Identification of Receptor-Binding Pharmacophores of Growth-Hormone-Releasing Factor in Rat Adenopituitary
Bibliographic record
Abstract
Previous structure-activity studies on growth-hormone-releasing factor (GRF) have mainly been carried out in pituitary cell culture assays. In such systems, the molecular features necessary to increase GRF receptor affinity cannot be fully distinguished from those that improve proteolytic resistance. To assess the affinity of GRF analogues, we have recently characterized [125I-Tyr10]hGRF(l-44)NH<sub>2</sub> binding to rat adenopituitary, developing a reliable binding assay in which GRF-carboxamide-related peptides are stable. In the present study, we have determined the binding affinity of two series of analogues in which the entire sequence of hGRF(1-29)NH2 was scanned with D-amino acid and alanine substitutions. To further document their potency, we have evaluated the ability of representative candidates of each series to stimulate cAMP production. In the first series, a D-amino acid substitution at Ala4, He5, Phe6, Thr7, Val13, Gin16, Leu17, Ala19, Arg20 and He26 decreased drastically the binding affinity of hGRF(1-29)NH<sub>2</sub> while it induced a smaller decrease at Tyr1, Asp3, Ser9, Tyr10, Arg11, Lys12, Leu14, Ala15, Ser18, Lys21, Leu22, Leu23, Gin24, Met27 and Ser28. Interestingly, a D-substitution in position 8 generated an analogue exhibiting a significantly greater binding affinity than hGRF(1-29)NH<sub>2</sub>, while it had no influence on hGRF(1-29)NH<sub>2</sub> affinity at Ala2, Asp25 and Arg29. Adenylate cyclase activities of [D-Tyr1], [D-Tyr10] and [D-Arg20]hGRF(1-29)NH<sub>2</sub> correlate with their binding affinity. In the second series, the largest decrease of binding affinity was observed with an alanine substitution at Tyr1, Asp3, He5, Phe6, Tyr10, Arg11, Lys12, Leu14, Leu17, Arg20 and Lys21. Replacement of Thr7, Ser9, Val13, Gin16, Ser18, Leu23, Gin24, Asp25, He26, Met27 or Arg29 by an Ala residue induced a smaller decrease of binding affinity, while it generated analogues showing a comparable affinity to hGRF(1-29)NH<sub>2</sub> at Leu22 or Ser28. An Ala-substitution significantly increased the binding affinity of hGRF(1-29)NH<sub>2</sub> at Ala8 and Ala15. Adenylate cyclase activities of [Ala9], [Ala16], [Ala17], [Ala18], [Ala20], [Ala22], [Ala26] and [Ala28]hGRF(1-29)NH<sub>2</sub> were also in agreement with their binding affinities. Altogether these results demonstrate that residues Ala4, Val13, Ala19 and He26 are involved in the maintenance of a hGRF(1-29)NH<sub>2</sub> structure for optimal receptor binding while residues Tyr1, Asp3, Tyr10, Arg11, Lys12, Leu14, Leu17, Arg20 and Lys21 are responsible for receptor contacts. Since the binding affinities are low for both a D- and Ala-substitution at He5 and Phe6, these two amino acids are probably important for structural and receptor contact roles. In addition, it suggests that residues involved in receptor binding are also required for receptor activation.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".