*4.4. <sup>35</sup>S-GTPγS Coupling*

The <sup>35</sup>S-GTPγS coupling assay was also performed as in our previous work (e.g., [18,31,35]). Briefly, 15 µg of cell membrane protein was combined with 0.1 nM of <sup>35</sup>S-GTPγS (PerkinElmer, #NEG030H250UC) and concentration curves of ligand and SNC80 (see the figure legends for details) in a 200 µL reaction volume in the presence of 40 µM GDP. The reactions were incubated at 30 ◦C for 1 h; then, they were collected and read as above. The resulting data were normalized to stimulation caused by vehicle (0%) and 10 µM SNC80 (100%) and fit to a 3-variable (Hill Slope = 1) agonist model using GraphPad Prism 9.0.

#### *4.5. cAMP Accumulation Assay*

This assay was also carried out as reported in our previous work [31]. First, 20,000 cells/well were plated in a 96-well plate in growth medium as above for 24 h. Then, cells were serum-starved in DMEM/F12 for 4 h and then incubated with 500 µM IBMX for 20 min. Stimulation buffer contained 500 µM IBMX and 50 µM forskolin, which is a known cAMP inducer. Serial dilutions of SNC80, a known reference DOR agonist, or test compounds were added in stimulation buffer for 10 min. Then, incubation mixtures were halted by adding ice-cold assay buffer and heating the plate at 80 ◦C for 10 min. The plate was centrifuged at 4,000 rpm for 10 min at 4 ◦C; then, supernatants were transferred into a new 96-well plate. The supernatants were co-incubated with 1 pmol of <sup>3</sup>H-cAMP (PerkinElmer #NET1161250UC) and 7 µg of bovine protein kinase A (Sigma-Aldrich, St. Louis, MO, USA) in 0.05% bovine serum albumin for 1 h at room temperature. Then, the reactions were collected and measured as above. The data were normalized to cAMP suppression caused by vehicle (0%) or 10 µM SNC80 (100%) and fit to a 3-variable (Hill Slope = 1) agonist curve by GraphPad Prism 9.0.

#### *4.6. Data Analysis*

The data generated by the above pharmacological analyses include binding affinity (K<sup>I</sup> ) and functional potency/efficacy (EC50/EMAX). Each experiment was performed as 3 independent experiments using separate plates, drug dilutions, etc. (N = 3). The above values were calculated separately for each independent experiment and then reported as the mean ± SEM of the N = 3 set. Statistical comparisons are not typical for this sort of pharmacological characterization and were not employed here.

**Author Contributions:** P.T. performed all biological experiments, analyzed the data, collaborated on project design, and wrote the first draft of the manuscript. V.P. synthesized and prepared all novel compounds for testing. S.Z. performed receptor/ligand modeling studies that informed the rational design of compounds. O.M.-C. and C.E.A.-S. supervised V.P. and S.Z. in the performance of their work, and C.E.A.-S. secured funding for the experiments. J.M.S. supervised P.T. in the performance of the experiments, analyzed some data, collaborated on project design, wrote subsequent drafts of the manuscript, and secured funding for the experiments. All authors had editorial input into the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by R01DA038635 and R01DA038635-S1 to C.E.A.-S. and J.M.S.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data sets are available from the Corresponding Author upon request.

**Conflicts of Interest:** J.M.S. has an equity stake in *Botanical Results, LLC* and is a co-founder of *Teleport Pharmaceuticals, LLC*; no company products or interests were tested in this study. The authors have no other relevant conflicts of interest to declare.

**Sample Availability:** Samples of the compounds are available from the authors.
