Sistema ETD

Archivio digitale delle tesi discusse presso l'Università di Pisa


Tesi etd-01092018-164301

Tipo di tesi
Tesi di laurea magistrale LM5
Design and synthesis of a series of isoindoline-1,3-diones as AMPK activators
Corso di studi
relatore Prof.ssa La Motta, Concettina
relatore Dott. Quattrini, Luca
Parole chiave
  • AMPK
  • activators
  • ADaM site
  • isoindoline-1
  • 3-diones
Data inizio appello
Data di rilascio
Riassunto analitico
The AMP-activated protein kinase (AMPK) is an αβγ heterotrimer responsible for the regulation of intracellular energy homeostasis, autophagy, mitochondrial biogenesis and anti-inflammatory immune defence. [1] It is a Thr/Ser kinase able to sense the energy status of the cell and thus it can promote the catabolic pathways and inhibit the anabolic functions when needed. AMPK is sensitive to variations in intracellular AMP:ATP and ADP:ATP ratios and it is activated by their rising. [2] In fact, in depletion of energy reserves, AMPK is activated through a direct phosphorylation on the α-Thr172 made by several kinases (LKB1, CaMKKβ and TAK1) [3] or through allosteric activation mediated by the binding of adenosine nucleotides to the sites (1-4) of γ subunit. Since AMPK, as a metabolic regulator, has the master role in energy sensing, drugs able to modulate AMPK activity could be exploited in therapeutic treatments of numerous pathologies such as diabetes, inflammatory diseases and cancer. As a matter of fact, anti-diabetic drugs like metformin have been supposed to owe their therapeutic effects partially thanks to an indirect AMPK activation. In particular, metformin represents a promising anti-cancer treatment even though its efficacy still has to be demonstrated. [4]In addition, it seems that metformin is also able to decrease the production of IL-17 by Th17 cells and thus it may exert an anti-inflammatory effect [5], just like AICAR, a synthetic prodrug miming AMP, which causes allosteric activation of AMPK by binding its γ subunit. [6]
Recently, it has been discovered another binding site, denominated “ADaM site”, situated at the interface between the small N-terminal lobe of α subunit and the carbohydrate-binding module (CBM) of β subunit. At this level, the interaction of molecules like salicylate, compound 991 and A-769662 causes the activation of the kinase, and this gives the opportunity of taking advantage of this activation mechanism in order to design new synthetic modulators of AMPK. For this purpose, in 2006 Abbott laboratories have found the lead A-592107 by means of high throughput screening researches and they have obtained the compound A- 769662 as product of the optimization process that, although it has a good activity in vitro, it still does not show a poor oral bioavailability. [7]
In order to extend the class of AMPK activators targeting the ADaM site, our research group decided to design a novel set of modulators bearing the isoindoline-1,3-dione nucleus (Fig.1). My research team has carried out docking studies exploiting the crystallized structure of the AMPK with A-769662 and found the main residues involved in the interaction of compound 1 (Fig.2), the first molecule of the series to be synthesized, with the ADaM site. These studies suggest that the two carbonyl groups of the isoindoline-1,3-dione core and the carbonyl group of the aryl amide function of 1 act as hydrogen bond acceptors in the interplay with α-Lys31, α-Lys29 and α-Asn48 residues of the binding pocket. In addition, thanks to the collaboration with the Research Institute of Pharmaceutical Sciences of the National University in Seoul, 1 has been shown to have good results on AMPK activation in vitro biological assays (Fig. 3). Therefore, according to the collected data, the aim of my work of thesis has been to synthetize a series of analogues using 1 as the hit compound.
Firstly, we decided to synthesize new molecules bearing the same isoindoline-1,3-dione scaffold of the hit compound substituted in position 5 with an aryl amide function, changing the pendant portion on the imide nitrogen and the substituents on the phenyl ring of the aromatic amide residue (Fig.4). Secondly, the synthetic research has been focused on the development of analogues substituted with the amide function in position 4, in order to evaluate in which way the position of the amide residue can influence the activity on the AMPK (Fig.5). Lastly, with the aim of better filling the cleft forming the ADaM site, the introduction of several substituents on the benzyl ring attached to the imide portion have led to the synthesis of compounds (in Fig.6).
To date, the Research Institute in Seoul is conducting in vitro biological assays to disclose the potency of these compounds as AMPK modulators. Once obtained, these results could allow us to build a structure-activity relationship for this series of compounds.

[1] J. Li, S. Li, F. Wang, and F. Xin, “Structural and biochemical insights into the allosteric activation mechanism of AMP-activated protein kinase,” Chem. Biol. Drug Des., vol. 89, no. 5, pp. 663–669, May 2017.
[2] D. G. Hardie and S. A. Hawley, “AMP-activated protein kinase: the energy charge hypothesis revisited,” BioEssays, vol. 23, no. 12, pp. 1112–1119, Dec. 2001.
[3] C. F. García-Prieto, M. Gil-Ortega, I. Aránguez, M. Ortiz-Besoain, B. Somoza, and M. S. Fernández-Alfonso, “Vascular AMPK as an attractive target in the treatment of vascular complications of obesity,” Vascul. Pharmacol., vol. 67–69, pp. 10–20, Jun. 2015.
[4] B. Faubert, E. E. Vincent, M. C. Poffenberger, and R. G. Jones, “The AMP-activated protein kinase (AMPK) and cancer: Many faces of a metabolic regulator,” Cancer Lett., vol. 356, no. 2, Part A, pp. 165–170, Jan. 2015.
[5] S.-Y. Lee et al., “Metformin Ameliorates Inflammatory Bowel Disease by Suppression of the STAT3 Signaling Pathway and Regulation of the between Th17/Treg Balance,” PLOS ONE, vol. 10, no. 9, p. e0135858, Sep. 2015.
[6] A. Bai et al., “Novel Anti-Inflammatory Action of 5-Aminoimidazole-4-carboxamide Ribonucleoside with Protective Effect in Dextran Sulfate Sodium-Induced Acute and Chronic Colitis,” J. Pharmacol. Exp. Ther., vol. 333, no. 3, pp. 717–725, Jun. 2010.
[7] M. Miglianico, G. A. F. Nicolaes, and D. Neumann, “Pharmacological Targeting of AMP-Activated Protein Kinase and Opportunities for Computer-Aided Drug Design,” J. Med. Chem., vol. 59, no. 7, pp. 2879–2893, Apr. 2016.