Structures of ferroportin in complex with its specific inhibitor vamifeport
Curation statements for this article:-
Curated by eLife
eLife assessment
This important study reports cryo-EM structures of human ferroportin (FPN), a protein essential for iron transport in humans. This manuscript will be of interest to researchers studying membrane transport mechanisms as well as to those interested in drug design. The structures detail interactions between FPN and the small-molecule inhibitor vamifeport, which is currently in clinical trials for sickle cell disease, and ta new (occluded) protein conformation that is stabilized by a sybody (a nanobody selected from a synthetic library) is identified. Evidence for the mechanism of inhibition by vamifeport is convincing, but evidence for the physiological relevance of the occluded conformation is still incomplete.
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (eLife)
- Biochemistry and Chemical Biology (eLife)
- Reading list (BiophysicsColab)
Abstract
A central regulatory mechanism of iron homeostasis in humans involves ferroportin (FPN), the sole cellular iron exporter, and the peptide hormone hepcidin, which inhibits Fe 2+ transport and induces internalization and degradation of FPN. Dysregulation of the FPN/hepcidin axis leads to diverse pathological conditions, and consequently, pharmacological compounds that inhibit FPN-mediated iron transport are of high clinical interest. Here, we describe the cryo-electron microscopy structures of human FPN in complex with synthetic nanobodies and vamifeport (VIT-2763), the first clinical-stage oral FPN inhibitor. Vamifeport competes with hepcidin for FPN binding and is currently in clinical development for β-thalassemia and sickle cell disease. The structures display two distinct conformations of FPN, representing outward-facing and occluded states of the transporter. The vamifeport site is located in the center of the protein, where the overlap with hepcidin interactions underlies the competitive relationship between the two molecules. The introduction of point mutations in the binding pocket of vamifeport reduces its affinity to FPN, emphasizing the relevance of the structural data. Together, our study reveals conformational rearrangements of FPN that are of potential relevance for transport, and it provides initial insight into the pharmacological targeting of this unique iron efflux transporter.
Article activity feed
-
-
eLife assessment
This important study reports cryo-EM structures of human ferroportin (FPN), a protein essential for iron transport in humans. This manuscript will be of interest to researchers studying membrane transport mechanisms as well as to those interested in drug design. The structures detail interactions between FPN and the small-molecule inhibitor vamifeport, which is currently in clinical trials for sickle cell disease, and ta new (occluded) protein conformation that is stabilized by a sybody (a nanobody selected from a synthetic library) is identified. Evidence for the mechanism of inhibition by vamifeport is convincing, but evidence for the physiological relevance of the occluded conformation is still incomplete.
-
Reviewer #1 (Public Review):
The study by Lehmann et al. reports novel structures of the human ferroportin (SLC40A1), which is responsible for iron transport in the body. Specifically, ferroportin controls the plasma concentration of iron by transporting Fe2+ out of the cell. To regulate plasma iron concentrations, the liver releases hepcidin, a peptide-based hormone that inhibits ferroportin activity. Specific inhibitors of ferroportin are being developed to treat thalassemia and sickle cell disease, which are diseases that result in reduced red blood cell function.
The present study reports the structure of human ferroportin in complex with one such inhibitor, vamifeport, which is currently in clinical trials for sickle cell disease. The authors use their structures to suggest a mechanism for vamifeport binding to ferroportin and …
Reviewer #1 (Public Review):
The study by Lehmann et al. reports novel structures of the human ferroportin (SLC40A1), which is responsible for iron transport in the body. Specifically, ferroportin controls the plasma concentration of iron by transporting Fe2+ out of the cell. To regulate plasma iron concentrations, the liver releases hepcidin, a peptide-based hormone that inhibits ferroportin activity. Specific inhibitors of ferroportin are being developed to treat thalassemia and sickle cell disease, which are diseases that result in reduced red blood cell function.
The present study reports the structure of human ferroportin in complex with one such inhibitor, vamifeport, which is currently in clinical trials for sickle cell disease. The authors use their structures to suggest a mechanism for vamifeport binding to ferroportin and support the structural data with in vitro binding assays to study the specific interactions made in the binding site. In addition, one of the structures obtained was a novel protein conformation, an occluded state. This is the first occluded state observed for ferroportin, enabling the authors to discuss the implications for understanding the transport mechanism. However, this appears to have resulted in a slightly confusing analysis.
Overall the study is well presented, although in several places appears overly wordy and might benefit from being edited to focus on the main points the authors wish to highlight. For example, the title focuses on the new insights gained from the vamifeport complex. Yet, the discussion section focuses almost entirely on the transport mechanism, with little additional analysis of the mechanism of vamifeport inhibition. In my view, the paper suffers from this disconnect, as the functional data support the vamifeport structure, not the transport mechanism. Yet, the discussion focuses heavily on the transport mechanism, with little reference to the results. Rather, the discussion relies on an in-depth understanding of secondary active transport literature (MFS, NRAMP, etc.).
The data is high quality, and the conclusions drawn about the orientation of the drug in the binding site are sound. This study represents an important advance in understanding iron homeostasis in the human body and current methods to modulate iron transport to treat human disease.
-
Reviewer #2 (Public Review):
This manuscript by Lehman et al. details the structural characterization of human Ferroportin, which builds on the previous structural characterisation of this protein. Here, through the use of synthetic nanobodies, the authors capture the protein in the outward-facing state that has been obtained previously, and a new conformation in an occluded state, information which would advance understanding of the Ferroportin transport mechanism. In addition, the authors capture Ferroportin in complex with the first clinical-stage Ferroportin inhibitor, Vamifeport, which provides insight that could be used to improve inhibitor efficacy to treat human disease. The structural data is very well supported by clear, well-executed, and informative binding and transport studies. These data reveal that the purified protein …
Reviewer #2 (Public Review):
This manuscript by Lehman et al. details the structural characterization of human Ferroportin, which builds on the previous structural characterisation of this protein. Here, through the use of synthetic nanobodies, the authors capture the protein in the outward-facing state that has been obtained previously, and a new conformation in an occluded state, information which would advance understanding of the Ferroportin transport mechanism. In addition, the authors capture Ferroportin in complex with the first clinical-stage Ferroportin inhibitor, Vamifeport, which provides insight that could be used to improve inhibitor efficacy to treat human disease. The structural data is very well supported by clear, well-executed, and informative binding and transport studies. These data reveal that the purified protein is functionally active, able to interact with the peptide-based inhibitor hepcidin in addition to Vamifeport and that hepcidin and vamifeport bind competitively. Site-directed mutagenesis and binding assays were used to convincingly validate the Vamifeport binding site.
Overall, the conclusions in this manuscript are well supported by the data, in particular those relating to inhibitor binding. However, as the authors point out, the occluded state captured here contains an unexpectedly large aqueous cavity compared to the size of the transported substrate. With this peculiar observation in mind, the requirement for the presence of Sy3 nanobody to capture this state and the positioning of the nanobody in between the 2 lobes of the transporter, raises the question of whether this conformation is physiologically relevant, or whether its formation is merely a consequence of Sy3 binding.
-
Reviewer #3 (Public Review):
To determine how the clinical-stage inhibitor vamifeport interacts with ferroportin, the authors used cryogenic electron microscopy (cryo-EM) to determine several structures of ferroportin in complex with newly isolated sybodies. They found that the highest resolution structure shows an occluded state of the transporter bound to sybody 3 and vamifeport. The inhibitor occupies a small portion of a large occluded cavity, interacts with both the N and the C lobe of the transporter, and overlaps with the binding site for both hepcidin and the iron ion binding site 2. The authors also use binding assays and mutagenesis to confirm that the residues in the vamifeport binding site are important for binding.
As the authors point out, the vamifeport inhibitor can readily be modeled in two orientations. The authors …
Reviewer #3 (Public Review):
To determine how the clinical-stage inhibitor vamifeport interacts with ferroportin, the authors used cryogenic electron microscopy (cryo-EM) to determine several structures of ferroportin in complex with newly isolated sybodies. They found that the highest resolution structure shows an occluded state of the transporter bound to sybody 3 and vamifeport. The inhibitor occupies a small portion of a large occluded cavity, interacts with both the N and the C lobe of the transporter, and overlaps with the binding site for both hepcidin and the iron ion binding site 2. The authors also use binding assays and mutagenesis to confirm that the residues in the vamifeport binding site are important for binding.
As the authors point out, the vamifeport inhibitor can readily be modeled in two orientations. The authors provide a reasonable argument that one orientation provides more specific interactions, but the case would be stronger if the structure had a high enough resolution to distinguish between the two orientations, or if the authors could provide some complementary supporting evidence. Still, the manuscript provides convincing evidence to explain how the compound inhibits ion transport and the similarities and differences between this inhibitor and the endogenous regulatory protein hepcidin.
The authors describe the occluded conformation that they resolve with bound sybody 3 and vamifeport as "on the transport pathway". However, this occluded conformation was captured in the presence of two ligands that are not on-pathway, the inhibitor and the sybody. It seems plausible (maybe even likely?) that the conformation is off-pathway and trapped by these additional ligands. The study would therefore benefit from additional evidence as to whether this conformation is indeed on-pathway.
-
