Cryo-EM reveals an unprecedented binding site for NaV1.7 inhibitors enabling rational design of potent hybrid inhibitors
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eLife assessment
This fundamental study describes the structure-based design of novel hybrid inhibitors targeting a human sodium channel which is a pain target. Exceptionally strong evidence for key claims was produced with a structural biological pipeline for iterative structural determination of drugs complexed with an engineered sodium channel. This work is expected to be of interest to biophysicists, drug developers, neurobiologist, and pain researchers.
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Abstract
The voltage-gated sodium (Na V ) channel Na V 1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available Na V channel-blocking drugs are not selective among the nine Na V channel subtypes, Na V 1.1–Na V 1.9. Moreover, the two currently known classes of Na V 1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure–activity relationships of the acylsulfonamide class of Na V 1.7 inhibitors, exemplified by the clinical development candidate GDC-0310 , has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the Na V 1.7 channel, we pursued high-resolution ligand-bound Na V 1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the Na V 1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in Na V channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective Na V channel modulators targeting VSD4.
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Author Response
Reviewer #1 (Public Review):
The manuscript by Kschonsak et al. describes the rational structure-based design of novel hybrid inhibitors targeting human Nav1.7 channel. CryoEM structure of arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex confirmed binding pose observed in x-ray structure GX-936 - VSD4 Nav1.7-NavAb channel. Remarkably, cryoEM structure of acylsulfonamide (GDC-0310) - VSD4 NaV1.7-NaVPas channel complex revealed a novel binding pocket between the S3 and S4 helices, with the S3 segment adopting a distinct conformation compared to the arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex. Creatively, the authors designed a novel class of hybrid inhibitors that simultaneously occupy both the aryl- and acylsulfonamide binding pockets. This study underscores the power of …
Author Response
Reviewer #1 (Public Review):
The manuscript by Kschonsak et al. describes the rational structure-based design of novel hybrid inhibitors targeting human Nav1.7 channel. CryoEM structure of arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex confirmed binding pose observed in x-ray structure GX-936 - VSD4 Nav1.7-NavAb channel. Remarkably, cryoEM structure of acylsulfonamide (GDC-0310) - VSD4 NaV1.7-NaVPas channel complex revealed a novel binding pocket between the S3 and S4 helices, with the S3 segment adopting a distinct conformation compared to the arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex. Creatively, the authors designed a novel class of hybrid inhibitors that simultaneously occupy both the aryl- and acylsulfonamide binding pockets. This study underscores the power of structure-guided drug design to target transmembrane proteins and will be useful to develop safer and more effective therapeutics.
We thank this Reviewer for the very positive feedback and for highlighting the importance of our work in utilizing structure-based drug design to target key membrane targets.
Reviewer #2 (Public Review):
In this manuscript, the authors identify a critical unmet need for the (structure-based) drug design of human Nav channels, which are of clinical interest. They cleverly rationalized a hybrid strategy for developing target-specific small molecule inhibitors, which integrate binding mechanisms of two drug candidates that act orthogonally on the VSD4 of Nav 1.7. Thus, the authors illustrate a promising outlook on pharmaceutical intervention on Nav channels.
Overall, the cryo-EM structures of the ligand-bound Nav channels are convincing, with a clear indication of the site-specific, distinct density of the small molecules. At the moment, it is difficult to tell how innovative the pipeline is compared to conventional cryo-EM structure determination.
We thank this Reviewer for this positive comments and for the very helpful suggestions. We are addressing the concerns regarding our cryoEM pipeline.
Reviewer #3 (Public Review):
This is an excellent manuscript, describing a few lines of discoveries:
- Establishment of a structural biological pipeline for iterative structural determination of an engineered Nav1.7;
- Illumination of the novel compound binding mode;
- Structure-based development of the hybrid compounds, which led to the novel Nav1.7 inhibitor;
The cryo-EM study on the engineered Nav1.7 consistently reveals the map at the mid to low 2 Å range, which is unprecedented and impressive, thus, demonstrating the high value of this workflow. The further strength of this study is that the authors were able to develop a new compound by combining structural information gained from the two Nav1.7 structures complexed to two different compounds with different binding modes. Overall, the depth and quality of this study are excellent.
We thank this Reviewer for highlighting the importance of this manuscript and specifically recognizing our accomplishments in enabling iterative high-resolution structure for this target which allowed us to perform SBDD and design a new series of hybrid compounds. We are also grateful for indicating the excellence of our studies.
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eLife assessment
This fundamental study describes the structure-based design of novel hybrid inhibitors targeting a human sodium channel which is a pain target. Exceptionally strong evidence for key claims was produced with a structural biological pipeline for iterative structural determination of drugs complexed with an engineered sodium channel. This work is expected to be of interest to biophysicists, drug developers, neurobiologist, and pain researchers.
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Reviewer #1 (Public Review):
The manuscript by Kschonsak et al. describes the rational structure-based design of novel hybrid inhibitors targeting human Nav1.7 channel. CryoEM structure of arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex confirmed binding pose observed in x-ray structure GX-936 - VSD4 Nav1.7-NavAb channel. Remarkably, cryoEM structure of acylsulfonamide (GDC-0310) - VSD4 NaV1.7-NaVPas channel complex revealed a novel binding pocket between the S3 and S4 helices, with the S3 segment adopting a distinct conformation compared to the arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex. Creatively, the authors designed a novel class of hybrid inhibitors that simultaneously occupy both the aryl- and acylsulfonamide binding pockets. This study underscores the power of structure-guided drug design to …
Reviewer #1 (Public Review):
The manuscript by Kschonsak et al. describes the rational structure-based design of novel hybrid inhibitors targeting human Nav1.7 channel. CryoEM structure of arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex confirmed binding pose observed in x-ray structure GX-936 - VSD4 Nav1.7-NavAb channel. Remarkably, cryoEM structure of acylsulfonamide (GDC-0310) - VSD4 NaV1.7-NaVPas channel complex revealed a novel binding pocket between the S3 and S4 helices, with the S3 segment adopting a distinct conformation compared to the arylsulfonamide (GNE-3565) - VSD4 NaV1.7-NaVPas channel complex. Creatively, the authors designed a novel class of hybrid inhibitors that simultaneously occupy both the aryl- and acylsulfonamide binding pockets. This study underscores the power of structure-guided drug design to target transmembrane proteins and will be useful to develop safer and more effective therapeutics.
-
Reviewer #2 (Public Review):
In this manuscript, the authors identify a critical unmet need for the (structure-based) drug design of human Nav channels, which are of clinical interest. They cleverly rationalized a hybrid strategy for developing target-specific small molecule inhibitors, which integrate binding mechanisms of two drug candidates that act orthogonally on the VSD4 of Nav 1.7. Thus, the authors illustrate a promising outlook on pharmaceutical intervention on Nav channels.
Overall, the cryo-EM structures of the ligand-bound Nav channels are convincing, with a clear indication of the site-specific, distinct density of the small molecules. At the moment, it is difficult to tell how innovative the pipeline is compared to conventional cryo-EM structure determination.
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Reviewer #3 (Public Review):
This is an excellent manuscript, describing a few lines of discoveries:
1. Establishment of a structural biological pipeline for iterative structural determination of an engineered Nav1.7;
2. Illumination of the novel compound binding mode;
3. Structure-based development of the hybrid compounds, which led to the novel Nav1.7 inhibitor;The cryo-EM study on the engineered Nav1.7 consistently reveals the map at the mid to low 2 Å range, which is unprecedented and impressive, thus, demonstrating the high value of this workflow. The further strength of this study is that the authors were able to develop a new compound by combining structural information gained from the two Nav1.7 structures complexed to two different compounds with different binding modes. Overall, the depth and quality of this study are …
Reviewer #3 (Public Review):
This is an excellent manuscript, describing a few lines of discoveries:
1. Establishment of a structural biological pipeline for iterative structural determination of an engineered Nav1.7;
2. Illumination of the novel compound binding mode;
3. Structure-based development of the hybrid compounds, which led to the novel Nav1.7 inhibitor;The cryo-EM study on the engineered Nav1.7 consistently reveals the map at the mid to low 2 Å range, which is unprecedented and impressive, thus, demonstrating the high value of this workflow. The further strength of this study is that the authors were able to develop a new compound by combining structural information gained from the two Nav1.7 structures complexed to two different compounds with different binding modes. Overall, the depth and quality of this study are excellent.
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