Poster Number-58 - Update on NTM-006, a Novel Orally-Active Non-Opioid Analgesic: Molecular-Modeling Consistent with Allosteric Modulation of the Adenosine A3 Receptor

Update on NTM-006, a Novel Orally-Active Non-Opioid Analgesic: Molecular-Modeling Consistent with Allosteric Modulation of the Adenosine A3 Receptor

Purpose: Neumentum NTM-006 emerged as a lead candidate from a Johnson & Johnson drug-discovery program seeking novel orally-active, non-opioid, non-NSAID analgesic agents having good efficacy, but absent the usual problems associated with current major classes of analgesics. Based on in vitro assay screens, the compound is non-opioid, and non-NSAID (does not inhibit COX-1 or COX-2 cyclooxygenase isozymes) at doses that produce its anti-nociceptive and anti-pyretic effects in a variety of animal models predictive of analgesic efficacy against acute pain conditions. But NTM-006’s preclinical and clinical analgesic efficacy seems to exceed that of the original target (e.g., it is active in a preclinical model of neuropathic pain). Based on radioligand binding data and the in vivo profile, we speculated that NTM-006 allosterically modulates the Adenosine A3 receptor, but had no further data to support such a mechanism of action.

Methods:

A homology model of the Adenosine A3 receptor was built using tools from the YASARA molecular modeling program. The sequence of human adenosine A3 was retrieved from the UniProt Knowledge base. Structures of Adenosine receptors containing bound adenosine were downloaded from the RCSB database and used as custom templates in the model building. The final YASARA hybrid model was used for docking and molecular dynamics simulations.

 

NTM-006 was built using YASARA’s small molecule building tool. Docking was performed using AutoDock VINA as implemented in YASARA using default parameters. The top 16 poses were retained for each docking result. Constructing the docking cell was done by aligning the receptor homology model with receptor structures containing bound adenosine (orthosteric), MIPS521 (transmembrane) or 6FA (cytostolic) using the Smith&Waterman method. NTM-006 was then superimposed on the known ligand and the cell centered on NTM-006.  For the N-terminal site, NTM-006 was placed based on interactions described for VCD171 in the A1 receptor.

 

Molecular Dynamics were done by constructing a membrane around the receptor, adding water and then neutralizing the cell by addition of Na and Cl ions. Parameters for NTM-006 were assigned by YASARA. Charges were assigned using the GAFF/AM1BCC method. The AMBER14 force field was used with periodic boundary conditions and an NPT ensemble (298K and 1 atm). Simulations were run for 100 ns after an initial minimization to remove clashes.

Results:

Molecular modeling studies suggest a novel combination of binding modes for NTM-006 with the AdenosineA3 receptor. The modeling to-date predicts that: (1) NTM-006 binds with low affinity to the site where endogenous Adenosine binds (Adenosine itself also binds with relatively low affinity to this 'orthosteric' binding site); (2) NTM-006 also binds to an N-terminal extracellular site suggested by prior molecular dynamics calculation as an 'allosteric' binding site; (3) NTM-006 also fits a cytostolic binding site suggested based on analogy to allosteric modulators of other G protein-coupled receptors, and (4) NTM-006 also fits a pocket in the transmembrane domain of the Adenosine-3 receptor identified in a cryo-EM structure as an allosteric binding site in the Adenosine-1 receptor. In this case NTM-006 leaves the pocket and migrates upward out of the lipid membrane. As it transits, the conformation of Adenosine in the orthosteric binding pocket changes.

Conclusion: NTM-006 was speculated to exert its primary action at Adenosine A3 receptors, possibly as an allosteric modulator. The results of the molecular modeling reported here supports this hypothesis. The combination of binding processes would be consistent with NTM-006's in vitro, animal model, and clinical characteristics of broad-spectrum and pronounced analgesic efficacy, and long-duration of analgesic action.

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