The TRUPATH screening platform has been developed as an alternative to cAMP second messenger assays to minimize signal overamplification35. Intriguingly, in contrast to U50,488 and MP1104, but similar to the mixed-action KOR agonist pentazocine, DNCP--NalA(1) exhibited KOR partial agonism at Gi2, Gi3, GoA, GoB and Ggastducin subtypes with Emax values ranging from 48% to 77%, whereas it elicited full agonism at Gi1 and Gz with Emax values of 81% and 101%, respectively. The highest potency in the picomolar range was observed at Gz subtype for DNCP--NalA(1), U50,488 and MP1104, while pentazocine revealed the least variation in potency and efficacy across the transducerome.

The TRUPATH screening platform has been developed as an alternative to cAMP second messenger assays to minimize signal overamplification35. Intriguingly, in contrast to U50,488 and MP1104, but similar to the mixed-action KOR agonist pentazocine, DNCP--NalA(1) exhibited KOR partial agonism at Gi2, Gi3, GoA, GoB and Ggastducin subtypes with Emax values ranging from 48% to 77%, whereas it elicited full agonism at Gi1 and Gz with Emax values of 81% and 101%, respectively. The highest potency in the picomolar range was observed at Gz subtype for DNCP--NalA(1), U50,488 and MP1104, while pentazocine revealed the least variation in potency and efficacy across the transducerome.

The TRUPATH screening platform has been developed as an alternative to cAMP second messenger assays to minimize signal overamplification35. Intriguingly, in contrast to U50,488 and MP1104, but similar to the mixed-action KOR agonist pentazocine, DNCP--NalA(1) exhibited KOR partial agonism at Gi2, Gi3, GoA, GoB and Ggastducin subtypes with Emax values ranging from 48% to 77%, whereas it elicited full agonism at Gi1 and Gz with Emax values of 81% and 101%, respectively. The highest potency in the picomolar range was observed at Gz subtype for DNCP--NalA(1), U50,488 and MP1104, while pentazocine revealed the least variation in potency and efficacy across the transducerome.

The TRUPATH screening platform has been developed as an alternative to cAMP second messenger assays to minimize signal overamplification35. Intriguingly, in contrast to U50,488 and MP1104, but similar to the mixed-action KOR agonist pentazocine, DNCP--NalA(1) exhibited KOR partial agonism at Gi2, Gi3, GoA, GoB and Ggastducin subtypes with Emax values ranging from 48% to 77%, whereas it elicited full agonism at Gi1 and Gz with Emax values of 81% and 101%, respectively. The highest potency in the picomolar range was observed at Gz subtype for DNCP--NalA(1), U50,488 and MP1104, while pentazocine revealed the least variation in potency and efficacy across the transducerome.

The TRUPATH screening platform has been developed as an alternative to cAMP second messenger assays to minimize signal overamplification35. Intriguingly, in contrast to U50,488 and MP1104, but similar to the mixed-action KOR agonist pentazocine, DNCP--NalA(1) exhibited KOR partial agonism at Gi2, Gi3, GoA, GoB and Ggastducin subtypes with Emax values ranging from 48% to 77%, whereas it elicited full agonism at Gi1 and Gz with Emax values of 81% and 101%, respectively. The highest potency in the picomolar range was observed at Gz subtype for DNCP--NalA(1), U50,488 and MP1104, while pentazocine revealed the least variation in potency and efficacy across the transducerome.

The TRUPATH screening platform has been developed as an alternative to cAMP second messenger assays to minimize signal overamplification35. Intriguingly, in contrast to U50,488 and MP1104, but similar to the mixed-action KOR agonist pentazocine, DNCP--NalA(1) exhibited KOR partial agonism at Gi2, Gi3, GoA, GoB and Ggastducin subtypes with Emax values ranging from 48% to 77%, whereas it elicited full agonism at Gi1 and Gz with Emax values of 81% and 101%, respectively. The highest potency in the picomolar range was observed at Gz subtype for DNCP--NalA(1), U50,488 and MP1104, while pentazocine revealed the least variation in potency and efficacy across the transducerome.

The TRUPATH screening platform has been developed as an alternative to cAMP second messenger assays to minimize signal overamplification35. Intriguingly, in contrast to U50,488 and MP1104, but similar to the mixed-action KOR agonist pentazocine, DNCP--NalA(1) exhibited KOR partial agonism at Gi2, Gi3, GoA, GoB and Ggastducin subtypes with Emax values ranging from 48% to 77%, whereas it elicited full agonism at Gi1 and Gz with Emax values of 81% and 101%, respectively. The highest potency in the picomolar range was observed at Gz subtype for DNCP--NalA(1), U50,488 and MP1104, while pentazocine revealed the least variation in potency and efficacy across the transducerome.

We obtained four DNCP--NalA conjugates (1-4); the conjugation of DNCP (35) and DNCP (36) was unsuccessful. DNCP--NalA conjugates (1-4) were pharmacologically characterized via radioligand binding and functional cAMP assays to investigate their affinity, potency and efficacy at the mouse KOR. The DNCP--NalA conjugates exhibited affinities in the low nanomolar range, with DNCP--NalA(1) being the strongest binder at KOR with a Ki value of 3.9 nM, as compared to -NalA, which has an affinity Ki of 72 nM. All four DNCP--NalA conjugates were full agonists at KOR (Emax = 89-101%), while -NalA alone was only a partial agonist with an EC50 of 130 nM and Emax of 61%. The most potent conjugates were DNCP--NalA(1) and (4) with EC50 values of 2.0 nM and 1.0 nM, respectively. DNCP--NalA(2) and DNCP--NalA(3) showed agonist activities at KOR with EC50 values of 7.5 and 14 nM in cAMP assay, respectively, while their Ki values were 31 and 24 nM in radioligand binding assay, respectively. Receptor reserve may account for this discrepancy between potency and affinity values of DNCP--NalA(2) and DNCP--NalA(4) which can be observed in functional GPCR assays with opioid receptors30. The higher potency and efficacy of the conjugates at KOR compared to -NalA can be attributed to interactions of the macrocycles with the ECL2 region as described later.

Next, we determined the opioid receptor subtype selectivity profile of DNCP--NalA(1) in radioligand binding assays with membrane preparations from HEK293 cells stably expressing the mouse MOR and DOR, and CHO cells stably expressing human nociceptin (NOP) receptor. Herein, DNCP--NalA(1) bound to mouse MOR and DOR with Ki values of 5.4 and 318 nM, respectively, supporting an ~80-fold selectivity for KOR over DOR whereas it bound to the human NOP with a Ki value of ~1.3 μM, thus having an ~330-fold selectivity for KOR over NOP receptor. In the functional cAMP assay, DNCP--NalA(1) was inactive at both mouse MOR and DOR up to 10 μM. This was confirmed at the human MOR and DOR in the [³⁵S]GTPγS binding assay. We next determined the mechanism of antagonism of DNCP--NalA(1) by measuring adenylyl cyclase-mediated cAMP inhibition and [³⁵S]GTPγS binding at mouse and human MOR, respectively, using Schild regression analysis. The MOR expressed in HEK293 and CHO cells was activated by DAMGO in the absence and presence of increasing concentrations of DNCP--NalA(1). We observed a rightward shift of the concentration-response curves of DAMGO in cAMP and [³⁵S]GTPγS binding assays. Schild analysis of DNCP--NalA(1) exhibited linear regression slopes of 0.9 and 1.5 and pA2 values of 9.1 and 7.9 in cAMP and [³⁵S]GTPγS binding assays, respectively, which corresponds to an average functional affinity of 0.8 and 13 nM, respectively, thus demonstrating the competitive antagonism of the DNCP--NalA(1) at MOR.

We obtained four DNCP--NalA conjugates (1-4); the conjugation of DNCP (35) and DNCP (36) was unsuccessful. DNCP--NalA conjugates (1-4) were pharmacologically characterized via radioligand binding and functional cAMP assays to investigate their affinity, potency and efficacy at the mouse KOR. The DNCP--NalA conjugates exhibited affinities in the low nanomolar range, with DNCP--NalA(1) being the strongest binder at KOR with a Ki value of 3.9 nM, as compared to -NalA, which has an affinity Ki of 72 nM. All four DNCP--NalA conjugates were full agonists at KOR (Emax = 89-101%), while -NalA alone was only a partial agonist with an EC50 of 130 nM and Emax of 61%. The most potent conjugates were DNCP--NalA(1) and (4) with EC50 values of 2.0 nM and 1.0 nM, respectively. DNCP--NalA(2) and DNCP--NalA(3) showed agonist activities at KOR with EC50 values of 7.5 and 14 nM in cAMP assay, respectively, while their Ki values were 31 and 24 nM in radioligand binding assay, respectively. Receptor reserve may account for this discrepancy between potency and affinity values of DNCP--NalA(2) and DNCP--NalA(4) which can be observed in functional GPCR assays with opioid receptors30. The higher potency and efficacy of the conjugates at KOR compared to -NalA can be attributed to interactions of the macrocycles with the ECL2 region as described later.

We obtained four DNCP--NalA conjugates (1-4); the conjugation of DNCP (35) and DNCP (36) was unsuccessful. DNCP--NalA conjugates (1-4) were pharmacologically characterized via radioligand binding and functional cAMP assays to investigate their affinity, potency and efficacy at the mouse KOR. The DNCP--NalA conjugates exhibited affinities in the low nanomolar range, with DNCP--NalA(1) being the strongest binder at KOR with a Ki value of 3.9 nM, as compared to -NalA, which has an affinity Ki of 72 nM. All four DNCP--NalA conjugates were full agonists at KOR (Emax = 89-101%), while -NalA alone was only a partial agonist with an EC50 of 130 nM and Emax of 61%. The most potent conjugates were DNCP--NalA(1) and (4) with EC50 values of 2.0 nM and 1.0 nM, respectively. DNCP--NalA(2) and DNCP--NalA(3) showed agonist activities at KOR with EC50 values of 7.5 and 14 nM in cAMP assay, respectively, while their Ki values were 31 and 24 nM in radioligand binding assay, respectively. Receptor reserve may account for this discrepancy between potency and affinity values of DNCP--NalA(2) and DNCP--NalA(4) which can be observed in functional GPCR assays with opioid receptors30. The higher potency and efficacy of the conjugates at KOR compared to -NalA can be attributed to interactions of the macrocycles with the ECL2 region as described later.

Next, we determined the opioid receptor subtype selectivity profile of DNCP--NalA(1) in radioligand binding assays with membrane preparations from HEK293 cells stably expressing the mouse MOR and DOR, and CHO cells stably expressing human nociceptin (NOP) receptor. Herein, DNCP--NalA(1) bound to mouse MOR and DOR with Ki values of 5.4 and 318 nM, respectively, supporting an ~80-fold selectivity for KOR over DOR whereas it bound to the human NOP with a Ki value of ~1.3 μM, thus having an ~330-fold selectivity for KOR over NOP receptor. In the functional cAMP assay, DNCP--NalA(1) was inactive at both mouse MOR and DOR up to 10 μM. This was confirmed at the human MOR and DOR in the [³⁵S]GTPγS binding assay. We next determined the mechanism of antagonism of DNCP--NalA(1) by measuring adenylyl cyclase-mediated cAMP inhibition and [³⁵S]GTPγS binding at mouse and human MOR, respectively, using Schild regression analysis. The MOR expressed in HEK293 and CHO cells was activated by DAMGO in the absence and presence of increasing concentrations of DNCP--NalA(1). We observed a rightward shift of the concentration-response curves of DAMGO in cAMP and [³⁵S]GTPγS binding assays. Schild analysis of DNCP--NalA(1) exhibited linear regression slopes of 0.9 and 1.5 and pA2 values of 9.1 and 7.9 in cAMP and [³⁵S]GTPγS binding assays, respectively, which corresponds to an average functional affinity of 0.8 and 13 nM, respectively, thus demonstrating the competitive antagonism of the DNCP--NalA(1) at MOR.