New Hybrid Pyrazole and Imidazopyrazole Antinflammatory Agents Able to Reduce ROS Production in Different Biological Targets

Several anti-inflammatory agents based on pyrazole and imidazopyrazole scaffolds and a large library of substituted catechol PDE4D inhibitors were reported by us in the recent past. To obtain new molecules potentially able to act on different targets involved in inflammation onset we designed and synthesized a series of hybrid compounds by linking pyrazole and imidazo-pyrazole scaffolds to differently decorated catechol moieties through an acylhydrazone chain. Some compounds showed antioxidant activity, inhibiting reactive oxygen species (ROS) elevation in neutrophils, and a good inhibition of phosphodiesterases type 4D and, particularly, type 4B, the isoform most involved in inflammation. In addition, most compounds inhibited ROS production also in platelets, confirming their ability to exert an antiinflammatory response by two independent mechanism. Structure-activity relationship (SAR) analyses evidenced that both heterocyclic scaffolds (pyrazole and imidazopyrazole) and the substituted catechol moiety were determinant for the pharmacodynamic properties, even if hybrid molecules bearing to the pyrazole series were more active than the imidazopyrazole ones. In addition, the pivotal role of the catechol substituents has been analyzed. In conclusion the hybridization approach gave a new serie of multitarget antiinflammatory compounds, characterized by a strong antioxidant activity in different biological targets

Polyamines and eIF5A Hypusination Modulate Mitochondrial Respiration and Macrophage Activation

How cells adapt metabolism to meet demands is an active area of interest across biology. Among a broad range of functions, the polyamine spermidine is needed to hypusinate the translation factor eukaryotic initiation factor 5A (eIF5A). We show here that hypusinated eIF5A (eIF5AH) promotes the efficient expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (OXPHOS). Several of these proteins have mitochondrial targeting sequences (MTSs) that in part confer an increased dependency on eIF5AH. In macrophages, metabolic switching between OXPHOS and glycolysis supports divergent functional fates stimulated by activation signals. In these cells, hypusination of eIF5A appears to be dynamically regulated after activation. Using in vivo and in vitro models, we show that acute inhibition of this pathway blunts OXPHOS-dependent alternative activation, while leaving aerobic glycolysis-dependent classical activation intact. These results might have implications for therapeutically controlling macrophage activation by targeting the polyamine-eIF5A-hypusine axis

Acetate Promotes T Cell Effector Function during Glucose Restriction.

Competition for nutrients like glucose can metabolically restrict T cells and contribute to their hyporesponsiveness during cancer. Metabolic adaptation to the surrounding microenvironment is therefore key for maintaining appropriate cell function. For instance, cancer cells use acetate as a substrate alternative to glucose to fuel metabolism and growth. Here, we show that acetate rescues effector function in glucose-restricted CD8+ T cells. Mechanistically, acetate promotes histone acetylation and chromatin accessibility and enhances IFN-γ gene transcription and cytokine production in an acetyl-CoA synthetase (ACSS)-dependent manner. Ex vivo acetate treatment increases IFN-γ production by exhausted T cells, whereas reducing ACSS expression in T cells impairs IFN-γ production by tumor-infiltrating lymphocytes and tumor clearance. Thus, hyporesponsive T cells can be epigenetically remodeled and reactivated by acetate, suggesting that pathways regulating the use of substrates alternative to glucose could be therapeutically targeted to promote T cell function during cancer

Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

Mutations of genes within the phosphatidylinositol-3-kinase (PI3K)-AKT-MTOR pathway are well known causes of brain overgrowth (megalencephaly) as well as segmental cortical dysplasia (such as hemimegalencephaly, focal cortical dysplasia and polymicrogyria). Mutations of the AKT3 gene have been reported in a few individuals with brain malformations, to date. Therefore, our understanding regarding the clinical and molecular spectrum associated with mutations of this critical gene is limited, with no clear genotype–phenotype correlations. We sought to further delineate this spectrum, study levels of mosaicism and identify genotype–phenotype correlations of AKT3-related disorders. We performed targeted sequencing of AKT3 on individuals with these phenotypes by molecular inversion probes and/or Sanger sequencing to determine the type and level of mosaicism of mutations. We analysed all clinical and brain imaging data of mutation-positive individuals including neuropathological analysis in one instance. We performed ex vivo kinase assays on AKT3 engineered with the patient mutations and examined the phospholipid binding profile of pleckstrin homology domain localizing mutations. We identified 14 new individuals with AKT3 mutations with several phenotypes dependent on the type of mutation and level of mosaicism. Our comprehensive clinical characterization, and review of all previously published patients, broadly segregates individuals with AKT3 mutations into two groups: patients with highly asymmetric cortical dysplasia caused by the common p.E17K mutation, and patients with constitutional AKT3 mutations exhibiting more variable phenotypes including bilateral cortical malformations, polymicrogyria, periventricular nodular heterotopia and diffuse megalencephaly without cortical dysplasia. All mutations increased kinase activity, and pleckstrin homology domain mutants exhibited enhanced phospholipid binding. Overall, our study shows that activating mutations of the critical AKT3 gene are associated with a wide spectrum of brain involvement ranging from focal or segmental brain malformations (such as hemimegalencephaly and polymicrogyria) predominantly due to mosaic AKT3 mutations, to diffuse bilateral cortical malformations, megalencephaly and heterotopia due to constitutional AKT3 mutations. We also provide the first detailed neuropathological examination of a child with extreme megalencephaly due to a constitutional AKT3 mutation. This child has one of the largest documented paediatric brain sizes, to our knowledge. Finally, our data show that constitutional AKT3 mutations are associated with megalencephaly, with or without autism, similar to PTEN-related disorders. Recognition of this broad clinical and molecular spectrum of AKT3 mutations is important for providing early diagnosis and appropriate management of affected individuals, and will facilitate targeted design of future human clinical trials using PI3K-AKT pathway inhibitors

Consensus protocol for EEG and amplitude-integrated EEG assessment and monitoring in neonates

The aim of this work is to establish inclusive guidelines on electroencephalography (EEG) applicable to all neonatal intensive care units (NICUs). Guidelines on ideal EEG monitoring for neonates are available, but there are significant barriers to their implementation in many centres around the world. These include barriers due to limited resources regarding the availability of equipment and technical and interpretive round-the-clock personnel. On the other hand, despite its limitations, amplitude-integrated EEG (aEEG) (previously called Cerebral Function Monitor [CFM]) is a common alternative used in NICUs. The Italian Neonatal Seizure Collaborative Network (INNESCO), working with all national scientific societies interested in the field of neonatal clinical neurophysiology, performed a systematic literature review and promoted interdisciplinary discussions among experts (neonatologists, paediatric neurologists, neurophysiologists, technicians) between 2017 and 2020 with the aim of elaborating shared recommendations. A consensus statement on videoEEG (vEEG) and aEEG for the principal neonatal indications was established. The authors propose a flexible frame of recommendations based on the complementary use of vEEG and aEEG applicable to the various neonatal units with different levels of complexity according to local resources and specific patient features. Suggestions for promoting cooperation between neonatologists, paediatric neurologists, and neurophysiologists, organisational restructuring, and teleneurophysiology implementation are provided

N,N,N-Tris(tert-butoxycarbonyl)-L-arginine: five isoforms whose obtainment depends on procedure and a scrupulous NMR confirmation of their structures.

L-arginine is often covalently linked to delivery systems for increasing their transfection activity and reducing toxicity and its basic nitrogen atoms need protection for example with tert-butoxycarbonyl group. Following three reported protocols which assured the goal of obtaining the widely cited \u3b1N,\u3c9N,\u3c9\u2019N-tris(tert-butyloxycarbonyl)-L-arginine, surprisingly we achieved also other four isoforms (Figure 1). With the first selected procedure [1] \u3b1N,\u3c9N,\u3c9\u2019N-Tris(tert-butyloxycarbonyl)-L-arginine was never obtained. The second one [2] provided the desired compound but as a mixture of geometric isomers E/Z while the third [3] protocol led to a single very pure isoform in high yield but with an unreported symmetrical structure. Since BOC protection is transient this discovery would seem of poor interest but results obtained from following investigations about the behavior of each one of the isoforms obtained in the esterification reactions of our interest shown that their reactivity depends on their structure. With this work we reported a detailed description of this unexpected results and the NMR investigation performed with particular care for double bonds geometry and position which confirmed the structures. [1] H. Konno, K. Kubo, H. Makabe, E. Toshiro, N. Hinoda, K. Nosakaa, K: Akaji, Tetrahedron 63, 9502 (2007) doi:10.1016/j.tet.2007.06.082 [2] J. Izdebski, T. Gers, D. Kunce, P. Markovsky, J. Pept. Sci. 11, 60 (2005) doi: 10.1002/psc.585 [3] M. A. Jones, A. D. Hislop, J. S. Snaith, Org. Biomol. Chem. 4, 3769 (2006) doi: 10.1039/b611170

How five different isoforms of N,N,N-Tris(tert-butoxycarbonyl)-L-arginine whose reactivity in esterification reactions was subsequently investigated were obtained

L-arginine, nowadays, is more and more used to functionalize scaffolds for producing delivery systems with high transfection activity and low toxicity. For these purposes it is better to be protected to basic nitrogen atoms. A well known protective residue is tert-butoxycarbonyl group (BOC) and three protocols of BOC-protection selected by us assured that the widely cited (E)-\u3b1N,\u3c9N,\u3c9\u2019N-tris(tert-butyloxycarbonyl)-L-arginine would be the only product obtainable. Surprisingly we achieved also other four isoforms (Figure 1) [1]. With the first tested procedure [2] \u3b1N,\u3c9N,\u3c9\u2019N-Tris(tert-butyloxycarbonyl)-L-arginine was never obtained. The second one [3] provided the goal compound but in mixture with the Z rotamer while the third protocol [4] led to a single very pure isoform in high yield but with an unreported symmetrical structure. Since BOC protection is transient this discovery would seem of poor interest but investigations about the behavior of each one of the isoforms obtained in esterification reactions, whose results have been described in details in another work by us presented in this context, shown that their reactivity depends on their structure. With this work we reported a thorough description of this unexpected results and the meticulous NMR investigation performed with particular care for double bonds geometry and position which confirmed the structures. References: 1. S. Alfei, S. Castellaro, Res. Chem. Intermediat. 44, 1811 (2018) DOI: 10.1007/s11164-017-3199-6. 2. H. Konno, K. Kubo, H. Makabe, E. Toshiro, N. Hinoda, K. Nosakaa, K: Akaji, Tetrahedron 63, 9502 (2007) doi:10.1016/j.tet.2007.06.082.3. J. Izdebski, T. Gers, D. Kunce, P. Markovsky, J. Pept. Sci. 11, 60 (2005) doi: 10.1002/psc.585.4. M. A. Jones, A. D. Hislop, J. S. Snaith, Org. Biomol. Chem. 4, 3769 (2006) doi: 10.1039/b611170