1. Calculate molecular properties of compound libraries including ADME –tox in silico studies
2. Measure molecular properties (pK, logP, solubility, membrane permeability, Ligand efficiency, promiscuous inhibition properties, albumin binding…).
3. Data analysis, scoring the experimental versus in silico data, comparative scoring, software implementation through appropriate algorithm.

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WP4
Library design and compound selection (virtual screening, scaffold selection, library design)
The aim is to design compounds to interfere with TS dimerization by binding to the TS monomer-monomer interface.

The design strategy will be two-pronged:
1. Design of peptides and peptidomimetics based on knowledge that peptides from the sequence of TS can affect dimerization and bind to the TS monomer. These peptides will be further optimized for binding to the TS monomer and libraries of peptidomimetics will be designed.
2. Fragment based de novo ligand design to identify low-affinity ligand/fragments by cysteine tethering and to optimize these to obtain high-affinity ligands.

To aid the ligand design, modelling and simulation will also be performed, for the interaction of TS with its mRNA and the effect of small ligands on this interaction. All ligand design, will be accompanied by in silico ADME screening (see WP4).
All design studies will target the human TS, and involve comparison with the homologous TSs from microorganisms.

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WP5
Scaffold chemistry validation. In parallel/combichem synthesis and new synthetic tools.

1. Scaffold synthesis evaluation for scaffold validation for one to two derivatization points.
2. Library synthesis and synthetic cycle refinement.
3. Peptide/peptide mimetic library synthesis
4. Development of synthetic strategies for selected compounds and preparation of the same
5. Lead optimisation and synthesis of drug candidates
6. Filing of product and process patent application(s)

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WP6
SH-labelling and ligand-protein interaction studies

1. Identification of the highest affinity hits and low affinity ligands in solution. Protein (monomeric form/dimeric form-wt and mutant) fishing out of the best affinity inhibitor.
2. Structural characterization of protein-ligand complexes with compounds. SH-labelling from WP6 for structure-based drug design. Covalent and non covalent complexes.
3. Characterization of the energetics of the interaction small molecule hit-protein, lead-protein (TS monomeric/dimeric form-wild type and mutant).

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WP7
Cellular/molecular pharmacology. In vivo evaluation of the monomer-monomer interface inhibitors against enzymes biolibrary and wt and resistant ovarian carcinoma cell line.

1. Enzyme bio-library preparation, enzyme purification
2. Compounds library rapid screening assay
3. Examination of cytotoxic effects of compounds designed to limit the intracellular levels of TS protein in cDDP-sensitive and cDDP-resistant cell lines and in a non-carcinoma cell line.
4. Evaluation of cell growth effects of TS monomer-monomer interface inhibitors in combination with cisplatin.
5. Characterization of the intracellular molecular mechanisms involved in the action of inhibitors.
6. Evaluation of transcription levels of the folate dependent enzymes in ovarian human cell lines (ATCC).
7. Identification of molecules competing with TS for their binding to mRNA.