Intracellular quantitative detection of human thymidylate synthase engagement with an unconventional inhibitor using tetracysteine-diarsenical-probe technology

Abstract

Demonstrating a candidate drug’s interaction with its target protein in live cells is of pivotal relevance to the successful outcome of the drug discovery process. Although thymidylate synthase (hTS) is an important anticancer target protein, the efficacy of the few anti-hTS drugs currently used in clinical practice is limited by the development of resistance. Hence, there is an intense search for new, unconventional anti-hTS drugs; there are approximately 1600 ongoing clinical trials involving hTS-targeting drugs, both alone and in combination protocols. We recently discovered new, unconventional peptidic inhibitors of hTS that are active against cancer cells and do not result in the overexpression of hTS, which is a known molecular source of resistance. Here, we propose an adaptation of the recently proposed tetracysteine-arsenic-binding-motif technology to detect and quantitatively characterize the engagement of hTS with one such peptidic inhibitor in cell lysates. This new model can be developed into a test for high-throughput screening studies of intracellular target-protein/small-molecule binding.

A Peptidic Thymidylate-Synthase Inhibitor Loaded on Pegylated Liposomes Enhances the Antitumour Effect of Chemotherapy Drugs in Human Ovarian Cancer Cells

“…The novel mechanism of action involves binding at the monomer-monomer interface of the enzyme, rather than at the catalytic pocket, with stabilization of the inactive conformation and decrease of the abundance of the active form of the protein. Among these, the LR (LSCQLYQR) peptide inhibited the growth of ovarian carcinomas (OCs) following transfection by means of a commercial peptide delivery system, SAINT-PhD, while it left the TS cellular levels essentially unchanged [19,20]. More recently, among the peptides developed by modifying the LR lead to improve TS inhibition and the anticancer effect, the D-glutamine-modified peptide at position 4 ([D-Gln 4 ]LR) displayed the best growth inhibition of both cDDP-sensitive and -resistant OC cell lines [21]; it was more active than LR and 5-FU and affected the TS/DHFR expression pattern similarly to LR.…”

Cyclic Peptides Acting as Allosteric Inhibitors of Human Thymidylate Synthase and Cancer Cell Growth

“…To this aim, we previously identified some small peptidic inhibitors that mime a portion of the monomer–monomer interfacial region in the h TS dimer and bind therein [17,18,19,20,21]. More precisely, from a parent peptide, C20, whose sequence came from the interface region of h TS (Figure 1A), we synthesized a series of octapeptides.…”

“…Fluorescence resonance energy transfer (FRET)-based target engagement experiments proved the binding of LR to TS in cells with good target specificity. In lysates of HEK293T cells, 2.5 micromoles/L of LR bound h TS, whereas only 30 nanomoles/L of the inhibitor interacted with other targets [19]. Proteomic experiments allowed the characterization of some proteins that are modulated by the peptides and that characterize the mechanism of action.…”

“…Closely connected with the intracellular activity is the low metabolic stability of the LR peptide [23]. Although a relatively fast proteolytic degradation did not prevent it from engaging the h TS target [19] and exhibiting an effect on cancer cell growth, we still expect higher activity from an increase in biostability.…”

Fluorometric detection of protein-ligand engagement: The case of phosphodiesterase5

“…Site-selective labelling of proteins in living cells can be achieved with this method that has been applied to address a variety of issues involving detection of proteinprotein interactions [10][11][12]. This molecular technology has also been recently employed to detect and quantitatively characterize the engagement of a small-molecule inhibitor with its target enzyme in a cell lysate [13] but, to our knowledge, it has never be applied to purified enzyme. We have expressed the catalytic domain of the PDE5A2 (identifier: O76074-2) with a genetically encoded 6xHis tag at the N-terminus and the 6-amino-acid motif CCPGCC (TC) at the C terminus.…”