Aiello, Daniele

Costi, Maria Paola

Falchi, Federico

Malpezzi, Giulia

Ponterini, Glauco

Saporito, Giulia

Tagliazucchi, Lorenzo

Venturelli, Alberto

English

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

 Abstract del contributo 1682ID: 1682Comunicazione posterDivisione SCI: Divisione di Chimica FarmaceuticaOptimization of chemical-biological tools for characterizing the activity of dissociative inhibitor of humanthymidylate synthaseSaporito, Giulia1; Aiello, Daniele1; Malpezzi, Giulia1; Tagliazucchi, Lorenzo1; Venturelli, Alberto1; Falchi, Federico2; Ponterini,Glauco1; Costi, Maria Paola11Università di Modena e Reggio Emilia, Italia; 2Università di Bologna , Italia; gsaporit@unimore.itHuman thymidylate synthase (hTS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) to deoxythymidinemonophosphate (dTMP) which requires methylene tetrahydrofolate (mTHF) as a cofactor [1]. This enzyme has been targeted in cancertreatment because of its crucial role. Drugs such as 5-fluorouracil (5-FU) and folate analogues like raltitrexed can bind covalently to theactive site of hTS. However, this mechanism often leads to an increase in the expression of hTS within the cell, resulting in drugresistance. Over the years, it was shown that the hTS is a homodimer of 76 kDa consisting of two identical subunits, each weighing 37kDa. Each subunit contributes to the formation of two activesites and can exist in different states. The dimer can switch between an active state and an inactive state, the dimer is in equilibrium withits monomeric form with a Kd around 6-8 nM. All these states can contribute to compose the broad interactome with different biomoleculartargets within the cell and trigger eventually different specific mechanism. To overcome drug resistance, our research group has focusedon finding new ways to switch the dimer to monomer equilibrium and reduce the enzyme catalytic activity causing its overexpression [2].We aim to target the interface of the dimer to shift the equilibrium towards the inactive monomeric form of hTS. In order to characterize thedissociative character of these newly synthesizedcompounds to shift the equilibrium toward the monomeric form of the protein and inactivate it, different chemical-biological tools were setup: fluorescence-resonance energy tarnsfer (FRET) experiments, kinetic assays with dedicate algorithms, site specific mutagenesis andmore recently dialysis experiments. The present work present the results from the different assays for some representative inhibitors andfocus specifically on the recent results on the dialysis experiments. By conjugating the protein with a fluorescent probe we were able tomonitor its movement in the dialysis device and detect some interesting events such as the dissociative effect of the dimer destabilizer.Inconclusion , we succeed in developing a new chemical biological tools to characterize the dissociative capacity of these newly shythesizedinhibitor , which rapresent a promisimg Medicinal Chemistry strategy .References:[1] Carreras CW, Santi DV. The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem. 1995, 64:721.[2] Costantino, L. et al. Destabilizers of the Thymidylate Synthase Homodimer Accelerate Its Proteasomal Degradation and Inhibit CancerGrowth. Elife 2022, 11, 1–57 

Optimization of chemical-biological tools for characterizing the activity of dissociative inhibitor of human thymidylate synthase

https://iris.unimore.it/bitstream/11380/1337347/1/SCI%202024%20abstract2.pdf

Optimization of chemical-biological tools for characterizing the activity of dissociative inhibitor of human thymidylate synthase

Abstract

Similar works

Full text

Available Versions

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia