A Single-Point Slight Alteration Set as a Tool for Structure−Activity Relationship Studies of Ovine Corticotropin Releasing Factor

Beyermann, Michael; Fechner, Klaus; Furkert, Jens; Krause, Eberhard; Bienert, Michael

doi:10.1021/jm960116z

Cited by 53 publications

(69 citation statements)

References 50 publications

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“…, and Thr 10 abolished activity. Consistent with our truncation study (section 3.1.4) and with prior studies on full length CRH (Beyermann et al 1996;Kornreich et al 1992), the side chains of these three residues seem to be essential for CRHR 1 activation. Ser 4 could be advantageously substituted for several amino acids.…”

Section: G-proteinsupporting

confidence: 90%

“…In particular, Rivier et al have performed alanine and D-amino acid scans of the whole CRH sequence (Kornreich et al 1992;Rivier et al 1993). As CRH and UCN share a high degree of homology in the activation segment (e.g., the fragments 4-7 in UCN and the corresponding 5-8 in CRH are only slightly different, the Ser 6 is conserved while Leu residues are exchanged by Ile residues and vice versa; the UCN 8-15 fragment is completely conserved in CRH [9][10][11][12][13][14][15][16] ; (Beyermann et al 1996). The mentioned SAR studies on the full length CRH required the laborious synthesis of the full length hormone to investigate the role of residues located in the N-terminal portion, thus greatly limiting the number of analogues within a library.…”

Section: Interestingly the Sermentioning

confidence: 99%

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Biomimetic Screening of Class-B G Protein-Coupled Receptors

et al. 2011

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The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF1R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF1 receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure–activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC50 = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.

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Section: G-proteinsupporting

confidence: 90%

Section: Interestingly the Sermentioning

confidence: 99%

Biomimetic Screening of Class-B G Protein-Coupled Receptors

et al. 2011

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“…Replacement of polar residues Ser 6 , Asp 8 , and Thr 10 abolished activity. Consistent with our truncation study (section 3.1.4) and with prior studies on full length CRH (Beyermann et al 1996;Kornreich et al 1992), the side chains of these three residues seem to be essential for CRHR 1 activation. Ser 4 could be advantageously substituted for several amino acids.…”

Section: G-proteinsupporting

confidence: 90%

“…In particular, Rivier et al have performed alanine and D-amino acid scans of the whole CRH sequence (Kornreich et al 1992;Rivier et al 1993). As CRH and UCN share a high degree of homology in the activation segment (e.g., the fragments 4-7 in UCN and the corresponding 5-8 in CRH are only slightly different, the Ser 6 is conserved while Leu residues are exchanged by Ile residues and vice versa; the UCN 8-15 fragment is completely conserved in CRH 9-16 ; Table 2 (Beyermann et al 1996). The mentioned SAR studies on the full length CRH required the laborious synthesis of the full length hormone to investigate the role of residues located in the N-terminal portion, thus greatly limiting the number of analogues within a library.…”

Section: G-proteinmentioning

confidence: 99%

Biomimetic screening of class B G protein-coupled receptors

et al. 2011

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“…Preparation of Peptides-Peptides were synthesized automatically (MilliGen 9050 peptide synthesizer) by the solid-phase method using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry in the continuous flow mode as described previously for the synthesis of CRF analogs (20). Purification was carried out by preparative HPLC to give final products of Ͼ95% purity by reverse phase-HPLC analysis.…”

Section: Methodsmentioning

confidence: 99%

A Role for a Helical Connector between Two Receptor Binding Sites of a Long-chain Peptide Hormone

Beyermann

Rothemund

Heinrich

et al. 2000

Journal of Biological Chemistry

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The conformational freedom of single-chain peptide hormones, such as the 41-amino acid hormone corticotropin releasing factor (CRF), is a major obstacle to the determination of their biologically relevant conformation, and thus hampers insights into the mechanism of ligand-receptor interaction. Since N-and C-terminal truncations of CRF lead to loss of biological activity, it has been thought that almost the entire peptide is essential for receptor activation. Here we show the existence of two segregated receptor binding sites at the N and C termini of CRF, connection of which is essential for receptor binding and activation. Connection of the two binding sites by highly flexible ⑀-aminocaproic acid residues resulted in CRF analogues that remained full, although weak agonists (EC 50 : 100 -300 nM) independent of linker length. Connection of the two sites by an appropriate helical peptide led to a very potent analogue, which adopted, in contrast to CRF itself, a stable, monomer conformation in aqueous solution. Analogues in which the two sites were connected by helical linkers of different lengths were potent agonists; their significantly different biopotencies (EC 50 : 0.6 -50 nM), however, suggest the relative orientation between the two binding sites rather than the maintenance of a distinct distance between them to be essential for a high potency.

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A Single-Point Slight Alteration Set as a Tool for Structure−Activity Relationship Studies of Ovine Corticotropin Releasing Factor

Cited by 53 publications

References 50 publications

Biomimetic Screening of Class-B G Protein-Coupled Receptors

Biomimetic Screening of Class-B G Protein-Coupled Receptors

Biomimetic screening of class B G protein-coupled receptors

A Role for a Helical Connector between Two Receptor Binding Sites of a Long-chain Peptide Hormone

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