Low glycemic index diet restrains epileptogenesis in a gender-specific fashion.

Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2-3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.

New Class of Benzodiazepinone Derivatives as Pro-Death Agents Targeting BIR Domains in Cancer Cells.

Inhibitor of Apoptosis Proteins (IAPs) are validated targets for cancer therapy, and the deregulation of their activities within the NF-κB pathway correlates with chemoresistance events, even after treatment with IAPs-antagonists in the clinic (Smac-mimetics). The molecule FC2 was identified as a NF-κB pathway modulator in MDA-MB-231 adenocarcinoma cancer cells after virtual screening of the Chembridge library against the Baculoviral IAP Repeat 1 (BIR1) domain of cIAP2 and XIAP. An improved cytotoxic effect is observed when FC2 is combined with Smac-mimetics or with the cytokine Tumor Necrosis Factor (TNF). Here, we propose a library of 22 derivatives of FC2, whose scaffold was rationally modified starting from the position identified as R1. The cytotoxic effect of FC2 derivatives was evaluated in MDA-MB-231 and binding to the cIAP2- and XIAP-BIR1 domains was assessed in fluorescence-based techniques and virtual docking. Among 22 derivatives, 4m and 4p display improved efficacy/potency in MDA-MB-231 cells and low micromolar binding affinity vs the target proteins. Two additional candidates (4b and 4u) display promising cytotoxic effects in combination with TNF, suggesting the connection between this class of molecules and the NF-κB pathway. These results provide the rationale for further FC2 modifications and the design of novel IAP-targeting candidates supporting known therapies.

Virtual Screening in the Identification of Sirtuins' Activity Modulators.

Sirtuins are NAD+-dependent deac(et)ylases with different subcellular localization. The sirtuins' family is composed of seven members, named SIRT-1 to SIRT-7. Their substrates include histones and also an increasing number of different proteins. Sirtuins regulate a wide range of different processes, ranging from transcription to metabolism to genome stability. Thus, their dysregulation has been related to the pathogenesis of different diseases. In this review, we discussed the pharmacological approaches based on sirtuins' modulators (both inhibitors and activators) that have been attempted in in vitro and/or in in vivo experimental settings, to highlight the therapeutic potential of targeting one/more specific sirtuin isoform(s) in cancer, neurodegenerative disorders and type 2 diabetes. Extensive research has already been performed to identify SIRT-1 and -2 modulators, while compounds targeting the other sirtuins have been less studied so far. Beside sections dedicated to each sirtuin, in the present review we also included sections dedicated to pan-sirtuins' and to parasitic sirtuins' modulators. A special focus is dedicated to the sirtuins' modulators identified by the use of virtual screening.

First Evidence of Anti-Steatotic Action of Macrotympanain A1, an Amphibian Skin Peptide from Odorrana macrotympana.

Many different amphibian skin peptides have been characterized and proven to exert various biological actions, such as wound-healing, immunomodulatory, anti-oxidant, anti-inflammatory and anti-diabetic effects. In this work, the possible anti-steatotic effect of macrotympanain A1 (MA1) (FLPGLECVW), a skin peptide isolated from the Chinese odorous frog Odorrana macrotympana, was investigated. We used a well-established in vitro model of hepatic steatosis, consisting of lipid-loaded rat hepatoma FaO cells. In this model, a 24 h treatment with 10 µg/mL MA1 exerted a significant anti-steatotic action, being able to reduce intracellular triglyceride content. Accordingly, the number and diameter of cytosolic lipid droplets (LDs) were reduced by peptide treatment. The expression of key genes of hepatic lipid metabolism, such as PPARs and PLINs, was measured by real-time qPCR. MA1 counteracted the fatty acid-induced upregulation of PPARγ expression and increased PLIN3 expression, suggesting a role in promoting lipophagy. The present data demonstrate for the first time a direct anti-steatotic effect of a peptide from amphibian skin secretion and pave the way to further studies on the use of amphibian peptides for beneficial actions against metabolic diseases.

Differential modulation of SIRT6 deacetylase and deacylase activities by lysine-based small molecules.

Sirtuin 6 (SIRT6) is an NAD+-dependent deacetylase regulating important functions: modulators of its enzymatic activity have been considered as possible therapeutic agents. Besides the deacetylase activity, SIRT6 also has NAD+-dependent deacylase activity, whereby it regulates the secretion of cytokines and proteins. We identified novel SIRT6 modulators with a lysine-based structure: compound 1 enhances SIRT6 deacylase while inhibiting the deacetylase activity. As expected based on the biological effects of SIRT6 deacetylase activity, compound 1 increased histone 3 lysine 9 acetylation and the activity of glycolytic enzymes. Moreover, the fact that compound 1 enhanced SIRT6 deacylase activity was accompanied by an increased TNF-α release. In conclusion, new SIRT6 modulators with a lysine-like structure were identified, with differential effects on specific SIRT6 activities. The novel SIRT6 modulator concomitantly inhibits deacetylase and enhances deacylase activity.

Molecular Docking and QSAR Studies as Computational Tools Exploring the Rescue Ability of F508del CFTR Correctors.

Cystic fibrosis (CF) is the autosomal recessive disorder most recurrent in Caucasian populations. Different mutations involving the cystic fibrosis transmembrane regulator protein (CFTR) gene, which encodes the CFTR channel, are involved in CF. A number of life-prolonging therapies have been conceived and deeply investigated to combat this disease. Among them, the administration of the so-called CFTR modulators, such as correctors and potentiators, have led to quite beneficial effects. Recently, based on QSAR (quantitative structure activity relationship) studies, we reported the rational design and synthesis of compound 2, an aminoarylthiazole-VX-809 hybrid derivative exhibiting promising F508del-CFTR corrector ability. Herein, we explored the docking mode of the prototype VX-809 as well as of the aforementioned correctors in order to derive useful guidelines for the rational design of further analogues. In addition, we refined our previous QSAR analysis taking into account our first series of in-house hybrids. This allowed us to optimize the QSAR model based on the chemical structure and the potency profile of hybrids as F508del-CFTR correctors, identifying novel molecular descriptors explaining the SAR of the dataset. This study is expected to speed up the discovery process of novel potent CFTR modulators.

A Bioactive Olive Pomace Extract Prevents the Death of Murine Cortical Neurons Triggered by NMDAR Over-Activation.

We have recently demonstrated that bioactive molecules, extracted by high pressure and temperature from olive pomace, counteract calcium-induced cell damage to different cell lines. Here, our aim was to study the effect of the same extract on murine cortical neurons, since the preservation of the intracellular Ca2+-homeostasis is essential for neuronal function and survival. Accordingly, we treated neurons with different stimuli in order to evoke cytotoxic glutamatergic activation. In these conditions, the high-pressure and temperature extract from olive pomace (HPTOPE) only abolished the effects of N-methyl-d-aspartate (NMDA). Particularly, we observed that HPTOPE was able to promote the neuron rescue from NMDA-induced cell death. Moreover, we demonstrated that HPTOPE is endowed with the ability to maintain the intracellular Ca2+-homeostasis following NMDA receptor overactivation, protecting neurons from Ca2+-induced adverse effects, including aberrant calpain proteolytic activity. Moreover, we highlight the importance of the extraction conditions used that, without producing toxic molecules, allow us to obtain protecting molecules belonging to proanthocyanidin derivatives like procyanidin B2. In conclusion, we can hypothesize that HPTOPE, due to its functional and nontoxic properties on neuronal primary culture, can be utilized for future therapeutic interventions for neurodegeneration.

Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Sirtuin 6 (SIRT6) is a sirtuin family member involved in a wide range of physiologic and disease processes, including cancer and glucose homeostasis. Based on the roles played by SIRT6 in different organs, including its ability to repress the expression of glucose transporters and glycolytic enzymes, inhibiting SIRT6 has been proposed as an approach for treating type 2 diabetes mellitus (T2DM). However, so far, the lack of small-molecule Sirt6 inhibitors has hampered the conduct of in vivo studies to assess the viability of this strategy. We took advantage of a recently identified SIRT6 inhibitor, compound 1, to study the effect of pharmacological Sirt6 inhibition in a mouse model of T2DM (i.e., in high-fat-diet-fed animals). The administration of the Sirt6 inhibitor for 10 d was well tolerated and improved oral glucose tolerance, it increased the expression of the glucose transporters GLUT1 and -4 in the muscle and enhanced the activity of the glycolytic pathway. Sirt6 inhibition also resulted in reduced insulin, triglycerides, and cholesterol levels in plasma. This study represents the first in vivo study of a SIRT6 inhibitor and provides the proof-of-concept that targeting SIRT6 may be a viable strategy for improving glycemic control in T2DM.-Sociali, G., Magnone, M., Ravera, S., Damonte, P., Vigliarolo, T., Von Holtey, M., Vellone, V. G., Millo, E., Caffa, I., Cea, M., Parenti, M. D., Del Rio, A., Murone, M., Mostoslavsky, R., Grozio, A., Nencioni, A., Bruzzone S. Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model.

Elicited ROS Scavenging Activity, Photoprotective, and Wound-Healing Properties of Collagen-Derived Peptides from the Marine Sponge Chondrosia reniformis.

Recently, the bioactive properties of marine collagen and marine collagen hydrolysates have been demonstrated. Although there is some literature assessing the general chemical features and biocompatibility of collagen extracts from marine sponges, no data are available on the biological effects of sponge collagen hydrolysates for biomedical and/or cosmetic purposes. Here, we studied the in vitro toxicity, antioxidant, wound-healing, and photoprotective properties of four HPLC-purified fractions of trypsin-digested collagen extracts-marine collagen hydrolysates (MCHs)-from the marine sponge C. reniformis. The results showed that the four MCHs have no degree of toxicity on the cell lines analyzed; conversely, they were able to stimulate cell growth. They showed a significant antioxidant activity both in cell-free assays as well as in H2O2 or quartz-stimulated macrophages, going from 23% to 60% of reactive oxygen species (ROS) scavenging activity for the four MCHs. Finally, an in vitro wound-healing test was performed with fibroblasts and keratinocytes, and the survival of both cells was evaluated after UV radiation. In both experiments, MCHs showed significant results, increasing the proliferation speed and protecting from UV-induced cell death. Overall, these data open the way to the use of C. reniformis MCHs in drug and cosmetic formulations for damaged or photoaged skin repair.

Speeding Up the Identification of Cystic Fibrosis Transmembrane Conductance Regulator-Targeted Drugs: An Approach Based on Bioinformatics Strategies and Surface Plasmon Resonance.

Cystic fibrosis (CF) is mainly caused by the deletion of Phe 508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR) protein that is thus withheld in the endoplasmic reticulum and rapidly degraded by the ubiquitin/proteasome system. New drugs able to rescue ΔF508-CFTR trafficking are eagerly awaited. An integrated bioinformatics and surface plasmon resonance (SPR) approach was here applied to investigate the rescue mechanism(s) of a series of CFTR-ligands including VX809, VX770 and some aminoarylthiazole derivatives (AAT). Computational studies tentatively identified a large binding pocket in the ΔF508-CFTR nucleotide binding domain-1 (NBD1) and predicted all the tested compounds to bind to three sub-regions of this main pocket. Noticeably, the known CFTR chaperone keratin-8 (K8) seems to interact with some residues located in one of these sub-pockets, potentially interfering with the binding of some ligands. SPR results corroborated all these computational findings. Moreover, for all the considered ligands, a statistically significant correlation was determined between their binding capability to ΔF508-NBD1 measured by SPR and the pockets availability measured by computational studies. Taken together, these results demonstrate a strong agreement between the in silico prediction and the SPR-generated binding data, suggesting a path to speed up the identification of new drugs for the treatment of cystic fibrosis.

Effects on Energy Metabolism of Two Guanidine Molecules, (Boc)2 -Creatine and Metformin.

Several enzymes are involved in the energy production, becoming a possible target for new anti-cancer drugs. In this paper, we used biochemical and in silico studies to evaluate the effects of two guanidine molecules, (Boc)2 -creatine and metformin, on creatine kinase, an enzyme involved in the regulation of intracellular energy levels. Our results show that both drugs inhibit creatine kinase activity; however, (Boc)2 -creatine displays a competitive inhibition, while metformin acts with a non-competitive mechanism. Moreover, (Boc)2 -creatine is able to inhibit the activity of hexokinase with a non-competitive mechanism. Considering that creatine kinase and hexokinase are involved in energy metabolism, we evaluated the effects of (Boc)2 -creatine and metformin on the ATP/AMP ratio and on cellular proliferation in healthy fibroblasts, human breast cancer cells (MDA-MB-468), a human neuroblastoma cell line (SH-SY5Y), a human Hodgkin lymphoma cell line (KMH2). We found that healthy fibroblasts were only partially affected by (Boc)2 -creatine, while both ATP/AMP ratio and viability of the three cancer cell lines were significantly decreased. By inhibiting both creatine kinase and hexokinase, (Boc)2 -creatine appears as a promising new agent in anticancer treatment. Further research is needed to understand what types of cancer cells are most suitable to treatment by this new compound. J. Cell. Biochem. 118: 2700-2711, 2017. © 2017 Wiley Periodicals, Inc.

Abscisic acid transport in human erythrocytes.

Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic ß-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [(3)H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [(3)H]ABA and [(35)S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone.

Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds.

Effects of amide creatine derivatives in brain hippocampal slices, and their possible usefulness for curing creatine transporter deficiency.

The creatine/phosphocreatine system carries ATP from production to consumption sites and buffers the intracellular content of ATP at times of energy deprivation. The creatine transporter deficiency syndrome is an X-linked disease caused by a defective creatine transporter into the central nervous system. This disease is presently untreatable because creatine lacking its carrier cannot cross neither the blood-brain barrier nor the cell plasma membranes. Possible strategies to cure this condition are to couple creatine to molecules which have their own carrier, to exploit the latter to cross biological membranes or to modify the creatine molecule to make it more lipophilic, in such a way that it may more easily cross lipid-rich biological membranes. Such molecules could moreover be useful for treatment of stroke or other ischemic brain syndromes of normal (transporter working) tissue. In this paper we tested four molecules in in vitro hippocampal slices experiments to investigate whether or not they had a neuroprotective effect similar to that of creatine. On two of them we also performed biochemical measurements to investigate whether or not they were able to increase the creatine and phosphocreatine content of the hippocampal slices with and without block of the transporter. We found that these molecules increase levels of creatine after block of the transporter, and significantly increased the levels of phosphocreatine. Both significantly increased the total creatine content in both conditions of active and blocked transporter. This shows that these molecules are capable of entering cells through biological membranes without using the creatine transporter. By contrast, neither of them was able to delay synaptic block during anoxia of normal (transporter functioning) tissue. We conclude that these compounds might possibly be useful for therapy of creatine transporter deficiency, but further research is needed to understand their possible role in anoxia/ischemia of normal tissue.

Trastuzumab quantification in serum: a new, rapid, robust ELISA assay based on a mimetic peptide that specifically recognizes trastuzumab.

Trastuzumab, a humanized monoclonal antibody directed against the epidermal growth factor receptor 2 (HER2), is a milestone in the treatment of HER2-overexpressing breast cancer patients. An enzyme-linked immunosorbent assay (ELISA) for trastuzumab has been developed for routine use in the laboratory to support clinical and pharmacokinetic studies to optimize therapy. The method relies on an antigen peptide linked to a 96-well plate via the streptavidin/biotin system. The peptide sequence mimics the extracellular portion of the HER2 receptor that is recognized by trastuzumab. The calibration range of the assay is 10 to 360 ng/mL per well, corresponding to a trastuzumab serum concentration from 5 to 180 µg/mL with a lower limit of quantification of 10 µg/mL. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum using this assay. The procedure was also tested in sera obtained from breast cancer patients to evaluate trastuzumab serum levels, confirming the applicability of method that could be a valid assay to use in daily laboratory practice.

Recent Advances in the Discovery of SIRT1/2 Inhibitors via Computational Methods: A Perspective.

Sirtuins (SIRTs) are classified as class III histone deacetylases (HDACs), a family of enzymes that catalyze the removal of acetyl groups from the ε-N-acetyl lysine residues of histone proteins, thus counteracting the activity performed by histone acetyltransferares (HATs). Based on their involvement in different biological pathways, ranging from transcription to metabolism and genome stability, SIRT dysregulation was investigated in many diseases, such as cancer, neurodegenerative disorders, diabetes, and cardiovascular and autoimmune diseases. The elucidation of a consistent number of SIRT-ligand complexes helped to steer the identification of novel and more selective modulators. Due to the high diversity and quantity of the structural data thus far available, we reviewed some of the different ligands and structure-based methods that have recently been used to identify new promising SIRT1/2 modulators. The present review is structured into two sections: the first includes a comprehensive perspective of the successful computational approaches related to the discovery of SIRT1/2 inhibitors (SIRTIs); the second section deals with the most interesting SIRTIs that have recently appeared in the literature (from 2017). The data reported here are collected from different databases (SciFinder, Web of Science, Scopus, Google Scholar, and PubMed) using "SIRT", "sirtuin", and "sirtuin inhibitors" as keywords.

Cyclic diacyl thioureas enhance activity of corrector Lumacaftor on F508del-CFTR.

Cystic fibrosis is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. In the search of novel series of CFTR modulators, a library of mono and diacyl thioureas were prepared by sequential synthesis. When tested alone, the obtained compounds 5 and 6 poorly affected F508del-CFTR conductance but, in combination with Lumacaftor, selected derivatives showed the ability to increase the activity of the approved modulator. Analogue 6 i displayed the most marked enhancing effect and acylthioureas 6 d and 6 f were also able to improve efficacy of Lumacaftor. All compounds proved to be non-cytotoxic against different cancer cell lines. Good pharmacokinetic properties were predicted for derivatives 5 and 6, thus supporting the value of these compounds for the development of novel modulators potentially useful for cystic fibrosis.

Comparative analysis of cancer vaccine settings for the selection of an effective protocol in mice.

BACKGROUND: Cancer vaccines are considered a promising therapeutic approach. However, their clinical results are not yet satisfactory. This may be due to the the difficulty of selection of an efficient tumor associated antigen (TAA) and immunization protocol. Indeed, the weak antigenicity of many TAA impairs the design of robust procedures, therefore a systematic analysis to identify the most efficient TAA is mandatory. Here, we performed a study to compare different gp100 vaccination strategies to identify the best strategy to provide a 100% protection against experimental melanoma in a reproducible manner. METHODS: C57BL/6J mice were challenged subcutaneously with B16F10 melanoma cells, after vaccination with: a) mouse or human gp10025-33 peptide plus CpG adjuvant; b) mouse or human gp100 gene; c) mouse or human gp10025-33 peptide-pulsed dendritic cells (DC). Alternatively, a neutralizing anti-IL-10 monoclonal antibody (mAb) was subcutaneously administered at the site of tumor challenge to counteract regulatory cells. Finally, combinatorial treatment was performed associating human gp10025-33 peptide-pulsed DC vaccination with administration of the anti-IL-10 mAb. RESULTS: Vaccination with human gp10025-33 peptide-pulsed DC was the most effective immunization protocol, although not achieving a full protection. Administration of the anti-IL-10 mAb showed also a remarkable protective effect, replicated in mice challenged with a different tumor, Anaplastic Large Cell Lymphoma. When immunization with gp10025-33 peptide-pulsed DC was associated with IL-10 counteraction, a 100% protective effect was consistently achieved. The analysis on the T-cell tumor infiltrates showed an increase of CD4+granzyme+ T-cells and a decreased number of CD4+CD25+Foxp3+ Treg elements from mice treated with either gp10025-33 peptide-pulsed DC vaccination or anti-IL-10 mAb administration. These data suggest that processes of intratumoral re-balance between effector and regulatory T cell subpopulations may play a critical protective role in immunotherapy protocols. CONCLUSIONS: Here we demonstrate that, in the setting of a cancer vaccine strategy, a comparative analysis of different personalized approaches may favour the unveiling of the most effective protocol. Moreover, our findings suggest that counteraction of IL-10 activity may be critical to revert the intratumoral environment promoting Treg polarization, thus increasing the effects of a vaccination against selected TAA.

A new method to synthesize creatine derivatives.

Creatine is an amino acid that has a pivotal role in energy metabolism of cells. Creatine acts as an "ATP shuttle", carrying ATP to the sites where it is utilized, through its reversible phosphorylation by creatine kinase. Moreover, the creatine-phosphocreatine system delays ATP depletion during anoxia or ischemia, thus exerting a neuroprotective role during those pathological conditions. Thus, its administration has been advocated as a treatment or prevention of several conditions involving the central nervous system. However, creatine crosses poorly the blood-brain barrier and the cell plasma membrane, thus its administration has but a limited effect. The use of more lipophilic creatine derivatives has thus been suggested. However, such a synthesis is complicated by the intrinsic characteristics of the creatine molecule that hardly reacts with other molecules and easily cyclizes to creatinine. We obtained amide derivatives from creatine starting from a new protected creatine molecule synthesized by us, the so-called (Boc)2-creatine. We used a temporary protection only on the creatine guanidine group while allowing a good reactivity on the carboxylic group. This temporary protection ensured efficient creatine dissolution in organic solvents and offered simultaneous protection of creatine toward intramolecular cyclization to creatinine. In this manner, it was possible to selectively conjugate molecules on the carboxylic group. The creatine guanidine group was easily released from the protection at the end of the reaction, thus obtaining the desired creatine derivative.

The high-resolution crystal structure of periplasmic Haemophilus influenzae NAD nucleotidase reveals a novel enzymatic function of human CD73 related to NAD metabolism.

Haemophilus influenzae is a major pathogen of the respiratory tract in humans that has developed the capability to exploit host NAD(P) for its nicotinamide dinucleotide requirement. This strategy is organized around a periplasmic enzyme termed NadN (NAD nucleotidase), which plays a central role by degrading NAD into adenosine and NR (nicotinamide riboside), the latter being subsequently internalized by a specific permease. We performed a biochemical and structural investigation on H. influenzae NadN which determined that the enzyme is a Zn2+-dependent 5'-nucleotidase also endowed with NAD(P) pyrophosphatase activity. A 1.3 Å resolution structural analysis revealed a remarkable conformational change that occurs during catalysis between the open and closed forms of the enzyme. NadN showed a broad substrate specificity, recognizing either mono- or di-nucleotide nicotinamides and different adenosine phosphates with a maximal activity on 5'-adenosine monophosphate. Sequence and structural analysis of H. influenzae NadN led us to discover that human CD73 is capable of processing both NAD and NMN, therefore disclosing a possible novel function of human CD73 in systemic NAD metabolism. Our data may prove to be useful for inhibitor design and disclosed unanticipated fascinating evolutionary relationships.