Discovery of CDC42 Inhibitors with a Favorable Pharmacokinetic Profile and Anticancer In Vivo Efficacy.

We previously reported trisubstituted pyrimidine lead compounds, namely, ARN22089 and ARN25062, which block the interaction between CDC42 with its specific downstream effector, a PAK protein. This interaction is crucial for the progression of multiple tumor types. Such inhibitors showed anticancer efficacy in vivo. Here, we describe a second class of CDC42 inhibitors with favorable drug-like properties. Out of the 25 compounds here reported, compound 15 (ARN25499) stands out as the best lead compound with an improved pharmacokinetic profile, increased bioavailability, and efficacy in an in vivo PDX tumor mouse model. Our results indicate that these CDC42 inhibitors represent a promising chemical class toward the discovery of anticancer drugs, with ARN25499 as an additional lead candidate for preclinical development.

Hazard assessment of hexagonal boron nitride and hexagonal boron nitride reinforced thermoplastic polyurethane composites using human skin and lung cells.

Hexagonal boron nitride (hBN) is an emerging two-dimensional material attracting considerable attention in the industrial sector given its innovative physicochemical properties. Potential risks are associated mainly with occupational exposure where inhalation and skin contact are the most relevant exposure routes for workers. Here we aimed at characterizing the effects induced by composites of thermoplastic polyurethane (TPU) and hBN, using immortalized HaCaT skin keratinocytes and BEAS-2B bronchial epithelial cells. The composite was abraded using a Taber® rotary abraser and abraded TPU and TPU-hBN were also subjected to photo-Fenton-mediated degradation mimicking potential weathering across the product life cycle. Cells were exposed to the materials for 24 h (acute exposure) or twice per week for 4 weeks (chronic exposure) and evaluated with respect to material internalization, cytotoxicity, and proinflammatory cytokine secretion. Additionally, comprehensive mass spectrometry-based proteomics and metabolomics (secretomics) analyses were performed. Overall, despite evidence of cellular uptake of the material, no significant cellular and/or protein expression profiles alterations were observed after acute or chronic exposure of HaCaT or BEAS-2B cells, identifying only few pro-inflammatory proteins. Similar results were obtained for the degraded materials. These results support the determination of hazard profiles associated with cutaneous and pulmonary hBN-reinforced polymer composites exposure.

Tezacaftor is a direct inhibitor of sphingolipid delta-4 desaturase enzyme (DEGS).

BACKGROUND: We recently demonstrated that 48 h exposure of primary human bronchial epithelial (hBE) cells, obtained from both CF (F508del homozygous) and non-CF subjects, to the triple drug combination Elexacaftor/Tezacaftor/Ivacaftor (ETI) results in a CFTR genotype-independent modulation of the de novo synthethic pathway of sphingolipids, with an accumulation of dihydroceramides (dHCer). Since dHCer are converted into ceramides (Cer) by the action of a delta-4 sphingolipid desaturase (DEGS) enzyme, we aimed to better understand this off-target effect of ETI (i.e., not related to CFTR rescue) METHODS: hBE cells, both F508del and wild-type, were cultured to create fully differentiated bronchial epithelia. We analyzed Cer and dHCer using an LC-MS based method previously developed by our lab. DEGS expression levels in differentiated hBE cells lysates were quantified by western blot analysis. RESULTS: We demonstrated that 1) dHCer accumulate in hBE with time following prolonged ETI exposure, that 2) similar inhibition occurs in wild-type primary human hepatocytes and that 3) this does not result in an alteration of DEGS expression. We then proved that 4) ETI is a direct inhibitor of DEGS, that 5) Tezacaftor is the molecule responsible for this effect, that 6) the inhibition is concentration dependent. Finally, after repeated oral administration of ETI to naïve, non-CF, mice, we observed a slight accumulation of dHCer in the brain. CONCLUSIONS: We believe that further investigations on Tezacaftor should be envisaged, particularly for the use of ETI during pregnancy, breastfeeding and in the early stages of development. DEGS dysfunction and dHCer accumulation causes impairment in the development of the nervous system, due to a derangement in myelin formation and maintenance.

Sources of biases in the in vitro testing of nanomaterials: the role of the biomolecular corona.

The biological fate of nanomaterials (NMs) is driven by specific interactions through which biomolecules, naturally adhering onto their surface, engage with cell membrane receptors and intracellular organelles. The molecular composition of this layer, called the biomolecular corona (BMC), depends on both the physical-chemical features of the NMs and the biological media in which the NMs are dispersed and cells grow. In this work, we demonstrate that the widespread use of 10% fetal bovine serum in an in vitro assay cannot recapitulate the complexity of in vivo systemic administration, with NMs being transported by the blood. For this purpose, we undertook a comparative journey involving proteomics, lipidomics, high throughput multiparametric in vitro screening, and single molecular feature analysis to investigate the molecular details behind this in vivo/in vitro bias. Our work indirectly highlights the need to introduce novel, more physiological-like media closer in composition to human plasma to produce realistic in vitro screening data for NMs. We also aim to set the basis to reduce this in vitro-in vivo mismatch, which currently limits the formulation of NMs for clinical settings.

Fungal and bacterial species richness in biodeteriorated seventeenth century Venetian manuscripts.

Historical paper documents are susceptible to complex degradation processes, including biodeterioration, which can progressively compromise their aesthetic and structural integrity. This study analyses seventeenth century handwritten historical letters stored at the Correr Museum Library in Venice, Italy, exhibiting pronounced signs of biodeterioration. The techniques used encompassed traditional colony isolation on agar plates and proteomics analyses, employing nanoscale liquid chromatography coupled with high-resolution mass spectrometry (nano-LC-MS). Fluorescence microscopy was used for the first time in the historical paper biodeterioration context to supplement the conventional stereoscopic, optical, and scanning electron microscopic imaging techniques. This method enables the visualisation of microorganisms beyond and beneath the paper's surface through their natural intrinsic autofluorescence in a non-invasive and non-destructive way. The results demonstrate a diverse, complex, and abundant microbiota composed of coexisting fungal and bacterial species (Ascomycota, Mucoromycota, Basidiomycota, Proteobacteria, and Actinobacteria), along with mite carcasses, insects, parasites, and possibly protists. Furthermore, this study reveals certain species that were not previously documented in the biodeterioration of historical paper, including human pathogens, such as Histoplasma capsulatum, Brucella, Candida albicans, and species of Aspergillus (A. flavus, A. fumigatus, A. oryzae, A. terreus, A. niger) known to cause infections or produce mycotoxins, posing substantial risk to both artefacts and humans.

Environmental and Health Impacts of Graphene and Other Two-Dimensional Materials: A Graphene Flagship Perspective.

Two-dimensional (2D) materials have attracted tremendous interest ever since the isolation of atomically thin sheets of graphene in 2004 due to the specific and versatile properties of these materials. However, the increasing production and use of 2D materials necessitate a thorough evaluation of the potential impact on human health and the environment. Furthermore, harmonized test protocols are needed with which to assess the safety of 2D materials. The Graphene Flagship project (2013-2023), funded by the European Commission, addressed the identification of the possible hazard of graphene-based materials as well as emerging 2D materials including transition metal dichalcogenides, hexagonal boron nitride, and others. Additionally, so-called green chemistry approaches were explored to achieve the goal of a safe and sustainable production and use of this fascinating family of nanomaterials. The present review provides a compact survey of the findings and the lessons learned in the Graphene Flagship.

Few-layered graphene increases the response of nociceptive neurons to irritant stimuli.

The unique properties of few-layered graphene (FLG) make it interesting for a variety of applications, including biomedical applications, such as tissue engineering and drug delivery. Although different studies focus on applications in the central nervous system, its interaction with the peripheral nervous system has been so far overlooked. Here, we investigated the effects of exposure to colloidal dispersions of FLG on the sensory neurons of the rat dorsal root ganglia (DRG). We found that the FLG flakes were actively internalized by sensory neurons, accumulated in large intracellular vesicles, and possibly degraded over time, without major toxicological concerns, as neuronal viability, morphology, protein content, and basic electrical properties of DRG neurons were preserved. Interestingly, in our electrophysiological investigation under noxious stimuli, we observed an increased functional response upon FLG treatment of the nociceptive subpopulation of DRG neurons in response to irritants specific for chemoreceptors TRPV1 and TRPA1. The observed effects of FLG on DRG neurons may open-up novel opportunities for applications of these materials in specific disease models.

Biomolecular Corona of Gold Nanoparticles: The Urgent Need for Strong Roots to Grow Strong Branches.

Gold nanoparticles (GNPs) are largely employed in diagnostics/biosensors and are among the most investigated nanomaterials in biology/medicine. However, few GNP-based nanoformulations have received FDA approval to date, and promising in vitro studies have failed to translate to in vivo efficacy. One key factor is that biological fluids contain high concentrations of proteins, lipids, sugars, and metabolites, which can adsorb/interact with the GNP's surface, forming a layer called biomolecular corona (BMC). The BMC can mask prepared functionalities and target moieties, creating new surface chemistry and determining GNPs' biological fate. Here, the current knowledge is summarized on GNP-BMCs, analyzing the factors driving these interactions and the biological consequences. A partial fingerprint of GNP-BMC analyzing common patterns of composition in the literature is extrapolated. However, a red flag is also risen concerning the current lack of data availability and regulated form of knowledge on BMC. Nanomedicine is still in its infancy, and relying on recently developed analytical and informatic tools offers an unprecedented opportunity to make a leap forward. However, a restart through robust shared protocols and data sharing is necessary to obtain "stronger roots". This will create a path to exploiting BMC for human benefit, promoting the clinical translation of biomedical nanotools.

Galantamine-memantine hybrids for Alzheimer's disease: The influence of linker rigidity in biological activity and pharmacokinetic properties.

Neurodegenerative processes characterizing Alzheimer's disease (AD) are strictly related to the impairment of cholinergic and glutamatergic neurotransmitter systems which provoke synaptic loss. These experimental evidences still represent the foundation of the actual standard-of-care treatment for AD, albeit palliative, consisting on the coadministration of an acetylcholinesterase inhibitor and the NMDAR antagonist memantine. In looking for more effective treatments, we previously developed a series of galantamine-memantine hybrids where compound 1 (ARN14140) emerged with the best-balanced action toward the targets of interest paired to neuroprotective efficacy in a murine AD model. Unfortunately, it showed a suboptimal pharmacokinetic profile, which required intracerebroventricular administration for in vivo studies. In this work we designed and synthesized new hybrids with fewer rotatable bonds, which is related to higher brain exposure. Particularly, compound 2, bearing a double bond in the tether, ameliorated the biological profile of compound 1 in invitro studies, increasing cholinesterases inhibitory potencies and selective antagonism toward excitotoxic-related GluN1/2B NMDAR over beneficial GluN1/2A NMDAR. Furthermore, it showed increased plasma stability and comparable microsomal stability in vitro, paired with lower half-life and faster clearance in vivo. Remarkably, pharmacokinetic evaluations of compound 2 showed a promising increase in brain uptake in comparison to compound 1, representing the starting point for further chemical optimizations.

Catalytic Bioswitch of Platinum Nanozymes: Mechanistic Insights of Reactive Oxygen Species Scavenging in the Neurovascular Unit.

Oxidative stress is known to be the cause of several neurovascular diseases, including neurodegenerative disorders, since the increase of reactive oxygen species (ROS) levels can lead to cellular damage, blood-brain barrier leaking, and inflammatory pathways. Herein, we demonstrate the therapeutic potential of 5 nm platinum nanoparticles (PtNPs) to effectively scavenge ROS in different cellular models of the neurovascular unit. We investigated the mechanism underlying the PtNP biological activities, analyzing the influence of the evolving biological environment during particle trafficking and disclosing a key role of the protein corona, which elicited an effective switch-off of the PtNP catalytic properties, promoting their selective in situ activity. Upon cellular internalization, the lysosomal environment switches on and boosts the enzyme-like activity of the PtNPs, acting as an intracellular "catalytic microreactor" exerting strong antioxidant functionalities. Significant ROS scavenging was observed in the neurovascular cellular models, with an interesting protective mechanism of the Pt-nanozymes along lysosomal-mitochondrial axes.

Design, Synthesis, In Vitro and In Vivo Characterization of CDC42 GTPase Interaction Inhibitors for the Treatment of Cancer.

CDC42 GTPases (RHOJ, CDC42, and RHOQ) are overexpressed in multiple tumor types and activate pathways critical for tumor growth, angiogenesis, and metastasis. Recently, we reported the discovery of a novel lead compound, ARN22089, which blocks the interaction of CDC42 GTPases with specific downstream effectors. ARN22089 blocks tumor growth in BRAF mutant mouse melanoma models and patient-derived xenografts (PDXs) in vivo. ARN22089 also inhibits tumor angiogenesis in three-dimensional vascularized microtumor models in vitro. Notably, ARN22089 belongs to a novel class of trisubstituted pyrimidines. Based on these results, we describe an extensive structure-activity relationship of ∼30 compounds centered on ARN22089. We discovered and optimized two novel inhibitors (27, ARN25062, and 28, ARN24928), which are optimal back-up/follow-up leads with favorable drug-like properties and in vivo efficacy in PDX tumors. These findings further demonstrate the potential of this class of CDC42/RHOJ inhibitors for cancer treatment, with lead candidates ready for advanced preclinical studies.

The combination elexacaftor/tezacaftor/ivacaftor (ETI) modulates the de novo synthethic pathway of ceramides in a genotype-independent manner.

We report here how the triple combination of drugs elexacaftor/tezacaftor/ivacaftor (ETI) alters the balance of the de-novo synthethic pathway of sphingolipids in primary cells of human bronchial epithelium. The treatment with ETI roughly doubles the levels of dihydrosphingolipids, possibly by modulating the delta(4)-desaturase enzymes that convert dihydroceramides into ceramides. This appears to be an off-target effect of ETI, since it occurs in a genotype-independent manner, for both cystic fibrosis (CF) and non-CF subjects.

Interactions of Graphene Oxide and Few-Layer Graphene with the Blood-Brain Barrier.

Thanks to their biocompatibility and high cargo capability, graphene-based materials (GRMs) might represent an ideal brain delivery system. The capability of GRMs to reach the brain has mainly been investigated in vivo and has highlighted some controversy. Herein, we employed two in vitro BBB models of increasing complexity to investigate the bionano interactions with graphene oxide (GO) and few-layer graphene (FLG): a 2D murine Transwell model, followed by a 3D human multicellular assembloid, to mimic the complexity of the in vivo architecture and intercellular crosstalk. We developed specific methodologies to assess the translocation of GO and FLG in a label-free fashion and a platform applicable to any nanomaterial. Overall, our results show good biocompatibility of the two GRMs, which did not impact the integrity and functionality of the barrier. Sufficiently dispersed subpopulations of GO and FLG were actively uptaken by endothelial cells; however, the translocation was identified as a rare event.

Boosting the Potential of Chemotherapy in Advanced Breast Cancer Lung Metastasis via Micro-Combinatorial Hydrogel Particles.

Breast cancer cell colonization of the lungs is associated with a dismal prognosis as the distributed nature of the disease and poor permeability of the metastatic foci challenge the therapeutic efficacy of small molecules, antibodies, and nanomedicines. Taking advantage of the unique physiology of the pulmonary circulation, here, micro-combinatorial hydrogel particles (µCGP) are realized via soft lithographic techniques to enhance the specific delivery of a cocktail of cytotoxic nanoparticles to metastatic foci. By cross-linking short poly(ethylene glycol) (PEG) chains with erodible linkers within a shape-defining template, a deformable and biodegradable polymeric skeleton is realized and loaded with a variety of therapeutic and imaging agents, including docetaxel-nanoparticles. In a model of advanced breast cancer lung metastasis, µCGP amplified the colocalization of docetaxel-nanoparticles with pulmonary metastatic foci, prolonged the retention of chemotoxic molecules at the diseased site, suppressed lesion growth, and boosted survival beyond 20 weeks post nodule engraftment. The flexible design and modular architecture of µCGP would allow the efficient deployment of complex combination therapies in other vascular districts too, possibly addressing metastatic diseases of different origins.

Computer-aided identification, synthesis, and biological evaluation of DNA polymerase η inhibitors for the treatment of cancer.

In cancer cells, Pol η allows DNA replication and cell proliferation even in the presence of chemotherapeutic drug-induced damages, like in the case of platinum-containing drugs. Inhibition of Pol η thus represents a promising strategy to overcome drug resistance and preserve the effectiveness of chemotherapeutic drugs. Here, we report the discovery of a novel class of Pol ƞ inhibitors, with 35 active close analogs. Compound 21 (ARN24964) stands out as the best inhibitor, with an IC50 value of 14.7 µM against Pol η and a good antiproliferative activity when used in combination with cisplatin - with a synergistic effect in three different cancer cell lines (A375, A549, OVCAR3). Moreover, it is characterized by a favorable drug-like profile in terms of its aqueous kinetic solubility, plasma and metabolic stability. Thus, ARN24964 is a promising compound for further structure-based drug design efforts toward developing drugs to solve or limit the issue of drug resistance to platinum-containing drugs in cancer patients.

Design, synthesis, docking, and biochemical characterization of non-nucleoside SARS-CoV-2 RdRp inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic. The identification of effective antiviral drugs remains an urgent medical need. In this context, here we report 17 new 1,4-benzopyrone derivatives, which have been designed, synthesized, and characterized for their ability to block the RNA-dependent RNA polymerase (RdRp) enzyme, a promising target for antiviral drug discovery. This compound series represents a good starting point for developing non-nucleoside inhibitors of RdRp. Compounds 4, 5, and 8 were the most promising drug-like candidates with good potency in inhibiting RdRp, improved in vitro pharmacokinetics compared to the initial hits, and no cytotoxicity effects on normal cell (HEK-293). Compound 8 (ARN25592) stands out as the most promising inhibitor. Our results indicate that this new chemical class of 1,4-benzopyrone derivatives deserves further exploration towards novel and potent antiviral drugs for the treatment of SARS-CoV-2 and potentially other viruses.

Treatment with THI, an inhibitor of sphingosine-1-phosphate lyase, modulates glycosphingolipid metabolism and results therapeutically effective in experimental models of Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disorder with no effective cure currently available. Over the past few years our research has shown that alterations in sphingolipid metabolism represent a critical determinant in HD pathogenesis. In particular, aberrant metabolism of sphingosine-1-phosphate (S1P) has been reported in multiple disease settings, including human postmortem brains from HD patients. In this study, we investigate the potential therapeutic effect of the inhibition of S1P degradative enzyme SGPL1, by the chronic administration of the 2-acetyl-5-tetrahydroxybutyl imidazole (THI) inhibitor. We show that THI mitigated motor dysfunctions in both mouse and fly models of HD. The compound evoked the activation of pro-survival pathways, normalized levels of brain-derived neurotrophic factor, preserved white matter integrity, and stimulated synaptic functions in HD mice. Metabolically, THI restored normal levels of hexosylceramides and stimulated the autophagic and lysosomal machinery, facilitating the reduction of nuclear inclusions of both wild-type and mutant huntingtin proteins.

Wool Keratin Nanoparticle-Based Micropatterns for Cellular Guidance Applications.

The waste stream of low-grade wool is an underutilized source of keratin-rich materials with appropriate methods for upcycling into high value-added products still being an open challenge. In the present work, keratins were precipitated from their water solution to produce hierarchical keratin particles via isoelectric precipitation. Matrix-assisted laser desorption/ionization coupled with time-of-flight tandem mass spectrometry analysis (MALDI-TOF/TOF MS/MS) showed the presence of the amino acid sequence leucine-aspartic acid-valine (LDV) in the extracted keratin. This well-known cell adhesion motif is recognized by the cell adhesion molecule α4ß1 integrin. We showed that keratin particles had this tripeptide exposed on the surface and that it could be leveraged, via patterns obtained with microcontact printing, to support and facilitate dermal fibroblast cell adhesion and direct their growth orientation. The zeta potential, isoelectric point, morphological structures, chemical composition, and biocompatibility of keratin particles and the influence of the surfactant sodium dodecyl sulfate (SDS) were investigated. An appropriate ink for microcontact printing of the keratin particles was developed and micron-sized patterns were obtained. Cells adhered preferentially to the patterns, showing how this strategy could be used to functionalize biointerfaces.

The Mechanistic Understanding of RAD51 Defibrillation: A Critical Step in BRCA2-Mediated DNA Repair by Homologous Recombination.

The cytotoxic action of anticancer drugs can be potentiated by inhibiting DNA repair mechanisms. RAD51 is a crucial protein for genomic stability due to its critical role in the homologous recombination (HR) pathway. BRCA2 assists RAD51 fibrillation and defibrillation in the cytoplasm and nucleus and assists its nuclear transport. BRC4 is a peptide derived from the fourth BRC repeat of BRCA2, and it lacks the nuclear localization sequence. Here, we used BRC4 to (i) reverse RAD51 fibrillation; (ii) avoid the nuclear transport of RAD51; and (iii) inhibit HR and enhance the efficacy of chemotherapeutic treatments. Specifically, using static and dynamic light scattering, transmission electron microscopy, and microscale thermophoresis, we show that BRC4 eroded RAD51 fibrils from their termini through a "domino" mechanism and yielded monomeric RAD51 with a cumulative nanomolar affinity. Using cellular assays (BxPC-3, pancreatic cancer), we show that a myristoylated BRC4 (designed for a more efficient cell entry) abolished the formation of nuclear RAD51 foci. The present study provides a molecular description of RAD51 defibrillation, an essential step in BRCA2-mediated homologous recombination and DNA repair.

ß-Glucocerebrosidase Deficiency Activates an Aberrant Lysosome-Plasma Membrane Axis Responsible for the Onset of Neurodegeneration.

ß-glucocerebrosidase is a lysosomal hydrolase involved in the catabolism of the sphingolipid glucosylceramide. Biallelic loss of function mutations in this enzyme are responsible for the onset of Gaucher disease, while monoallelic ß-glucocerebrosidase mutations represent the first genetic risk factor for Parkinson's disease. Despite this evidence, the molecular mechanism linking the impairment in ß-glucocerebrosidase activity with the onset of neurodegeneration in still unknown. In this frame, we developed two in vitro neuronal models of ß-glucocerebrosidase deficiency, represented by mouse cerebellar granule neurons and human-induced pluripotent stem cells-derived dopaminergic neurons treated with the specific ß-glucocerebrosidase inhibitor conduritol B epoxide. Neurons deficient for ß-glucocerebrosidase activity showed a lysosomal accumulation of glucosylceramide and the onset of neuronal damage. Moreover, we found that neurons react to the lysosomal impairment by the induction of their biogenesis and exocytosis. This latter event was responsible for glucosylceramide accumulation also at the plasma membrane level, with an alteration in lipid and protein composition of specific signaling microdomains. Collectively, our data suggest that ß-glucocerebrosidase loss of function impairs the lysosomal compartment, establishing a lysosome-plasma membrane axis responsible for modifications in the plasma membrane architecture and possible alterations of intracellular signaling pathways, leading to neuronal damage.