SIGMA 1 RECEPTOR (S1R) MODULATORS AS A THERAPEUTIC STRATEGY FOR PROMOTING NEUROPLASTICITY. DESIGN AND SYNTHESIS OF NOVEL MONO- AND BI-VALENT LIGANDS FOR SRS

During my three-years-project I focused on the development of novel mono- and bi-valent Sigma1 Receptor (S1R) modulators to address two main objectives: (i) the obtainment of multitarget-directed ligands (MTDLs) endowed with therapeutic potential for the treatment of neurodegenerative diseases; (ii) the preparation of a series of bivalent compounds to be used for the study of S1R oligomerization process. These two major topics are briefly discussed hereafter. (i) Neurodegeneration is a key event in many challenging disorders (e.g. Alzheimers diseases, Parkinsons disease, multiple sclerosis). Such pathologies involve the alteration of several molecular pathways, making the multi-target paradigm a promising strategy for new effective therapies. Among the numerous molecular targets that have been correlated with neurodegenerative disorders, S1R has gained great attention from the scientific community, and S1R agonists are considered viable pharmacological tools for their neuroprotective activity. Hence, we reasoned that coupling S1R agonism with modulation of other selected targets might afford new molecular entities more effective in counteracting neuropathies. The additional targets of our MTDLs include N-Methyl-D-Aspartate (NMDA) receptor, which plays a relevant role in synaptic plasticity, and acetylcholinesterase (AChE), which regulates acetylcholine levels in central nervous system. A structurally focused compound library was prepared through a divergent synthesis. The so-obtained compounds were tested for a preliminary biological evaluation, evaluating their affinity and selectivity towards S1R and NMDA receptor, the AChE inhibition and their antioxidant properties, since oxidative stress is considered a hallmark of neurodegeneration. A number of promising compounds, endowed with effective multitarget profile, was identified. These results will pave the way for further biological investigation and structure optimization in order to achieve viable tools for the treatment of neurodegenerative diseases. (ii) In the last decade numerous studies have supported the hypothesis that S1R can exist in multiple oligomeric forms. In detail, agonists seem to stabilize S1R monomers and dimers that act as chaperones, whereas antagonists bind to higher oligomer complexes, maintaining them in repository forms. Moreover, the recently disclosed crystal of S1R was obtained as a trimer. Nevertheless, the mechanism of generation, as well as the precise biological function of S1R oligomers, are still unknown. Accordingly, a series of homo- and hetero-bivalent S1R ligands was designed and synthetized to investigate S1R oligomerization process. Since S1R agonists are known to exert neuroprotective effects, and S1R can form homo-dimeric structures upon interaction with agonists, we reasoned that promoting dimerization through bivalent agonists might enhance ligands activity. The designed bivalent compounds consist in two units of (R)-RC-33 (a potent and selective S1R agonist developed by our group) joined by a linker. Different lengths, polarities and spatial constraints were explored for the linker. The key precursor of the synthesis is (R)-RC-33A, an aminic derivative of RC-33. For the obtainment of enantiopure (R)-RC-33A, three different synthetic approaches have been explored, resulting in the identification of an efficient pathway to access (R)-RC-33 derivatives with high yield and chiral purity. Once the designed ligands were obtained in sufficient amount and purity, they were tested in binding assays to assess their S1R affinity. Moreover, computational studies were performed on both mono- and bi-valent S1R modulators. In detail, docking into the crystals binding pocket served as basis for the development of a 3D-QSAR model and for the rationalization of experimental results. Molecular dynamics studies are ongoing, and future functional assays will contribute to shed light on the S1R oligomeric states

Setup and Validation of a Reliable Docking Protocol for the Development of Neuroprotective Agents by Targeting the Sigma-1 Receptor (S1R)

Sigma-1 receptor (S1R) is a promising molecular target for the development of novel effective therapies against neurodegenerative diseases. To speed up the discovery of new S1R modulators, herein we report the development of a reliable in silico protocol suitable to predict the affinity of small molecules against S1R. The docking method was validated by comparing the computational calculated Ki values of a test set of new aryl-aminoalkyl-ketone with experimental determined binding affinity. The druggability profile of the new compounds, with particular reference to the ability to cross the blood–brain barrier (BBB) was further predicted in silico. Moreover, the selectivity over Sigma-2 receptor (S2R) and N-methyl-d-aspartate (NMDA) receptor, another protein involved in neurodegeneration, was evaluated. 1-([1,1’-biphenyl]-4-yl)-4-(piperidin-1-yl)butan-1-one (12) performed as the best compound and was further investigated for acetylcholinesterase (AchE) inhibitor activity and determination of antioxidant activity mediated by aquaporins (AQPs). With a good affinity against both S1R and NMDA receptor, good selectivity over S2R and favorable BBB penetration potential together with its AChE inhibitory activity and its ability to exert antioxidant effects through modulation of AQPs, 12 represents a viable candidate for further development as a neuroprotective agent

Synthesis of a drug discovery library for the identification of sigma receptors modulators

Synthesis of a drug discovery library for the identification of sigma receptors modulator

Synthesis of a drug discovery library for the identification of sigma receptors modulators

Synthesis of a drug discovery library for the identification of sigma receptors modulator

PEG 400/Cerium Ammonium Nitrate Combined with Microwave-Assisted Synthesis for Rapid Access to Beta-Amino Ketones. An Easy-to-Use Protocol for Discovering New Hit Compounds

Compound libraries are important requirement in target-based drug discovery. In the present work, a small focused compound library based on β-aminoketone scaffold has been prepared combining microwave-assisted organic synthesis (MAOS) with polymer-assisted solution phase synthesis (PASPS) and replacing reaction workup standard purification procedures with solid phase extraction (SPE). Specifically, the effects of solvent, such as dioxane, dimethylformamide (DMF), polyethylene glycol 400 (PEG 400), temperature, irradiation time, stoichiometric ratio of reagents, and catalysts (HCl, acetic acid, cerium ammonium nitrate (CAN)) were investigated to maximize both conversion and yield. The optimized protocol generally afforded the desired products in satisfying yields and purities. The designed library is a part of our current research on sigma 1 receptor modulators, a valuable tool for the identification of novel potential hit compounds

Sigma-1 receptor antagonists: promising players in fighting neuropathic pain

Sigma-1 receptors (S1Rs) are strongly correlated to neuropathic pain (NP), since their inactivation may decrease allodynia or dysesthesia, promoting analgesic effects. In the recent patent landscape, S1R antagonists endowed with nanomolar S1Rs affinity emerged as potent antinociceptive agents. So far, three patented compounds have been proposed for counteracting NP. Particularly PV 752 and AV1066, disclosed by the University of Pavia (Italy) and Anavex, respectively, showed good analgesic activity in preclinical studies. Moreover, E 52862 developed by Esteve (Spain) has been proved to be effective, both in preclinical and Phase II clinical trials, against several symptoms of NP. These patents ascertain S1R antagonists as potential drugs, alone or in combination with other analgesic drugs, for managing NP in humans

Biophysical Assays for Investigating Modulators of Macromolecular Complexes: An Overview

: Drug discovery is a lengthy and intricate process, and in its early stage, crucial steps are the selection of the therapeutic target and the identification of novel ligands. Most targets are dysregulated in pathogenic cells; typically, their activation or deactivation leads to the desired effect, while in other cases, interfering with the target-natural binder complex achieves the therapeutic results. Biophysical assays are a suitable strategy for finding new ligands or interferent agents, being able to evaluate ligand-protein interactions and assessing the effect of small molecules (SMols) on macromolecular complexes. This mini-review provides a detailed analysis of widely used biophysical methods, including fluorescence-based approaches, circular dichroism, isothermal titration calorimetry, microscale thermophoresis, and NMR spectroscopy. After a brief description of the methodologies, examples of interaction and competition experiments are described, together with an analysis of the advantages and disadvantages of each technique. This mini-review provides an overview of the most relevant biophysical technologies that can help in identifying SMols able not only to bind proteins but also to interfere with macromolecular complexes

Sigma-1 Receptor Agonists Acting on Aquaporin-Mediated H2O2 Permeability: New Tools for Counteracting Oxidative Stress

Sigma1 Receptor (S1R) is involved in oxidative stress, since its activation is triggered by oxidative or endoplasmic reticulum stress. Since specific aquaporins (AQP), called peroxiporins, play a relevant role in controlling H2O2 permeability and ensure reactive oxygen species wasted during oxidative stress, we studied the effect of S1R modulators on AQP-dependent water and hydrogen peroxide permeability in the presence and in the absence of oxidative stress. Applying stopped-flow light scattering and fluorescent probe methods, water and hydrogen peroxide permeability in HeLa cells have been studied. Results evidenced that S1R agonists can restore water permeability in heat-stressed cells and the co-administration with a S1R antagonist totally counteracted the ability to restore the water permeability. Moreover, compounds were able to counteract the oxidative stress of HeLa cells specifically knocked down for S1R. Taken together these results support the hypothesis that the antioxidant mechanism is mediated by both S1R and AQP-mediated H2O2 permeability. The finding that small molecules can act on both S1R and AQP-mediated H2O2 permeability opens a new direction toward the identification of innovative drugs able to regulate cell survival during oxidative stress in pathologic conditions, such as cancer and degenerative diseases