Please can you give a brief introduction to Nucleoside Reverse Transcriptase Inhibitors (NRTIs) and the different diseases they have been used to treat?
NRTIs are compounds which were originally developed in the 1960s as anti-cancer agents. They are similar in structure to the bases which make up DNA, and it was hoped that they would interfere with DNA replication in fast-growing cancer cells, slowing down or stopping tumour growth.
Unfortunately, they did not work as expected, and the compounds were shelved until, in the 1980s, they were found to be effective against human immunodeficiency virus (HIV).
As part of its life-cycle, HIV must transcribe, or copy, its genetic material from RNA to DNA using an enzyme called reverse transcriptase, and NRTIs inhibit this process, preventing HIV from making new copies of itself.
The first NRTi, AZT (azidothymidine), was approved by the US Food and Drug Administration in 1987 for the treatment of HIV. Since then, NRTIs have also been shown to be effective in other viral diseases, including Hepatitis B.
What prompted you to consider researching the effect of NRTIs on age-related macular degeneration (AMD)?
My field of expertise is the structure and function of a family of proteins called P2X receptors. One member of this family, P2X7, plays an important role in inflammation by binding to a danger signal, ATP, which is released when cells are infected or injured.
When activated by ATP binding, P2X7 changes shape, which opens a channel in the cell membrane, allowing positively charged ions (including calcium ions) to enter the cell, transmitting the danger signal to the inside of the cell, and initiating an inflammatory signalling cascade.
In healthy individuals, this is a natural response to infection or injury, but in many inflammatory diseases (including AMD) this signalling pathway is over-active.
I was aware that P2X7 signalling was involved in AMD, but I did not know that NRTIs and AMD were connected until I was contacted by the lead author of our recently published study in Science, Professor Jayakrishna Ambati of the University of Kentucky, who told me that his laboratory had found some exciting results testing NRTIs on a mouse model of AMD.
AMD is caused by the toxic (inflammatory) effects of a molecule called Alu RNA, which, like HIV, requires reverse transcriptase for its life-cycle.
Prof. Ambati hypothesised that NRTIs might block Alu RNA in the same way that they blocked HIV, but instead he found that they acted through the P2X7 receptor signalling pathway.
At this point we did not know where the NRTIs were acting, and we reasoned that they may be stopping P2X7 from being able to form a channel in the membrane.
In my laboratory, we tested the action of NRTIs on P2X7 function, finding that they had no effect on ion channel formation, meaning that the NRTIs were acting on the inflammatory signalling pathway downstream of P2X7 receptor activation.
How do the two types of AMD differ and what treatment options are available for wet and dry AMD?
AMD affects part of the eye called the macula, a small area of the retina which is important for seeing detail, colour and objects directly in front of you.
Both wet and dry AMD can lead to loss of central vision (a blank patch in the centre of vision in both eyes). This means that AMD sufferers can find it difficult or impossible to recognise faces.
In wet AMD, which affects 10-15% of sufferers, signalling by a molecule called vascular endothelial growth factor (VEGF) leads to over-growth of blood vessels underneath the macula, which causes bleeding, scarring and damage to the macula.
Wet AMD can progress at a faster rate than dry AMD, and if left unchecked the damage is permanent, but several effective treatments designed to stop the abnormal vessel growth, by inhibiting the action of VEGF, are now available.
Dry AMD, which affects 85-90% of sufferers, is caused by the loss of retinal pigment epithelial (RPE) cells in the macula, which are vital for the support of photoreceptor cells essential for vision.
The onset of dry AMD is slow, occurring over several years, and it is characterised by the build-up of yellow deposits (drusen) in the macula.
An increased number and size of drusen correlates with the likelihood of contracting dry AMD, for which there is currently no cure, although there is some evidence that taking supplements of the antioxidants lutein and zeaxanthin may slow disease progression.
How did you test whether NRTIs would be effective in treating dry AMD?
In order to test the effectiveness of NRTIs on dry AMD, we needed an appropriate model for the disease. For this, we used mice, inducing dry AMD using Alu RNA.
We then imaged the progression of dry AMD and the loss of RPE cells in the retinas of control mice, mice treated with Alu RNA, and mice treated with both Alu RNA and NRTIs.
What did your research find?
Our research found that a range of different NRTI compounds were capable of completely blocking the damage to RPE cells that was induced by Alu RNA.
Following this discovery, our goal was to determine how the NRTIs were blocking dry AMD progression, which we initially assumed was due to inhibition of reverse transcriptase.
Was the preventative effect due to the drug inhibiting reverse transcriptase or was another mechanism involved?
We tested if reverse transcriptase was involved in the mechanism of action of NRTIs by making a modified version of an NRTI molecule that could not interact with the reverse transcriptase enzyme.
We found that this modified NRTI was still capable of blocking damage to RPE cells, showing that NRTIs must work through a different mechanism.
Because we knew that Alu RNA (the cause of AMD) causes inflammation, via the activation of the P2X7 receptor and its inflammatory signalling pathway, this led us to study the effects of NRTIs on P2X7-dependent inflammatory signalling.
We found that NRTIs stop the inflammation induced by the activation of P2X7, but not by blocking the P2X7 receptor itself – instead they block the signalling pathway downstream of receptor activation.
Is it likely that NRTIs would also be effective in treating wet AMD?
We tested this hypothesis using a mouse model of wet AMD, where abnormal blood vessel growth has been induced in the macula.
We found that NRTIs reduced abnormal blood vessel growth, indicating that NRTIs would also be effective in treating wet AMD.
Furthermore, we showed that NRTIs did not reduce abnormal vessel growth in genetically modified mice which lacked the P2X7 receptor, showing that P2X7 signalling is also involved in wet AMD progression.
How many other conditions do you think NRTIs could be used to treat and do you have any plans to research these?
In our work we tested NRTIs on dry AMD, wet AMD, and two other inflammatory diseases; graft-versus-host disease (a problem for transplant patients, where cells from the transplant attack the host) and sterile liver inflammation (a problem in drug-induced liver injury).
We found that NRTIs were effective in each of the four conditions, raising our hopes that NRTIs might be capable of treating a broad range of inflammatory conditions.
I am interested in the role of P2X7 receptors in inflammation, in particular the proteins that interact with the receptor (the P2X7 Interactome) and I would like to understand more precisely how NRTIs affect P2X7 inflammatory signalling, and whether or not they have any benefit in inflammatory diseases such as rheumatoid arthritis.
Where can readers find more information?
The publication in Science describing our results can be found here: http://www.sciencemag.org/content/346/6212/1000.full
This is a good site from the RNIB describing age-related macular degeneration: https://www.rnib.org.uk/eye-health-eye-conditions-z-eye-conditions/age-related-macular-degeneration-amd
Dr Mark Young lab webpage: http://www.cardiff.ac.uk/biosi/contactsandpeople/stafflist/u-z/young-mark-dr-overview_new.html
About Dr Mark Young
Mark T Young is a Lecturer in the School of Biosciences at Cardiff University. Mark received his PhD in Biochemistry from The University of Bristol, investigating the structure and function of membrane transporter proteins, and his research focuses on the 3D structure and signalling functions of P2X receptor ion channels.
He is currently working on the BBSRC-funded P2X7 Interactome project (www.p2x7.co.uk), which seeks to understand the molecular basis of P2X7 signalling in inflammation.