Different drug discovery-approaches for neuroprotection: design, synthesis and biological evaluation of pro-autophagic enhancers

Autophagy is one of the main biological processes involved in maintaining the cellular homeostasis. Indeed, autophagy dysregulation represents a common feature in many pathologies, such as cancer, metabolic disorders and neurodegenerative diseases (NDDs). Importantly, it is demonstrated that autophagy restoration is linked to neuroprotective effects on in vitro and in vivo models. Therefore, the aim of this research project is to develop novel pro-autophagic compounds endowed with neuroprotective activity to counteract neurodegeneration. To do so, I applied three different drug discovery approaches, namely target-based, phenotype-based and natural product-inspired approaches. The target-based approach focused on developing novel allosteric AMPK activator. AMPK is an heterotrimeric kinase tightly involved in autophagy initiation, and so it represents an attractive protein to target for this research. Thanks to an in silico study we designed three series of benzimidazole-based structures capable of binding the allosteric pocket of AMPK. Nineteen compounds were synthesized and characterized. Subsequently, the obtained products were assessed for their early ADME-Tox properties. The panel of assays included cytotoxicity (A549 and HEK293 cell lines, 24h), potential cardiotoxicity (hERG-binding assay), off-target and epigenetic liability (HDAC6, HDAC8 inhibition assay) and preliminary ecotoxicity (Aliivibrio fischeri toxicity). Furthermore, with the aim of evaluating their ability to activate AMPK, I established an enzymatic protocol to apply in an in vitro assay. A small group of these small molecules were also tested in a cellular model able to detect the modulation of the autophagy flux. The phenotype-based approach focused on modifying the structure of a diarylmethane derivative that demonstrated neuroprotective and pro-autophagic effects on in vitro and in vivo models, namely SG2. In fact, SG2 also showed a warning probability of cardiotoxicity during a preliminary ADME-Tox assessment. This result brought us to design novel diarylmethane derivatives inspired by SG2. Twelve new analogues were synthesized and characterized together with other two compounds which were already obtained, namely SG22 and SG23. All compounds have been tested for their ADME-Tox profile, also to evaluate their potential cardiotoxicity. A small group of the new derivatives were selected to be tested for their neuroprotective activities on two phenotype-based models for Alzheimer’s Disease (AD). Furthermore, due to a contraposition of in vitro and in vivo results of SG22, an in vivo uptake study was performed on a AD model of Caenorhabditis elegans. Besides this, since other NDDs are linked to autophagy disruption, a drug screening on a Danio rerio model for neuronal ceroid lipofuscinosis was run as well. The natural product-inspired approach focused on designing and synthesizing novel analogues of urolithin A (UA). UA is a gut metabolite of ellagic acid, a polyphenol provided by natural sources, such as pomegranate, berries and nuts. UA is known for its multiple biological activities, including anti-inflammatory, antioxidant and pro-autophagic properties. Therefore, we selected UA’s scaffold as starting point of this drug design. The urolithin structure was combined with other pharmacophores or molecules capable of direct or indirect neuroprotective effects, namely short-chain fatty acids, polyamines, and the carbamate moiety of rivastigmine. Moreover, a small group of UA analogues was developed following an in silico screening with the aim of selectively targeting SIRT1, a known autophagy promoter. The biological evaluation of UA-inspired compounds included early ADME-Tox profiling, ecotoxicity activity, and both in vitro and in vivo neuroprotection. Furthermore, the most promising compound was evaluated for its ability to overcome the blood brain barrier. The techniques and the protocol applied for both synthesis and biological evaluation of all the small molecules developed within this project are described in detail in the related PhD thesis.