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. 2011 Aug 23;108(34):E542-9.
doi: 10.1073/pnas.1104829108. Epub 2011 Jul 27.

Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Daniela Cardinale  1 , Giambattista Guaitoli, Donatella Tondi, Rosaria Luciani, Stefan Henrich, Outi M H Salo-Ahen, Stefania Ferrari, Gaetano Marverti, Davide Guerrieri, Alessio Ligabue, Chiara Frassineti, Cecilia Pozzi, Stefano Mangani, Dimitrios Fessas, Remo Guerrini, Glauco Ponterini, Rebecca C Wade, M Paola Costi
Affiliations

Protein-protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Daniela Cardinale et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2011 Sep 20;108(38):16133

Abstract

Human thymidylate synthase is a homodimeric enzyme that plays a key role in DNA synthesis and is a target for several clinically important anticancer drugs that bind to its active site. We have designed peptides to specifically target its dimer interface. Here we show through X-ray diffraction, spectroscopic, kinetic, and calorimetric evidence that the peptides do indeed bind at the interface of the dimeric protein and stabilize its di-inactive form. The "LR" peptide binds at a previously unknown binding site and shows a previously undescribed mechanism for the allosteric inhibition of a homodimeric enzyme. It inhibits the intracellular enzyme in ovarian cancer cells and reduces cellular growth at low micromolar concentrations in both cisplatin-sensitive and -resistant cells without causing protein overexpression. This peptide demonstrates the potential of allosteric inhibition of hTS for overcoming platinum drug resistance in ovarian cancer.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Peptide design and characterization. (A) Interface location of peptide sequences in the crystal structure of hTS. For clarity, only subunit A of hTS (shown as a gray surface with ribbons) plus the regions corresponding to the hC20 and C8 peptides of subunit B (shown as rainbow and violet colored highlighted ribbons, respectively) are shown. (B) Seven octameric peptides (referred to by their first and last residues) were designed to cover the complete sequence of hC20 with an overlap of six residues. The coloring scheme follows Fig. 1A and measured percentage inhibition values are given. (C) Dixon plot, obtained from kinetic inhibition studies, showing mixed-type inhibition of hTS in the presence of different concentrations of the LR peptide (I, x-axis) and different concentrations of dUMP substrate for the differently coloured lines. (D) The CD spectra of the inhibitory peptides (colored lines) show secondary structure, whereas the spectra of the inactive peptides (black lines) do not. The spectrum for the YS peptide is shown in gray because, although it shows some inhibitory activity, its spectrum revealed little secondary structure.
Fig. 2.
Fig. 2.
X-ray crystal structure of the ht-hTS-LR complex. (A) Stereo view of the whole molecule with the LR peptide (ball and stick representation: carbon atoms are coral, nitrogen is blue, oxygen is red and sulfur is yellow) bound to the hTS dimer interface. LR is surrounded by the omit map (blue) contoured at 2.5 σ. Subunits A and B are represented as ribbons, with color ramping from green to red and from violet to blue, respectively. (B) Stereo view reporting a close up of the omit map (coral wire) contoured at 2.5 σ and superimposed to a ball and stick model of the LR peptide (ball and stick representation as above). The two independent subunits of hTS are represented as ribbons and cylinders (subunit A: magenta; subunit B: light blue).
Fig. 3.
Fig. 3.
hTS-LR peptide binding thermodynamics and proposed mechanism of hTS inhibition. (A) Top: Raw data from ITC experiments at 25 °C showing the heat flux recorded for each titration. Bottom: ΔH-vs-r plot where ΔH is the cumulative enthalpy (sum of the peak areas in the top panel) expressed per mole of protein and r is the concentration ratio of total titrated ligand to total protein. The red line represents the best fit of the experimental data (circles) using a thermodynamic model with two species of the protein with a constant population ratio, one of which presents two consecutive binding sites. A fixed stoichiometry of one ligand per binding site was assumed. Errors are within 10% of the reported values. The thermodynamic data for the first and second binding sites, respectively, are: Kb1 = 6.6 × 106 M-1(Kd1 = 151 nM), ΔH1 = -12.3 kJ/mol, TΔS1 = 26.6 kJ/mol, ΔG° = -38.9 kJ/mol1, and Kb2 = 7.8 × 105 M-1(Kd2 = 1.3 μM), ΔH2 = -3.1 kJ/mol, TΔS2 = 30.5 kJ/mol, ΔG° = -33.6 kJ/mol2. (B) Schematic diagram of the proposed mechanism of hTS inhibition in which the dimeric protein is represented by active (light blue) and inactive (magenta) subunits that interact with the substrate (S ≡ dUMP, green) and the inhibitor (L ≡ peptide, orange). The fractions of free monomers, of di-active protein with both active sites occupied by a substrate, and of di-inactive protein with two L molecules bound are assumed to be negligible.
Fig. 4.
Fig. 4.
Inhibition of cell growth by the LR peptide and 5FU for cDDP-sensitive and -resistant cells. (A) 2008 and C13* cells; (B) A2780 and A2780/CP cells. The concentration of LR (red) or 5-FU (blue) is given on the x-axis in μM. The LR Peptide was transfected into cells via a peptide delivery system. Cell survival percentages are the mean ± S.E.M. of at least three separate experiments performed in duplicate. Results were analyzed with Student t tests. *P < 0.05 compared with control cells.
Fig. 5.
Fig. 5.
Effects of LR and 5-FU on hTS and DHFR protein levels, hTS mRNA levels and hTS activity in two ovarian cancer cell lines. (A) Western immunoblot analysis of hTS (above) and DHFR (below) protein levels from 2008 and C13* cells treated for 72 h with the indicated concentrations of 5-FU (blue) and LR peptide (red). The latter was administered via a peptide delivery system. The 35-kDa hTS monomer, with or without its ternary complex, and the 21-kDa hDHFR monomer are reported below the bar graphs of their respective densitometric analyses. Western blot analyses were performed on cytosolic extracts from cells in the exponential phase of growth using anti-hTS and DHFR monoclonal antibodies. Each experiment was carried out three times, and a representative result is shown. An antihuman ß-tubulin mouse antibody was used to verify equal protein loading in the gel. (B) RT-PCR analysis of hTS mRNA levels. hTS expression in 2008 and C13* cells extracts was determined after treatment with the LR peptide or 5-FU for 72 h. The amount of hTS mRNA was normalized by the mRNA of glyceraldehyde-3-phosphate dehydrogenase. The results shown are the means ± S.E.M. of four separate experiments performed in duplicate. (C) Inhibition of intracellular TS activity.
Fig. P1.
Fig. P1.
Identification of peptides that inhibit hTS by binding the protein interface. (A) Some octapeptides from the interface of the dimeric hTS were found to inhibit the hTS protein. (B) The crystal structure of the complex of hTS with one of the peptides showed that the peptide bound at the dimer interface of the inactive form of hTS. (C) From analysis of the experimental data and computational modeling, a model was proposed for the inhibition mechanism involving the stabilization of the di-inactive form of the enzyme by binding of the peptide inhibitor at the dimer interface. The peptides were shown to have effective inhibitory properties in both cisplatin-sensitive and cisplatin-resistant ovarian cancer cell lines.
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