Structural activity relationship analysis (SAR) and in vitro testing reveal the anti-ageing potential activity of acetyl aspartic acid

Synopsis Background Acetyl aspartic acid (A-A-A) was discovered through gene array analysis with corresponding connectivity mapping (Cmap), aiming for identification of new compounds with anti-ageing properties. Objective The aim of this study was to use structural activity relationship (SAR) analysis to identify a predictive mechanism of action of A-A-A. The findings from SAR will be further characterized by in vitro activity testing. Furthermore, we aimed to investigate the role of polymerized filamentous F-actin in ageing fibroblasts and to evaluate the effect of A-A-A on this model. Methods To predict the mode of action of A-A-A, we used the PASS computer program as a SAR model. In vitro, scratch motility tests with immortalized keratinocytes were used as a model for wound healing potential. Matrix metalloproteinase 1-3 (MMP 1-3) was analysed using multiplex protein assays (Luminex), and polymerized actin was detected by phalloidin staining in dermal fibroblasts (HDF). Results SAR analysis predicted that A-A-A would possess both epidermal and dermal activities with identification of wound healing and MMP inhibition potential. Further in vitro studies confirmed the wound healing potential using keratinocyte scratch motility assays. We were also able to confirm the dermal activities predicted by inhibition of MMP (MMP 1–3) in HDF by A-A-A. In addition, we found a positive relationship between age and F-actin expression. We also discovered that stimulation of HDF with A-A-A for 72 h significantly reduced the polymerized cytoskeletal network as visualized by inhibition of F-actin expression. In fact, A-A-A leveraged the expression of F-actin in middle-aged female fibroblasts (50 years of age) to the level of young female fibroblasts (30 years of age), corresponding to a 40% reduction in F-actin expression. Conclusion Using an in silico and in vitro approach, we were able to demonstrate that A-A-A has the capacity to target different compartments of the skin through keratinocyte regeneration, MMP inhibition and relief in fibroblasts stiffness by reduction of F-actin cytoskeletal network in HDF. Resume Contexte L'acide aspartique acetyle (A-A-A) a ete identifie en utilisant la technologie de puces a ADN combinee a une analyse en ‘connectivity mapping’ (Cmap), pour ces proprietes potentielles anti-âge. Ces proprietes ont ete testees par une approche in silico et in vitro sur modeles cellulaires humains. Methodes La prediction in silico, utilise un logiciel de relation ‘Structure - Activite’, qui permet d'orienter le design des experimentations in vitro. Ces tests ont realises sur keratinocytes pour les tests de reparation celullaire et sur fibroblastes pour l’etude de l'inhibition de metallo-proteinases ainsi que l'expression de l'actine-F, mesuree en fonction de l’âge puis apres traitement avec A-A-A. Resultats Les predictions in silico ont permis de determiner que A-A-A presentent de capacites de reparation cellulaire et d'inhibition des metallo-proteinases (MMP). La validation de ces activites a ete confirmee respectivement via un test in vitro de migration cellulaire sur keratinocytes et des activites MMP sur fibroblastes. L'augmentation significative de la mobilite cellulaire apres stimulation avec A-A-A, nous a conduit a tester l'expression de l'actine –F tout d'abord en demontrant une augmentation de son expression en fonction de l′âge, induisant une rigidite mecanique du fibroblaste. Nous avons ensuite confirme que la stimulation of fibroblastes âges avec A-A-A durant 72 heures reduisait significativement l'expression de ce marqueur de rigidite. En l'occurrence, A-A-A reduit l'expression de l'actine-F sur de fibroblastes de 50 ans de 40% ce qui equivaut a une expression de l'actine-F de fibroblastes de 30 ans. Conclusion En combinant une approche in silico et in vitro, nous avons confirme le potentiel de A-A-A a ameliorer des marqueurs cellulaires du viellissement dans les compartiments epidermique et dermique via la regeneration cellulaire sur keratinocytes, l'inhibition des metalloproteinases et la reduction de l'expresssion de l'actine-F, comme marqueur de rigidite cellulaire liee a l'âge sur fibroblastes.