Bone-Targeting Drugs: From vesicle to cytosol

Drugs called bisphosphonates are used to treat a range of bone diseases, but how do they reach the enzymes that are their target?
  1. Michael J Rogers  Is a corresponding author
  2. Marcia A Munoz
  1. Garvan Institute of Medical Research, Australia

Despite its appearance, bone is a highly metabolic and dynamic tissue that is composed of a vast network of cells called osteocytes that are embedded in a matrix made mostly of collagen and various salts of calcium and phosphate. These osteocytes sense regions of damaged or weakened bone, and 'instruct' bone-destroying cells (called osteoclasts) and bone-forming cells (osteoblasts) to, respectively, remove old bone and deposit new bone (Figure 1). Hence, like a team of road-repairers, the osteocytes, osteoclasts and osteoblasts work together to repair bone and maintain our skeleton in good health (Crockett et al., 2011).

How bisphosphonates act in bone.

(A) Old and damaged bone is constantly being broken down by cells called osteoclasts in a process called resorption (top left), while new bone is deposited by cells called osteoblasts (top right). Cells called osteocytes (bottom) influence both of these processes through a spidery system of tiny canals called canaliculi. After entering the circulation, the drug bisphosphonate (green) binds very effectively to calcium ions on the bone mineral surface. During resorption, the bisphosphonate on the bone surface is released into the acidic extracellular space beneath the osteoclast. (B) The bisphosphonate in the extracellular space is engulfed into osteoclasts via a process called endocytosis (1). The resulting endosomes mature to form structures called lysosomes, and two proteins, SLC37A3 and ATRAID, then interact in the membrane of the lysosome to allow the bisphosphonate to enter the cytosol (2). Once in the cytosol, the nitrogen-containing bisphosphonates inhibit an enzyme called FDPS and prevent the osteoclast from breaking down bone (3). BP: bisphosphonate; FDPS: farnesyl diphosphate synthase; SLC37A3: solute carrier family 37 member A3.

In young adult life there is usually a balance between the amount of old bone broken down and the amount of new bone formed by this repair process, so there is no net gain or loss of bone mass. However, in diseases that affect the skeleton, such as post-menopausal osteoporosis or cancers growing in bone, this delicate balance can be disturbed by osteoclasts being over-active, which leads to excessive bone destruction and fractures. Drugs called bisphosphonates – which inhibit osteoclasts – have been used for more than three decades to treat such diseases and protect the skeleton from potentially catastrophic bone loss, although researchers still do not fully understand how they work. Now, in eLife, Erin O'Shea of Harvard University and the Howard Hughes Medical Institute (HHMI) and colleagues – including Zhou Yu as first author – report the answer to one of the remaining questions about these drugs (Yu et al., 2018).

Bisphosphonates are synthetic molecules that closely resemble the chemical structure of pyrophosphate, which is a natural by-product of numerous metabolic reactions. Importantly, bisphosphonates have two negatively-charged phosphonate groups that enable them to bind calcium ions very effectively, and hence to localize rapidly to any exposed calcium on the bone surface (Rogers et al., 2011). The mechanisms used by bisphosphonates to inhibit osteoclasts remained a mystery for several decades after they were first used in the clinic, but this did not stop the development of improved versions of the drugs (Russell et al., 2008). Eventually it was discovered that nitrogen-containing bisphosphonates (N-BPs), which are now widely used to treat osteoporosis and other bone diseases, work by inhibiting an enzyme called FDPS inside the osteoclasts (Luckman et al., 1998; Bergstrom et al., 2000; Dunford et al., 2001). The N-BP molecules displace the lipid substrates that the FDPS enzyme usually acts on, locking the enzyme in an inactive state (Kavanagh et al., 2006; Rondeau et al., 2006). Without FDPS activity, osteoclasts are no longer able to degrade bone (Rogers et al., 2011).

However, one question remained: how do the N-BPs and other bisphosphonates actually reach the FDPS enzyme, which is in the cytosol of the osteoclasts? There was little or no evidence that a receptor on the plasma membrane was involved (Thompson et al., 2006). Studies with fluorescently-tagged bisphosphonates showed that they first entered the osteoclasts via endocytosis – a process that involves the cell membrane folding inwards and then pinching off to create a vesicle inside the cell (Coxon et al., 2008). But how do the drugs leave these vesicles – which are enclosed by a membrane – to enter the cytosol? Bisphosphonate molecules have a large negative charge, which rules out passive diffusion across the vesicle membrane, which in turn suggests the possibility of a hitherto unidentified transport mechanism (Thompson et al., 2006).

Yu et al. – who are based at Harvard, HHMI, UCSF, MIT, the Broad, Koch and Whitehead Institutes, and Washington University – report that they have identified a protein called SLC37A3 that is required for the release of N-BP molecules from vesicles into the cytosol. Using a CRISPR-based approach to screen for genes that, when missing, confer resistance to bisphosphonates, they identified SLC37A3 as the gene with the strongest effect. Although the exact function of the SLC37A3 protein remains to be clarified, related members of this protein family are involved in the transport of charged molecules across membranes (Cappello et al., 2018).

Yu et al. found that SLC37A3 interacts and co-localizes with a protein called ATRAID at the vesicle membrane (Figure 1). Importantly, vesicles isolated from cells that did not express SLC37A3 or ATRAID appeared unable to release N-BP molecules, and these cells were much less sensitive to the pharmacological effect of N-BPs.

This new transport mechanism identified by Yu et al. raises interesting questions about how the SLC37A3/ATRAID complex specifically recognizes N-BP molecules, and how it transports them across the membrane of the vesicle. It will also be worthwhile to determine whether differences in the expression of SLC37A3 or ATRAID account for the different sensitivity of osteoclasts and other cell types to N-BP molecules, or whether variants in these genes affect the clinical responsiveness of patients to these drugs. Nevertheless, these elegant studies explain how negatively-charged N-BP molecules can gain access to the cell cytosol after endocytosis and, as a result, go on to benefit huge numbers of patients with potentially devastating bone diseases.

References

    1. Dunford JE
    2. Thompson K
    3. Coxon FP
    4. Luckman SP
    5. Hahn FM
    6. Poulter CD
    7. Ebetino FH
    8. Rogers MJ
    (2001)
    Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates
    Journal of Pharmacology and Experimental Therapeutics 296:235–242.

Article and author information

Author details

  1. Michael J Rogers

    Michael J Rogers is in the Bone Biology Division, Garvan Institute of Medical Research, Darlinghurst, Australia

    For correspondence
    m.rogers@garvan.org.au
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1818-9249
  2. Marcia A Munoz

    Marcia A Munoz is in the Bone Biology Division, Garvan Institute of Medical Research, Darlinghurst, Australia

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7603-0351

Publication history

  1. Version of Record published: June 27, 2018 (version 1)

Copyright

© 2018, Rogers et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,155
    Page views
  • 128
    Downloads
  • 5
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Michael J Rogers
  2. Marcia A Munoz
(2018)
Bone-Targeting Drugs: From vesicle to cytosol
eLife 7:e38847.
https://doi.org/10.7554/eLife.38847

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Dirk H Siepe, Lukas T Henneberg ... Kenan Christopher Garcia
    Tools and Resources

    Secreted proteins, which include cytokines, hormones and growth factors, are extracellular ligands that control key signaling pathways mediating cell-cell communication within and between tissues and organs. Many drugs target secreted ligands and their cell-surface receptors. Still, there are hundreds of secreted human proteins that either have no identified receptors ('orphans') and are likely to act through cell surface receptors that have not yet been characterized. Discovery of secreted ligand-receptor interactions by high-throughput screening has been problematic, because the most commonly used high-throughput methods for protein-protein interaction (PPI) screening do not work well for extracellular interactions. Cell-based screening is a promising technology for definition of new ligand-receptor interactions, because multimerized ligands can enrich for cells expressing low affinity cell-surface receptors, and such methods do not require purification of receptor extracellular domains. Here, we present a proteo-genomic cell-based CRISPR activation (CRISPRa) enrichment screening platform employing customized pooled cell surface receptor sgRNA libraries in combination with a magnetic bead selection-based enrichment workflow for rapid, parallel ligand-receptor deorphanization. We curated 80 potentially high value orphan secreted proteins and ultimately screened 20 secreted ligands against two cell sgRNA libraries with targeted expression of all single-pass (TM1) or multi-pass (TM2+) receptors by CRISPRa. We identified previously unknown interactions in 12 of these screens, and validated several of them using surface plasmon resonance and/or cell binding. The newly deorphanized ligands include three receptor tyrosine phosphatase (RPTP) ligands and a chemokine like protein that binds to killer cell inhibitory receptors (KIR's). These new interactions provide a resource for future investigations of interactions between the human secreted and membrane proteomes.

    1. Biochemistry and Chemical Biology
    Meiling Wu, Anda Zhao ... Dongyun Shi
    Research Article

    Antioxidant intervention is considered to inhibit reactive oxygen species (ROS) and alleviates hyperglycemia. Paradoxically, moderate exercise can produce ROS to improve diabetes. The exact redox mechanism of these two different approaches remains largely unclear. Here, by comparing exercise and antioxidants intervention on type 2 diabetic rats, we found moderate exercise upregulated compensatory antioxidant capability and reached a higher level of redox balance in the liver. In contrast, antioxidant intervention achieved a low-level redox balance by inhibiting oxidative stress. Both of these two interventions could promote glucose catabolism and inhibit gluconeogenesis through activation of hepatic AMPK signaling, therefore ameliorating diabetes. During exercise, different levels of ROS generated by exercise have differential regulations on the activity and expression of hepatic AMPK. Moderate exercise-derived ROS promoted hepatic AMPK glutathionylation activation. However, excessive exercise increased oxidative damage and inhibited the activity and expression of AMPK. Overall, our results illustrate that both exercise and antioxidant intervention improve blood glucose in diabetes by promoting redox balance, despite different levels of redox balance. These results indicate that the AMPK signaling activation, combined with oxidative damage markers, could act as a sensitive biomarker, reflecting the threshold of redox balance defining effective treatment in diabetes. These findings provide theoretical evidence for the precise treatment of diabetes by antioxidants and exercise.