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Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL.

Schieferdecker A, Voigt M, Riecken K, Braig F, Schinke T, Loges S, Bokemeyer C, Fehse B, Binder M - Oncotarget (2014)

Bottom Line: The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far.Mutational analysis confirmed the core residues as critical for this interaction.We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Hematology, BMT with section Pneumology, Hubertus Wald Tumorzentrum / UCCH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

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3-dimensional model of the RANK-RANKL-OPG-denosumab interactionA: Lateral view (left) and bottom-up view (right) of the human RANKL trimer (light grey) in complex with three OPG monomers (dark grey). This model was adapted from Luan et al. [pdb 3URF] [3]. B: Dimer of human RANKL (left) and its complex with OPG (right). Amino acid residues of OPG binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Luan et al. [pdb 3URF] [3]. In red, the denosumab epitope is shown. C: Dimer of murine RANKL (left) and its complex with murine RANK (right). Amino acid residues of RANK binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Nelson et al. [pdb 4GIQ] [4]. In red, the denosumab epitope is shown.
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Figure 3: 3-dimensional model of the RANK-RANKL-OPG-denosumab interactionA: Lateral view (left) and bottom-up view (right) of the human RANKL trimer (light grey) in complex with three OPG monomers (dark grey). This model was adapted from Luan et al. [pdb 3URF] [3]. B: Dimer of human RANKL (left) and its complex with OPG (right). Amino acid residues of OPG binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Luan et al. [pdb 3URF] [3]. In red, the denosumab epitope is shown. C: Dimer of murine RANKL (left) and its complex with murine RANK (right). Amino acid residues of RANK binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Nelson et al. [pdb 4GIQ] [4]. In red, the denosumab epitope is shown.

Mentions: Since RANKL has been crystallized in complex with OPG (structural model shown in Figure 3A) and the two binding sites of this natural antagonist of RANKL are known, we wished to explore the spatial relationship between the denosumab epitope and the OPG binding site on RANKL. We therefore displayed both OPG interaction sites on a 3-dimensional model of RANKL (Figure 3B). Interestingly, we found that the denosumab epitope overlapped with the major binding site (binding site II) of OPG on RANKL which corresponds to the major binding site of the RANK receptor on RANKL (Figure 3C). This suggested that denosumab mimics the natural decoy receptor OPG at the molecular level and thereby prevents RANK binding and subsequent bone loss.


Denosumab mimics the natural decoy receptor osteoprotegerin by interacting with its major binding site on RANKL.

Schieferdecker A, Voigt M, Riecken K, Braig F, Schinke T, Loges S, Bokemeyer C, Fehse B, Binder M - Oncotarget (2014)

3-dimensional model of the RANK-RANKL-OPG-denosumab interactionA: Lateral view (left) and bottom-up view (right) of the human RANKL trimer (light grey) in complex with three OPG monomers (dark grey). This model was adapted from Luan et al. [pdb 3URF] [3]. B: Dimer of human RANKL (left) and its complex with OPG (right). Amino acid residues of OPG binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Luan et al. [pdb 3URF] [3]. In red, the denosumab epitope is shown. C: Dimer of murine RANKL (left) and its complex with murine RANK (right). Amino acid residues of RANK binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Nelson et al. [pdb 4GIQ] [4]. In red, the denosumab epitope is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4196153&req=5

Figure 3: 3-dimensional model of the RANK-RANKL-OPG-denosumab interactionA: Lateral view (left) and bottom-up view (right) of the human RANKL trimer (light grey) in complex with three OPG monomers (dark grey). This model was adapted from Luan et al. [pdb 3URF] [3]. B: Dimer of human RANKL (left) and its complex with OPG (right). Amino acid residues of OPG binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Luan et al. [pdb 3URF] [3]. In red, the denosumab epitope is shown. C: Dimer of murine RANKL (left) and its complex with murine RANK (right). Amino acid residues of RANK binding site I (green) and binding site II (blue) on RANKL are displayed. Model and residues were adapted from Nelson et al. [pdb 4GIQ] [4]. In red, the denosumab epitope is shown.
Mentions: Since RANKL has been crystallized in complex with OPG (structural model shown in Figure 3A) and the two binding sites of this natural antagonist of RANKL are known, we wished to explore the spatial relationship between the denosumab epitope and the OPG binding site on RANKL. We therefore displayed both OPG interaction sites on a 3-dimensional model of RANKL (Figure 3B). Interestingly, we found that the denosumab epitope overlapped with the major binding site (binding site II) of OPG on RANKL which corresponds to the major binding site of the RANK receptor on RANKL (Figure 3C). This suggested that denosumab mimics the natural decoy receptor OPG at the molecular level and thereby prevents RANK binding and subsequent bone loss.

Bottom Line: The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far.Mutational analysis confirmed the core residues as critical for this interaction.We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Hematology, BMT with section Pneumology, Hubertus Wald Tumorzentrum / UCCH, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Bone homeostasis critically relies on the RANKL-RANK-OPG axis which can be targeted by the fully human monoclonal antibody denosumab in conditions with increased bone resporption such as bone metastases. The binding site and therefore the molecular mechanism by which this antibody inhibits RANKL has not been characterized so far. Here, we used random peptide phage display library screenings to identify the denosumab epitope on RANKL. Alignments of phage derived peptide sequences with RANKL suggested that this antibody recognized a linear epitope between position T233 and Y241. Mutational analysis confirmed the core residues as critical for this interaction. The spatial localization of this epitope on a 3-dimensional model of RANKL showed that it overlapped with the major binding sites of OPG and RANK on RANKL. We conclude that denosumab inhibits RANKL by both functional and molecular mimicry of the natural decoy receptor OPG.

Show MeSH
Related in: MedlinePlus