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...

Many bioactive peptides, presenting an unstructured conformation in aqueous solution, are made resistant to degradation by posttranslational modifications. Here, we describe how molecular oligomerization in aqueous solution can generate a still unknown transport form for amphipathic peptides, which is more compact and resistant to proteases than forms related to any possible monomer. This phenomenon emerged from 3D structure, function, and degradation properties of distinctin, a heterodimeric antimicrobial compound consisting of two peptide chains linked by a disulfide bond. After homodimerization in water, this peptide exhibited a fold consisting of a symmetrical full-parallel four-helix bundle, with a well secluded hydrophobic core and exposed basic residues. This fold significantly stabilizes distinctin against proteases compared with other linear amphipathic peptides, without affecting its antimicrobial, hemolytic, and ion-channel formation properties after membrane interaction. This full-parallel helical orientation represents a perfect compromise between formation of a stable structure in water and requirement of a drastic structural rearrangement in membranes to elicit antimicrobial potential. Thus, distinctin can be claimed as a prototype of a previously unrecognized class of antimicrobial derivatives. These results suggest a critical revision of the role of peptide oligomerization whenever solubility or resistance to proteases is known to affect biological properties.NMR structure ͉ oligomerization ͉ pore-forming peptide ͉ disulfide