In vitro potency and NEP resistance for CNP variants
Data are expressed ± S.D. values as applicable.
| Description | Molecular Mass | Potencya (EC50) | NEP Resistanceb |
|---|---|---|---|
| kDa | nM | % Intact | |
| CNP22 | 2.2 | 13 ± 5.4 | 2.4 ± 1.8 |
| CNP22, K4Rc | 2.2 | 12 ± 1.4 | <5 |
| CNP27, K4R, K5R, K9Rc | 2.8 | 8.7 ± 1.8 | <5 |
| ANP28 | 3.1 | >2000 | NT |
| CNP22, Cys6-methylened | 2.2 | 44 ± 6.2 | <5 |
| CNP22, N-methyl-Phe7d | 2.2 | 860 ± 380 | <5 |
| CNP22, 20 kDa PEG | 22 | >2000 | 100 |
| CNP22, 5 kDa PEG | 7.2 | >2000 | 84 |
| CNP22, 2 kDa PEG | 4.2 | >2000 | 100 |
| CNP22, 1 kDa PEO24 | 3.2 | 640 ± 320 | 90 |
| CNP22, 0.6 kDa PEO12 | 2.8 | 210 ± 30 | 40 |
| CNP27, 2 kDa PEG | 4.8 | >2000 | 100 |
| CNP27, 1 kDa PEO24 | 3.8 | 16 ± 2.8 | 103 ± 2.7 |
| CNP27, 0.6 kDa PEO12 | 3.4 | 7.8 ± 1.4 | 69 ± 1.6 |
| CNP30 | 3.1 | 8.4 ± 3.9 | 36 ± 1.9 |
| CNP33 | 3.5 | 11 ± 0.1 | 99 ± 1.2 |
| CNP36 | 3.8 | 5.8 ± 3.5 | 98 ± 2.2 |
| CNP37 | 3.9 | 11 ± 2.0 | 97 ± 8.3 |
| CNP38 | 4.1 | 6.8 ± 0.4 | 105 ± 7.7 |
| CNP39 | 4.2 | 17 ± 1.6 | 95 ± 6.8 |
| CNP40 | 4.3 | 10 ± 2.6 | 101 ± 6.3 |
| CNP53 | 5.8 | 7.1 ± 0.5 | 106 ± 20 |
| BMN 1B2 | 4.0 | 8.7 ± 0.5 | 110 ± 0.02 |
| BMN 1B2(QQ) | 4.0 | 130 ± 20 | 102 |
| BMN 111e | 4.1 | 4.9 ± 1.5 | 99 ± 0.6 |
| HSA(231–245)-CNP22f | 3.9 | 11 ± 3.2 | 20 ± 0.6 |
| IgG1(224–237)-CNP22f | 3.7 | 72 ± 5.9 | 75 |
| IgG1(224–233)-CNP27(QQ)f | 3.9 | 920 ± 50 | 40 |
| HSA(27–36)-CNP27f | 4.0 | 6.9 ± 2.1 | 105 ± 6.4 |
NT, not tested.
↵a Mean EC50 (n ≥ 2) of cGMP production in murine NIH3T3 fibroblasts after 15-minute exposure to CNP variants (10−10 M to 10−5 M), with nonlinear curve fit using the Hill equation (Erithacus Software).
↵b NEP resistance was determined by measuring the amount of intact peptide remaining after exposure to human NEP for 140 minutes in PBS at 37°C (n = 2, for variants with near native potency; n = 1 for all other variants). Peptide digests were analyzed by liquid chromatography/mass spectrometry.
↵c Peptides used for PEGylation variants.
↵d Non-native Cys6-Phe7 peptide bond analogs were synthesized based on reported initial NEP cleavage site (Watanabe et al., 1997).
↵e Biologic synthesis (all other analogs in this table were prepared by chemical synthesis).
↵f Chimeric sequences were synthesized on the amino terminus of CNP (IgG, Ac P01857, PDB ID 2IWG; HSA Ac P02768, PDB ID 1BM0).