Biologically active cyclic (lactam) analogs of growth hormone-releasing factor: Effect of ring size and location on conformation and biological activity

Félix, Arthur; Wang, Ching-Tso; Campbell, Robert M.; Toome, V.; Fry, David C.

doi:10.1007/978-94-011-2264-1_23

Cited by 12 publications

(17 citation statements)

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“…The observation that 11 Ala-substituted analogues (mostly replacing hydrophilic residues) exhibited potency equal to or greater than that of the parent compound suggests that overall R-helicity and/or hydrophobicity are important factors in receptor binding. Other studies mentioned earlier [61][62][63] showed optimal lactam sizes of 20-and 21-membered rings for retention or gain in biological potencies. In contrast, we saw maximal potencies for a range of ring sizes from 18-to 20-membered [i-(i+4)] and 18-membered [i-(i+3)] lactam analogues in the C-terminus of GHRH(1-29)-NH 2 .…”

Section: Discussionmentioning

confidence: 94%

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH₂: Systematic Structure−Activity Relationship Studies

et al. 1998

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Two complete and two partial structure-activity relationship scans of the active fragment of human growth hormone-releasing hormone, [Nle27]-hGHRH(1-29)-NH2, have identified potent agonists in vitro. Single-point replacement of each amino acid by alanine led to the identification of [Ala8]-, [Ala9]-, [Ala15]- (Felix et al. Peptides 1986 1986, 481), [Ala22]-, and [Ala28, Nle27]-hGHRH(1-29)-NH2 as being 2-6 times more potent than hGHRH(1-40)-OH (standard) in vitro. Nearly complete loss of potency was seen for [Ala1], [Ala3], [Ala5], [Ala6], [Ala10], [Ala11], [Ala13], [Ala14], and [Ala23], whereas [Ala16], [Ala18], [Ala24], [Ala25], [Ala26], and [Ala29] yielded equipotent analogues and [Ala7], [Ala12], [Ala17], [Ala20], [Ala21], and [Ala27] gave weak agonists with potencies 15-40% that of the standard. The multiple-alanine-substituted peptides [MeTyr1,Ala15,22,Nle27]-hGHRH(1-29)-NH2 (29) and [MeTyr1,Ala8,9,15,22,28,Nle 27]-hGHRH(1-29)-NH2 (30) released growth hormone 26 and 11 times, respectively, more effectively than the standard in vitro. Individual substitution of the nine most potent peptides identified from the Ala series with the helix promoter alpha-aminoisobutyric acid (Aib) produced similar results, except for [Aib8] (doubling vs [Ala8]), [Aib9] (having vs [Ala9]), and [Aib15] (10-fold decrease vs [Ala15]). A series of cyclic analogues was synthesized having the general formula cyclo(25-29)[MeTyr1,-Ala15,Xaa25,Nle27,Yaa29+ ++]-GHRH(1-29)-NH2, where Xaa and Yaa represent the bridgehead residues of a side-chain cystine or [i-(i + 4)] lactam ring. The ring size, bridgehead amino acid chirality, and side-chain amide bond location were varied in this partial series in an attempt to maximize potency. Application of lactam constraints in the C-terminus of GHRH(1-29)-NH2 identified cyclo(25-29)[MeTyr1,Ala15,DAsp25,Nle27,Orn29+ ++]-hGHRH(1-29)-NH2 (46) as containing the optimum bridging element (19-membered ring) in this region of the molecule. This analogue (46) was 17 times more potent than the standard. Equally effective was an [i-(i + 3)] constraint yielding the 18-membered ring cyclo(25-28)[MeTyr1,Ala15,Glu25,Nle,27Lys28]- hGHRH-(1-29)-NH2 (51) which was 14 times more potent than the standard. A complete [i-(i + 3)] scan of cyclo(i,i + 3)[MeTyr1,Ala15,Glui,Lys(i + 3),Nle27]-hGHRH(1-29)-NH2 was then produced in order to test the effects of a Glu-to-Lys lactam bridge at all points in the peptide. Of the 26 analogues in the series, 11 had diminished potencies of less than 10% that of the agonist standard, 4 were weak agonists (15-40% relative potency), and 4 analogues were equipotent to the standard. The 7 most potent analogues ranged in potency from 3 to 14 times greater than that of the standard and contained the [i-(i + 3)] cycles between residues 4-7, 5-8, 9-12, 16-19, 21-24, 22-25, and 25-28. The combined results from these systematic studies allowed for an analysis of structural features in the native peptide that are important for receptor activation. Reinforcement of the characteristics of amphiphilicity,...

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Section: Discussionmentioning

confidence: 94%

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH₂: Systematic Structure−Activity Relationship Studies

et al. 1998

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“…Substitution of Asp17 for Asn17 was maintained and the side chain of the α,ω‐diamino acid residue substituting for Thr21 was shortened systematically. The 20‐member ring between residues i and i + 4 of 1 had originally been chosen [22] based on the well‐described α helix‐stabilizing properties of such structures when applied to sequences with an α helix‐forming potential [38–45]. However, our molecular modelling studies indicated that a smaller ring structure might be more suitable for β turn/antiparallel β sheet stabilization.…”

Section: Discussionmentioning

confidence: 99%

Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues

Kapurniotu

Kayed

Taylor

et al. 1999

European Journal of Biochemistry

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Calcitonin is known for its hypocalcaemic effect and the inhibition of bone resorption, and is used therapeutically for the treatment of osteoporosis and Paget's disease. Our studies on the conformational features of human calcitonin (hCt) bioactivity have led to the conformationally constrained hCt analogue cyclo17,21-[Asp17,Lys21]hCt (1), which had a 5±10 times higher in vivo hypocalcaemic potency than hCt [Kapurniotu, A. & Taylor, J.W. (1995) J. Med. Chem. 38, 836±847]. We hypothesized that a stabilized, possibly type I b turn/b sheet conformation between residues 17 and 21 could play a crucial role in hCt bioactivity. Here, we designed, synthesized and studied the conformation and bioactivity of 19-member to 17-member ring-size analogues of 1 with the structure cyclo17,21-[Asp17,XX21]hCt with XX = Orn (2), Dab (3) and Dap (4), of the control peptide [Asp17,Orn21]hCt (5), and of the 19-member cyclo17,21-[Glu17,Dab21]hCt (6). Analyses of the far-UV CD spectra indicated increased type I b turn and antiparallel b sheet content in the bicyclic analogues compared with hCt. In the in vivo hypocalcaemic assay, cyclo17,21-[Asp17,Orn21]hCt (2) was found to have a 400-fold higher potency than hCt and was fourfold more potent than salmon calcitonin (sCt), which has been the most potent known Ct. Analogue 3 had a 30-fold higher potency than hCt, whereas the highly constrained analogue 4 was as potent as hCt. Bioactivity was not enhanced for the nonbridged compound [Asp17, Orn21]hCt (5), whereas cyclo17,21-[Glu17,Dab21]hCt (6) showed the same bioactivity as 1. This study identifies 2 as exhibiting the highest in vivo potency among currently known Cts, while it differs in only one amino acid residue from hCt, strongly suggesting that the introduced constraint may have served in`freezing' hCt in a bioactive conformation. Our findings provide evidence for the first time that a b turn/b sheet conformation in region 17±21 of hCt and the topological features of the side chain of Asn17 are strongly associated with in vivo bioactivity, and offer a novel lead structure for a hCt-based drug for the treatment of osteoporosis and other bonedisorder-related diseases.

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“…In addition, the studies in 50% TFE confirmed our earlier observations [14] that the 20‐membered Asp17 to Lys21 lactam bridge had an α helix‐destabilizing and a β sheet‐stabilizing effect on hCt. It has been reported that ( i , i + 4) Asp/Lys bridges may result in both stabilization [54] and destabilization [55] of α helices. Together with these reports, our results suggest that the effect of such bridges on α helix stabilization strongly depends on the particular peptide sequence the bridges are being introduced into [14].…”

Section: Resultsmentioning

confidence: 99%

Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt

Kazantzis

Waldner

Taylor

et al. 2002

European Journal of Biochemistry

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Calcitonin (Ct) is a 32-residue peptide hormone that is mainly known for its hypocalcemic effect and the inhibition of bone resorption. Our previous studies have led to ]hCt (4), corresponding partial sequence peptides containing the lactam-bridged region 16-22, and nonbridged control peptides. Only 1 showed a higher Ct receptor binding affinity than hCt, whereas analogues 2-4 had similar receptor affinities to hCt.In the in vivo hypocalcemic assay, 3 and 4 were as potent as 1, whereas 2 completely lost the high potency of 1, suggesting that type I (and II¢) b turn-promoting substituents are fully compatible with in vivo bioactivity. CD spectroscopy showed that analogues 1-4 were markedly b sheet-stabilized compared to hCt and indicated the presence of distinct b turn conformeric populations in each of the analogues. Unexpectedly, the D-amino acid-or Aib-containing cyclic analogues 2-4 but not 1 or hCt self-associated into SDS denaturation-stable dimers. Our results demonstrate a crucial role of the conformational and topological features of the residues in sequence 17-21 and in particular of residues 18 and 19 for human Ct receptor binding and in vivo bioactivity and also for the self association state of hCt. These results may assist to delineate the structure-function relationships of hCt and to design novel hCt agonists for the treatment of osteoporosis and other bone-disorder-related diseases.

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Biologically active cyclic (lactam) analogs of growth hormone-releasing factor: Effect of ring size and location on conformation and biological activity

Cited by 12 publications

References 1 publication

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH₂: Systematic Structure−Activity Relationship Studies

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH₂: Systematic Structure−Activity Relationship Studies

Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues

Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt

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Biologically active cyclic (lactam) analogs of growth hormone-releasing factor: Effect of ring size and location on conformation and biological activity

Cited by 12 publications

References 1 publication

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH2: Systematic Structure−Activity Relationship Studies

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH2: Systematic Structure−Activity Relationship Studies

Rational design, conformational studies and bioactivity of highly potent conformationally constrained calcitonin analogues

Conformationally constrained human calcitonin (hCt) analogues reveal a critical role of sequence 17–21 for the oligomerization state and bioactivity of hCt

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Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH₂: Systematic Structure−Activity Relationship Studies

Human Growth Hormone-Releasing Hormone hGHRH(1−29)-NH₂: Systematic Structure−Activity Relationship Studies