Structural insights into the interaction of a monoclonal antibody and Nodal peptides by STD-NMR spectroscopy

Calvanese, Luisa; Focà, Annalia; Sandomenico, Annamaria; Focà, Giuseppina; Caporale, Andrea; Doti, Nunzianna; Iaccarino, Emanuela; Leonardi, Antonio; D’Auria, Gabriella; Ruvo, Menotti; Falcigno, Lucia

doi:10.1016/j.bmc.2017.10.036

Cited by 7 publications

(4 citation statements)

References 46 publications

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“…As the intensity loss of the STD-NMR signal has an r –6 distance dependence, the experiment allows the determination of ligand binding modes via group epitope mapping (GEM), and can therefore be employed to interpret the proximities of ligand protons to the receptor protein. STD-NMR-based GEM is well established for mapping binding epitopes of small molecules, including peptides, − to protein targets.…”

Section: Introductionmentioning

confidence: 99%

Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1

Bumbak

Thomas

Noonan-Williams

et al. 2020

ACS Pharmacol. Transl. Sci.

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Cell–cell communication via endogenous peptides and their receptors is vital for controlling all aspects of human physiology and most peptides signal through G protein-coupled receptors (GPCRs). Disordered peptides bind GPCRs through complex modes for which there are few representative crystal structures. The disordered peptide neurotensin (NT) is a neuromodulator of classical neurotransmitters such as dopamine and glutamate, through activation of neurotensin receptor 1 (NTS1). While several experimental structures show how NT binds NTS1, details about the structural dynamics of NT during and after binding NTS1, or the role of peptide dynamics on receptor activation, remain obscure. Here saturation transfer difference (STD) NMR revealed that the binding mode of NT fragment NT10-13 is heterogeneous. Epitope maps of NT10-13 at NTS1 suggested that tyrosine 11 (Y11) samples other conformations to those observed in crystal structures of NT-bound NTS1. Molecular dynamics (MD) simulations confirmed that when NT is bound to NTS1, residue Y11 can exist in two χ1 rotameric states, gauche plus (g+) or gauche minus (g–). Since only the g+ Y11 state is observed in all the structures solved to date, we asked if the g– state is important for receptor activation. NT analogues with Y11 replaced with 7-OH-Tic were synthesized to restrain the dynamics of the side chain. P(OH-TIC)IL bound NTS1 with the same affinity as NT10-13 but did not activate NTS1, instead acted as an antagonist. This study highlights that flexibility of Y11 in NT may be required for NT activation of NTS1.

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Section: Introductionmentioning

confidence: 99%

Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1

Bumbak

Thomas

Noonan-Williams

et al. 2020

ACS Pharmacol. Transl. Sci.

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“…More interestingly, in vivo, the reduction in pSMAD2 levels is also appreciable in mouse xenografts of C8161 and A375SM human melanoma cell lines, with a sensitive decrease in tumor volume, when compared to a control IgG [94,95]. The biological effects of this Ab are due to the fact that the region of Nodal targeted by the 3D1 is the pre-helix loop and the H3 helix of Nodal, which is the fundamental helix that interacts with CR-1 [96].…”

Section: Monoclonal Antibodiesmentioning

confidence: 97%

New Insights into Cancer Targeted Therapy: Nodal and Cripto-1 as Attractive Candidates

Arboretto

Cillo

Leonardi

2021

IJMS

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The transforming growth factor beta (TGF-β) signaling is fundamental for correct embryonic development. However, alterations of this pathway have been correlated with oncogenesis, tumor progression and sustaining of cancer stem cells (CSCs). Cripto-1 (CR-1) and Nodal are two embryonic proteins involved in TGF-β signaling. Their expression is almost undetectable in terminally differentiated cells, but they are often re-expressed in tumor cells, especially in CSCs. Moreover, cancer cells that show high levels of CR-1 and/or Nodal display more aggressive phenotypes in vitro, while in vivo their expression correlates with a worse prognosis in several human cancers. The ability to target CSCs still represents an unmet medical need for the complete eradication of certain types of tumors. Given the prognostic role and the selective expression of CR-1 and Nodal on cancer cells, they represent archetypes for targeted therapy. The aim of this review is to clarify the role of CR-1 and Nodal in cancer stem populations and to summarize the current therapeutic strategy to target CSCs using monoclonal antibodies (mAbs) or other molecular tools to interfere with these two proteins.

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“…Based on these considerations and given the active role played by Nodal and Cripto-1 as oncogenes and immunotherapeutic targets for CSC [ 20 ], we developed neutralizing mAbs that target specific Nodal [ 21 , 22 , 23 ] and Cripto-1 [ 24 , 25 ] hot spots mediating the interactions with the receptors involved in their activity. Such mAbs, as well as engineered variants, may become powerful agents for cancer immunotherapy or for specifically accumulating other drugs on cancer tissues and cells expressing them.…”

Section: Introductionmentioning

confidence: 99%

“…A previously described anti-Nodal murine monoclonal antibody, named 3D1 against the pre-helix loop of the protein, showed anti-tumor effects in in vitro and in vivo models of melanoma [ 21 , 22 ]. The antibody recognizes specific residues (E49 and E50) of a protein loop involved in the binding to Cripto-1 and prevents their interaction [ 21 , 23 ].…”

Section: Introductionmentioning

confidence: 99%

Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein

et al. 2023

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Drug development in recent years is increasingly focused on developing personalized treatments based on blocking molecules selective for therapeutic targets specifically present in individual patients. In this perspective, the specificity of therapeutic targets and blocking agents plays a crucial role. Monoclonal antibodies (mAbs) and their surrogates are increasingly used in this context thanks to their ability to bind therapeutic targets and to inhibit their activity or to transport bioactive molecules into the compartments in which the targets are expressed. Small antibody-like molecules, such as Fabs, are often used in certain clinical settings where small size and better tissue penetration are required. In the wake of this research trend, we developed a murine mAb (3D1) neutralizing the activity of Nodal, an oncofetal protein that is attracting an ever-increasing interest as a selective therapeutic target for several cancer types. Here, we report the preparation of a recombinant Fab of 3D1 that has been humanized through a computational approach starting from the sequence of the murine antibody. The Fab has been expressed in bacterial cells (1 mg/L bacterial culture), biochemically characterized in terms of stability and binding properties by circular dichroism and bio-layer interferometry techniques and tested in vitro on Nodal-positive cancer cells.

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Structural insights into the interaction of a monoclonal antibody and Nodal peptides by STD-NMR spectroscopy

Cited by 7 publications

References 46 publications

Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1

Conformational Changes in Tyrosine 11 of Neurotensin Are Required to Activate the Neurotensin Receptor 1

New Insights into Cancer Targeted Therapy: Nodal and Cripto-1 as Attractive Candidates

Production in Bacteria and Characterization of Engineered Humanized Fab Fragment against the Nodal Protein

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