Inherited retinal dystrophy (IRD) is a group of phenotypes caused by mutations in visual pathways-related genes, mostly occurring at photoreceptors. This heterogeneous group includes retinitis pigmentosa (RP) recognised by bone spicule at the peripheral retina and the other is Stargardt with macular pisiform flecks. In this study, a 20- year-old male patient with RP symptoms was accompanied by a yellowish pisiform flex in the macula. However, his brother, mother and aunty have typical Stargardt disease. This study involved four persons, two males (cases 1 and 2), their mother (case 3) and aunt (case 4). Initially, cases 1 and 2 came to the clinic, case 1 was diagnosed as RP and macular dystrophy, and case 2 was diagnosed as Stargardt disease. On the follow-up, cases 1 and 2 as well as their father, mother and other family members underwent comprehensive eye examination, including fundus, Snellen, OCT, OCT-A and HFA, and found an uncommon macular abnormality besides typical RP appearance in case 1. The father is healthy while the mother and one of his aunties were diagnosed as Stargardt. A genetics analysis was conducted in case 1, finding various mutations associated with IRD mutation at the cone protein-encoded gene that concentrated at the central and rod protein-encoded gene concentrated at the peripheral retina. Whether the combination of multiple or the same mutations is responsible for this RP phenotype needs further analysis and validation. Cases 2 and 3 genetic analysis showed similar mutation results but with a healthy peripheral retina and only represented Stargardt. Case 1 is considered as RP with macular dystrophy, while cases 2, 3 and 4 are confirmed as Stargardt.
Previous studies have reported that compounds bearing an arylamide linked to a heterocyclic planar ring have successfully inhibited the hemopexin-like domain (PEX9) of matrix metalloproteinase 9 (MMP9). PEX9 has been suggested to be more selectively targeted than MMP9's catalytic domain in a degrading extracellular matrix under some pathologic conditions, especially in cancer. In this study, we aim to synthesize and evaluate 10 arylamide compounds as MMP9 inhibitors through an enzymatic assay as well as a cellular assay. The mechanism of inhibition for the most active compounds was investigated via molecular dynamics simulation (MD). Molecular docking was performed using AutoDock4.0 with PEX9 as the protein model to predict the binding of the designed compounds. The synthesis was carried out by reacting aniline derivatives with 3-bromopropanoyl chloride using pyridine as the catalyst at room temperature. The MMP9 assay was conducted using the FRET-based MMP9 kits protocol and gelatin zymography assay. The cytotoxicity assay was done using the MTT method, and the MD simulation was performed using AMBER16. Assay on MMP9 demonstrated activities of three compounds (2, 7, and 9) with more than 50% inhibition. Further inhibition on MMP9 expressed by 4T1 showed that two compounds (7 and 9) inhibited its gelatinolytic activity more than 50%. The cytotoxicity assay against 4T1 cells results in the inhibition of the cell growth with an EC50 of 125 μM and 132 μM for 7 and 9, respectively. The MD simulation explained a stable interaction of 7 and 9 in PEX9 at 100 ns with a free energy of binding of -8.03 kcal/mol and -6.41 kcal/mol, respectively. Arylamides have potential effects as selective MMP9 inhibitors in inhibiting breast cancer cell progression.