Interfering in the production of FGFR3: a potential strategy to treat achondroplasia

In several of the previous articles of this blog we have reviewed concepts such as what is DNA and how proteins like the fibroblast growth factor receptor 3 (FGFR3) are produced. However, as this time the topic is exactly a strategy that aims to inhibit the expression (production) of FGFR3, I think it will worth to see it again. 

The reader should keep in mind that the text is like a panoramic view of the subject, with many details being taken off on purpose, to make it easier to understand. We will be talking only about FGFR3 and the growth plate chondrocyte, since things can occur slightly differently in other body tissues or conditions.

For some of the seventeen readers who follow this blog, the text might sound repetitive, with already known information. However, when the subject has already been seen before, I try to include a piece more of detail, so we will together be capable to increase our knowledge. And finally, for this text I prepared a simple glossary located at the end, before the references. Words in italic will have a simple definition there so, if things seem to be hard to understand I hope it would help a bit. Some of these terms will also have hyperlinks to other sites containing more information about them.

 How FGFR3 is made

We have reviewed this process in a series of three articles published in the beginning of 2012 and you can find them by clicking on the English tab on the top of the page. There, look at the articles mentioning FGFR3 production and the RNA world.

In brief

FGFR3 is an active protein (an enzyme), a large molecule made of smaller molecules called amino acids. You can think on FGFR3 as a long chain of Lego blocks (amino acids), each one bearing distinct electrical charges. When the final block disposition is attained, this receptor enzyme will gain its functional electric properties. Making a functional enzyme, however, is a long assembly process.

The DNA is a kind of vault where all the instructions needed to create proteins are saved. When the chondrocyte receives a chemical signal instructing the cell to produce more FGFR3, a multi-step process begins by opening the vault.

First, transcription factors bind a specific site upstream of the DNA region where the FGFR3 gene is located. When a transcription factor does this, other local nuclear proteins are triggered and form a complex that will start to read the gene sequence (transcription) to produce a RNA chain that mirrors what is written in the DNA. This RNA chain is called pre messenger RNA, or simply pre-mRNA. You can watch these short videos (animation 1 and animation 2) to have an idea of how it works.

The RNA chain created in the transcription process contains all the information existent in the gene DNA sequence, so it carries not only those nucleotide sequences that generates the proteins (called exons) but also those that are made of non-coding sequences (called introns). Thus, before the mRNA can be read and translated to a protein, it needs to be cleared of those useless parts.

After this step, the mature mRNA is driven to the cytoplasm where it will be engaged by another set of proteins within the ribosomes, starting the process called translation, which leads to the assembly of the FGFR3 protein. 

This short but very nice animation from Nature will help you to understand the translation process. Pay attention to the last 20 seconds. The small molecules binding to the mRNA in the ribosome will bring the right amino acid according to the nucleotide sequence in the mRNA. Did you notice they look like three sting forks? There is a reason for that. Each amino acid is chosen according to groups comprised of three nucleotides, which are called codons.

In achondroplasia, a change of a single nucleotide (located in the position 1138) in one of those codons of the FGFR3 gene provokes the change of one amino acid in the position 380 of the FGFR3 protein. This is enough to cause the clinical features of achondroplasia. In this previous article of the blog there is a more detailed explanation of the mutation behind achondroplasia.

However, transcription is a strongly controlled process and some additional steps take place before FGFR3 is produced and released to exert its actions. One of these steps is ruled by another kind of RNA molecule, called micro-RNA, or simply miRNA. Micro-RNAs' main action is to regulate the expression of proteins and they do this through binding specific sequences in the mRNA, thus interfering with and interrupting its reading and consequently the protein assembly.(1) It is supposed that miRNA function is related to balance the expression of proteins which, if otherwise were continuously produced, could cause diseases.There is at least one miRNA already identified which has FGFR3 as a natural target.(2)

Potential therapies for achondroplasia

When we look at all the current potential strategies publicly available for achondroplasia, we conclude that all are directed against the receptor after it is assembled or against the chemical cascades linked to FGFR3 activity. This includes tyrosine kinase inhibitors, CNP, PTH/PThrP, aptamers, peptides, ligand traps and antibodies. With the possible exception of the still unproven hypothesis that MK-4 can inhibit the production of FGFR3 in chondrocytes, none of these strategies are directed against FGFR3 production.

But what if there is someone already looking at this? A couple of weeks ago, during a search I performed when I was writing the blog's previous article about aptamers, I found a patent registry which immediately caught my attention. 

Interfering with FGFR3 production

The American biotechnology company called Marina Biotech has patented a group of molecules designed to interfere with FGFR3 production, through the processs called RNA interference. (3) RNA interference is shown in this very nice animation provided by the journal Nature.

Visiting the company's website was a bit frustrating since nothing is mentioned regarding these compounds there, but the patent description contains interesting information about tests already made in FGFR3-related cancer models where the compounds were able to reduce cancer growth.

Unfortunately, the patent, although citing the possibility of using this invention as a therapy for achondroplasia seven times, doesn't show any test made in an achondroplasia model. Since nothing is disclosed, we can think this looks like another case of a potential therapy waiting for an interested investigator to be explored. The same case of the peptides, ligand traps and antibodies we have reviewed here in the last months.

RNA interference against FGFR3 is possible and has already been accomplished in some studies, such as in the one by Drs. Guzman, Pintor and Legeai-Mallet.(4) 

The good news is that we are witnessing an increasing number of therapeutic options becoming available for testing. It makes me think that, in the words of a top investigator in the field, it is likely that in the near future the question will not be if there will be a therapy for achondroplasia but which one we will choose to treat our children. 

Glossary

Amino acids: molecules with several distinct chemical properties that are combined in multiple sequences to generate proteins.

Codon: sets of three nucleotides that contain the information needed to match the right amino acid during the protein assembly. A nucleotide mismatch in the position 1138 of the FGFR3 gene leads to the placement of the wrong amino acid in the FGFR3 protein chain and causes achondroplasia.

 (from BioGem.Org)

Chondrocytes: the cells within the cartilage growth plates, the masters of bone growth. In achondroplasia, due to the excessive function of the FGFR3 mutation G380R, they are forced to reduce their multiplication and maturation rates, which in turn causes a significant loss of bone growth.

Exons and introns: exons are the parts of the gene sequence containing the codons that will be translated in amino acids to form proteins. Introns are the parts of the gene that do not have codons.

(from Wikipedia)

Fibroblast growth factor receptor 3 (FGFR3): an enzyme responsible for the control of chondrocyte proliferation and maturation speed, working like a natural brake. When it works too much due to an activating mutation, it causes bone growth arrest. Achondroplasia, the most common cause of dwarfism, is caused by an activating mutation of FGFR3.

Nucleotides: they are the four basic molecules which, combined in sequences of three (the codons), will encode the information needed to the production of the proteins.

(from http://cs.boisestate.edu/~amit/teaching/342/lab/structure.html)

Transcription: the process through which a gene is read and a messenger RNA is created. It is the first main step for a protein to be produced.

Transcription factors: proteins capable to identify certain nucleotide sequences in the DNA and bind them.

Translation: This is the process through which a sequence of nucleotides present in the mRNA chain is translated into an amino acid sequence, creating a new protein.

Upstream sites: They mean sequences of nucleotides located before the gene sequence. There are at least two of these regions located before the gene sequence, the promoter and the enhancer sequences and they are very important to allow gene expression, like beacons for the protein complex that will read the gene (see text).

References

1. Gibson G and Asahara H. microRNAs and cartilage. J Orthop Res. 2013 Jun 10. doi: 10.1002/jor.22397. [Epub ahead of print].

2. Oneyama C et al. MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor
growth induced by Src-related oncogenic pathways. Oncogene 2011;30:3489–501.

3. Marina Biotech Patent WO 2011139842 A2. Nucleic acid compounds for inhibiting fgfr3 gene expression and uses thereof.

4. Guzman-Aránguez A, Legeai‐Mallet L, Pintor J. Fibroblast growth factor receptor 3 inhibition by small interfering RNAs in achondroplasia. Anales Real Acad Nac Farm 2011; 77(1).