The Hippo Signalling cascade is an emerging target in tumour suppression regulation, neoplastic hypertrophy, and regenerative medicine. The pathway is activated by circulating anti-proliferative signals which leads to the phosphorylation of Yes Associated Protein (hYAP1) on Ser127/381, thus 14-3-3σ mediated cytosolic retention. Genetic alterations or exogenous factor may cause YAP nuclear migration and association to TEAD1-4 (Transcription Enhancer Associated Domain), triggering up-regulation of anti-apoptotic genes [1]. hTEAD is an enhancer that activates the nuclear transcription of genes as EMT’s, EGFR and cyclins, and promotes the synthesis of survivin, tyrosine kinase HER3, and mitochondrial Bcl-xL involved in cell proliferation. TEAD binds a palmitic (palm) or myristic (myr) acids, tethered at Cys367 pocket, however its biological role is still not well known. hTEAD isoform-4 is the most represented of its family in solid tumours and its overexpression or mutation leads to cancer development and metastasis. Recent studies have considered hTEAD a promising target for anticancer drugs. Its inhibition strategy includes the disruption/prevention of YAP1:TEAD4 complex formation [2]. With the aim to develop a specific cysteine-directed inhibition strategy, we studied Cys on the protein surface and investigated their reactivity. Hence, our studies focus on characterizing the recombinant hTEAD4-ybd (aa217-434) surface though the analysis of the reactivity of its four Cys thiols (Cys310, Cys335, Cys367, Cys410), all close to YAP binding area. First, myr-Cys-367 was investigated to confirm the auto-myristoilation of the E. coli recombinant hTEAD4 through RP-chromatography on UHPLC-Orbitrap Q-Ex (ThermoFisher™) by multicharged TIC deconvolution, and the total myr-TEAD was assessed around 25%. Myristate position was confirmed by FASP protein tryptic hydrolysis and tandem-MS peptide analysis. We studied hTEAD binding of a small disulphides and thiols library with different chemical properties through the exposed cysteines residues in presence of different concentration of reducing agent [3]. Top8 DDA (HCD)-MS/MS scan on the tryptic peptides suggested the ligands’ high selectivity towards Cys335. Cys367 was never found conjugated, even in the non-Myr fraction, hinting the low accessibility to the lipid pocket. The number of surface reactive Cys was confirmed by a reverse-titration of the protein against increasing amount of thiophenol; excess of unreacted thiophenol was measured by HPLC-UV-ELSD (Agilent™ 1260), suggesting a 1:1 stoichiometry. We confirmed hTEAD-ybd ligand ratio by fluoresceine labelling with absorption and fluorescence differential spectroscopy. The ongoing work engages the screening of a larger compound library to study YAP:TEAD interaction with a ligand displacement assay of labelled TEAD to a rhodamine-tagged peptidomimetic probe to achieve structural information of the heterodimer interface and to start a hit-optimization programme. REFERENCES [1] Santucci M, Vignudelli T, et al. The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment. J Med Chem. 2015 Jun 25;58(12):4857-73. [2] Elisi G.M, Santucci M, et al. Repurposing of Drugs Targeting YAP-TEAD Functions. Cancers 2018, 10, 329. [3] Malpezzi G MSc Degree Thesis, Solvent exposure, and reactivity of the cysteines of Transcription Enhancer Associate Domain (TEAD), a potential anticancer target, 2021. University of Pavia – University of Modena and Reggio Emilia.

Designing selective Cys-ligands to unpair the binding of the Human Transcription Enhancer Associated Domain 4 (hTEAD-4) with its modulators to halt cancer cell growth / Tagliazucchi, Lorenzo; Malpezzi, Giulia; Pozzi, Cecilia; Lopresti, Ludovica; Venturelli, Alberto; D'Arca, Domenico; Marverti, Gaetano; Cecconi, Ciro; Ponterini, Glauco; Costi, Maria Paola. - (2021). ((Intervento presentato al convegno XX Giornata della Chimica dell'Emilia Romagna 2021 (XX GdC-ER 2021) tenutosi a Università degli Studi di Ferrara nel 17/12/2021.

Designing selective Cys-ligands to unpair the binding of the Human Transcription Enhancer Associated Domain 4 (hTEAD-4) with its modulators to halt cancer cell growth

Lorenzo Tagliazucchi
;
Giulia Malpezzi;Alberto Venturelli;Domenico d'Arca;Gaetano Marverti;Ciro Cecconi;Glauco Ponterini;Maria Paola Costi
2021

Abstract

The Hippo Signalling cascade is an emerging target in tumour suppression regulation, neoplastic hypertrophy, and regenerative medicine. The pathway is activated by circulating anti-proliferative signals which leads to the phosphorylation of Yes Associated Protein (hYAP1) on Ser127/381, thus 14-3-3σ mediated cytosolic retention. Genetic alterations or exogenous factor may cause YAP nuclear migration and association to TEAD1-4 (Transcription Enhancer Associated Domain), triggering up-regulation of anti-apoptotic genes [1]. hTEAD is an enhancer that activates the nuclear transcription of genes as EMT’s, EGFR and cyclins, and promotes the synthesis of survivin, tyrosine kinase HER3, and mitochondrial Bcl-xL involved in cell proliferation. TEAD binds a palmitic (palm) or myristic (myr) acids, tethered at Cys367 pocket, however its biological role is still not well known. hTEAD isoform-4 is the most represented of its family in solid tumours and its overexpression or mutation leads to cancer development and metastasis. Recent studies have considered hTEAD a promising target for anticancer drugs. Its inhibition strategy includes the disruption/prevention of YAP1:TEAD4 complex formation [2]. With the aim to develop a specific cysteine-directed inhibition strategy, we studied Cys on the protein surface and investigated their reactivity. Hence, our studies focus on characterizing the recombinant hTEAD4-ybd (aa217-434) surface though the analysis of the reactivity of its four Cys thiols (Cys310, Cys335, Cys367, Cys410), all close to YAP binding area. First, myr-Cys-367 was investigated to confirm the auto-myristoilation of the E. coli recombinant hTEAD4 through RP-chromatography on UHPLC-Orbitrap Q-Ex (ThermoFisher™) by multicharged TIC deconvolution, and the total myr-TEAD was assessed around 25%. Myristate position was confirmed by FASP protein tryptic hydrolysis and tandem-MS peptide analysis. We studied hTEAD binding of a small disulphides and thiols library with different chemical properties through the exposed cysteines residues in presence of different concentration of reducing agent [3]. Top8 DDA (HCD)-MS/MS scan on the tryptic peptides suggested the ligands’ high selectivity towards Cys335. Cys367 was never found conjugated, even in the non-Myr fraction, hinting the low accessibility to the lipid pocket. The number of surface reactive Cys was confirmed by a reverse-titration of the protein against increasing amount of thiophenol; excess of unreacted thiophenol was measured by HPLC-UV-ELSD (Agilent™ 1260), suggesting a 1:1 stoichiometry. We confirmed hTEAD-ybd ligand ratio by fluoresceine labelling with absorption and fluorescence differential spectroscopy. The ongoing work engages the screening of a larger compound library to study YAP:TEAD interaction with a ligand displacement assay of labelled TEAD to a rhodamine-tagged peptidomimetic probe to achieve structural information of the heterodimer interface and to start a hit-optimization programme. REFERENCES [1] Santucci M, Vignudelli T, et al. The Hippo Pathway and YAP/TAZ-TEAD Protein-Protein Interaction as Targets for Regenerative Medicine and Cancer Treatment. J Med Chem. 2015 Jun 25;58(12):4857-73. [2] Elisi G.M, Santucci M, et al. Repurposing of Drugs Targeting YAP-TEAD Functions. Cancers 2018, 10, 329. [3] Malpezzi G MSc Degree Thesis, Solvent exposure, and reactivity of the cysteines of Transcription Enhancer Associate Domain (TEAD), a potential anticancer target, 2021. University of Pavia – University of Modena and Reggio Emilia.
XX Giornata della Chimica dell'Emilia Romagna 2021 (XX GdC-ER 2021)
Università degli Studi di Ferrara
17/12/2021
Tagliazucchi, Lorenzo; Malpezzi, Giulia; Pozzi, Cecilia; Lopresti, Ludovica; Venturelli, Alberto; D'Arca, Domenico; Marverti, Gaetano; Cecconi, Ciro; Ponterini, Glauco; Costi, Maria Paola
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