Periodic Reporting for period 1 - Clockstop (A Fast Throughput Drosophila Model to Identify Drug Treatments for Age-related Hearing Loss (ARHL))
Periodo di rendicontazione: 2019-06-01 al 2021-05-31
Sintesi del contesto e degli obiettivi generali del progetto
Hearing loss affects 5% of the population according to World Health Organisation (WHO) and by 2050 it is expected that 1 in every 10 people will have disabling hearing loss. Hearing loss can be caused by disease, noise, or heredity; however, the age-related hearing loss (ARHL) amounts for the majority of cases. Approximately 1 in 3 people between the ages of 65 and 74 are affected by ARHL, which can lead to various mental health issues, ranging from anxiety to depression. Also, recently the research has shown that hearing loss can be associated with cognitive decline and dementia.
ARHL is progressive and irreversible condition, for which there is no effective treatment nor cure available. However, the quality of life can be improved with assistive devices, such as hearing aids or in more severe situations – cochlear implants. The difficulty in researching and finding appropriate treatments for ARHL is associated with the complexity of the hearing process and the age-dependent decline. The fruit fly, Drosophila melanogaster, has been efficiently used in drug discovery and in lifespan studies. Over past two decades fruit flies served as a good model for hearing and deafness, where the genes involved in human deafness syndromes were also evolutionarily conserved in flies. Recently, our group showed that flies are prone to ARHL. The circadian profiling of the fly’s ear also allowed us to discover the homeostatic genes, including kinases, which were previously linked to the noise induced hearing loss in mice. Hence, our hypothesis was that kinases might be involved in the machinery of the ear’s daily maintenance and that the breakdown of this process might lead to ARHL.
Many molecular pathways are conserved between the ears of humans and Drosophila, hence the latter has become a powerful tool to study human hearing and deafness. One such example of the homology is found between a gene called atonal, which was first described in the fruit fly, and its human homologue ATOH1 , which was found to be also crucial for human ear development.
Here, two kinase-related pathways, previously linked to ageing, were of particular interest to us – namely mTOR and ERK/MAPK. mTOR is evolutionarily conserved; it can be found not only in yeast, plants, nematodes, and mammals. In the recent decade, the mTOR pathway has been extensively researched - pharmacologically inhibiting TOR by Rapamycin in some model organisms has been reported to lead to an increase in lifespan. ERK/MAPK play crucial roles in various cellular processes, such as proliferation, apoptosis, stress response, as well as in the survival and development of tumour cells. Moreover, many kinase inhibitor (KI) compounds, inhibiting key kinase pathways, are under development, and many have successfully completed phase 1 and/or 2 of clinical trials or are already approved for the patient use by US Food and Drug Administration. By using complex bioinformatics analyses predicting downstream regulated targets of the recently identified key ARHL master regulatory genes, several kinase signalling pathways were identified. In order to inhibit the key components of these signalling pathways, we studied kinase inhibitor databases and the best suitable candidates were shortlisted based on their biding affinity to the fruit fly kinase orthologue regions and based on their status for use in patients (successfully completed phase 2/3 or FDA approved). Our main objective was to establish a successful drug-discovery pipeline in a Drosophila ARHL model.
Our strategy of exploring KIs is illustrated in the diagram below. Briefly, target kinases were selected following three important parameters: first, the kinase of interest should be expressed in the fly’s ear and/or expression should change throughout the fly’s age, for which data was extracted from RNA sequencing analyses carried out by our lab; second, kinases should be conserved in humans; third, kinase inhibitors (KIs) should be predicted to efficiently inhibit the human orthologue of the fruit fly kinase and the available KIs should be post Phase I clinical trials or already FDA-approved (with known targets and approved safety profiles).
To achieve an efficient (medium)-throughput experimental regime, we tested flies in an accelerated ARHL (aARHL) paradigm that reduced experimental time, and amount of compound needed. The fly’s hearing in the aARHL conditions was tested using our established Laser Doppler vibrometer (LDV) setup, which allows for quantifying the integrity and sensitivity of the flies’ antennal ears. The ultimate goal of this project was to find compounds that would merit further validations in mouse models and could eventually lead to clinical trials in humans.
In summary, during the first half of the project (i.e. first 12 months) we tested 18 compounds, of which 7 accelerated the flies’ ARHL, 2 were toxic and led to lethality, 5 caused minor improvements of individual hearing parameters and 4 had no statistically detectable effects on the flies’ hearing.
Three new kinases, not previously linked to hearing or deafness, were found to be expressed in the neurons of the Johnston’s organ located in the fly’s ear. By genetically targeting kinases of interest in Johnston’s organ neurons (all tested kinases were found to be expressed in neurons), we were able to identify how overexpressing or suppressing these proteins affects hearing. This allowed to compare, reproduce (and validate) effects observed after pharmacological application of KI compounds. The effects of the genetic manipulation often differed from the phenotypes observed after compound administration. One of the reasons lies in the (non-specific) nature of the kinase inhibitor compounds, which typically target several kinases, with different affinities.
In summary, our pilot project provided two very important outcomes: (i) A new drug-discovery pipeline has been successfully established in an accelerated ARHL fruit fly model; and (ii) new signalling kinase pathways never previously linked to hearing (or hearing loss) have been identified and are now available to further scientific investigation. We will ourselves use the newly created fruit fly model to advance the scientific analysis of kinase function in hearing, auditory homeostasis and deafness. We are also preparing a manuscript to share this model - and our first findings - with the wider scientific community.
ARHL is progressive and irreversible condition, for which there is no effective treatment nor cure available. However, the quality of life can be improved with assistive devices, such as hearing aids or in more severe situations – cochlear implants. The difficulty in researching and finding appropriate treatments for ARHL is associated with the complexity of the hearing process and the age-dependent decline. The fruit fly, Drosophila melanogaster, has been efficiently used in drug discovery and in lifespan studies. Over past two decades fruit flies served as a good model for hearing and deafness, where the genes involved in human deafness syndromes were also evolutionarily conserved in flies. Recently, our group showed that flies are prone to ARHL. The circadian profiling of the fly’s ear also allowed us to discover the homeostatic genes, including kinases, which were previously linked to the noise induced hearing loss in mice. Hence, our hypothesis was that kinases might be involved in the machinery of the ear’s daily maintenance and that the breakdown of this process might lead to ARHL.
Many molecular pathways are conserved between the ears of humans and Drosophila, hence the latter has become a powerful tool to study human hearing and deafness. One such example of the homology is found between a gene called atonal, which was first described in the fruit fly, and its human homologue ATOH1 , which was found to be also crucial for human ear development.
Here, two kinase-related pathways, previously linked to ageing, were of particular interest to us – namely mTOR and ERK/MAPK. mTOR is evolutionarily conserved; it can be found not only in yeast, plants, nematodes, and mammals. In the recent decade, the mTOR pathway has been extensively researched - pharmacologically inhibiting TOR by Rapamycin in some model organisms has been reported to lead to an increase in lifespan. ERK/MAPK play crucial roles in various cellular processes, such as proliferation, apoptosis, stress response, as well as in the survival and development of tumour cells. Moreover, many kinase inhibitor (KI) compounds, inhibiting key kinase pathways, are under development, and many have successfully completed phase 1 and/or 2 of clinical trials or are already approved for the patient use by US Food and Drug Administration. By using complex bioinformatics analyses predicting downstream regulated targets of the recently identified key ARHL master regulatory genes, several kinase signalling pathways were identified. In order to inhibit the key components of these signalling pathways, we studied kinase inhibitor databases and the best suitable candidates were shortlisted based on their biding affinity to the fruit fly kinase orthologue regions and based on their status for use in patients (successfully completed phase 2/3 or FDA approved). Our main objective was to establish a successful drug-discovery pipeline in a Drosophila ARHL model.
Our strategy of exploring KIs is illustrated in the diagram below. Briefly, target kinases were selected following three important parameters: first, the kinase of interest should be expressed in the fly’s ear and/or expression should change throughout the fly’s age, for which data was extracted from RNA sequencing analyses carried out by our lab; second, kinases should be conserved in humans; third, kinase inhibitors (KIs) should be predicted to efficiently inhibit the human orthologue of the fruit fly kinase and the available KIs should be post Phase I clinical trials or already FDA-approved (with known targets and approved safety profiles).
To achieve an efficient (medium)-throughput experimental regime, we tested flies in an accelerated ARHL (aARHL) paradigm that reduced experimental time, and amount of compound needed. The fly’s hearing in the aARHL conditions was tested using our established Laser Doppler vibrometer (LDV) setup, which allows for quantifying the integrity and sensitivity of the flies’ antennal ears. The ultimate goal of this project was to find compounds that would merit further validations in mouse models and could eventually lead to clinical trials in humans.
In summary, during the first half of the project (i.e. first 12 months) we tested 18 compounds, of which 7 accelerated the flies’ ARHL, 2 were toxic and led to lethality, 5 caused minor improvements of individual hearing parameters and 4 had no statistically detectable effects on the flies’ hearing.
Three new kinases, not previously linked to hearing or deafness, were found to be expressed in the neurons of the Johnston’s organ located in the fly’s ear. By genetically targeting kinases of interest in Johnston’s organ neurons (all tested kinases were found to be expressed in neurons), we were able to identify how overexpressing or suppressing these proteins affects hearing. This allowed to compare, reproduce (and validate) effects observed after pharmacological application of KI compounds. The effects of the genetic manipulation often differed from the phenotypes observed after compound administration. One of the reasons lies in the (non-specific) nature of the kinase inhibitor compounds, which typically target several kinases, with different affinities.
In summary, our pilot project provided two very important outcomes: (i) A new drug-discovery pipeline has been successfully established in an accelerated ARHL fruit fly model; and (ii) new signalling kinase pathways never previously linked to hearing (or hearing loss) have been identified and are now available to further scientific investigation. We will ourselves use the newly created fruit fly model to advance the scientific analysis of kinase function in hearing, auditory homeostasis and deafness. We are also preparing a manuscript to share this model - and our first findings - with the wider scientific community.