripeptides were designed and optimized for specific interactions with the target surface. The resulting candidates were subsequently assessed through a series of biophysical assays to evaluate their binding affinity, selectivity, and potential therapeutic activity. Complementary computational simulations were employed to gain atomistic insight into the dynamics of peptide–protein recognition. This integrated computational–experimental strategy led to the identification of two D‐tripeptides, RYR and VWR, that bind Gadd45β at a biologically relevant site, illustrating a general framework for early‐stage peptide ligand discovery.

The intron retention (IR) is a phenomenon utilized by cells to allow diverse fates at the same mRNA, leading to a different pattern of synthesis of the same protein. In this study, we analyzed the modulation of phosphoinositide‐specific phospholipase C (PI‐PLC) enzymes by Harpagophytum procumbens extract (HPE) in synoviocytes from joins of osteoarthritis (OA) patients. In some samples, the PI‐PLC γ1 isoform mature mRNA showed the IR and, in these synoviocytes, the HPE treatment increased the phenomenon. Moreover, we highlighted that as a consequence of IR, a lower amount of PI‐PLC γ1 was produced. The decrease of PI‐PLC γ1 was associated with the decrease of metalloprotease‐3 (MMP‐3), and MMP‐13, and ADAMTS‐5 after HPE treatment. The altered expression of MMPs is a hallmark of the onset and progression of OA, thus substances able to decrease their expression are very desirable. The interesting outcomes of this study are that 35% of analyzed synovial tissues showed the IR phenomenon in the PI‐PLC γ1 mRNA and that the HPE treatment increased this phenomenon. For the first time, we found that the decrease of PI‐PLC γ1 protein in synoviocytes interferes with MMP production, thus affecting the pathways involved in the MMP expression. This finding was validated by the silencing of PI‐PLC γ1 in synoviocytes where the IR phenomenon was not present. Our results shed new light on the biochemical mechanisms involved in the degrading enzyme production in the joint of OA patients, suggesting a new therapeutic target and highlighting the importance of personalized medicine.