Many topics we review here are very technical. To make them easier for more readers (you see, I hope some day will be more than seventeen) I usually introduce the theme in parts, aiming to give an appropriate background to then go to the main subject for the particular article. In this one, I found it would be good to increase our view of one of the most important way the fibroblast growth factor receptor 3 (FGFR3) uses to exert its effects in the growth plate, the mitogen-activated protein kinase (MAPK) enzymatic cascade.
A relevant aspect of molecules like NC-2 is their size and as this is a key property for any potential therapy aiming to reach the chondrocytes in the growth plate, we will be also briefly revisiting the growth plate.
The Rasopathies
The Rasopathies are a family of genetic disorders that share a common feature: all of them are caused by disturbances in the mitogen activated protein kinase (MAPK) signaling pathway, associated to mutations in proteins of this signaling cascade or in proteins that regulate its activity (Figure 1). No matter where the mutation is located, it leads to excessive MAPK signaling, which in turn is the cause of the different clinical aspects found in the several associated syndromes. (1)
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| An outline of the RAS-MAKP signalling pathway. Syndromes caused by mutations in the genes which control the production of certain signal proteins. From Socialstyrelsen.se. |
For more information about the MAPK pathway, read the last article published in the blog. The blog also has a number of previous articles talking about the MAPK cascade, accessible through your preferential language index page (link located on the top of this page).
MAPK signaling impairs bone growth
A common feature that individuals with RASopathies share is short stature. Since that the MAPK pathway is key in the growth plate development (2), it is reasonable to think that the excessive MAPK activity found in RASopathies is also influencing chondrocytes in the growth plate of affected individuals. However, there is little specific information about the consequences of MAPK excessive signaling in the context of this group of disorders.
MAPK signaling impairs bone growth
A common feature that individuals with RASopathies share is short stature. Since that the MAPK pathway is key in the growth plate development (2), it is reasonable to think that the excessive MAPK activity found in RASopathies is also influencing chondrocytes in the growth plate of affected individuals. However, there is little specific information about the consequences of MAPK excessive signaling in the context of this group of disorders.
That's why, aiming to understand the mechanism of short stature in one of the RASopathies, neurofibromatosis type 1, on April 2013 the group of Dr. Florent Elefteriou published an elegant and insightful study in which they explored the roles of the MAPK pathway in growth plate chondrocytes in an animal model where they could turn off neurofibromin in those cells. (3)
We have already reviewed their study at that time in a previous article, but I am returning to it to talk more about NC-2, the C-type natriuretic peptide (CNP) analogue used in their work. After looking for it for some time, I finally found the patent describing NC-2 and the many results of the tests already done with this compound. The patent claims that NC-2 could be used to treat achondroplasia.(4) Furthermore, NC-2 seems to be now under development by Alexion, a known biotech, which increases the chance that it could progress to clinical studies. However, before going to the patent, let's start from the basics, talking a bit about the study by Ono et al., neurofibromin, FGFR3 and CNP.
Neurofibromin and CNP in the growth plate
Neurofibromin works by inactivating the protein RAS in the top of the MAPK cascade (Figure 2). Ono et al. developed a mouse model where neurofibromin was absent in growth plates. The affected animals had patterns similar to what is found in FGFR3-related dysplasias, including short bones and shorter growth plates, with reduced proliferative and hypertrophic zones. The absence of neurofibromin made RAS staying active more time, thus keeping MAPK more active than normal, in a mechanism resembling what happens in achondroplasia.
Figure 2. Receptor enzymes-RAS-MAPK pathway and neurofibromin interactions
NC-2 rescues the growth impairment caused by the absence of neurofibromin
Ono et al. gave NC-2 to mice with no neurofibromin in chondrocytes and found that this CNP analogue was capable to rescue the cell growth in the exposed animals, counteracting the effects of the loss of neurofibromin. One merit of this study is that it could solidly demonstrate the kind of consequences defects in neurofibromin can cause to the growth plate and also brought insights for novel therapeutic strategies to treat the RASopathies and FGFR3-related dysplasias. Think about a molecule that could interact with RAS in the same way neurofibromin does...
We have already reviewed their study at that time in a previous article, but I am returning to it to talk more about NC-2, the C-type natriuretic peptide (CNP) analogue used in their work. After looking for it for some time, I finally found the patent describing NC-2 and the many results of the tests already done with this compound. The patent claims that NC-2 could be used to treat achondroplasia.(4) Furthermore, NC-2 seems to be now under development by Alexion, a known biotech, which increases the chance that it could progress to clinical studies. However, before going to the patent, let's start from the basics, talking a bit about the study by Ono et al., neurofibromin, FGFR3 and CNP.
Neurofibromin and CNP in the growth plate
Neurofibromin works by inactivating the protein RAS in the top of the MAPK cascade (Figure 2). Ono et al. developed a mouse model where neurofibromin was absent in growth plates. The affected animals had patterns similar to what is found in FGFR3-related dysplasias, including short bones and shorter growth plates, with reduced proliferative and hypertrophic zones. The absence of neurofibromin made RAS staying active more time, thus keeping MAPK more active than normal, in a mechanism resembling what happens in achondroplasia.
Figure 2. Receptor enzymes-RAS-MAPK pathway and neurofibromin interactions
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The function of neurofibromin in the RAS–MAPK pathway
Binding of growth factors to RTKs results in dimerisation and auto-phosphorylation. Grb2, an adaptor protein, binds to the receptor and forms a complex with SOS, stimulating GDP/GTP exchange on RAS. Neurofibromin has RAS-GTPase activity and inactivates RAS. GTP-bound RAS activates RAF, leading to phosphorylation and activation of MEK and then ERK: an extracellular signal regulated kinase that can enter the nucleus and phosphorylate transcription factors. RAS also activates PI3K, AKT, and mTOR. RTKs=receptor tyrosin kinases. P=phosphorylation. Grb2=growth factor receptor bound 2. SOS=mammalian homolog of the Drosophila son of sevenless. RAS=rat sarcoma viral oncogene homologue. GDP=guanosine diphosphate. GTP=guanosine triphosphate. RAF=murine sarcoma viral oncogene homologue. MEK=MAPK-ERK kinase. PI3K=phosphatidylinositol-3–kinase. AKT=V-akt murine thymoma viral oncogene homologue 1. mTOR=mammalian target of rapamycin.
From: Brems H et al. Lancet Oncol 2009;10 (5):508-15. N.A.: In the context of the chondrocyte, think on growth factors as FGFs and RTK as FGFR3. Disclosure: figure freely available through Google Images, reproduced here for information purposes only.
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Ono et al. gave NC-2 to mice with no neurofibromin in chondrocytes and found that this CNP analogue was capable to rescue the cell growth in the exposed animals, counteracting the effects of the loss of neurofibromin. One merit of this study is that it could solidly demonstrate the kind of consequences defects in neurofibromin can cause to the growth plate and also brought insights for novel therapeutic strategies to treat the RASopathies and FGFR3-related dysplasias. Think about a molecule that could interact with RAS in the same way neurofibromin does...
CNP in chondrocytes
Researchers have learned that CNP signaling works directly on the protein RAF of the MAPK cascade, reducing the activity of MAPK. Since MAPK is considered a key driver of FGFR3 signaling in chondrocytes, CNP is thought to be a modulator of this receptor. (Figure 3). (5) In other words, while FGFR3 negatively influence bone growth, CNP has a positive effect.
Figure 3. FGFR3 and CNP pathway interaction
NC-2, another CNP analogue
In the study by Ono and coworkers, NC-2 is described as a molecule made by the fusion of an active form of CNP, called CNP 22, with the Fc part of an immunoglobulin (antibody). Let's make a stop here to briefly explain what is the Fc fragment of an antibody.
Immunoglobulins are proteins created by the body defense system (immune system) to beat foreign molecules or life forms (virus, etc.). Basically, they are composed by two parts: a) the Fc part is common to all immunoglobulins, like a scaffold; and b) the Fab part, which is tailored by the cells of the immune system to attack a specific target (Figure 4). Both parts are integrated to become an active antibody. The Fc part of an antibody can be linked to other molecules to increase their half-life, transport and targeting capability. There are several examples available, such as a molecule that fuse the Fc fragment with interferon. (6)
Figure 4. Very simple representation of an immunoglobulin
The Fc fragments usually have a molecular weight of around 50 kiloDaltons (kDa), according to the books. This size could represent a problem for NC-2, in the context of the growth plate. Let's see why.
The cartilage growth plate as a barrier
The growth plate is a dense tissue, also called extracellular matrix, where chondrocytes are surrounded by proteins of the collagen family and other large molecules. The matrix not only is dense but also electrically charged and does not receive direct blood flow uniformly. All of these features work as chemical and physical barriers for large molecules to traffic inside it. This probably serves as a very ancient evolutionary defense mechanism in the growing organism: the core of the bone growth machinery had to be very well protected against invaders under the risk of having the growth program disturbed. By the other side, reducing the entry of part of the body agents might have also contributed to modulate the growth rate. Most of the body agents with developmental roles are positive agents for growth.
Some researchers studied how the traffic of molecules within the growth plate is and they showed that it is unlikely that molecules with 40 kDa or more would have free pass within the cartilage. (7, 8)
This finding may explain why PRO-001, the first specific anti-FGFR3 antibody never progressed to clinical development for achondroplasia. PRO-001 was developed by Dr. Avner Yayon's group (9) more than a decade ago and showed promising results in the first tests, but we presume it wasn't successful due to its large size: a typical antibody will have around 150 kDa.
On the other hand, we saw above that the growth plate has always been described as an avascular environment, but there is evidence that this concept might be only partially correct.(10)
Furthermore, in view of the natural properties of the growth plate, how can we interpret the results from the study by Ono et al. and those from the Dr Gouze's group (11), recently reviewed here? In both cases, the molecules weigh more than that threshold of 40 kDa, yet they exerted the expected effects in the growth plate.
One could speculate that in these cases the researchers performed their tests using doses of their compounds to obtain the desired outcomes that would be too high to be proportionally used in human patients safely. However, as informed by the Dr Gouze's group, toxicological tests were perfomed in the exposed animals and nothing of concern was found. (11)
Immunoglobulins are proteins created by the body defense system (immune system) to beat foreign molecules or life forms (virus, etc.). Basically, they are composed by two parts: a) the Fc part is common to all immunoglobulins, like a scaffold; and b) the Fab part, which is tailored by the cells of the immune system to attack a specific target (Figure 4). Both parts are integrated to become an active antibody. The Fc part of an antibody can be linked to other molecules to increase their half-life, transport and targeting capability. There are several examples available, such as a molecule that fuse the Fc fragment with interferon. (6)
Figure 4. Very simple representation of an immunoglobulin
The Fc fragments usually have a molecular weight of around 50 kiloDaltons (kDa), according to the books. This size could represent a problem for NC-2, in the context of the growth plate. Let's see why.
The cartilage growth plate as a barrier
The growth plate is a dense tissue, also called extracellular matrix, where chondrocytes are surrounded by proteins of the collagen family and other large molecules. The matrix not only is dense but also electrically charged and does not receive direct blood flow uniformly. All of these features work as chemical and physical barriers for large molecules to traffic inside it. This probably serves as a very ancient evolutionary defense mechanism in the growing organism: the core of the bone growth machinery had to be very well protected against invaders under the risk of having the growth program disturbed. By the other side, reducing the entry of part of the body agents might have also contributed to modulate the growth rate. Most of the body agents with developmental roles are positive agents for growth.
Some researchers studied how the traffic of molecules within the growth plate is and they showed that it is unlikely that molecules with 40 kDa or more would have free pass within the cartilage. (7, 8)
This finding may explain why PRO-001, the first specific anti-FGFR3 antibody never progressed to clinical development for achondroplasia. PRO-001 was developed by Dr. Avner Yayon's group (9) more than a decade ago and showed promising results in the first tests, but we presume it wasn't successful due to its large size: a typical antibody will have around 150 kDa.
On the other hand, we saw above that the growth plate has always been described as an avascular environment, but there is evidence that this concept might be only partially correct.(10)
Furthermore, in view of the natural properties of the growth plate, how can we interpret the results from the study by Ono et al. and those from the Dr Gouze's group (11), recently reviewed here? In both cases, the molecules weigh more than that threshold of 40 kDa, yet they exerted the expected effects in the growth plate.
One could speculate that in these cases the researchers performed their tests using doses of their compounds to obtain the desired outcomes that would be too high to be proportionally used in human patients safely. However, as informed by the Dr Gouze's group, toxicological tests were perfomed in the exposed animals and nothing of concern was found. (11)
It looks like that we still need to learn more about the growth plate properties.
Conclusion
NC-2 is a very interesting molecule. We do not know today if it is still being evaluated as a potential candidate for clinical development by Alexion. Since it is not common that a drug developer share a strong clinical development candidate for external researchers working independently, the fact that NC-2 has been used in such a way may imply that this analogue might have been retired. Even if this is the case, the concept is still compelling and shows how researchers can be creative looking for smart solutions to tackle clinical conditions with clear medical needs.
If you are interested in knowing more about CNP and the growth plate just visit these previous blog's articles:
The last stronghold: the growth plate cartilage
The last stronghold: the growth plate cartilage
References
2. Foldynova-Trantirkova S et al. Sixteen years and counting: the current understanding of fibroblast growth factor receptor 3 (FGFR3) signaling in skeletal dysplasias. Hum Mutat 2012; 33:29–41. Free access.
3. Ono K et al. The ras-GTPase activity of neurofibromin restrains ERK-dependent FGFR signaling during endochondral bone formation. Hum Mol Genet 2013;22(15):3048-62.
4. Alexion Pharma International. Compositions comprising natriuretic peptides and methods of use thereof. Patent US 20120164142 A1. Jun, 28th 2012. Free access.
5. Krejci P et al. Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis. J Cell Sci 2006; 118: 5089-00. Free access.
6. Jones TD et al. The development of a modified human IFN-alpha2b linked to the Fc portion of human IgG1 as a novel potential therapeutic for the treatment of hepatitis C virus infection. J Interferon Cytokine Res 2004;24(9):560-72.
7. Farnum CE et al. In vivo delivery of fluoresceinated dextrans to the murine growth plate: imaging of three vascular routes by multiphoton microscopy. Anat Rec A Discov Mol Cell Evol Biol 2006;288(1):91-103. Free access.
8. Williams RM et al. Solute transport in growth plate cartilage: in vitro and in vivo.
Biophys J 2007;93(3):1039-50. Free access.
9. Aviezer D et al. Fibroblast growth factor receptor-3 as a therapeutic target for Achondroplasia--genetic short limbed dwarfism. Curr Drug Targets 2003;4(5):353-65.
10. Wirth T et al. The
blood supply of the growth plate and the epiphysis: a comparative
scanning electron microscopy and histological experimental study in
growing sheep. Calcif Tissue Int 2002;70(4):312-9.
11. Garcia S et al. Postnatal soluble FGFR3 therapy rescues achondroplasia symptoms and restores bone growth in mice. Sci Transl Med 2013;5:203ra124.
11. Garcia S et al. Postnatal soluble FGFR3 therapy rescues achondroplasia symptoms and restores bone growth in mice. Sci Transl Med 2013;5:203ra124.
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