Converting the Highly Amyloidogenic Human Calcitonin into a Powerful Fibril Inhibitor by Three-dimensional Structure Homology with a Non-amyloidogenic Analogue

Abstract

Irreversible aggregation limits bioavailability and therapeutic activity of protein-based drugs. Here we show that an aggregation-resistant mutant can be engineered by structural homology with a non-amyloidogenic analogue and that the aggregation-resistant variant may act as an inhibitor. This strategy has successfully been applied to the amyloidogenic human calcitonin (hCT). Including only five residues from the non-amyloidogenic salmon calcitonin (sCT), we obtained a variant, polar human calcitonin (phCT), whose solution structure was shown by CD, NMR, and calculations to be practically identical to that of sCT. phCT was also observed to be a potent amyloidogenesis inhibitor of hCT when mixed with it in a 1:1 ratio. Fibrillation studies of phCT and the phCT-hCT mixture mimicked the sCT behavior in the kinetics and shapes of the fibrils with a dramatic reduction with respect to hCT. Finally, the effect of phCT alone and of the mixture on the intracellular cAMP level in T47D cells confirmed for the mutant and the mixture their calcitonin-like activity, exhibiting stimulation effects identical to those of sCT, the current therapeutic form. The strategy followed appears to be suitable to develop new forms of hCT with a striking reduction of aggregation and improved activity. Finally, the inhibitory properties of the aggregation-resistant analogue, if confirmed for other amyloidogenic peptides, may favor a new strategy for controlling fibril formation in a variety of human diseases.The intrinsic propensity of peptides and proteins to irreversibly aggregate limits the development of protein-based drugs because aggregation compromises their bioavailability and therapeutic activity and increases the risk of immunogenic reactions (1, 2). Possible strategies for overcoming these problems involve the design of specific analogues in which the physicochemical properties of the molecule are changed through mutations of a small number of amino acids (3) or the development of safe inhibitors such as small peptide fragments (4 -6). However, because at the moment large molar excesses of inhibitors are used, their pharmacological efficacy appears to be of limited relevance.A good example of a bioactive peptide with limited pharmaceutical potential due to a high tendency to aggregate is human calcitonin (hCT).3 It is a 32-residue hormone synthesized and secreted by the C cells of the thyroid and involved in calcium regulation and bone dynamics (7). In its common form it presents an N-terminal disulfide bridge between positions 1 and 7 and a C-terminal proline amide residue. Only eight residues are common to all species so far studied, and these are clustered at the two ends of the molecule. The salmon variant (sCT) is widely used in the treatment of osteoporosis and Paget disease as well as malignancy-caused hypercalcemia and musculoskeletal pain (8,9). This is because hCT shows an extremely high tendency to form amyloid fibrils both in vivo in patients with medullar carcinoma of the thyroid (10) and in vitro in preparations desi...

How Glycosaminoglycans Promote Fibrillation of Salmon Calcitonin

“…At low pH, hCT fibrillation is slowed by Asp 15 protonation (32). The decreased fibrillation propensity of sCT is attributed to the increased number of polar amino acids compared with hCT (33). Our results show that this barrier to fibrillation can be overcome by introducing GAG as an electrostatic binding partner for excess positive charges.…”

“…We suggest sCT-NH 2 to have Lys 11 placed in the fibril interior in a face-to-face arrangement (pairing ␤-stands are found in the same conformation and arranged with the same residues in the interior of the structure) stabilized by a salt bridge to Glu 15 . Biological Implications-sCT is known to be less fibrillationprone than hCT because of the increased polarity of sCT (33). Lowering the pH increases the polarity of the peptide due to His protonation, and hence both hCT and sCT are expected to be less fibrillation-prone at low pH.…”

Lysine-Specific Molecular Tweezers Are Broad-Spectrum Inhibitors of Assembly and Toxicity of Amyloid Proteins

“…Based on the number, location, and relative abundance of Lys residues in each protein (Table 3 and Supplementary Figure S3), we expected that CLR01 would inhibit the aggregation of α-synuclein, insulin, β 2 m, TTR, and lysozyme, but not of IAPP, which contains a single Lys residue at position 1. CT also has only one Lys residue, but this residue, Lys18, is within a sequence previously reported to be important for amyloid formation 31–33 and therefore, we predicted that CLR01 might be able to inhibit CT aggregation. It was difficult to predict the impact of CLR01 on PrP(106–126), which has two Lys residues corresponding to 9.5% of its sequence, but are located in positions 1 and 5, away from the hydrophobic, amyloidogenic region at the C-terminus.…”

Structural Biology of Calcitonin: From Aqueous Therapeutic Properties to Amyloid Aggregation

“…The unstructured nature of the CT C‐terminus, along with the lack of sequence conservation between species, suggests a minimal role in receptor activation ,,. C‐terminally mutated hCT peptides have shown no decrease in receptor binding or hypocalcemic effect, calling into question whether or not the binding exhibited by Johansson and Lee was sufficient for receptor activation ,. The use of truncated versions of sCT and CTR by Johansson and Lee, along with the lack of a membrane environment and nonevaluation of activation also presents questions as to the biological relevance of the interactions observed ,.…”

“…Additionally, more of this α‐helix promoting hCT mutant remained soluble once aggregation was completed, compared with wild‐type, and the peptide modifications did not adversely affect receptor binding or the hypocalcemic effect. Another study by Andreotti and colleagues found that the mutation of five central and C‐terminally located residues in hCT to their sCT equivalents (Y12L, N17H, A26N, I27T, A31T) both decreased the rate of aggregation and maintained structure and biological function . This modified hCT also had the ability to inhibit the aggregation of wild‐type hCT when the two were coincubated .…”