Optimization of Peptides That Target Human Thymidylate Synthase to Inhibit Ovarian Cancer Cell Growth

Abstract

Thymidylate synthase (TS) is a target for pemetrexed and the prodrug 5-fluorouracil (5-FU) that inhibit the protein by binding at its active site. Prolonged administration of these drugs causes TS overexpression, leading to drug resistance. The peptide lead, LR (LSCQLYQR), allosterically stabilizes the inactive form of the protein and inhibits ovarian cancer (OC) cell growth with stable TS and decreased dihydrofolate reductase (DHFR) expression. To improve TS inhibition and the anticancer effect, we have developed 35 peptides by modifying the lead. The d-glutamine-modified peptide displayed the best inhibition of cisplatin-sensitive and -resistant OC cell growth, was more active than LR and 5-FU, and showed a TS/DHFR expression pattern similar to LR. Circular dichroism spectroscopy and molecular dynamics studies provided a molecular-level rationale for the differences in structural preferences and the enzyme inhibitory activities. By combining target inhibition studies and the modulation pattern of associated proteins, this work avenues a concept to develop more specific inhibitors of OC cell growth and drug leads.

Mass Spectrometric/Bioinformatic Identification of a Protein Subset That Characterizes the Cellular Activity of Anticancer Peptides

“…35 Figure 6 highlights the similar patterns observed after the LR and [D-Gln 4 ]LR treatments: this result is consistent with the structural similarity of the two peptides and their similar inhibition against purified hTS. 11,12 As expected based on our previous observations, 11,12 hTS and DHFR expression levels remained essentially unchanged or decreased after LR treatment. Interesting results were also obtained for two differentially expressed proteins that were directly or indirectly altered by LR: TRAP1 and HSP90AA1.…”

“…9,10 In an effort to overcome drug resistance induced by these drugs, we have recently identified a new class of peptidic compounds that inhibit hTS with a novel mechanism of action and reduce cancer cell growth. 11,12 Among these, the peptide with the best activity profile, LR (LSCQLYQR), inhibits the intracellular enzyme in cisplatin-sensitive and cisplatin-resistant OC cell lines without causing the typical protein overexpression associated with acquired resistance to PMX, thus showing the potential of this innovative hTS inhibition strategy. 11 These peptides specifically target the hTS dimer interface and stabilize its di-inactive form as shown in the crystallographic structure deposited in the Protein Data Bank (PDB 1HVY) ( Figure 2A,B) and through biophysical studies.…”

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

“…The failure of hTS monomers to regulate hTS mRNA with ensuing hTS overexpression is one the mechanisms of the onset of resistance to TS-targeted drugs 13 14 15 . Following the identification of hotspot residues at the intermonomer interface that are crucial for the stability of the dimeric assembly 16 , we discovered several octapeptidic inhibitors that, differently from classical hTS inhibitors, bind the protein dimer at the monomer/monomer interface and stabilize its inactive conformation 17 18 . Among these inhibitors, peptide LSCQLYQR (LR) was shown to accumulate in cells to steady-state concentrations of several tens of micromoles/litre 19 and was able to inhibit hTS and cancer cell growth without causing the overexpression of the enzyme 17 20 .…”

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

“…The [D-Gln4]LR peptide and its lead, LR, have exhibited cancer cell-growth inhibitory activity by mainly reducing the abundance of the active form of hTS, and, unlike 5-FU and PMX, without inducing overexpression of the enzyme [19,21], but even by down-modulating the expression of other folate pathway genes, DHFR and AICAR transformylase (ATIC) [22]. Cells that acquire resistance to classical TS inhibitors because of an enhanced TS expression exhibit general cross-resistance with platinum-based drugs [3] and display cross-resistance to antifolates such as RTX [27,28].…”

“…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.…”