Saturday, April 27, 2013

New evidence of the mechanism of action of CNP in the FGFR3 pathway

FGFR3 and CNP in the cartilage growth plate

In the last two years, people all over the world interested in finding therapeutic alternatives for fibroblast growth factor receptor type 3 (FGFR3) related chondrodysplasias, and especially achondroplasia, have been paying strong attention to the C-type natriuretic peptide (CNP) analogue BMN-111, which is currently under clinical development as a potential therapy for achondroplasia.

FGFR3 has been showed to work by reducing the capacity of chondrocytes to proliferate and mature to a state called hypertrophy (1,2; also reviewed here). It does so by turning on a series of controlled chemical reactions inside the cell, which we use to call cascades or pathways (figure).  

CNP works by inhibiting one of the most important chemical cascades triggered by the activation of FGFR3, the Ras-Raf-MEK-ERK pathway (1,2; also reviewed here). These acronyms stand for a group of proteins with active chemical properties and therefore called enzymes (the mitogen-activated protein kinase or MAPK enzymes).

The MAPK cascade, when turned on by FGFR3 in chondrocytes, the core cell of the cartilage growth plate, acts like a brake in the bone growth process, so FGFR3 is a natural negative controller of bone growth. When FGFR3 is overactive, chondrocytes can’t multiply and mature normally and less bony tissue is created, resulting in shorter bones, specially the apendicular ones (arms and legs). So, you can figure out how having a mutation in FGFR3 making it more active than normal causes the bone growth impairment seen in achondroplasia. Now, let’s see in more detail how FGFR3 and CNP cascades interact inside the chondrocyte.

When CNP binds to its receptor called NPR-b at the chondrocyte cell membrane it triggers another chemical cascade that will block the MAPK cascade at the level of the enzyme Raf (figure). In this case, as the MAPK enzymes had their activity reduced, the other mechanisms that control the bone growth are at least partially restored, the chondrocyte is again able to multiply and mature in increased numbers and the bone growth is rescued. This is true in mice and presumably in the other animal models which have been exposed to BMN-111. Currently there is no paper describing the effect of CNP administration in larger animals but we know that, during a public presentation where the BMN-111 program was announced, the developer of this CNP analogue revealed that exposed monkeys grew more than expected.

The bone growth mechanism is complex

The bone growth is a very complex process, where the chondrocytes within the growth plate cartilage will endure a fast and extremely dynamic program determined by the effects of reactions caused by local and systemic molecules (most of the times proteins or peptides). When you read local this means that the protein or the agent is produced by the cells in the growth plate or surroundings (for instance, FGFR3, CNP, PTHrP; look at previous articles of the blog for more details). Systemic means the agent is produced far away from the growth plate, such as the pituitary hormones or the parathyroid hormone.

A study shows another view of CNP effects in bone growth

A recent study, authored by Ono and colleagues (3), and published in Human Molecular Genetics adds more complexity to this process. The authors describe the function of a common protein present in many cells and called neurofibromin, in the chondrocyte. Its role in the chondrocyte is not completely understood as well its relevance for the appropriate development of the growth plate. The authors tested a large number of possible connections this protein could have with some of the other local growth players, among them FGFR3, CNP, IHH and PTHrP.

What is relevant in this study, when thinking in achondroplasia?

They found that neurofibromin exerts its regulatory role in the endochondral development (this is how scientists name the process by which the long bones grow, through the cartilage growth plate development) acting as an inhibitor of the FGFR3-activated MAPK cascade. Neurofibromin has some properties similar to other enzymes called GTPases (the suffix ase describes an enzyme) that naturally reduce the activity of Ras, the first enzyme triggered by FGFR3 in the MAPK cascade. In this study, the researchers showed that neurofibromin action resembles the effect of CNP in the MAPK cascade (reviewed here). When they took out neurofibromin, the chondrocytes reduced their proliferation and maturation rates and the bones grew less, similarly to what happens in FGFR3 overactive mutations. When they injected CNP in the animals lacking neurofibromin, the bone growth was rescued.

Furthermore, they observed that in mutant chondrocytes (no neurofibromin), there was also less expression (production) of IHH, another fundamental factor for cartilage and bone growth, which in turn is also important for the generation of PTHrP . We know that IHH and PTHrP are very important for the proper development of the growth plate (4; also reviewed in this article of the blog).

One could think that overexpression (or superproduction) of neurofibromin could rescue the bone growth in achondroplasia, but this would be very difficult, since neurofibomin is an universal protein and its disregulation (up or down) could bring undesired effects in the treated individual. However, this study brings to light more valuable information on how MAPK acts in the chondrocyte.

In conclusion, this study examines and confirms among other relevant findings, how CNP works in the growth plate, giving more evidence for its use in potential therapies for achondroplasia.

Other analogues of CNP in the horizon?

Another relevant information given in this study is related to the kind of CNP the researchers used to perform their tests. They chose a distinct CNP analogue which they called NC-2. NC-2 has a very long half life compared to CNP (~20h vs. ~3 min) and is the result of the assemblage of part of the CNP molecule to the basic part of an antibody (what is called the Fc part of an immunoglobulin G, IgG). I couldn’t find more information about this compound beyond what is described in the paper by Ono and colleagues, so there are many questions to ask about it. But at least two points are interesting: with the structure described in their work, it looks like NC-2 is a large molecule (larger than the natural CNP). Since it showed to work in their tests, one could ask if the researchers noticed any toxic effects in the tested animals or any other evidence of undesired effects under the long exposure to NC-2. For instance, we must take in account that the other known CNP analogue BMN-111 has a half-life of 20 min and causes mild effects in the blood pressure (5).

The other point is that the compound called FP-1039 (6), which I mentioned in the previous article of the blog, is also composed by a Fc part of the IgG, so also a larger molecule. Since NC-2 seems to reach the growth plate, it would be interesting to learn if FP-1039 also reached the growth plate in the experiments done during the tests with this compound.

Doors are opening.

  1. Horton W. Molecular pathogenesis of achondroplasia. GGH 2006; 22 (4): 49-54
  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.
  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. doi: 10.1093/hmg/ddt162.
  4. Kronemberg H. Developmental regulation of the growth plate. Nature 2003; 423: 332-6.
  5. Biomarin press release.  
  6. Harding TC et al. Blockade of nonhormonal fibroblast growth factors by FP-1039 inhibits growth of multiple types of cancer. Sci Transl Med 2013;5:178ra39. 


No comments:

Post a Comment