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Identifier

000466068

Title

Exploring SecA conformation dynamics for the development of effective antibiotics

Alternative Title

Διερεύνηση της δυναμικής διαμόρφωσης της SecA για την ανάπτυξη αποτελεσματικών αντιβιοτικών

Author

Γιώτας, Εμμανουήλ Π.

Thesis advisor

Ελευθεριάδης, Νικόλαος

Reviewer

Παυλίδης, Ιωάννης
Γανωτάκης, Δημήτριος

Abstract

SecA is a motor protein in prokaryotic cells, which participates in the secretion mechanism of pre-proteins outside the cell. It is a crucial partner to Sec pathway and essential for bacteria survival. The inhibition of SecA’ s activity is believed to have a pivotal role in the discovery of new antibiotics. SecA is a promising drug target as it is vital for the bacteria and there is no human counterpart. In this thesis, SecA was produced and purified, and its ATPase activity was used to identify potent inhibitors. A high throughput protocol based on Malachite Green was developed and used to screen more than 100 novel compounds with different properties. The inhibitory concentration was measured for the four most promising compounds and the IC50 values varied from 160 μΜ to 2 mM . The most effective compound, MZ102, used for kinetic studies to evaluate the inhibition type, which found to be competitive. Also, molecular modelling experiments were performed to find the interactions of the best compound inside the ATP binding pocket of the protein, which shown that MZ102 and ATP occupy the same space between NBD1 and NBD2. SmFRET was used to study protein dynamics, and previous findings verified the protocol that was used. Finally, the effect of MZ102 in protein dynamics was investigated, using smFRET, which seemed to slightly affect the monomer state of the protein.

Language

English

Subject

Enzyme kinetics

Inhibitors

Novel antibiotics

smFRET

Αναστολείς

Καινοτόμα αντιβιοτικά

Κινητική ενζύμων

Issue date

2024-07-19