Phenotypic high throughput screening highlights promising molecules for the treatment of SLC7A8 dependent age-related hearing loss

Age-related hearing loss (ARHL) is the most common sensory impairment affecting 30% of the individuals over 65 years old. A complex multifactorial etiology characterizes ARHL, thus environmental and genetics factors affects its onset, progression and severity. However, regarding the genetic contributions, few causative genes have been highlighted resulting in a lack of molecular target identification for tailored therapy development. A promising ARHL-candidate gene is SLC7A8, which encodes for the catalytic subunit of an amino acid transporter. A previous study (Espino G.M. et al., 2018) proved that Slc7a8 expression in mice’s cochlea increased with aging, and knock-out mice displayed ARHL. Furthermore, four damaging heterozygous missense variants (c.904G>A, p.(V302I); c.1205C>T, p.(T402M); c.1252C>T, p.(R418C); c.1379T>A, p.(V460E)) were identified in subjects affected by ARHL. The functional characterization of all the variants confirmed their pathogenetic effect, demonstrating a reduced ability of the protein to carry out its function as an amino acid transporter. These evidences support the idea that SLC7A8 should be further investigated as a possible therapeutic target. Considering these preliminary findings, the first aim of this thesis has been to better define the prevalence of SLC7A8 variant in the Italian population. For this purpose, whole genome sequencing data of 162 individuals coming from Italian genetic isolates with a careful phenotypical evaluation were employed. The analysis allowed the identification of a novel rare damaging variant c.734T>C; p.(Y245C) within the gene in a subject affected by ARHL and absent in controls. The variant was characterized proving that it significantly impairs the amino acid transport activity of the protein, in line with what was demonstrated for the variants described in the work of Espino G.M. et al. This new evidence confirms that a significant percentage (i.e., ~3%) of ARHL patients is a carrier of damaging SLC7A8 variants. Thus, it is possible to hypothesize that a treatment directed against SLC7A8 may have a high impact in the population. Considering that all the identified variants cause the production of a protein with reduced functionality, the second aim of this work has been to perform a high throughput screening (HTS) searching for SLC7A8 transcription regulators. The strategy was defined hypothesizing that a treatment aimed at recovering the normal levels of functional SLC7A8 might be beneficial to ARHL-affected patients. The CRISPR/Cas9 technology was employed to create a reporter line expressing the luciferase gene under the transcriptional control of the SLC7A8 promoter. In a high throughput process, the reporter line was incubated 48 hours with a library of 2236 Food and Drug Administration (FDA)-approved drugs, 960 drug-like compounds, and 44 molecules selected as putative SLC7A8 transcription regulators. A total of 68 positive hit compounds were detected (48 are already approved drugs). The most efficient molecules were tested in a second independent HTS evaluating in parallel luciferase activity and cell viability, and the activity of almost all molecules was confirmed. Furthermore, three hit compounds were further selected following literature data highlighting their association with hearing function, or the activation of pathways that lead to the increased expression of SLC7A8. A qRT-PCR was performed for those compounds, confirming that the treatment with a concentration of 1.5µM significantly increased SLC7A8 expression in the analyzed cell lines. In conclusion, this study has allowed us to clarify with greater precision the prevalence of variants of the SLC7A8 gene in the Italian population and to identify several molecules to be proposed as potential treatments for presbycusis. These results, therefore, may represent the starting point for developing the first targeted therapy for the treatment of ARHL.