TRG-TCC2-3

With nearly 700 structures solved and a growing number of customized structure prediction algorithms being developed at a fast pace, G protein-coupled receptors (GPCRs) are an optimal test case for validating new approaches for the prediction of receptor active state and ligand bioactive conformation complexes. In this study, we leveraged the availability of hundreds of peptide GPCRs in the active state and both classical homology and artificial intelligence (AI) based protein modeling combined with docking and AI-based peptide structure prediction approaches to predict the nociceptin/orphanin FQ-NOP receptor active state complex (N/OFQ-NOPa). The In Silico generated hypotheses were validated via the design, synthesis, and pharmacological characterization of novel linear N/OFQ(1-13)-NH₂ analogues, leading to the discovery of a novel antagonist (3B; pK_B = 6.63) bearing a single ring-constrained residue in place of the Gly²-Gly³ motif of the N/OFQ message sequence (FGGF). While the experimental validation was ongoing, the availability of the Cryo-EM structure of the predicted complex enabled us to unambiguously validate the generated hypotheses. To the best of our knowledge, this is the first example of a peptide-GPCR complex predicted with atomistic accuracy (full complex Cα RMSD < 1.0 Å) and of the N/OFQ message moiety being successfully modified with a rigid scaffold.

The authors investigated the synthesis and opioid receptor-binding affinity of enkephalin analogs, peptidomimetics containing a stereogenic trifluoroethylamine moiety in place of Gly²-Gly³ and Gly³-Phe⁴ peptide bonds.Thus, peptidomimetics that are 1:1 mixture of Leu¹, Leu² epimers for Leu-enkephalin and 1:1 mixture of Met¹, Met² epimers for Met-enkephalin were prepared and their binding affinity for μ-, δ- and κ-opioid receptors were measured.Similarly, peptidomimetics containing trifluoroethylamine moiety in place of Gly³-Phe⁴ peptide bond (Leu³, Leu⁴ and Met³, Met⁴ analogs) were prepared and their binding affinity for μ-, δ- and κ-opioid receptors were measured.In addition, the biol. activity of above enkephalin analogs were examined in mouse antinoception assays.

The charge of heterogeneity of antibodies to DNP-glycylglycylglycine (DNP-Gly-3) and DNP-p-aminobenzoylglutamate (DNP-pABG) has been investigated using preparative liquid isoelectric focusing techniques. The focusing profiles of the two antibody preparations are qualitatively similar, each containing four or five major peaks. The results are also similar to the profiles obtained earlier for DNP antibodies. The binding properties of refocused fractions from the electro-focusing separation of the two DNP-specific antibodies were investigated. Both the unfocused and refocused DNP-pABG antibody fractions were found to be functionally less heterogeneous than the corresponding DNP-Gly-3 fractions, suggesting that the site-filling capacity of the haptens contributed to the observed functional heterogeneity. A marked increase in K0 with increasing pI of the focused antibody fraction was observed for the DNP-pABG antibody preparation. A much smaller increase of K0 with pI was observed for DNP-Gly-3 antibodies and no observed change was found for DNP antibodies. These differences probably reflect the difference in the net charge on the three DNP hapten groups. The isoelectric focusing properties of the separated light and heavy chains from a DNP-antibody preparation and a DNP-pABG antibody preparation were also determined. The light chains in both instances focused as one or two major bands, while the profile of the heavy chain in both cases exhibited five to six major bands. These results are discussed in terms of the model proposed by Haselkorn, Friedman, Givol and Pecht (1974).