Morgillo, Carmine Marco (2016) Computational methods applied to drug discovery: the rational design of dual inhibitors of FAAH and COX. [Tesi di dottorato]


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Item Type: Tesi di dottorato
Lingua: English
Title: Computational methods applied to drug discovery: the rational design of dual inhibitors of FAAH and COX
Morgillo, Carmine
Date: 31 March 2016
Number of Pages: 113
Institution: Università degli Studi di Napoli Federico II
Department: Farmacia
Scuola di dottorato: Scienze farmaceutiche
Dottorato: Scienza del farmaco
Ciclo di dottorato: 28
Coordinatore del Corso di dottorato:
D'Auria, Maria
Catalanotti, BrunoUNSPECIFIED
Date: 31 March 2016
Number of Pages: 113
Uncontrolled Keywords: endocannabinoid system, molecular dynamics, drug design
Settori scientifico-disciplinari del MIUR: Area 03 - Scienze chimiche > CHIM/08 - Chimica farmaceutica
Date Deposited: 06 May 2016 12:43
Last Modified: 11 May 2019 01:00


The search for effective and safe drugs in pain-relief treatment represents a great challenge for medicinal chemists. Lipid derived mediators, such as endocannabinoids, may have different roles as agonists of cannabinoid receptors, relieving pain, or as substrates of cyclooxygenase (COX), generating the pro-inflammatory prostamides. Moreover, the tissue-protective endocannabinoid anandamide is metabolised by fatty acid amide hydrolase (FAAH). Therefore, a new challenging approach in pain-relief might be the development of dual action FAAH/COX inhibitors. The purpose of this thesis is to apply computational methods in drug discovery to assist medicinal chemistry studies targeting the rational design of novel FAAH/COX inhibitors, and to exploit structural studies relative to two side projects on other biological targets. The wider project of this thesis explores the mechanism of action and the rational design of novel FAAH/COX dual inhibitors. The reversible mixed type inhibitors Flu-AM1 and Ibu-AM5, derivatives of flurbiprofen and ibuprofen, respectively, retain similar COX inhibitory properties and are more potent FAAH inhibitors than the parent compounds. Applying a combination of molecular docking, MD simulations and free energy evaluation of the ligand-receptor complex, the binding mode of the enantiomer forms of Flu-AM1 and Ibu-AM5 has been found in the substrate access channel of FAAH and has been supported by studies of site-directed mutagenesis. The substitution of the isobutyl group of Ibu-AM5 with 4-(2-(trifluoromethyl)pyridin-4-yl)amino group led to the design of TPA5 derivative, which showed an inhibitory activity (IC50 = 0.59 μM) similar to the lead compound (Ibu-AM5, IC50 = 0.52 μM). Kinetic studies of TPA5 revealed that it is a pure competitive inhibitor of rat FAAH and molecular modeling studies supported a binding mode that overlap the anandamide analog MAFP. Among TPA5 derivatives, compound TPA27 exhibited a 10-fold enhancement in the inhibitory profile against FAAH (IC50 = 0.058 μM). Thermodynamic Integration calculations performed to complete the transformation of TPA5 in TPA27 yielded a free energy difference of 0.3 kcal/mol, which indicates a slight lower affinity of TPA27 with respect to TPA5, in the competitive binding site. Kinetics studies showed that TPA27 could be considered the first non-competitive reversible FAAH inhibitor reported so far, and that it more likely binds to an allosteric site. Differences in the inhibitory potency against rat and mouse FAAH for all compounds studied suggested different aminoacid composition of both competitive and non-competitive binding sites. This information was used as criteria of selection for a putative allosteric site found between the cytosolic port and the interface of the FAAH monomers. Computational studies in the allosteric site allowed the definition of the binding mode of Ibu-AM5 and TPA27. Nevertheless, a series of derivatives of Ibu-AM5 and Flu-AM1 were designed in order to get more information on the structure-activity relationships, leading to the identification of novel derivatives with improved activity against FAAH (Ibu-AM56, IC50 = 0.08 μM; Ibu-AM57, IC50 = 0.1 μM; Flu-AM3, IC50 = 0.02 μM. Finally, the thesis also reports the results of two other projects: i) the design of potential anticancer peptides that interfere in the formation of the tetrameric complex hUbA1/UbcH10/Ub2, key intermediate of the ubiquitination cascade.; ii) structural studies on the hybridization of PNA of different length with miR-509-3p, involved in regulating the expression of the CFTR gene, as a way to validate a potential new strategy for the treatment of Cystic Fibrosis.

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