Chemistry as an Expanding Resource in Protein Science: Fully Synthetic and Fully Active Human Parathyroid Hormone‐Related Protein (1–141)

Human parathyroid hormone related protein (hPTHrP), originally isolated from lung cancer cell lines in 1987, [1] is a 141-amino acid polypeptide widely found in both normal and tumor tissue cells. The N-terminal region of hPTHrP possesses a high degree of structural homology with human parathyroid hormone (hPTH), and both hormones effect the elevation of calcium levels in the blood.[2] Although PTH and PTHrP act through binding to the same receptor, the PTH-receptor type-1 (PTHR1), in vitro studies suggest that the two ligands may differ in the precise molecular modes of their receptor interactions.[3,4] Under normal conditions, hPTHrP, which is widely expressed in the tissues of embryos and adults, plays an essential role in a range of functions related to development and growth, including: fostering of the cartilaginous growth plate, [5] bone anabolism, [6] development of mammary gland, [7] transport of calcium ions across the placenta, [8] relaxation of smooth muscle, or vasodilatation, [9] and eruption of tooth.[10] In analogy to the related anti-osteoporosis therapeutic agent, PTH, researchers have found that PTHrP, administered daily, may induce anabolic effects on the skeleton. Interestingly, the risk of hypercalcemia associated with PTH–based therapeutics may be lowered with the use of PTHrP. These findings raise the possibility that PTHrP and/or congeners, thereof could offer an advantage over currently used PTH peptides in therapeutics related to osteoporosis. A growing understanding of the role that hPTHrP may play in mediating the progression of cancer further enhances interest in this polypeptide. An intriguing property of hPTHrP is the finding that it exhibits anti-apoptotic and proliferation–promoting effects on tumor cells.[11–14] Recent studies have shown that antagonists of PTHR1 are able to remarkably inhibit the growth of tumors.[15–17] The development of an efficient synthetic route to homogeneous hPTHrP, and analogs thereof, would facilitate the systematic study of the interaction between hPTHrP and its receptor, PTHR1. Such research would offer important insights into the structure-activity relationship (SAR) of the polypeptide, and could well facilitate the development of practical PTHR1 antagonists, to suppress the growth of tumors, or agonists, for the treatment of osteoporosis.[18] Certainly, one could imagine that a wisely crafted hPTHrP lookalike could have exploitable antiproliferative properties. In our judgment, the synthesis of protein targets offers significant learning opportunities at the interface of chemistry, biology and medicine.[19] The advantage of pursuing chemistry based approaches to protein targets arises from the fact that this forum uniquely allows for the versatile design of unnatural probe structures possessing defined alterations of amino sequence and structure, including the incorporation of non-proteogenic amino acids.[20–22] Notwithstanding impressive accomplishments in protein engineering, which were enabled by spectacular advances in molecular biology, we have felt that chemical based synthesis, in principle, also has much to offer in terms of reaching a specific protein target, in reasonable research–level quantities (usually several milligrams), above all with very high levels of homogeneity. Thus, the purposes of this research were several. First, we hoped to reach hPTHrP by purely chemical means, and to show that it manifests full biological function. With this accomplished, the basis for an SAR program, involving alterations of primary structure (proteogenic and non-proteogenic amino acid substitutions) and molecular constraints, would be solidly in place. More broadly, we would be exploring, albeit in only a preliminary fashion, prospects for using chemistry as a major resource in protein discovery science.[23] The field of protein chemical synthesis was greatly advanced with the discovery of cysteine-based native chemical ligation (NCL), by Kent and co-workers.[24–26] More recently, the scope of NCL has been expanded to encompass a wide range of non-cysteine amino acids, through methods developed in our laboratory and others.[27–33] As outlined in Figure 1, the general non-cysteine based NCL strategy adopted by our group involves the installation of a temporary thiol functionality on the N-terminal amino acid residue at the site of ligation. Following amide bond formation, the polypeptide or glycopeptide is exposed to mild, metal-free dethiylation conditions, resulting in the selective removal of the extraneous thiol functionality. Figure 1 In a demonstration of the applicability of this ligation strategy to the assembly of challenging polypeptides lacking Cys residues, we recently disclosed total syntheses of hPTH, [34] and analogs thereof. Using these methods, we have now achieved the de novo total synthesis of hPTHrP (1–141).[35] We describe herein the synthesis and demonstration of biological activity of our synthetic hPTHrP (1– 141) polypeptide and a truncated analog, hPTHrP (1–37).