Bisello A, Nakamoto C, Rosenblatt M, Chorev M
Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
Biochemistry. 1997 Mar 18;36(11):3293-9. doi: 10.1021/bi9619029.
The bioactive conformation of parathyroid hormone-related protein (PTHrP), a single-chain linear peptide structurally similar to parathyroid hormone (PTH), is of considerable interest because PTH and PTHrP both recognize and bind to a shared G-protein-coupled receptor. Both hormones are thought to present a bioactive conformation to the receptor which is substantially alpha-helical in nature. To better characterize this putative biologically relevant conformation, we prepared a series of conformationally constrained analogs of PTHrP with enhanced alpha-helical stability. A combination of structural constraint and helix stabilization was achieved through side chain-to-side chain lactam ring formation between Lys(i) and Asp(i+4) residues (13-to-17 and 26-to-30) along the PTHrP sequence. Mono- and bicyclic analogs derived from the agonist PTHrP-(1-34)NH2 and the antagonist PTHrP-(7-34)NH2 were prepared and characterized in terms of receptor binding and stimulation (or antagonism) of PTH-stimulated adenylyl cyclase activity in osteoblast-like cells. The binding affinity of monocyclic [Lys13,Asp17]-(I) and bicyclic [Lys13,Asp17,Lys26,Asp30]PTHrP-(1-34)NH2 (III) agonists was in the low nanomolar range and similar to that of the parent linear peptide. Furthermore, their efficacy was in the sub-nanomolar range and about 10-fold higher than that of the corresponding linear parent peptide. Analogs I and III are the first cyclic PTH/PTHrP receptor agonists and amongst the most potent PTHrP analogs yet designed. The rank-order of potency in the cyclic antagonist series does not correlate with the binding affinities. In light of the positional dependence and the differential effects of lactam bridge formation on the biological activities of agonist vs antagonists, these analogs may provide insight regarding the biologically relevant conformations of PTHrP-derived ligands [Maretto et al. (1997) Biochemistry 36, 3300-3307].
甲状旁腺激素相关蛋白（PTHrP）是一种单链线性肽，其结构与甲状旁腺激素（PTH）相似，其生物活性构象备受关注，因为PTH和PTHrP都能识别并结合同一种G蛋白偶联受体。这两种激素都被认为向受体呈现出一种本质上基本为α螺旋的生物活性构象。为了更好地表征这种假定的生物学相关构象，我们制备了一系列具有增强α螺旋稳定性的PTHrP构象受限类似物。通过沿着PTHrP序列在Lys(i)和Asp(i + 4)残基（13至17以及26至30）之间形成侧链到侧链的内酰胺环，实现了结构限制和螺旋稳定的结合。制备了源自激动剂PTHrP-(1 - 34)NH2和拮抗剂PTHrP-(7 - 34)NH2的单环和双环类似物，并就其在成骨样细胞中对受体的结合以及对PTH刺激的腺苷酸环化酶活性的刺激（或拮抗）作用进行了表征。单环[Lys13,Asp17]-(I)和双环[Lys13,Asp17,Lys26,Asp30]PTHrP-(1 - 34)NH2（III）激动剂的结合亲和力处于低纳摩尔范围，与亲本线性肽相似。此外，它们的效力处于亚纳摩尔范围，比相应的线性亲本肽高约10倍。类似物I和III是首批环状PTH/PTHrP受体激动剂，也是迄今设计的最有效的PTHrP类似物之一。环状拮抗剂系列中的效力排序与结合亲和力不相关。鉴于内酰胺桥形成的位置依赖性以及对激动剂与拮抗剂生物活性的不同影响，这些类似物可能为了解PTHrP衍生配体的生物学相关构象提供见解[马雷托等人（1997年）《生物化学》36卷，3300 - 3307页] 。
