Periodic Reporting for period 1 - MTD4ALS (Development of inovative multi-target drugs for ALS) Reporting period: 2020-06-01 to 2022-05-31 Summary of the context and overall objectives of the project Motor Neuron Disorders (MNDs) are a heterogeneous group of diseases characterised by the progressive loss of motor neurons (MNs). Among them, Amyotrophic Lateral Sclerosis (ALS) is the most common form of adult-onset form of MND. ALS is characterized by the progressive loss of MNs responsible for the voluntary movements in the motor cortex and in the spinal cord of the patients and dia due to respiratory failure 3-5 years after diagnosis. Nevertheless, there is no cure available for the ALS, and the two only drugs approved for ALS treatment, Riluzole and Edaravone are only capable of, respectively, increasing patient survival (3-4 months) and delay the progression of symptoms in the initial stages of the disease. Therefore, is not surprising that due to the high social and economical burden of ALS that there is tremendous pressure from the patients and their families, and the global healthcare system for the development of more efficient therapies.ALS is a multifactorial disease characterised by defects in several biological mechanisms such as mitochondria homeostasis, antioxidant response or inflammation that contribute to the disease progression. Hence, to surpass the past failures the development of small molecules capable to modulate multiple pathways in ALS may provide prime tools for the development of new and better pharmacological therapies than the drugs currently accessible to the patients. Following this rationale in this action, we proposed the development of a therapeutic approach based on small molecules capable to modulate NF-kB transcription factor activity (a key player in neuroinflammation) and, with iron chelation properties to minimize the negative effects of the abnormal iron accumulation in the patient tissues (Fenton/Haber-Weiss reactions and ferroptosis) and trigger cell-survival mechanisms through mild HIF stabilization. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far In this action, we proposed the development of novel small molecules capable to inhibit IKKB which is a protein crucial for the activation of NF-κB, the key regulator of inflammation in glial cells of ALS patients, and at the same time presenting iron chelation properties to minimize the effect of the toxic accumulation of iron observed in the tissues of ALS patients and trigger the expression of pro-survival and neurotrophic genes in MNs. In this action, we started by designing a library of multitarget small molecules after applying two virtual screening filters to select the most promising compounds to interact with IKKB and with the most promising profile for BBB permeation we selected a library of 20 potential multitarget small molecules. The small library was then synthesised by newly developed synthetic routes and after evaluating their iron and antioxidant capacity we selected 16 compounds for our in-vitro cellular cytotoxicity and neuroprotective studies. From our cellular screening studies, 13 and 14 were selected as the most promising Lead. Additionally, our preliminary studies of some properties that may influence the 13 and 14 ADMET profiles revealed that the compounds present a % of HSA binding that suggests higher bioavailability than Edaravone, Riluzole and Deferiprone. The BBB permeability profile of 13 and 14 was also better than at least the one predicted for Edaravone and Deferiprone and worse than Riluzole's passive permeability profile through BBB. Several activities were conducted to ensure the efficient dissemination and exploitation of the results obtained throughout the action. Therefore, to share our results with our peers and receive their feedback the researcher participated in two international conferences where the results of his work were presented. The researcher was also invited to deliver two oral communications in the scientific meetings organized by the chemistry students from the UC and the UP. The researcher also acquired experience in the supervision of one undergraduate student project and participated in the co-supervision of one Master Student (ongoing) project during which some results of this project were acquired and then presented in the form of a final report. Furthermore, the researcher also submitted one review that is currently in a peer review process where he described the potential of developing drugs for ALS for the targets highlighted in this action. The work developed in this action is still ongoing and already revealed that our discoveries may have major scientific impacts therefore, shortly at least 2 more papers describing the results of our work will be submitted for peer review. The work and experience gathered during this action encouraged the researcher to establish new collaborations and to apply for research funding to support projects related to the topic of this project. Hence, the researcher also explored the results of this work for a grant application for one exploratory project from FCT to captivate funding to further progress the project developed herein and to achieve the scientific goals not achieved during the action. Additionally, he also participated in the preparation of several grant proposals aiming to develop and/or validate novel therapeutic approaches for ALS under the MSCA-ITN, FCT and other granting schemes. Two of these grant applications were successful and the researcher is currently a member of the research teams of two collaborative projects funded by the FCT granting schemes. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) In this project, we developed of novel chemical entities that could lead to the development of novel therapeutics for ALS. In this action, we aimed to develop novel small molecules capable to modulate the key biological targets involved in inflammation and capable to protect neurons from the negative effects of iron metabolic dysregulation observed in ALS patients. Therefore, we designed and synthesised a novel family of multitarget small molecules and after in vitro screening of their cellular and ADMET profile, we identified two promising Lead compounds that may be optimized into potential candidates for novel therapies for ALS. From a personal point of view, the researcher involved in this project enhanced his scientific maturity and increased his data and project management skills. On the other hand, he also made contacts and discussed his research with his peers and was capable to start to establish a network to support that supported this project and his future career prospects. On the other hand, this project is expected to have a major scientific impact in the field of drug discovery since it addresses the development of novel chemical entities targeting key biological and druggability features common to the fields of neurosciences, oncology and immunology. Furthermore, the project is also aimed to create awareness of the disease and the urgency of the development of novel therapies for ALS. Additionally, the endpoint of this project is to provide new small molecules that could lead to innovative therapies and improve ALS patients' well-being and decrease the pressure on the global healthcare system and, therefore decrease the social burden on the patients and their families and decrease the economical costs involved with the patient´s palliative care. The researcher engaged the Portuguese Mirror group of the European Joint Programme for Rare Diseases (EJPRD) to engage the general public and create awareness of the disease and the benefits of our research advances in their daily lives by sharing the goals of our research. msca-summary-image-ps-pdf.jpg
