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1. Combined FAAH and COX inhibition by Flurbiprofen amide derivatives for the treatment of pain and inflammation

Author

Moraca F, Morgillo CM, Deplano A, Novellino E, Onnis V, Fowler CJ, Catalanotti B, Federica Moraca, Carmine Marco Morgillo, Alessandro Deplano, Ettore Novellino, Valentina Onnis, Christopher J. Fowler, Bruno Catalanotti, Moraca, F, Morgillo, Cm, Deplano, A, Novellino, E, Onnis, V, Fowler, Cj, and Catalanotti, B

Abstract

Nonsteroidal anti‐inflammatory drugs (NSAIDs) – such as ibuprofen and flurbiprofen – are non-selective COX inhibitors widely used to treat acute and chronic pain. Several studies have indicated that the analgesic effect of NSAIDs is enhanced when administered in combination with drugs that inhibit also the fatty acid amide hydrolase (FAAH) [1], an enzyme that degrades endocannabinoid anandamide (AEA), greatly decreasing the severity of GI side effects. From these evidences, arised the rational basis for the design of multi-target FAAH/COX inhibitors [2]. A series of ibuprofen and flurbiprofen amides derivatives have been previously designed as dual FAAH/COX inhibitors and, among them, ibu-am5 and flu-am1 revealed an interesting dual-action, retaining similar COX-inhibitory properties and an increased inhibition of FAAH than the parent compounds [3]. Here, we present the design, molecular modelling and in vitro and in vivo evaluation of a small series of flu-am1 analogs (Figure 1) with an increased dual FAAH/COX inhibition as promising compounds in the treatment of pain and inflammation.

Published
2019

2. Design and pharmacological evaluation of Ibuprofen amides derivatives as dual FAAH/COX inhibitors

Author

Moraca F, Morgillo CM, Deplano A, Novellino E, Onnis V, Fowler CJ, Catalanotti B, Federica Moraca, Carmine Marco Morgillo, Alessandro Deplano, Ettore Novellino, Valentina Onnis, Christopher J. Fowler, Bruno Catalanotti, Moraca, F, Morgillo, Cm, Deplano, A, Novellino, E, Onnis, V, Fowler, Cj, and Catalanotti, B

Abstract

Fatty acid amide hydrolase (FAAH) is a serine hydrolase enzyme responsible of the hydrolytic degradation of N-acylethanolamine endocannabinoids, such as the Arachidonoylethanolamide (anandamide, AEA), which it has been shown to alleviate pain and inflammation (1). In particular, the anti-nociceptive and anti-inflammatory effects of AEA could be enhanced by the simultaneous block of FAAH and COX enzymes (2). For this reason, several studies have been carried out in order to develop new FAAH/COX inhibitors (2). In 1997 it was reported that the NSAID ibuprofen inhibited FAAH, although with a modest potency (3), and successively the first dual inibhitor, the amide derivative of ibuprofen with a 2-amino-3-methylpyridine side chain (Ibu-AM5) was reported (4). -5). Benzylamides and piperazinoamides analogs of Ibuprofen have been also designed as less potent FAAH inhibitors than Ibu-AM5 (5). Here, I discuss the computational studies and the structure–activity relationships leading to the design, of novel Ibuprofen amide derivatives with a higher inhibition potency of FAAH and COX, which represent novel powerful anti-nociceptive agents.

Published
2019

4. Circular dichroism and thermal melting differentiation of Hoechst 33258 binding to the curved (A(4)T(4)) and straight (T(4)A(4)) DNA sequences

Author

Canzonetta C. 1, 2, Caneva R. 1, Savino M. 2, 3, Scipioni A. 4, Catalanotti B. 5, and Galeone A. 5

Abstract

The ability of the B-DNA minor groove ligand Hoechst 33258 to discriminate between prototype curved and straight duplex DNA sequences was investigated by circular dichroism (CD) titrations at the wavelengths of absorbance of the ligand. The sequences were studied either within the framework of the ligated decamers (CAAAATTTTG) n and (CTTTTAAAAG) n, or within that of the single dodecamers GCAAAATTTTGC and GCTTTTAAAAGC, to confirm and extend our earlier results based on fluorescence titrations of ligated decamers. A unique, strong binding site is invariantly present in both sequence units. The binding affinity of the drug for the site in the curved A4T4 sequence was found 3- to 4-fold higher compared to the straight sequence. All these features hold true irrespective of the sequence framework, thus confirming that they reflect specific properties of the binding to the two sequences. Ligand binding increases the thermal stability of straight and curved duplex dodecamers to the same extent, thus maintaining the melting temperature differential between the two sequences. However, the different melting patterns and the difference between [total ligand]:[site] ratios needed for site saturation in the two duplexes are in agreement with the difference between binding constants derived from CD measurements.

Published
2002

6. Specific Targeting Highly Conserved Residues in the HIV-1 Reverse Transcriptase Primer Grip Region. Design, Synthesis, and Biological Evaluation of Novel, Potent, and Broad Spectrum NNRTIs with Antiviral Activity

Author

Fattorusso, C., Gemma, S., Butini, S., Huleatt, P., Catalanotti, B., Persico, M., Angelis, M. De, Fiorini, I., Nacci, V., Ramunno, A., Rodriquez, M., Greco, G., Novellino, E., Bergamini, A., Marini, S., Coletta, M., Maga, G., Spadari, S., and Campiani, G.

Abstract

Pyrrolobenzoxazepinones (PBOs) represent a new class of human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) whose prototype is 5. Molecular modeling studies based on the X-ray structures of HIV-1 RT prompted the synthesis of novel analogues which were tested as anti-HIV agents. The PBO derivatives specifically designed to target the highly conserved amino acid residues within the β12-β13 hairpin, namely primer grip, proved to be very potent against the most common mutant enzymes, including the highly resistant K103N mutant strain. Structure−activity relationships (SARs) are discussed in terms of a possible interaction with the RT binding site, depending on the nature of the substituents at C-6. Among the pyrrolobenzoxazepines investigated, 15c appeared to be the most promising NNRTI of the series characterized by potent antiviral activity, broad spectrum, and low cytotoxicity. 15c showed synergistic antiviral activity with AZT.

Published
2005

7. Pyrrolo[1,5]benzoxa(thia)zepines as a New Class of Potent Apoptotic Agents. Biological Studies and Identification of an Intracellular Location of Their Drug Target

Author

Gee, M. M. Mc, Gemma, S., Butini, S., Ramunno, A., Zisterer, D. M., Fattorusso, C., Catalanotti, B., Kukreja, G., Fiorini, I., Pisano, C., Cucco, C., Novellino, E., Nacci, V., Williams, D. C., and Campiani, G.

Abstract

We have recently developed five novel pyrrolo-1,5-benzoxazepines as proapoptotic agents. Their JNK-dependent induction of apoptosis in tumor cells suggested their potential as novel anticancer agents. The core structure of the apoptotic agent 6 was investigated, and the SARs were expanded with the design and synthesis of several analogues. To define the apoptotic mechanism of the new compounds and the localization of their drug target, two analogues of 6 were designed and synthesized to delineate events leading to JNK activation. The cell-penetrating compound 16 induced apoptosis in tumor cells, while its nonpenetrating analogue, 17, was incapable of inducing apoptosis or activating JNK. Plasma membrane permeabilization of tumor cells resulted in 17-induced JNK activation, suggesting that the pyrrolo-1,5-benzoxazepine molecular target is intracellular. Interestingly, compound 6 displayed cytotoxic activity against a panel of human tumor cell lines but demonstrated negligible toxicity in vivo with no effect on the animals' hematology parameters.

Published
2005

8. Development of Molecular Probes for the Identification of Extra Interaction Sites in the Mid-Gorge and Peripheral Sites of Butyrylcholinesterase (BuChE). Rational Design of Novel, Selective, and Highly Potent BuChE Inhibitors

Author

Campiani, G., Fattorusso, C., Butini, S., Gaeta, A., Agnusdei, M., Gemma, S., Persico, M., Catalanotti, B., Savini, L., Nacci, V., Novellino, E., Holloway, H. W., Greig, N. H., Belinskaya, T., Fedorko, J. M., and Saxena, A.

Abstract

Tacrine heterobivalent ligands were designed as novel and reversible inhibitors of cholinesterases. On the basis of the investigation of the active site gorge topology of butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) and by using flexible docking procedures, molecular modeling studies formulated the hypothesis of extra interaction sites in the active gorge of hBuChE, namely, a mid-gorge interaction site and a peripheral interaction site. The design strategy led to novel BuChE inhibitors, balancing potency and selectivity. Among the compounds identified, the heterobivalent ligand 4m, containing an amide nitrogen and a sulfur atom at the 8-membered tether level, is one of the most potent and selective BuChE inhibitors described to date. The novel inhibitors, bearing postulated key features, validated the hypothesis of the presence of extra interaction sites within the hBuChE active site gorge.

Published
2005

9. Novel Atypical Antipsychotic Agents:  Rational Design, an Efficient Palladium-Catalyzed Route, and Pharmacological Studies

Author

Campiani, G., Butini, S., Fattorusso, C., Trotta, F., Gemma, S., Catalanotti, B., Nacci, V., Fiorini, I., Cagnotto, A., Mereghetti, I., Mennini, T., Minetti, P., Cesare, M. A. Di, Stasi, M. A., Serio, S. Di, Ghirardi, O., Tinti, O., and Carminati, P.

Abstract

Using rational drug design to develop atypical antipsychotic drug candidates, we generated novel and metabolically stable pyrrolobenzazepines with an optimized pKi 5-HT2A/D2 ratio. 5a, obtained by a new palladium-catalyzed three-step synthesis, was selected for further pharmacological and biochemical investigations and showed atypical antipsychotic properties in vivo. 5a was active on conditioned avoidance response at 0.56 mg/kg, it had low cataleptic potential and proved to be better than ST1899, clozapine, and olanzapine, representing a new clinical candidate.

Published
2005

10. Benzoxepin-Derived Estrogen Receptor Modulators:  A Novel Molecular Scaffold for the Estrogen Receptor

Author

Lloyd, D. G., Hughes, R. B., Zisterer, D. M., Williams, D. C., Fattorusso, C., Catalanotti, B., Campiani, G., and Meegan, M. J.

Abstract

We present and examine the efficacy of a novel benzoxepin-based scaffold for modulation of the human estrogen receptor. Receptor tolerance of this new molecular scaffold is examined through presentation of experimentally determined antiproliferative effects on human MCF-7 breast tumor cells and measured binding affinities. The effect of functional group substitution on the benzoxepin scaffold is explored through a brief computational structure−activity relationship investigation with molecular simulation.

Published
2004

11. Pyrrolo[1,3]benzothiazepine-Based Serotonin and Dopamine Receptor Antagonists. Molecular Modeling, Further Structure−Activity Relationship Studies, and Identification of Novel Atypical Antipsychotic Agents

Author

Campiani, G., Butini, S., Fattorusso, C., Catalanotti, B., Gemma, S., Nacci, V., Morelli, E., Cagnotto, A., Mereghetti, I., Mennini, T., Carli, M., Minetti, P., Cesare, M. A. Di, Mastroianni, D., Scafetta, N., Galletti, B., Stasi, M. A., Castorina, M., Pacifici, L., Vertechy, M., Serio, S. D., Ghirardi, O., Tinti, O., and Carminati, P.

Abstract

Recently we reported the pharmacological characterization of the 9,10-dihydropyrrolo[1,3]benzothiazepine derivative (S)-(+)-8 as a novel atypical antipsychotic agent. This compound had an optimum pKi 5-HT2A/D2 ratio of 1.21 (pKi 5-HT2A = 8.83; pKi D2 = 7.79). The lower D2 receptor affinity of (S)-(+)-8 compared to its enantiomer was explained by the difficulty in reaching the conformation required to optimally fulfill the D2 pharmacophore. With the aim of finding novel atypical antipsychotics we further investigated the core structure of (S)-(+)-8, synthesizing analogues with specific substituents; the structure−activity relationship (SAR) study was also expanded with the design and synthesis of other analogues characterized by a pyrrolo[2,1-b][1,3]benzothiazepine skeleton, substituted on the benzo-fused ring or on the pyrrole system. On the 9,10-dihydro analogues the substituents introduced on the pyrrole ring were detrimental to affinity for dopamine and for 5-HT2A receptors, but the introduction of a double bond at C-9/10 on the structure of (S)-(+)-8 led to a potent D2/5-HT2A receptor ligand with a typical binding profile (9f, pKi 5-HT2A/D2 ratio of 1.01, log Y = 8.43). Then, to reduce D2 receptor affinity and restore atypicality on unsaturated analogues, we exploited the effect of specific substitutions on the tricyclic system of 9f. Through a molecular modeling approach we generated a novel series of potential atypical antipsychotic agents, with optimized 5HT2A/D2 receptor affinity ratios and that were easier to synthesize and purify than the reference compound (S)-(+)-8. A number of SAR trends were identified, and among the analogues synthesized and tested in binding assays, 9d and 9m were identified as the most interesting, giving atypical log Y scores respectively 4.98 and 3.18 (pKi 5-HT2A/D2 ratios of 1.20 and 1.30, respectively). They had a multireceptor affinity profile and could be promising atypical agents. Compound 9d, whose synthesis is easier and whose binding profile is atypical (log Y score similar to that of olanzapine, 3.89), was selected for further biological investigation. Pharmacological and biochemical studies confirmed an atypical antipsychotic profile in vivo. The compound was active on conditioned avoidance response at 1.1 mg/kg, a dose 100-times lower than that required to cause catalepsy (ED50 >90 mg/kg), it induced a negligible increase of prolactin serum levels after single and multiple doses, and antagonized the cognitive impairment induced by phencyclidine. In conclusion, the pharmacological profile of 9d proved better than clozapine and olanzapine, making this compound a potential clinical candidate.

Published
2004

12. Synthesis and Pharmacological Evaluation of Potent and Highly Selective D<INF>3</INF> Receptor Ligands:  Inhibition of Cocaine-Seeking Behavior and the Role of Dopamine D<INF>3</INF>/D<INF>2</INF> Receptors

Author

Campiani, G., Butini, S., Trotta, F., Fattorusso, C., Catalanotti, B., Aiello, F., Gemma, S., Nacci, V., Novellino, E., Stark, J. A., Cagnotto, A., Fumagalli, E., Carnovali, F., Cervo, L., and Mennini, T.

Abstract

The synthesis, pharmacological evaluation, and structure−activity relationships (SARs) of a series of novel arylalkylpiperazines structurally related to BP897 (3) are described. In binding studies, the new derivatives were tested against a panel of dopamine, serotonin, and noradrenaline receptor subtypes. Focusing mainly on dopamine D3 receptors, SAR studies brought to light a number of structural features required for high receptor affinity and selectivity. Several heteroaromatic systems were explored for their dopamine receptor affinities, and combinations of synthesis, biology, and molecular modeling, were used to identify novel structural leads for the development of potent and selective D3 receptor ligands. Introduction of an indole ring linked to a dichlorophenylpiperazine system provided two of the most potent and selective ligands known to date (D3 receptor affinity in the picomolar range). The intrinsic pharmacological properties of a subset of potent D3 receptor ligands were also assessed in [35S]-GTPγS binding assays. Evidence from animal studies, in particular, has highlighted the dopaminergic system's role in how environmental stimuli induce drug-seeking behavior. We therefore tested two novel D3 receptor partial agonists and a potent D3-selective antagonist in vivo for their effect in the cocaine-seeking behavior induced by reintroduction of cocaine-associated stimuli after a long period of abstinence, and without any further cocaine. Compound 5g, a nonselective partial D3 receptor agonist with a pharmacological profile similar to 3, and 5p, a potent and selective D3 antagonist, reduced the number of active lever presses induced by reintroduction of cocaine-associated stimuli. However, 5q, a highly potent and selective D3 partial agonist, did not have any effect on cocaine-seeking behavior. Although brain uptake studies are needed to establish whether the compounds achieve brain concentrations comparable to those active in vitro on the D3 receptor, our experiments suggest that antagonism at D2 receptors might significantly contribute to the reduction of cocaine craving by partial D3 agonists.

Published
2003

13. Specific Targeting of Acetylcholinesterase and Butyrylcholinesterase Recognition Sites. Rational Design of Novel, Selective, and Highly Potent Cholinesterase Inhibitors

Author

Savini, L., Gaeta, A., Fattorusso, C., Catalanotti, B., Campiani, G., Chiasserini, L., Pellerano, C., Novellino, E., McKissic, D., and Saxena, A.

Abstract

Tacrine-based AChE and BuChE inhibitors were designed by investigating the topology of the active site gorge of the two enzymes. The homobivalent ligands characterized by a nitrogen-bridged atom at the tether level could be considered among the most potent and selective cholinesterase inhibitors described to date. The nitrogen-containing homobivalent ligands 3e,g and the sulfur-containing 3h validated the hypothesis of extra sites of interaction in the AChE and BuChE active site gorges.

Published
2003

14. Pyrrolo[1,3]benzothiazepine-Based Atypical Antipsychotic Agents. Synthesis, Structure−Activity Relationship, Molecular Modeling, and Biological Studies

Author

Campiani, G., Butini, S., Gemma, S., Nacci, V., Fattorusso, C., Catalanotti, B., Giorgi, G., Cagnotto, A., Goegan, M., Mennini, T., Minetti, P., Cesare, M. A. Di, Mastroianni, D., Scafetta, N., Galletti, B., Stasi, M. A., Castorina, M., Pacifici, L., Ghirardi, O., Tinti, O., and Carminati, P.

Abstract

The prototypical dopamine and serotonin antagonist (±)-7-chloro-9-(4-methylpiperazin-1-yl)-9,10-dihydropyrrolo[2,1-b][1,3]benzothiazepine (5) was resolved into its R and S enantiomers via crystallization of the diastereomeric tartaric acid salts. Binding studies confirmed that the (R)-(−)-enantiomer is a more potent D2 receptor antagonist than the (S)-(+)-enantiomer, with almost identical affinity at the 5-HT2 receptor ((S)-(+)-5, log Y = 4.7; (R)-(−)-5, log Y = 7.4). These data demonstrated a significant stereoselective interaction of 5 at D2 receptors. Furthermore, enantiomer (S)-(+)-5 (ST1460) was tested on a panel of receptors; this compound showed an intriguing binding profile characterized by high affinity for H1 and the α1 receptor, a moderate affinity for α2 and D3 receptors, and low affinity for muscarinic receptors. Pharmacological and biochemical investigation confirmed an atypical pharmacological profile for (S)-(+)-5. This atypical antipsychotic lead has low propensity to induce catalepsy in rat. It has minimal effect on serum prolactin levels, and it has been selected for further pharmacological studies. (S)-(+)-5 increases the extracellular levels of dopamine in the rat striatum after subcutaneous administration. By use of 5 as the lead compound, a novel series of potential atypical antipsychotics has been developed, some of them being characterized by a stereoselective interaction at D2 receptors. A number of structure−activity relationships trends have been identified, and a possible explanation is advanced in order to account for the observed stereoselectivity of the enantiomer of (±)-5 for D2 receptors. The molecular structure determination of the enantiomers of 5 by X-ray diffraction and molecular modeling is reported.

Published
2002

15. A Rational Approach to the Design of Selective Substrates and Potent Nontransportable Inhibitors of the Excitatory Amino Acid Transporter EAAC1 (EAAT3). New Glutamate and Aspartate Analogues as Potential Neuroprotective Agents

Author

Campiani, G., Angelis, M. De, Armaroli, S., Fattorusso, C., Catalanotti, B., Ramunno, A., Nacci, V., Novellino, E., Grewer, C., Ionescu, D., Rauen, T., Griffiths, R., Sinclair, C., Fumagalli, E., and Mennini, T.

Abstract

Two three-dimensional receptor interaction models for EAAT substrates and nontransportable inhibitors have been developed, and new glutamate (Glu) and aspartate (Asp) analogues have been synthesized. The analogues 1a and 3 represent novel lead compounds for the development of EAAT substrates and nontransportable inhibitors, selective for EAATs over iGluRs, as possible neuroprotective agents useful to minimize the progression of chronic or acute neurodegenerative diseases. The role played by the protonatable amine function in the interaction with EAATs has been discussed.

Published
2001

19. Development of potent and selective peripheral 5-HT3 receptor agonists and antagonists, keystones of a new therapeutic strategy to cardiac diseases

Author

Budriesi, R., Ioan, P., Chiarini, A., Campiani, G., Stefania Butini, Fattorusso, C., Morelli, E., Catalanotti, B., Mennini, T., Budriesi, R., Ioan, P., Chiarini, A., Campiani, G., Butini, S., Fattorusso, Caterina, Morelli, Elena, Catalanotti, Bruno, Mennini, T., R. Budriesi, P. Ioan, A. Chiarini, G. Campiani, S. Butini, C. Fattorusso, E. Morelli, B. Catalanotti, and T. Mennini

Subjects
Pharmacology & Pharmacy, Cardiac Diseases

20. The TBC1D31/praja2 complex controls primary ciliogenesis through PKA‐directed OFD1 ubiquitylation

Author

Corrado Garbi, Bianca Fiorillo, Eduard Stefan, Brunella Franco, Manuela Morleo, Daniela Intartaglia, Omar Torres-Quesada, Emanuela Senatore, Ivan Conte, Bruno Catalanotti, Federica Moraca, Laura Rinaldi, Giuliana Giamundo, Marcel Kwiatkowski, Alienke van Pijkeren, Antonio Feliciello, Rossella Delle Donne, Andrea Raffeiner, Francesco Chiuso, Giovanni Scala, Luciano Pirone, Emilia Pedone, Senatore, E, Chiuso, F, Rinaldi, L, Intartaglia, D, Delle Donne, R, Pedone, E, Catalanotti, B, Pirone, L, Fiorillo, B, Moraca, F, Giamundo, G, Scala, G, Raffeiner, A, Torres-Quesada, O, Stefan, E, Kwiatkowski, M, van Pijkeren, A, Morleo, M, Franco, B, Garbi, C, Conte, I, Feliciello, A, Senatore, E., Chiuso, F., Rinaldi, L., Intartaglia, D., Delle Donne, R., Pedone, E., Catalanotti, B., Pirone, L., Fiorillo, B., Moraca, F., Giamundo, G., Scala, G., Raffeiner, A., Torres-Quesada, O., Stefan, E., Kwiatkowski, M., van Pijkeren, A., Morleo, M., Franco, B., Garbi, C., Conte, I., and Feliciello, A.

Subjects
Ubiquitin-Protein Ligases, Oryzias, Ciliopathies, Article, General Biochemistry, Genetics and Molecular Biology, praja2, 03 medical and health sciences, 0302 clinical medicine, Ubiquitin, Two-Hybrid System Techniques, Ciliogenesis, medicine, Animals, Humans, PKA, Membrane & Intracellular Transport, Protein kinase A, Molecular Biology, 030304 developmental biology, 0303 health sciences, General Immunology and Microbiology, biology, General Neuroscience, Cilium, Ubiquitination, Post-translational Modifications, Proteolysis & Proteomics, Articles, medicine.disease, Cyclic AMP-Dependent Protein Kinases, Ubiquitin ligase, Cell biology, Ciliopathy, Centrosome, biology.protein, OFD1, 030217 neurology & neurosurgery, primary cilium, Signal Transduction
Abstract

The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders., OFD1 (oro‐facial digital type I syndrome protein) resides in a centrosomal complex that links GPCR signalling to ubiquitylation and degradation of OFD1, controlling cilium morphology and dynamics and vertebrate development.

Published
2021
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21. Ligand-based drug repurposing strategy identified SARS-CoV-2 RNA G-quadruplex binders

Author

Federica Moraca, Simona Marzano, Francesco D'Amico, Antonio Lupia, Silvia Di Fonzo, Eleonora Vertecchi, Erica Salvati, Anna Di Porzio, Bruno Catalanotti, Antonio Randazzo, Bruno Pagano, Jussara Amato, Moraca, F., Marzano, S., D'Amico, F., Lupia, A., Di Fonzo, S., Vertecchi, E., Salvati, E., Di Porzio, A., Catalanotti, B., Randazzo, A., Pagano, B., and Amato, J.

Subjects
G-quadruplex, SARS-CoV-2, Drug Repositioning, Metals and Alloys, RNA G-quadruplex, DNA, General Chemistry, Ligands, Antiviral Agents, Catalysis, COVID-19 Drug Treatment, Surfaces, Coatings and Films, Electronic, Optical and Magnetic Materials, Molecular Docking Simulation, G-Quadruplexes, Antiviral Agents, COVID-19, Drug Repositioning, Ligands, Molecular Docking Simulation, RNA, SARS-CoV-2, Materials Chemistry, Ceramics and Composites, Humans, RNA, Viral
Abstract

The single-stranded RNA genome of SARS-CoV-2 contains some G-quadruplex-forming G-rich elements which are putative drug targets. Here, we performed a ligand-based pharmacophore virtual screening of FDA approved drugs to find candidates targeting such RNA structures. Further in silico and in vitro assays identified three drugs as emerging SARS-CoV-2 RNA G-quadruplex binders.

Published
2022
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22. Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders

Author

Bianca Fiorillo, Rosalinda Roselli, Claudia Finamore, Michele Biagioli, Cristina di Giorgio, Martina Bordoni, Paolo Conflitti, Silvia Marchianò, Rachele Bellini, Pasquale Rapacciuolo, Chiara Cassiano, Vittorio Limongelli, Valentina Sepe, Bruno Catalanotti, Stefano Fiorucci, Angela Zampella, Fiorillo, B., Roselli, R., Finamore, C., Biagioli, M., di Giorgio, C., Bordoni, M., Conflitti, P., Marchiano, S., Bellini, R., Rapacciuolo, P., Cassiano, C., Limongelli, V., Sepe, V., Catalanotti, B., Fiorucci, S., and Zampella, A.

Subjects
General Chemical Engineering, General Chemistry
Abstract

Retinoic acid receptor-related orphan receptor γ-t (RORγt) and GPBAR1, a transmembrane G-protein-coupled receptor for bile acids, are attractive drug targets to develop clinically relevant small modulators as potent therapeutics for autoimmune diseases. Herein, we designed, synthesized, and evaluated several new bile acid-derived ligands with potent dual activity. Furthermore, we performed molecular docking and MD calculations of the best dual modulators in the two targets to identify the binding modes as well as to better understand the molecular basis of the inverse agonism of RORγt by bile acid derivatives. Among these compounds, 7 was identified as a GPBAR1 agonist (EC50 5.9 μM) and RORγt inverse agonist (IC50 0.107 μM), with excellent pharmacokinetic properties. Finally, the most promising ligand displayed robust anti-inflammatory activity in vitro and in vivo in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis.

Published
2023

23. GLP-1 Mediates Regulation of Colonic ACE2 Expression by the Bile Acid Receptor GPBAR1 in Inflammation

Author

Michele Biagioli, Silvia Marchianò, Rosalinda Roselli, Cristina Di Giorgio, Rachele Bellini, Martina Bordoni, Eleonora Distrutti, Bruno Catalanotti, Angela Zampella, Luigina Graziosi, Annibale Donini, Stefano Fiorucci, Biagioli, M., Marchiano, S., Roselli, R., Di Giorgio, C., Bellini, R., Bordoni, M., Distrutti, E., Catalanotti, B., Zampella, A., Graziosi, L., Donini, A., and Fiorucci, S.

Subjects
Inflammation, Tumor Necrosis Factor-alpha, SARS-CoV-2, ACE2, intestinal inflammation, bile acids, GPBAR1, GLP-1, General Medicine, Colitis, Receptors, G-Protein-Coupled, Bile Acids and Salts, Mice, Crohn Disease, Glucagon-Like Peptide 1, Animals, Humans, bile acid, Angiotensin-Converting Enzyme 2, RNA, Messenger, hormones, hormone substitutes, and hormone antagonists
Abstract

Background & Aims: ACE2, a carboxypeptidase that generates Ang-(1-7) from Ang II, is highly expressed in the lung, small intestine and colon. GPBAR1, is a G protein bile acid receptor that promotes the release of the insulinotropic factor glucagon-like peptide (GLP)-1 and attenuates intestinal inflammation. Methods: We investigated the expression of ACE2, GLP-1 and GPBAR1 in two cohorts of Crohn’s disease (CD) patients and three mouse models of colitis and Gpbar1−/− mice. Activation of GPBAR1 in these models and in vitro was achieved by BAR501, a selective GPBAR1 agonist. Results: In IBD patients, ACE2 mRNA expression was regulated in a site-specific manner in response to inflammation. While expression of ileal ACE2 mRNA was reduced, the colon expression was induced. Colon expression of ACE2 mRNA in IBD correlated with expression of TNF-α and GPBAR1. A positive correlation occurred between GCG and GPBAR1 in human samples and animal models of colitis. In these models, ACE2 mRNA expression was further upregulated by GPABR1 agonism and reversed by exendin-3, a GLP-1 receptor antagonist. In in vitro studies, liraglutide, a GLP-1 analogue, increased the expression of ACE2 in colon epithelial cells/macrophages co-cultures. Conclusions: ACE2 mRNA expression in the colon of IBD patients and rodent models of colitis is regulated in a TNF-α- and GLP-1-dependent manner. We have identified a GPBAR1/GLP-1 mechanism as a positive modulator of ACE2.

Published
2022
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24. Combinatorial targeting of G-protein-coupled bile acid receptor 1 and cysteinyl leukotriene receptor 1 reveals a mechanistic role for bile acids and leukotrienes in drug-induced liver injury

Author

Michele Biagioli, Silvia Marchianò, Cristina di Giorgio, Rosalinda Roselli, Martina Bordoni, Rachele Bellini, Bianca Fiorillo, Valentina Sepe, Bruno Catalanotti, Chiara Cassiano, Maria Chiara Monti, Eleonora Distrutti, Angela Zampella, Stefano Fiorucci, Biagioli, M., Marchiano, S., di Giorgio, C., Roselli, R., Bordoni, M., Bellini, R., Fiorillo, B., Sepe, Valentina, Catalanotti, B., Cassiano, C., Monti, M. C., Distrutti, E., Zampella, Angela, and Fiorucci, S.

Subjects
Hepatology
Abstract

Background and Aim: Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist. Approach and Results: RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl4. Conclusions: By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI.

Published
2022

25. Design, synthesis and in vitro and in vivo biological evaluation of flurbiprofen amides as new fatty acid amide hydrolase/cyclooxygenase-2 dual inhibitory potential analgesic agents

Author

Jessica Karlsson, Federica Moraca, Valentina Onnis, Alessandro Deplano, Mona Svensson, Bruno Catalanotti, Roberto Russo, Claudia Cristiano, Christopher J. Fowler, Carmine Marco Morgillo, Deplano, A., Karlsson, J., Moraca, F., Svensson, M., Cristiano, C., Morgillo, C. M., Fowler, C. J., Russo, R., Catalanotti, B., and Onnis, V.

Subjects
Amide, Male, Models, Molecular, Flurbiprofen, Pharmacology, 01 natural sciences, Mice, Inbred Strain, Rats, Sprague-Dawley, Mice, Fatty acid amide hydrolase, Drug Discovery, fatty acid amide hydrolase, non-steroidal anti-inflammatory drugs, Enzyme Inhibitor, Amidohydrolase, biology, Molecular Structure, Chemistry, Läkemedelskemi, General Medicine, Farmakologi och toxikologi, Endocannabinoid system, cyclooxygenase, Hyperalgesia, flurbiprofen amides, lipids (amino acids, peptides, and proteins), medicine.symptom, medicine.drug, Static Electricity, RM1-950, Flurbiprofen amides, Pharmacology and Toxicology, Inhibitory postsynaptic potential, Flurbiprofen amide, Structure-Activity Relationship, non-steroidal anti-inflammatory drug, In vivo, medicine, Rats, Wistar, allodynia, hyperalgesia, Dose-Response Relationship, Drug, 010405 organic chemistry, Animal, endocannabinoid, In vitro, 0104 chemical sciences, FAAH inhibition, 010404 medicinal & biomolecular chemistry, Cyclooxygenase 2, Drug Design, biology.protein, Quantum Theory, Rat, Analgesic, Cyclooxygenase, Therapeutics. Pharmacology, Medicinal Chemistry
Abstract

Compounds combining dual inhibitory action against FAAH and cyclooxygenase (COX) may be potentially useful analgesics. Here, we describe a novel flurbiprofen analogue, N-(3-bromopyridin-2-yl)-2-(2-fluoro-(1,1'-biphenyl)-4-yl)propanamide (Flu-AM4). The compound is a competitive, reversible inhibitor of FAAH with a Ki value of 13 nM and which inhibits COX activity in a substrate-selective manner. Molecular modelling suggested that Flu-AM4 optimally fits a hydrophobic pocket in the ACB region of FAAH, and binds to COX-2 similarly to flurbiprofen. In vivo studies indicated that at a dose of 10 mg/kg, Flu-AM4 was active in models of prolonged (formalin) and neuropathic (chronic constriction injury) pain and reduced the spinal expression of iNOS, COX-2, and NFκB in the neuropathic model. Thus, the present study identifies Flu-AM4 as a dual-action FAAH/substrate-selective COX inhibitor with anti-inflammatory and analgesic activity in animal pain models. These findings underscore the potential usefulness of such dual-action compounds.

Published
2021

26. Structural Basis for Developing Multitarget Compounds Acting on Cysteinyl Leukotriene Receptor 1 and G-Protein-Coupled Bile Acid Receptor 1

Author

Michele Biagioli, Angela Zampella, Pasquale Rapacciuolo, Vittorio Limongelli, Bianca Fiorillo, Silvia Marchianò, Paolo Conflitti, Bruno Catalanotti, Pasquale De Luca, Chiara Cassiano, Giuliana Baronissi, Rosalinda Roselli, Stefano Fiorucci, Valentina Sepe, Fiorillo, B., Sepe, V., Conflitti, P., Roselli, R., Biagioli, M., Marchiano, S., De Luca, P., Baronissi, G., Rapacciuolo, P., Cassiano, C., Catalanotti, B., Zampella, A., Limongelli, V., and Fiorucci, S.

Subjects
Drug, media_common.quotation_subject, Leukotriene D4, Receptors, G-Protein-Coupled, Mice, Structure-Activity Relationship, 03 medical and health sciences, 0302 clinical medicine, Drug Discovery, Animals, Humans, Receptor, 030304 developmental biology, G protein-coupled receptor, media_common, Receptors, Leukotriene, 0303 health sciences, Cellular process, Chemistry, Macrophages, Anti-Inflammatory Agents, Non-Steroidal, SUPERFAMILY, Colitis, Ligand (biochemistry), G protein-coupled bile acid receptor, 3. Good health, Molecular Docking Simulation, Cysteinyl leukotriene receptor 1, RAW 264.7 Cells, Biochemistry, 030220 oncology & carcinogenesis, Molecular Medicine, Protein Binding
Abstract

G-protein-coupled receptors (GPCRs) are the molecular target of 40% of marketed drugs and the most investigated structures to develop novel therapeutics. Different members of the GPCRs superfamily can modulate the same cellular process acting on diverse pathways, thus representing an attractive opportunity to achieve multitarget drugs with synergic pharmacological effects. Here, we present a series of compounds with dual activity toward cysteinyl leukotriene receptor 1 (CysLT1R) and G-protein-coupled bile acid receptor 1 (GPBAR1). They are derivatives of REV5901─the first reported dual compound─with therapeutic potential in the treatment of colitis and other inflammatory processes. We report the binding mode of the most active compounds in the two GPCRs, revealing unprecedented structural basis for future drug design studies, including the presence of a polar group opportunely spaced from an aromatic ring in the ligand to interact with Arg792.60 of CysLT1R and achieve dual activity.

Published
2021

27. Exploring the fatty acid amide hydrolase and cyclooxygenase inhibitory properties of novel amide derivatives of ibuprofen

Author

Mona Svensson, Jessica Karlsson, Valentina Onnis, Christopher J. Fowler, Bruno Catalanotti, Federica Moraca, Alessandro Deplano, Deplano, A, Karlsson, J, Svensson, M, Moraca, F, Catalanotti, B, Fowler, Cj, and Onnis, V

Subjects
Ibuprofen, 01 natural sciences, Rats, Sprague-Dawley, chemistry.chemical_compound, Fatty acid amide hydrolase, Amide, Drug Discovery, fatty acid amide hydrolase, Enzyme Inhibitors, biology, Molecular Structure, Chemistry, Ibuprofen amides, Läkemedelskemi, General Medicine, Farmakologi och toxikologi, Endocannabinoid system, Molecular Docking Simulation, cyclooxygenase, Biochemistry, lipids (amino acids, peptides, and proteins), medicine.drug, Research Paper, RM1-950, Pharmacology and Toxicology, Inhibitory postsynaptic potential, Amidohydrolases, Structure-Activity Relationship, medicine, Animals, Humans, Rats, Wistar, Pharmacology, Sulindac, Dose-Response Relationship, Drug, 010405 organic chemistry, endocannabinoid, Amides, digestive system diseases, 0104 chemical sciences, Rats, FAAH inhibition, 010404 medicinal & biomolecular chemistry, Cyclooxygenase 2, Ibuprofen amides, FAAH inhibition, fatty acid amide hydrolase, endocannabinoid, cyclooxygenase, biology.protein, Cyclooxygenase 1, Therapeutics. Pharmacology, Cyclooxygenase, Medicinal Chemistry
Abstract

Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting that a dual-action FAAH-cyclooxygenase (COX) inhibitor could have useful therapeutic properties. Here, we have investigated 12 novel amide analogues of ibuprofen as potential dual-action FAAH/COX inhibitors. N-(3-Bromopyridin-2-yl)−2-(4-isobutylphenyl)propanamide (Ibu-AM68) was found to inhibit the hydrolysis of [3H]anandamide by rat brain homogenates by a reversible, mixed-type mechanism of inhibition with a Ki value of 0.26 µM and an α value of 4.9. At a concentration of 10 µM, the compound did not inhibit the cyclooxygenation of arachidonic acid by either ovine COX-1 or human recombinant COX-2. However, this concentration of Ibu-AM68 greatly reduced the ability of the COX-2 to catalyse the cyclooxygenation of the endocannabinoid 2-arachidonoylglycerol. It is concluded that Ibu-AM68 is a dual-acting FAAH/substrate-selective COX inhibitor.

Published
2020

28. 3D GRID-based pharmacophore and Metadynamics approaches for the rational design of N-Methyl β-sheet breaker peptides as inhibitors of the Alzheimer's Aβ-amyloid fibrillogenesis

Author

F. Moraca, M. De Nisco, I. Vespoli, R. Gaglione, A. Arciello, G. Crescente, S. Pacifico, D. Mastroianni, S. Pedatella, B. Catalanotti, F. Moraca, M. De Nisco, I.Vespoli, R. Gaglione, A. Arciello, G. Crescente, S. Pacifico, D. Mastroianni, S. Pedatella, B. Catalanotti, Moraca, F., De Nisco, M., Vespoli, I., Gaglione, R., Arciello, A., Crescente, G., Pacifico, S., Mastroianni, D., Pedatella, S., and Catalanotti, B.

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the loss of the cognitive functions and dementia. Several scientific evidences report that a central role in the pathogenesis of AD is played by the brain deposition of insoluble aggregates of β-amyloid protein (Aβ) proteins, thus causing neuronal cell death [1]. For this reason, one of the promising approach is to inhibit the aggregation of Aβ peptides. Because Aβ is self-assembling, one possible strategy to prevent this process is to use short peptide fragments homologous to the full-length wild-type Aβ protein. From this consideration, several short synthetic peptides were designed as beta-sheet breakers (BSB) [2]. In particular, the pentapetide Ac-LPFFD-NH2 (iAβ5p) exhibited a certain capability to inhibit Aβ fibrillogenesis [3]. iAβ5p analogs [4] were, then, designed by introducing N-Methylation at the amide bond nitrogen were also promising BSB. Here, we describe the methodological approach, which combines 3D GRID-based pharmacophore peptide screening with Well-Tempered Metadynamics simulations aimed to the discovery of novel N-Methylated BSB. This approach led us to identify two promising, cell permeable, N-Methylated peptides that were further evaluated for their BSB properties showing a significant improvement of the fibrillogenesis inhibition with respect to the lead iAβ5p.

Published
2020

29. Hijacking SARS-CoV-2/ACE2 Receptor Interaction by Natural and Semi-synthetic Steroidal Agents Acting on Functional Pockets on the Receptor Binding Domain

Author

Bianca Fiorillo, Daniela Francisci, Bruno Catalanotti, Claudia Finamore, Eleonora Distrutti, Federica Moraca, Michele Biagioli, Adriana Carino, Angela Zampella, Silvia Marchianò, Stefano Fiorucci, Silvia Bozza, Valentina Sepe, Carino, A, Moraca, F, Fiorillo, B, Marchianò, S, Sepe, V, Biagioli, M, Finamore, C, Bozza, S, Francisci, D, Distrutti, E, Catalanotti, B, Zampella, A, and Fiorucci, S

Subjects
In silico, Druggability, 02 engineering and technology, Computational biology, 010402 general chemistry, spike protein, 01 natural sciences, lcsh:Chemistry, chemistry.chemical_compound, drug repurposing and repositioning, Binding site, Receptor, Original Research, bile acids, nutraceuticals, Virtual screening, biology, SARS-CoV-2, Obeticholic acid, COVID-19, General Chemistry, 021001 nanoscience & nanotechnology, virtual screening, Carboxypeptidase, 0104 chemical sciences, Chemistry, lcsh:QD1-999, chemistry, biology.protein, 0210 nano-technology, SARS-CoV-2, COVID-19, virtual screening, nutraceuticals, drug repurposing and repositioning, bile acids, spike protein, Binding domain
Abstract

The coronavirus disease 2019 (COVID-19) is a respiratory tract infection caused by the severe acute respiratory syndrome coronavirus (SARS)-CoV-2. In light of the urgent need to identify novel approaches to be used in the emergency phase, we have embarked on an exploratory campaign aimed at repurposing natural substances and clinically available drugs as potential anti-SARS-CoV2-2 agents by targeting viral proteins. Here we report on a strategy based on the virtual screening of druggable pockets located in the central β-sheet core of the SARS-CoV-2 Spike's protein receptor binding domain (RBD). By combining an in silico approach and molecular in vitro testing we have been able to identify several triterpenoid/steroidal agents that inhibit interaction of the Spike RBD with the carboxypeptidase domain of the Angiotensin Converting Enzyme (ACE2). In detail, we provide evidence that potential binding sites exist in the RBD of the SARS CoV-2 Spike protein and that occupancy of these pockets reduces the ability of the RBD to bind to the ACE2 consensus in vitro. Naturally occurring and clinically available triterpenoids such as glycyrrhetinic and oleanolic acids, as well as primary and secondary bile acids and their amidated derivatives such as glyco-ursodeoxycholic acid and semi-synthetic derivatives such as obeticholic acid reduces the RBD/ACE2 binding. In aggregate, these results might help to define novel approaches to COVID-19 based on SARS-CoV-2 entry inhibitors.

Published
2020

30. Feedback inhibition of cAMP effector signaling by a chaperone-assisted ubiquitin system

Author

Nicola Antonino Russo, Matthis Synofzik, Omar Torres-Quesada, Sonia Piccinin, Federica Moraca, Herbert Lindner, Bruno Catalanotti, Antonio Feliciello, Ulrich Stelzl, Verena Bachmann, Rossella Delle Donne, Laura Rinaldi, Corrado Garbi, Robert Nisticò, Antonella Scorziello, Lucio Annunziato, Francesco Chiuso, Eduard Stefan, Florian Enzler, Rinaldi, L., Delle Donne, R., Catalanotti, B., Torres-Quesada, O., Enzler, F., Moraca, F., Nistico', ROBERT GIOVANNI, Chiuso, Francesco, Piccinin, S., Bachmann, V., Lindner, H. H., Garbi, C., Scorziello, A., Russo, N. A., Synofzik, M., Stelzl, U., Annunziato, L., Stefan, Eduard, and Feliciello, A.

Subjects
0301 basic medicine, pathology [Spinocerebellar Ataxias], Molecular Chaperone, Ubiquitin-Protein Ligase, Ubiquitylation, Leupeptins, metabolism [Cyclic AMP-Dependent Protein Kinase Catalytic Subunits], benzyloxycarbonylleucyl-leucyl-leucine aldehyde, General Physics and Astronomy, 02 engineering and technology, drug effects [Feedback, Physiological], Hippocampus, Mice, Ubiquitin, HEK293 Cell, Cyclic AMP, genetics [Spinocerebellar Ataxias], antagonists & inhibitors [HSP70 Heat-Shock Proteins], Proteolysi, Phosphorylation, Receptor, lcsh:Science, metabolism [Molecular Chaperones], genetics [Ubiquitin-Protein Ligases], Feedback, Physiological, HSP70 Heat-Shock Protein, Multidisciplinary, biology, Chemistry, Effector, pharmacology [Purine Nucleosides], Settore BIO/14, 021001 nanoscience & nanotechnology, 3. Good health, Ubiquitin ligase, Cell biology, VER 155008, pharmacology [Leupeptins], Holoenzyme, drug effects [Protein Binding], Fibroblast, ddc:500, 0210 nano-technology, metabolism [Cyclic AMP], Stub1 protein, mouse, Human, Cell signalling, Protein Binding, Signal Transduction, drug effects [Signal Transduction], congenital, hereditary, and neonatal diseases and abnormalities, Science, Ubiquitin-Protein Ligases, Primary Cell Culture, Leupeptin, Purine Nucleoside, General Biochemistry, Genetics and Molecular Biology, metabolism [Holoenzymes], Article, 03 medical and health sciences, metabolism [Ubiquitin-Protein Ligases], Hippocampu, physiology [Signal Transduction], Animals, Humans, Spinocerebellar Ataxias, HSP70 Heat-Shock Proteins, Protein kinase A, STUB1 protein, human, Spinocerebellar Ataxia, Cyclic AMP-Dependent Protein Kinase Catalytic Subunits, Animal, HEK 293 cells, drug effects [Proteolysis], Ubiquitination, General Chemistry, Purine Nucleosides, Fibroblasts, physiology [Feedback, Physiological], Mice, Inbred C57BL, 030104 developmental biology, pathology [Hippocampus], HEK293 Cells, Ubiquitin ligases, Chaperone (protein), Proteolysis, biology.protein, lcsh:Q, Cyclic AMP-Dependent Protein Kinase Catalytic Subunit, Holoenzymes, physiology [Ubiquitination], Molecular Chaperones
Abstract

Activation of G-protein coupled receptors elevates cAMP levels promoting dissociation of protein kinase A (PKA) holoenzymes and release of catalytic subunits (PKAc). This results in PKAc-mediated phosphorylation of compartmentalized substrates that control central aspects of cell physiology. The mechanism of PKAc activation and signaling have been largely characterized. However, the modes of PKAc inactivation by regulated proteolysis were unknown. Here, we identify a regulatory mechanism that precisely tunes PKAc stability and downstream signaling. Following agonist stimulation, the recruitment of the chaperone-bound E3 ligase CHIP promotes ubiquitylation and proteolysis of PKAc, thus attenuating cAMP signaling. Genetic inactivation of CHIP or pharmacological inhibition of HSP70 enhances PKAc signaling and sustains hippocampal long-term potentiation. Interestingly, primary fibroblasts from autosomal recessive spinocerebellar ataxia 16 (SCAR16) patients carrying germline inactivating mutations of CHIP show a dramatic dysregulation of PKA signaling. This suggests the existence of a negative feedback mechanism for restricting hormonally controlled PKA activities., How intracellular cAMP activate PKA is well-characterized, but PKA inactivation remains poorly understood. Here, Rinaldi et al. show that CHIP/HSP70 ubiquitinates the catalytic subunit of PKA, with implications for the human disease spinocerebellar ataxia 16, as patients often have CHIP mutations.

Published
2019
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31. Discovery of a AHR pelargonidin agonist that counter-regulates Ace2 expression and attenuates ACE2-SARS-CoV-2 interaction

Author

Anna Gidari, Cristina Di Giorgio, Martina Bordoni, Michele Biagioli, Angela Zampella, Rosalinda Roselli, Gabriele Costantino, Bruno Catalanotti, Stefano Fiorucci, Rachele Bellini, Silvia Marchianò, Bianca Fiorillo, Eleonora Distrutti, Adriana Carino, Samuele Sabbatini, Daniela Francisci, Biagioli, M., Marchiano, S., Roselli, R., Di Giorgio, C., Bellini, R., Bordoni, M., Gidari, A., Sabbatini, S., Francisci, D., Fiorillo, B., Catalanotti, B., Distrutti, E., Carino, A., Zampella, A., Costantino, G., and Fiorucci, S.

Subjects
Anthocyanin, Male, 0301 basic medicine, Ahr, ACE2, Intestinal inflammation, Pharmacology, Biochemistry, Protein Structure, Secondary, Pelargonidin, Anthocyanins, Mice, chemistry.chemical_compound, 0302 clinical medicine, Fatty acid binding, Chlorocebus aethiops, Drug Discovery, NF-kB, Receptor, Mice, Knockout, biology, Hep G2 Cells, 030220 oncology & carcinogenesis, Angiotensin-converting enzyme 2, Angiotensin-Converting Enzyme 2, medicine.symptom, hormones, hormone substitutes, and hormone antagonists, Human, Antagonists & inhibitors, Hep G2 Cell, Mice, Transgenic, Inflammation, Chlorocebus aethiop, Gene Expression Regulation, Enzymologic, Article, 03 medical and health sciences, Downregulation and upregulation, medicine, Animals, Humans, Vero Cells, Dose-Response Relationship, Drug, Animal, SARS-CoV-2, Aryl hydrocarbon receptor, Protein Structure, Tertiary, Mice, Inbred C57BL, 030104 developmental biology, Receptors, Aryl Hydrocarbon, chemistry, TNF-α, Vero Cell, biology.protein
Abstract

Graphical abstract Pelargonidin down-regulates Ace2 expression and inhibits binding of the SARS-Cov-2 virus on the host cell ACE2 receptor. Inflammatory stimuli lead to the release of TNF-α which binds to its receptor present on epithelial cells of the colon. The activation of TNF-α receptor induces an activation of NF-kB that migrates to the nucleus where activates the transcription of several genes including Il-6 and Ace2. Pelargonidin exerts a protective effect through AHR which blocks NF-kB translocation into the nucleus (indicated with a red line because this mechanism has not been demonstrated in this study but in previous studies [57,58]) and thereby inhibiting the expression of Ace2. Pelargonidin also directly inhibits the binding of the SARS-Cov-2 virus to the ACE2 receptor., The severe acute respiratory syndrome (SARS)-CoV-2 is the pathogenetic agent of Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE)2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV-2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of Ace2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin activates the Aryl hydrocarbon Receptor (AHR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent TNBS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of Ace2 mRNA expression. Colon levels of Ace2 mRNA were directly correlated with Tnf-α mRNA levels. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV-2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV-2 Spike protein to ACE2 and reduces the SARS-CoV-2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.

Published
2021
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32. Targeting multiple G-quadruplex–forming DNA sequences: Design, biophysical and biological evaluations of indolo-naphthyridine scaffold derivatives

Author

Jussara Amato, Claudia Sissi, Federica Moraca, Lisa Dalla Via, Bruno Pagano, Antonio Lupia, Stefano Alcaro, Antonio Randazzo, Raffaella Catalano, Roberta Rocca, Riccardo Rigo, Giosuè Costa, Bruno Catalanotti, Annalisa Maruca, Camilla Cristofari, Anna Artese, Ettore Novellino, Catalano, R., Moraca, F., Amato, J., Cristofari, C., Rigo, R., Via, L. D., Rocca, R., Lupia, A., Maruca, A., Costa, G., Catalanotti, B., Artese, A., Pagano, B., Randazzo, A., Sissi, C., Novellino, E., and Alcaro, S.

Subjects
Models, Molecular, Biophysical characterization, Scaffold, Indoles, Ligands, Drug Screening Assays, 01 natural sciences, chemistry.chemical_compound, Biophysical characterization, G-quadruplex DNA, Ligands, Molecular modelling, Multi-target, Oncogene promoters, Models, Drug Discovery, Tumor Cells, Cultured, Multi-target, 0303 health sciences, Cultured, Molecular Structure, Chemistry, DNA, Neoplasm, General Medicine, Tumor Cells, G-quadruplex DNA, Molecular modelling, Oncogene promoters, Antineoplastic Agents, Base Sequence, Cell Proliferation, Dose-Response Relationship, Drug, Drug Screening Assays, Antitumor, G-Quadruplexes, Humans, Naphthyridines, Structure-Activity Relationship, Drug Design, Drug, G-quadruplex, DNA sequencing, Dose-Response Relationship, 03 medical and health sciences, Structure–activity relationship, Molecule, 030304 developmental biology, Pharmacology, 010405 organic chemistry, Organic Chemistry, Molecular, DNA, Antitumor, Combinatorial chemistry, 0104 chemical sciences, Förster resonance energy transfer, Duplex (building), Neoplasm
Abstract

It is well recognized that the non-canonical DNA structures known as G-quadruplexes (G4s) have a potential anticancer significance and several compounds have been discovered and evaluated as promising G4 binders with anticancer activity. Here, starting from a promising hit with an indolo-naphthyridine scaffold, a small series of five indolo-naphthyridine based derivatives have been designed and evaluated as G4-targeting compounds. FRET biophysical studies were performed on multiple DNA G4 structures, leading to the identification of a multi-target G4 stabilizer with a slight preference for the c-KIT1 and a good G4 over duplex selectivity. The good affinity of this compound against c-KIT1 G4 was also confirmed by SPR and MST experiments, while biological assays revealed its cytotoxic activity on tumour cells. Finally, Molecular Dynamics simulations helped to elucidate the stabilization effect of the selected compound against the c-KIT1 G4.

Published
2019

33. Neuronal High-Affinity Sodium-Dependent Glutamate Transporters (EAATs): Targets for the Development of Novel Therapeutics Against Neurodegenerative Diseases

Author

Ettore Novellino, Isabella Fiorini, Vito Nacci, Bruno Catalanotti, Stefania Butini, Meri De Angelis, Roberto Fattorusso, Giuseppe Campiani, Caterina Fattorusso, Campiani, G., Fattorusso, Caterina, DE ANGELIS, M., Catalanotti, Bruno, Butini, S., Fattorusso, R., Fiorini, I., Nacci, V., Novellino, Ettore, Fattorusso, C., Catalanotti, B., Fattorusso, Roberto, and Novellino, E.

Subjects
Synaptic cleft, Amino Acid Transport System X-AG, Molecular Conformation, Glutamic Acid, Biology, Neurotransmission, Ligands, Neuroprotection, Drug Discovery, Animals, Humans, Neurotoxin, Neurons, Pharmacology, Molecular Structure, Cell Membrane, Sodium, Glutamate receptor, Metabotropic glutamate receptor 6, Neurodegenerative Diseases, Neuroprotective Agents, Receptors, Glutamate, Biochemistry, Metabotropic glutamate receptor, Drug Design, NMDA receptor, Neuroscience
Abstract

L-Glutamate is the major excitatory neurotransmitter in mammalian central nervous system, and excitatory amino acid transporters (EAATs) are essential for terminating synaptic excitation and for maintaining extracellular glutamate concentration below toxic levels. Although the structure of these channel-like proteins has not been yet reported, their membrane topology has been hypothesised based on biochemical and protein sequence analyses. In the case of an inadequate clearance from synaptic cleft and from the extrasynaptic space, glutamate behaves as a potent neurotoxin, and it may be related to several neurodegenerative pathologies including epilepsy, ischemia, amyotrophic lateral sclerosis, and Alzheimer disease. The recent boom of glutamate is demonstrated by the enormous amount of publications dealing with the function of glutamate, with its role on modulation of synaptic transmission throughout the brain, mainly focusing: i). on the structure of its receptors, ii). on molecular biology and pharmacology of Glu transporters, and iii). on the role of glutamate uptake and reversal uptake in several neuropathologies. This review will deal with the recent and most interesting published results on Glu transporters membrane topology, Glu transporters physiopathological role and Glu transporters medicinal chemistry, highlighting the guidelines for the development of potential neuroprotective agents targeting neuronal high-affinity sodium-dependent glutamate transporters.

Published
2003

34. Repurposing FDA-approved drugs to target G-quadruplexes in breast cancer.

Author

Moraca F, Arciuolo V, Marzano S, Napolitano F, Castellano G, D'Aria F, Di Porzio A, Landolfi L, Catalanotti B, Randazzo A, Pagano B, Malfitano AM, and Amato J

Subjects
Humans, Female, Molecular Structure, Structure-Activity Relationship, Dose-Response Relationship, Drug, United States Food and Drug Administration, Cell Line, Tumor, United States, G-Quadruplexes drug effects, Drug Repositioning, Breast Neoplasms drug therapy, Breast Neoplasms pathology, Antineoplastic Agents pharmacology, Antineoplastic Agents chemistry, Antineoplastic Agents chemical synthesis, Cell Proliferation drug effects, Drug Screening Assays, Antitumor, Drug Approval
Abstract

Breast cancer, a leading cause of cancer-related mortality in women, is characterized by genomic instability and aberrant gene expression, often influenced by noncanonical nucleic acid structures such as G-quadruplexes (G4s). These structures, commonly found in the promoter regions and 5'-untranslated RNA sequences of several oncogenes, play crucial roles in regulating transcription and translation. Stabilizing these G4 structures offers a promising therapeutic strategy for targeting key oncogenic pathways. In this study, we employed a drug repurposing approach to identify FDA-approved drugs capable of binding and stabilizing G4s in breast cancer-related genes. Using ligand-based virtual screening and biophysical methods, we identified several promising compounds, such as azelastine, belotecan, and irinotecan, as effective G4 binders, with significant antiproliferative effects in breast cancer cell lines. Notably, belotecan and irinotecan exhibited a synergistic mechanism, combining G4 stabilization with their established topoisomerase I inhibition activity to enhance cytotoxicity in cancer cells. Our findings support the therapeutic potential of G4 stabilization in breast cancer, validate drug repurposing as an efficient strategy to identify G4-targeting drugs, and highlight how combining G4 stabilization with other established drug activities may improve anticancer efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Masson SAS. All rights reserved.)

Published
2025
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35. Microbial-derived bile acid reverses inflammation in IBD via GPBAR1 agonism and RORγt inverse agonism.

Author

Biagioli M, Di Giorgio C, Massa C, Marchianò S, Bellini R, Bordoni M, Urbani G, Roselli R, Lachi G, Morretta E, Piaz FD, Charlier B, Fiorillo B, Catalanotti B, Cari L, Nocentini G, Ricci P, Distrutti E, Festa C, Sepe V, Zampella A, Monti MC, and Fiorucci S

Subjects
Animals, Humans, Male, Mice, Female, Feces microbiology, Feces chemistry, Disease Models, Animal, Inflammation drug therapy, Inflammation metabolism, Macrophages metabolism, Macrophages drug effects, Middle Aged, Adult, Bile Acids and Salts metabolism, Inflammatory Bowel Diseases drug therapy, Inflammatory Bowel Diseases metabolism, Inflammatory Bowel Diseases microbiology, Gastrointestinal Microbiome drug effects, Nuclear Receptor Subfamily 1, Group F, Member 3 metabolism, Mice, Inbred C57BL, Receptors, G-Protein-Coupled agonists, Receptors, G-Protein-Coupled metabolism, Colitis drug therapy, Colitis metabolism, Colitis microbiology
Abstract

The interplay between the dysbiotic microbiota and bile acids is a critical determinant for development of a dysregulated immune system in inflammatory bowel disease (IBD). Here we have investigated the fecal bile acid metabolome, gut microbiota composition, and immune responses in IBD patients and murine models of colitis and found that IBD associates with an elevated excretion of primary bile acids while secondary, allo- and oxo- bile acids were reduced. These changes correlated with the disease severity, mucosal expression of pro-inflammatory cytokines and chemokines, and reduced inflow of anti-inflammatory macrophages and Treg in the gut. Analysis of bile acids metabolome in the feces allowed the identification of five bile acids: 3-oxo-DCA, 3-oxo-LCA, allo-LCA, iso-allo-LCA and 3-oxo-UDCA, whose excretion was selectively decreased in IBD patients and diseased mice. By transactivation assay and docking calculations all five bile acids were shown to act as GPBAR1 agonists and RORγt inverse agonists, skewing Th17/Treg ratio and macrophage polarization toward an M2 phenotype. In a murine model of colitis, administration of 3-oxo-DCA suffices to reverse colitis development and intestinal dysbiosis in a GPBAR1-dependent manner. In vivo administration of 3-oxo-DCA to colitic mice also reverses disease severity and RORγt activation induced by a RORγt agonist and IL-23, a Th17 inducing cytokine. These results demonstrated that intestinal excretion of 3-oxoDCA, a dual GPBAR1 agonist and RORγt inverse agonist, is reduced in IBD and in models of colitis and its restitution protects against colitis development, highlighting a potential role for this agent in IBD management., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Published
2024
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36. Design, Synthesis, and Pharmacological Evaluation of Dual FXR-LIFR Modulators for the Treatment of Liver Fibrosis.

Author

Rapacciuolo P, Finamore C, Giorgio CD, Fiorillo B, Massa C, Urbani G, Marchianò S, Bordoni M, Cassiano C, Morretta E, Spinelli L, Lupia A, Moraca F, Biagioli M, Sepe V, Monti MC, Catalanotti B, Fiorucci S, and Zampella A

Subjects
Humans, Animals, Male, Structure-Activity Relationship, Mice, Isoxazoles pharmacology, Isoxazoles chemistry, Isoxazoles chemical synthesis, Isoxazoles therapeutic use, Isoxazoles pharmacokinetics, Mice, Inbred C57BL, Receptors, Cytoplasmic and Nuclear agonists, Receptors, Cytoplasmic and Nuclear antagonists & inhibitors, Receptors, Cytoplasmic and Nuclear metabolism, Drug Design, Liver Cirrhosis drug therapy, Liver Cirrhosis pathology
Abstract

Although multiple approaches have been suggested, treating mild-to-severe fibrosis in the context of metabolic dysfunction associated with liver disease (MASLD) remains a challenging area in drug discovery. Pathogenesis of liver fibrosis is multifactorial, and pathogenic mechanisms are deeply intertwined; thus, it is well accepted that future treatment requires the development of multitarget modulators. Harnessing the 3,4,5-trisubstituted isoxazole scaffold, previously described as a key moiety in Farnesoid X receptor (FXR) agonism, herein we report the discovery of a novel class of hybrid molecules endowed with dual activity toward FXR and the leukemia inhibitory factor receptor (LIFR). Up to 27 new derivatives were designed and synthesized. The pharmacological characterization of this series resulted in the identification of 3a as a potent FXR agonist and LIFR antagonist with excellent ADME properties. In vitro and in vivo characterization identified compound 3a as the first-in-class hybrid LIFR inhibitor and FXR agonist that protects against the development of acute liver fibrosis and inflammation.

Published
2024
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38. Bile acids serve as endogenous antagonists of the Leukemia inhibitory factor (LIF) receptor in oncogenesis.

Author

Di Giorgio C, Morretta E, Lupia A, Bellini R, Massa C, Urbani G, Bordoni M, Marchianò S, Lachi G, Rapacciuolo P, Finamore C, Sepe V, Chiara Monti M, Moraca F, Natalizi N, Graziosi L, Distrutti E, Biagioli M, Catalanotti B, Donini A, Zampella A, and Fiorucci S

Subjects
Humans, Carcinogenesis, Leukemia Inhibitory Factor metabolism, Receptors, OSM-LIF, Interleukin-6, Receptors, Cytokine metabolism
Abstract

The leukemia inhibitory factor (LIF) is member of interleukin (IL)-6 family of cytokines involved immune regulation, morphogenesis and oncogenesis. In cancer tissues, LIF binds a heterodimeric receptor (LIFR), formed by a LIFRβ subunit and glycoprotein(gp)130, promoting epithelial mesenchymal transition and cell growth. Bile acids are cholesterol metabolites generated at the interface of host metabolism and the intestinal microbiota. Here we demonstrated that bile acids serve as endogenous antagonist to LIFR in oncogenesis. The tissue characterization of bile acids content in non-cancer and cancer biopsy pairs from gastric adenocarcinomas (GC) demonstrated that bile acids accumulate within cancer tissues, with glyco-deoxycholic acid (GDCA) functioning as negative regulator of LIFR expression. In patient-derived organoids (hPDOs) from GC patients, GDCA reverses LIF-induced stemness and proliferation. In summary, we have identified the secondary bile acids as the first endogenous antagonist to LIFR supporting a development of bile acid-based therapies in LIF-mediated oncogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2024
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39. Synthesis, biological evaluation and metadynamics simulations of novel N -methyl β-sheet breaker peptides as inhibitors of Alzheimer's β-amyloid fibrillogenesis.

Author

Moraca F, Vespoli I, Mastroianni D, Piscopo V, Gaglione R, Arciello A, De Nisco M, Pacifico S, Catalanotti B, and Pedatella S

Abstract

Several scientific evidences report that a central role in the pathogenesis of Alzheimer's disease is played by the deposition of insoluble aggregates of β-amyloid proteins in the brain. Because Aβ is self-assembling, one possible design strategy is to inhibit the aggregation of Aβ peptides using short peptide fragments homologous to the full-length wild-type Aβ protein. In the past years, several studies have reported on the synthesis of some short synthetic peptides called β-sheet breaker peptides (BSBPs). Herein, we present the synthesis of novel (cell-permeable) N -methyl BSBPs, designed based on literature information on the structural key features of BSBPs. Three-dimensional GRID-based pharmacophore peptide screening combined with PT-WTE metadynamics was performed to support the results of the design and microwave-assisted synthesis of peptides 2 and 3 prepared and analyzed for their fibrillogenesis inhibition activity and cytotoxicity. An HR-MS-based cell metabolomic approach highlighted their cell permeability properties., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Published
2024
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40. The leukemia inhibitory factor regulates fibroblast growth factor receptor 4 transcription in gastric cancer.

Author

Di Giorgio C, Bellini R, Lupia A, Massa C, Urbani G, Bordoni M, Marchianò S, Rosselli R, De Gregorio R, Rapacciuolo P, Sepe V, Morretta E, Monti MC, Moraca F, Cari L, Ullah KRS, Natalizi N, Graziosi L, Distrutti E, Biagioli M, Catalanotti B, Donini A, Zampella A, and Fiorucci S

Subjects
Humans, Cell Line, Tumor, Transcription, Genetic drug effects, Signal Transduction drug effects, Phosphorylation drug effects, Organoids metabolism, Organoids drug effects, Leukemia Inhibitory Factor Receptor alpha Subunit, Stomach Neoplasms genetics, Stomach Neoplasms metabolism, Stomach Neoplasms pathology, Receptor, Fibroblast Growth Factor, Type 4 metabolism, Receptor, Fibroblast Growth Factor, Type 4 genetics, Leukemia Inhibitory Factor metabolism, Leukemia Inhibitory Factor genetics, STAT3 Transcription Factor metabolism, Gene Expression Regulation, Neoplastic drug effects
Abstract

Purpose: The gastric adenocarcinoma (GC) represents the third cause of cancer-related mortality worldwide, and available therapeutic options remain sub-optimal. The Fibroblast growth factor receptors (FGFRs) are oncogenic transmembrane tyrosine kinase receptors. FGFR inhibitors have been approved for the treatment of various cancers and a STAT3-dependent regulation of FGFR4 has been documented in the H.pylori infected intestinal GC. Therefore, the modulation of FGFR4 might be useful for the treatment of GC., Methods: To investigate wich factors could modulate FGFR4 signalling in GC, we employed RNA-seq analysis on GC patients biopsies, human patients derived organoids (PDOs) and cancer cell lines., Results: We report that FGFR4 expression/function is regulated by the leukemia inhibitory factor (LIF) an IL-6 related oncogenic cytokine, in JAK1/STAT3 dependent manner. The transcriptomic analysis revealed a direct correlation between the expression of LIFR and FGFR4 in the tissue of an exploratory cohort of 31 GC and confirmed these findings by two external validation cohorts of GC. A LIFR inhibitor (LIR-201) abrogates STAT3 phosphorylation induced by LIF as well as recruitment of pSTAT3 to the promoter of FGFR4. Furthermore, inhibition of FGFR4 by roblitinib or siRNA abrogates STAT3 phosphorylation and oncogentic effects of LIF in GC cells, indicating that FGFR4 is a downstream target of LIF/LIFR complex. Treating cells with LIR-201 abrogates oncogenic potential of FGF19, the physiological ligand of FGFR4., Conclusions: Together these data unreveal a previously unregnized regulatory mechanism of FGFR4 by LIF/LIFR and demonstrate that LIF and FGF19 converge on the regulation of oncogenic STAT3 in GC cells., (© 2023. The Author(s).)

Published
2024
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41. Exploring the DNA 2 -PNA heterotriplex formation in targeting the Bcl-2 gene promoter: A structural insight by physico-chemical and microsecond-scale MD investigation.

Author

Falanga AP, Lupia A, Tripodi L, Morgillo CM, Moraca F, Roviello GN, Catalanotti B, Amato J, Pastore L, Cerullo V, D'Errico S, Piccialli G, Oliviero G, and Borbone N

Abstract

Peptide Nucleic Acids (PNAs) represent a promising tool for gene modulation in anticancer treatment. The uncharged peptidyl backbone and the resistance to chemical and enzymatic degradation make PNAs highly advantageous to form stable hybrid complexes with complementary DNA and RNA strands, providing higher stability than the corresponding natural analogues. Our and other groups' research has successfully shown that tailored PNA sequences can effectively downregulate the expression of human oncogenes using antigene, antisense, or anti-miRNA approaches. Specifically, we identified a seven bases-long PNA sequence, complementary to the longer loop of the main G-quadruplex structure formed by the bcl2midG4 promoter sequence, capable of downregulating the expression of the antiapoptotic Bcl-2 protein and enhancing the anticancer activity of an oncolytic adenovirus. Here, we extended the length of the PNA probe with the aim of including the double-stranded Bcl-2 promoter among the targets of the PNA probe. Our investigation primarily focused on the structural aspects of the resulting DNA 2 -PNA heterotriplex that were determined by employing conventional and accelerated microsecond-scale molecular dynamics simulations and chemical-physical analysis. Additionally, we conducted preliminary biological experiments using cytotoxicity assays on human A549 and MDA-MB-436 adenocarcinoma cell lines, employing the oncolytic adenovirus delivery strategy., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Published
2024
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42. Correction to "Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders".

Author

Fiorillo B, Roselli R, Finamore C, Biagioli M, di Giorgio C, Bordoni M, Conflitti P, Marchianò S, Bellini R, Rapacciuolo P, Cassiano C, Limongelli V, Sepe V, Catalanotti B, Fiorucci S, and Zampella A

Abstract

[This corrects the article DOI: 10.1021/acsomega.2c07907.]., (© 2023 The Authors. Published by American Chemical Society.)

Published
2023
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43. Molecular and Structural Aspects of Clinically Relevant Mutations of SARS-CoV-2 RNA-Dependent RNA Polymerase in Remdesivir-Treated Patients.

Author

Gratteri C, Ambrosio FA, Lupia A, Moraca F, Catalanotti B, Costa G, Bellocchi M, Carioti L, Salpini R, Ceccherini-Silberstein F, Frazia S, Malagnino V, Sarmati L, Svicher V, Bryant S, Artese A, and Alcaro S

Abstract

(1) Background: SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target to fight COVID-19, and many RdRp inhibitors nucleotide/nucleoside analogs, such as remdesivir, have been identified or are in clinical studies. However, the appearance of resistant mutations could reduce their efficacy. In the present work, we structurally evaluated the impact of RdRp mutations found at baseline in 39 patients treated with remdesivir and associated with a different degree of antiviral response in vivo. (2) Methods: A refined bioinformatics approach was applied to assign SARS-CoV-2 clade and lineage, and to define RdRp mutational profiles. In line with such a method, the same mutations were built and analyzed by combining docking and thermodynamics evaluations with both molecular dynamics and representative pharmacophore models. (3) Results: Clinical studies revealed that patients bearing the most prevalent triple mutant P323L+671S+M899I, which was present in 41% of patients, or the more complex mutational profile P323L+G671S+L838I+D738Y+K91E, which was found with a prevalence of 2.6%, showed a delayed reduced response to remdesivir, as confirmed by the increase in SARS-CoV-2 viral load and by a reduced theoretical binding affinity versus RdRp (ΔGbind WT = -122.70 kcal/mol; ΔGbind P323L+671S+M899I = -84.78 kcal/mol; ΔGbind P323L+G671S+L838I+D738Y+K91E = -96.74 kcal/mol). Combined computational approaches helped to rationalize such clinical observations, offering a mechanistic understanding of the allosteric effects of mutants on the global motions of the viral RNA synthesis machine and in the changes of the interactions patterns of remdesivir during its binding.

Published
2023
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44. Combinatorial targeting of G-protein-coupled bile acid receptor 1 and cysteinyl leukotriene receptor 1 reveals a mechanistic role for bile acids and leukotrienes in drug-induced liver injury.

Author

Biagioli M, Marchianò S, di Giorgio C, Roselli R, Bordoni M, Bellini R, Fiorillo B, Sepe V, Catalanotti B, Cassiano C, Monti MC, Distrutti E, Zampella A, and Fiorucci S

Subjects
Mice, Animals, Bile Acids and Salts metabolism, Arachidonate 5-Lipoxygenase metabolism, Endothelial Cells metabolism, Acetaminophen toxicity, Receptors, G-Protein-Coupled metabolism, Leukotrienes metabolism, GTP-Binding Proteins metabolism, Liver Diseases metabolism, Chemical and Drug Induced Liver Injury etiology, Chemical and Drug Induced Liver Injury metabolism
Abstract

Background and Aim: Drug-induced liver injury (DILI) is a common disorder that involves both direct liver cell toxicity and immune activation. The bile acid receptor, G-protein-coupled bile acid receptor 1 (GPBAR1; Takeda G-protein-coupled receptor 5 [TGR5]), and cysteinyl leukotriene receptor (CYSLTR) 1 are G-protein-coupled receptors activated by bile acids and leukotrienes, exerting opposite effects on cell-to-cell adhesion, inflammation, and immune cell activation. To investigate whether GPBAR1 and CYSLTR1 mutually interact in the development of DILI, we developed an orally active small molecule, CHIN117, that functions as a GPBAR1 agonist and CYSLTR1 antagonist., Approach and Results: RNA-sequencing analysis of liver explants showed that acetaminophen (APAP) intoxication positively modulates the leukotriene pathway, CYSLTR1, 5-lipoxygenase, and 5-lipoxygenase activating protein, whereas GPBAR1 gene expression was unchanged. In mice, acute liver injury induced by orally dosing APAP (500 mg/kg) was severely exacerbated by Gpbar1 gene ablation and attenuated by anti-Cysltr1 small interfering RNA pretreatment. Therapeutic dosing of wild-type mice with CHIN117 reversed the liver damage caused by APAP and modulated up to 1300 genes, including 38 chemokines and receptors, that were not shared by dosing mice with a selective GPBAR1 agonist or CYSLTR1 antagonist. Coexpression of the two receptors was detected in liver sinusoidal endothelial cells (LSECs), monocytes, and Kupffer cells, whereas combinatorial modulation of CYSLTR1 and GPBAR1 potently reversed LSEC/monocyte interactions. CHIN117 reversed liver damage and liver fibrosis in mice administered CCl 4 ., Conclusions: By genetic and pharmacological approaches, we demonstrated that GPBAR1 and CYSLTR1 mutually interact in the development of DILI. A combinatorial approach designed to activate GPBAR1 while inhibiting CYSLTR1 reverses liver injury in models of DILI., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Published
2023
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45. Ubiquitylation of BBSome is required for ciliary assembly and signaling.

Author

Chiuso F, Delle Donne R, Giamundo G, Rinaldi L, Borzacchiello D, Moraca F, Intartaglia D, Iannucci R, Senatore E, Lignitto L, Garbi C, Conflitti P, Catalanotti B, Conte I, and Feliciello A

Subjects
Animals, Protein Transport, Signal Transduction, Receptors, G-Protein-Coupled genetics, Ubiquitination, Cilia metabolism, Bardet-Biedl Syndrome genetics
Abstract

Bardet-Biedl syndrome (BBS) is a ciliopathy characterized by retinal degeneration, obesity, renal abnormalities, postaxial polydactyly, and developmental defects. Genes mutated in BBS encode for components and regulators of the BBSome, an octameric complex that controls the trafficking of cargos and receptors within the primary cilium. Although both structure and function of the BBSome have been extensively studied, the impact of ubiquitin signaling on BBSome is largely unknown. We identify the E3 ubiquitin ligase PJA2 as a novel resident of the ciliary compartment and regulator of the BBSome. Upon GPCR-cAMP stimulation, PJA2 ubiquitylates BBSome subunits. We demonstrate that ubiquitylation of BBS1 at lysine 143 increases the stability of the BBSome and promotes its binding to BBS3, an Arf-like GTPase protein controlling the targeting of the BBSome to the ciliary membrane. Downregulation of PJA2 or expression of a ubiquitylation-defective BBS1 mutant (BBS1 K143R ) affects the trafficking of G-protein-coupled receptors (GPCRs) and Shh-dependent gene transcription. Expression of BBS1 K143R in vivo impairs cilium formation, embryonic development, and photoreceptors' morphogenesis, thus recapitulating the BBS phenotype in the medaka fish model., (© 2023 The Authors. Published under the terms of the CC BY 4.0 license.)

Published
2023
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46. Discovery of BAR502, as potent steroidal antagonist of leukemia inhibitory factor receptor for the treatment of pancreatic adenocarcinoma.

Author

Di Giorgio C, Bellini R, Lupia A, Massa C, Bordoni M, Marchianò S, Rosselli R, Sepe V, Rapacciuolo P, Moraca F, Morretta E, Ricci P, Urbani G, Monti MC, Biagioli M, Distrutti E, Catalanotti B, Zampella A, and Fiorucci S

Abstract

Introduction: The leukemia inhibitory factor (LIF), is a cytokine belonging to IL-6 family, whose overexpression correlate with poor prognosis in cancer patients, including pancreatic ductal adenocarcinoma (PDAC). LIF signaling is mediate by its binding to the heterodimeric LIF receptor (LIFR) complex formed by the LIFR receptor and Gp130, leading to JAK1/STAT3 activation. Bile acids are steroid that modulates the expression/activity of membrane and nuclear receptors, including the Farnesoid-X-Receptor (FXR) and G Protein Bile Acid Activated Receptor (GPBAR1)., Methods: Herein we have investigated whether ligands to FXR and GPBAR1 modulate LIF/LIFR pathway in PDAC cells and whether these receptors are expressed in human neoplastic tissues., Results: The transcriptome analysis of a cohort of PDCA patients revealed that expression of LIF and LIFR is increased in the neoplastic tissue in comparison to paired non-neoplastic tissues. By in vitro assay we found that both primary and secondary bile acids exert a weak antagonistic effect on LIF/LIFR signaling. In contrast, BAR502 a non-bile acid steroidal dual FXR and GPBAR1 ligand, potently inhibits binding of LIF to LIFR with an IC 50 of 3.8 µM., Discussion: BAR502 reverses the pattern LIF-induced in a FXR and GPBAR1 independent manner, suggesting a potential role for BAR502 in the treatment of LIFR overexpressing-PDAC., Competing Interests: Authors AL and FM were employed by Net4Science srl. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Di Giorgio, Bellini, Lupia, Massa, Bordoni, Marchianò, Rosselli, Sepe, Rapacciuolo, Moraca, Morretta, Ricci, Urbani, Monti, Biagioli, Distrutti, Catalanotti, Zampella and Fiorucci.)

Published
2023
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47. Discovery of a Novel Class of Dual GPBAR1 Agonists-RORγt Inverse Agonists for the Treatment of IL-17-Mediated Disorders.

Author

Fiorillo B, Roselli R, Finamore C, Biagioli M, di Giorgio C, Bordoni M, Conflitti P, Marchianò S, Bellini R, Rapacciuolo P, Cassiano C, Limongelli V, Sepe V, Catalanotti B, Fiorucci S, and Zampella A

Abstract

Retinoic acid receptor-related orphan receptor γ-t (RORγt) and GPBAR1, a transmembrane G-protein-coupled receptor for bile acids, are attractive drug targets to develop clinically relevant small modulators as potent therapeutics for autoimmune diseases. Herein, we designed, synthesized, and evaluated several new bile acid-derived ligands with potent dual activity. Furthermore, we performed molecular docking and MD calculations of the best dual modulators in the two targets to identify the binding modes as well as to better understand the molecular basis of the inverse agonism of RORγt by bile acid derivatives. Among these compounds, 7 was identified as a GPBAR1 agonist (EC 50 5.9 μM) and RORγt inverse agonist (IC 50 0.107 μM), with excellent pharmacokinetic properties. Finally, the most promising ligand displayed robust anti-inflammatory activity in vitro and in vivo in a mouse model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis., Competing Interests: The authors declare the following competing financial interest(s): S. F. and A. Z. have filed a patent application in the name of PRECISION BIO-THERAPEUTICS S.R.L., a spinoff of the University of Perugia, on the same compounds described in this paper., (© 2023 The Authors. Published by American Chemical Society.)

Published
2023
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48. Molecular Basis for Non-Covalent, Non-Competitive FAAH Inhibition.

Author

Morgillo CM, Lupia A, Deplano A, Pirone L, Fiorillo B, Pedone E, Luque FJ, Onnis V, Moraca F, and Catalanotti B

Subjects
Humans, Amidohydrolases metabolism, Polyunsaturated Alkamides metabolism, Inflammation drug therapy, Molecular Dynamics Simulation, Endocannabinoids metabolism, Neuralgia drug therapy
Abstract

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.

Published
2022
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49. Repositioning Mifepristone as a Leukaemia Inhibitory Factor Receptor Antagonist for the Treatment of Pancreatic Adenocarcinoma.

Author

Di Giorgio C, Lupia A, Marchianò S, Bordoni M, Bellini R, Massa C, Urbani G, Roselli R, Moraca F, Sepe V, Catalanotti B, Morretta E, Monti MC, Biagioli M, Distrutti E, Zampella A, and Fiorucci S

Subjects
Pregnancy, Female, Humans, Receptors, OSM-LIF genetics, Mifepristone pharmacology, Mifepristone therapeutic use, Drug Repositioning, Hormone Antagonists pharmacology, Pancreatic Neoplasms drug therapy, Pancreatic Neoplasms genetics, Pancreatic Neoplasms pathology, Adenocarcinoma drug therapy, Adenocarcinoma genetics, Carcinoma, Pancreatic Ductal pathology
Abstract

Pancreatic cancer is a leading cause of cancer mortality and is projected to become the second-most common cause of cancer mortality in the next decade. While gene-wide association studies and next generation sequencing analyses have identified molecular patterns and transcriptome profiles with prognostic relevance, therapeutic opportunities remain limited. Among the genes that are upregulated in pancreatic ductal adenocarcinomas (PDAC), the leukaemia inhibitory factor (LIF), a cytokine belonging to IL-6 family, has emerged as potential therapeutic candidate. LIF is aberrantly secreted by tumour cells and promotes tumour progression in pancreatic and other solid tumours through aberrant activation of the LIF receptor (LIFR) and downstream signalling that involves the JAK1/STAT3 pathway. Since there are no LIFR antagonists available for clinical use, we developed an in silico strategy to identify potential LIFR antagonists and drug repositioning with regard to LIFR antagonists. The results of these studies allowed the identification of mifepristone, a progesterone/glucocorticoid antagonist, clinically used in medical abortion, as a potent LIFR antagonist. Computational studies revealed that mifepristone binding partially overlapped the LIFR binding site. LIF and LIFR are expressed by human PDAC tissues and PDAC cell lines, including MIA-PaCa-2 and PANC-1 cells. Exposure of these cell lines to mifepristone reverses cell proliferation, migration and epithelial mesenchymal transition induced by LIF in a concentration-dependent manner. Mifepristone inhibits LIFR signalling and reverses STAT3 phosphorylation induced by LIF. Together, these data support the repositioning of mifepristone as a potential therapeutic agent in the treatment of PDAC.

Published
2022
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50. Discovery of a Potent and Orally Active Dual GPBAR1/CysLT 1 R Modulator for the Treatment of Metabolic Fatty Liver Disease.

Author

Fiorucci S, Rapacciuolo P, Fiorillo B, Roselli R, Marchianò S, Di Giorgio C, Bordoni M, Bellini R, Cassiano C, Conflitti P, Catalanotti B, Limongelli V, Sepe V, Biagioli M, and Zampella A

Abstract

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are two highly prevalent human diseases caused by excessive fat deposition in the liver. Although multiple approaches have been suggested, NAFLD/NASH remains an unmet clinical need. Here, we report the discovery of a novel class of hybrid molecules designed to function as cysteinyl leukotriene receptor 1 (CysLT 1 R) antagonists and G protein bile acid receptor 1 (GPBAR1/TGR5) agonists for the treatment of NAFLD/NASH. The most potent of these compounds generated by harnessing the scaffold of the previously described CystLT 1 R antagonists showed efficacy in reversing liver histopathology features in a preclinical model of NASH, reshaping the liver transcriptome and the lipid and energy metabolism in the liver and adipose tissues. In summary, the present study described a novel orally active dual CysLT 1 R antagonist/GPBAR1 agonist that effectively protects against the development of NAFLD/NASH, showing promise for further development., Competing Interests: The authors declare the following competing financial interest(s): SF and AZ have filed the Italian patent application no. 102020000019210 and the corresponding PCT application (PCT/IB 2021/057,131) in the name of PRECISION BIO-THERAPEUTICS S.R.L, a spinoff of the University of Perugia, on same the compounds described in this paper. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fiorucci, Rapacciuolo, Fiorillo, Roselli, Marchianò, Di Giorgio, Bordoni, Bellini, Cassiano, Conflitti, Catalanotti, Limongelli, Sepe, Biagioli and Zampella.)

Published
2022
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