Sclerostin small molecule inhibitors promote osteogenesis by activating canonical Wnt and BMP pathways

  1. Sreedhara Sangadala
  2. Chi Heon Kim
  3. Lorenzo M Fernandes
  4. Pooja Makkara
  5. George R Beck
  6. Scott D Boden
  7. Hicham Drissi  Is a corresponding author
  8. Steven M Presciutti  Is a corresponding author
  1. Emory University, United States
  2. Panjab University, India
  3. Atlanta VA Medical Center, United States

Abstract

Background: The clinical healing environment after a posterior spinal arthrodesis surgery is one of the most clinically challenging bone healing environments across all orthopaedic interventions due to the absence of a contained space and the need to form de novo bone. Our group has previously reported that sclerostin in expressed locally at high levels throughout a developing spinal fusion. However, the role of sclerostin in controlling bone fusion remains to be established.

Methods: We computationally identified two FDA-approved drugs, as well as a single novel small molecule drug, for their ability to disrupt the interaction between sclerostin and its receptor, LRP5/6. The drugs were tested in several in vitro biochemical assays using murine MC3T3 and MSCs, assessing their ability to: (1) enhance canonical Wnt signaling, (2) promote the accumulation of the active (non-phosphorylated) form of b-catenin, and (3) enhance the intensity and signaling duration of BMP signaling. These drugs were then tested subcutaneously in rats as standalone osteoinductive agents on plain collagen sponges. Finally, the top drug candidates (called VA1 and C07) were tested in a rabbit posterolateral spine fusion model for their ability to achieve a successful fusion at 6 weeks.

Results: We show that by controlling GSK3b phosphorylation, our three SMIs simultaneously enhance canonical Wnt signaling and potentiate canonical BMP signaling intensity and duration. We also demonstrate that the SMIs produce dose-dependent ectopic mineralization in vivo in rats as well as significantly increase posterolateral spine fusion rates in rabbits in vivo, both as standalone osteogenic drugs and in combination with autologous iliac crest bone graft.

Conclusions: Few if any osteogenic small molecules possess the osteoinductive potency of BMP itself – that is, the ability to form de novo ectopic bone as a standalone agent. Herein, we describe two such SMIs that have this unique ability and were shown to induce de novo bone in a stringent in vivo environment. These SMIs may have the potential to be used in novel, cost-effective bone graft substitutes for either achieving spinal fusion or in the healing of critical-sized fracture defects.

Funding: This work was supported by a Veteran Affairs Career Development Award (IK2-BX003845).

Data availability

All data generated or analysed during this study are included in the manuscript and supporting file.

Article and author information

Author details

  1. Sreedhara Sangadala

    Department of Orthopaedics, Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Chi Heon Kim

    Department of Orthopaedics, Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Lorenzo M Fernandes

    Department of Orthopaedics, Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Pooja Makkara

    Department of Biotechnology, Panjab University, Chandigarh, India
    Competing interests
    The authors declare that no competing interests exist.
  5. George R Beck

    Department of Medicine, Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Scott D Boden

    Department of Medicine, Emory University, Atlanta, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Hicham Drissi

    Department of Medicine, Emory University, Atlanta, United States
    For correspondence
    hicham.drissi@emory.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3322-281X
  8. Steven M Presciutti

    Atlanta VA Medical Center, Decatur, United States
    For correspondence
    prescius33@gmail.com
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6547-9495

Funding

U.S. Department of Veterans Affairs (IK2-BX003845)

  • Lorenzo M Fernandes

U.S. Department of Veterans Affairs (I01BX001516)

  • George R Beck

U.S. Department of Veterans Affairs (I01 BX004708)

  • Hicham Drissi

U.S. Department of Veterans Affairs (I01 BX004878)

  • Hicham Drissi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All rat and rabbit surgeries and procedures were first approved (#VOOS-14 - 2016-020211) by the Atlanta VA Medical Center Institutional Animal Care and Use Committee (IACUC).

Copyright

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Metrics

  • 797
    views
  • 138
    downloads
  • 8
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Sreedhara Sangadala
  2. Chi Heon Kim
  3. Lorenzo M Fernandes
  4. Pooja Makkara
  5. George R Beck
  6. Scott D Boden
  7. Hicham Drissi
  8. Steven M Presciutti
(2023)
Sclerostin small molecule inhibitors promote osteogenesis by activating canonical Wnt and BMP pathways
eLife 12:e63402.
https://doi.org/10.7554/eLife.63402

Share this article

https://doi.org/10.7554/eLife.63402

Further reading

    1. Cell Biology
    2. Evolutionary Biology
    Paul Richard J Yulo, Nicolas Desprat ... Heather L Hendrickson
    Research Article

    Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

    1. Cell Biology
    2. Developmental Biology
    Pavan K Nayak, Arul Subramanian, Thomas F Schilling
    Research Article

    Mechanical forces play a critical role in tendon development and function, influencing cell behavior through mechanotransduction signaling pathways and subsequent extracellular matrix (ECM) remodeling. Here we investigate the molecular mechanisms by which tenocytes in developing zebrafish embryos respond to muscle contraction forces during the onset of swimming and cranial muscle activity. Using genome-wide bulk RNA sequencing of FAC-sorted tenocytes we identify novel tenocyte markers and genes involved in tendon mechanotransduction. Embryonic tendons show dramatic changes in expression of matrix remodeling associated 5b (mxra5b), matrilin1 (matn1), and the transcription factor kruppel-like factor 2a (klf2a), as muscles start to contract. Using embryos paralyzed either by loss of muscle contractility or neuromuscular stimulation we confirm that muscle contractile forces influence the spatial and temporal expression patterns of all three genes. Quantification of these gene expression changes across tenocytes at multiple tendon entheses and myotendinous junctions reveals that their responses depend on force intensity, duration and tissue stiffness. These force-dependent feedback mechanisms in tendons, particularly in the ECM, have important implications for improved treatments of tendon injuries and atrophy.