Structure–Activity Relationship Studies on Oxazolo[3,4-a]pyrazine Derivatives Leading to the Discovery of a Novel Neuropeptide S Receptor Antagonist with Potent In Vivo Activity

Neuropeptide S modulates important neurobiological functions including locomotion, anxiety, and drug abuse through interaction with its G protein-coupled receptor known as neuropeptide S receptor (NPSR). NPSR antagonists are potentially useful for the treatment of substance abuse disorders against which there is an urgent need for new effective therapeutic approaches. Potent NPSR antagonists in vitro have been discovered which, however, require further optimization of their in vivo pharmacological profile. This work describes a new series of NPSR antagonists of the oxazolo[3,4-a]pyrazine class. The guanidine derivative 16 exhibited nanomolar activity in vitro and 5-fold improved potency in vivo compared to SHA-68, a reference pharmacological tool in this field. Compound 16 can be considered a new tool for research studies on the translational potential of the NPSergic system. An in-depth molecular modeling investigation was also performed to gain new insights into the observed structure–activity relationships and provide an updated model of ligand/NPSR interactions.


SUPPORTING INFORMATION CONTENTS
Pag. Figures S1-S2: 1 H-NMR and NOE NMR analysis for compound 17 S2-S3 Table S1: Human GPCRs sharing with NPSR a sequence identity higher than 20%, a sequence coverage higher than 70%, and that were crystallized in their inactive states.

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S4 Table S1. Human GPCRs sharing with NPSR a sequence identity higher than 20%, a sequence coverage higher than 70%, and that were crystallized in their inactive states.  Figure S3. Phylogenetic tree of the human NPSR and the six selected human GPCRs used as template structures. Figure S4. Pairwise sequence alignment as calculated through the gpcrdb.org webserver between human M2 muscarinic receptor and the human neuropeptide S receptor.

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S7 Figure S5. Pairwise sequence alignment as calculated through the gpcrdb.org webserver between human type-2 angiotensin receptor and the human neuropeptide S receptor.
S8 Figure S6. Pairwise sequence alignment as calculated through the gpcrdb.org webserver between human C5a anaphylatoxin chemotactic receptor 1 and the human neuropeptide S receptor.
S9 Figure S7. Pairwise sequence alignment as calculated through the gpcrdb.org webserver between human neuropeptide Y Y1 receptor and the human neuropeptide S receptor. S10 Figure S8. Pairwise sequence alignment as calculated through the gpcrdb.org webserver between human  opioid receptor and the human neuropeptide S receptor. S11 Figure S9. Pairwise sequence alignment as calculated through the gpcrdb.org webserver between human orexin-1 receptor and the human neuropeptide S receptor.     The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand. S16 Figure S17. Schematic of detailed ligand atom interactions between 1 and NPSR during the MD simulation in BM1. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. S17 Figure S18. Plot of the ligand-protein interactions between 1 and NPSR in BM2 throughout the simulation. Protein-ligand interactions (or 'contacts') are categorized into four types: Hydrogen Bonds (green bars), Hydrophobic (violet bars), Ionic (magenta bars) and Water Bridges (blue bars). The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand. S18 Figure S19. Schematic of detailed ligand atom interactions between 1 and NPSR during the MD simulation in BM2. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. S19 Figure S20. Plot of the ligand-protein interactions between 16 and NPSR in BM1 throughout the simulation. Protein-ligand interactions (or 'contacts') are categorized into four types: Hydrogen Bonds (green bars), Hydrophobic (violet bars), Ionic (magenta bars) and Water Bridges (blue bars). The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand.
S20 Figure S21. Schematic of detailed ligand atom interactions between 16 and NPSR during the MD simulation in BM1. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. Figure S22. Plot of the ligand-protein interactions between 16 and NPSR in BM2 throughout the simulation. Protein-ligand interactions (or 'contacts') are categorized into four types: Hydrogen Bonds (green bars), Hydrophobic (violet bars), Ionic (magenta bars) and Water Bridges (blue bars). The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand.

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S22 Figure S23. Schematic of detailed ligand atom interactions between 16 and NPSR during the MD simulation in BM2. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. Figure S24. Plot of the ligand-protein interactions between 21 and NPSR in BM1 throughout the simulation. Protein-ligand interactions (or 'contacts') are categorized into four types: Hydrogen Bonds (green bars), Hydrophobic (violet bars), Ionic (magenta bars) and Water Bridges (blue bars).The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand.

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S24 Figure S25. Schematic of detailed ligand atom interactions between 21 and NPSR during the MD simulation in BM1. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown. Figure S26. Plot of the ligand-protein interactions between 21 and NPSR in BM2 throughout the simulation. Protein-ligand interactions (or 'contacts') are categorized into four types: Hydrogen Bonds (green bars), Hydrophobic (violet bars), Ionic (magenta bars) and Water Bridges (blue bars). The stacked bar charts are normalized over the course of the trajectory: for example, a value of 0.7 suggests that 70% of the simulation time the specific interaction is maintained. Values over 1.0 are possible as some protein residue may make multiple contacts of same subtype with the ligand. Figure S27. Schematic of detailed ligand atom interactions between 21 and NPSR during the MD simulation in BM2. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0.00 through 100.00 ns), are shown.