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

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...

Congenital disorders of glycosylation (CDG) are a group of genetic disorders that affect protein and lipid glycosylation and glycosylphosphatidylinositol synthesis. More than 100 different disorders have been reported and the number is rapidly increasing. Since glycosylation is an essential post-translational process, patients present a large range of symptoms and variable phenotypes, from very mild to extremely severe. Only for few CDG, potentially curative therapies are being used, including dietary supplementation (e.g., galactose for PGM1-CDG, fucose for SLC35C1-CDG, Mn2+ for TMEM165-CDG or mannose for MPI-CDG) and organ transplantation (e.g., liver for MPI-CDG and heart for DOLK-CDG). However, for the majority of patients, only symptomatic and preventive treatments are in use. This constitutes a burden for patients, care-givers and ultimately the healthcare system. Innovative diagnostic approaches, in vitro and in vivo models and novel biomarkers have been developed that can lead to novel therapeutic avenues aiming to ameliorate the patients’ symptoms and lives. This review summarizes the advances in therapeutic approaches for CDG.

The term “pharmacological chaperone” was introduced 20 years ago. Since then the approach with this type of drug has been proposed for several diseases, lysosomal storage disorders representing the most popular targets. The hallmark of a pharmacological chaperone is its ability to bind a protein specifically and stabilize it. This property can be beneficial for curing diseases that are associated with protein mutants that are intrinsically active but unstable. The total activity of the affected proteins in the cell is lower than normal because they are cleared by the quality control system. Although most pharmacological chaperones are reversible competitive inhibitors or antagonists of their target proteins, the inhibitory activity is neither required nor desirable. This issue is well documented by specific examples among which those concerning Fabry disease. Direct specific binding is not the only mechanism by which small molecules can rescue mutant proteins in the cell. These drugs and the properly defined pharmacological chaperones can work together with different and possibly synergistic modes of action to revert a disease phenotype caused by an unstable protein.

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHNalpha coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic alpha-helix from Leu9 to Phel6, followed by a type-I beta-turn between residues Phel6 and Phel9. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure-activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed.

Salmon calcitonin (sCT) forms an amphipathic helix in the region 9-19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161-174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu 23,Ala 24-sCT in which Pro 23 and Arg 24 were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic ␣-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9-19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.The most recognized action of calcitonin (CT) 4 is the inhibition of osteoclast-mediated bone resorption. This forms the basis for its primary clinical use in the treatment of bone-related disorders such as Paget disease, osteoporosis, and hypercalcemia of malignancy (1). CT activity also includes modulation of renal ion excretion, analgesia, inhibition of appetite, and gastric acid secretion as well as influence on reproduction via the effects on embryological implantation and sperm function (Ref. 1 and references therein). Recently, CT has been put forward as an ideal agent for treatment of osteoarthritis (2).CT is a single-chain polypeptide hormone of 32 amino acids with an N-terminal disulfide bridge between positions 1 and 7 and a C-terminal amidated proline. CT species so far studied can be subdivided into three major classes: human/rodent, artiodactyl, and teleost/avian; of these, the members of the teleost/avian group are generally the most potent, although relative potency varies in a species-and isoform-specific manner (3). The higher potency combined with a longer in vivo halflife has led to fish-like CT, exemplified by salmon CT (sCT), as the standard form of CT used for the clinical treatment of bone disorders (4). However, the usefulness of CT is limited by the development of clinical resistance. This can be due to development of circulating antibodies against non-human CT (5) and/or loss of responsiveness to CT, presumably via receptor down-regulation and inhibition of new receptor synthesis (6). ...

Background: Mutations in phosphomannomutase2 cause glycosylation disorder, a disease without a cure that will largely benefit from accurate ligand-bound models.Results: We obtained two models of phospomannomutase2 bound to glucose 1,6-bisphosphate and validated them with limited proteolysis.Conclusion: Ligand binding induces a large conformational transition in PMM2.Significance: We produce and validate closed-form models of PMM2 that represent a starting point for rational drug discovery.

Thermal shift assay can be used for the high-throughput screening of pharmacological chaperones. These drugs are small molecules that bind a mutant protein and stabilize it. We demonstrated the robustness, reproducibility and versatility of the method using two molecules that are in clinical trial for Fabry or Pompe disease, Deoxygalactonojirimycin and N-Butyldeoxynojirimycin, and their target enzymes, lysosomal alpha-galactosidaseA and alpha-glucosidase, as test cases. We assessed the influence of solvents and of scanning rate on the measures. We showed that a value that is equivalent to the melting temperature can be obtained by the first derivatives of raw data. We discuss the advantages of the method and the precaution to be taken in running the experiments. © 2015 The Authors Drug Testing and Analysis Published by John Wiley & Sons Ltd.A good share of the mutations associated with human diseases causes protein destabilization. [1] Pharmacological chaperones (PCs) stabilize proteins that retain the essential residues needed for activity, but have become unstable upon mutation and are degraded by the quality control system of the cell. Some PCs are already in clinical trial. [2] Mutations in the gene encoding lysosomal alpha-galactosidaseA and lysosomal alpha-glucosidase are associated with Fabry and Pompe diseases, respectively. Deoxygalactonojirimycin (DGJ) at low concentration effectively increases mutant lysosomal alphagalactosidaseA activity (for a review please consult Fabry_CEP [3] and references therein) while N-Butyldeoxynojirimycin (NB-DNJ) increases mutant lysosomal alpha-glucosidase activity. [4] PCs are often described as reversible inhibitors used at sub-inhibitory concentration. This description is deceptive, because the ability to reduce the activity of the target enzyme is neither required nor desirable for the therapeutic action. The first effective drugs, as DGJ or NB-DNJ, were indeed found among inhibitors, but this was due to practical reasons, specifically the lack of a systematic screening. The best definition for these medicines should be 'thermodynamic drugs' because altogether they are ligands that preferentially bind the folded state of a protein. For this reason a screening method based on protein stabilization is more appropriate to find PC than a method based on enzyme inhibition. Thermal shift assay (TSA), which has been described by several authors, [5][6][7][8][9][10] has found different applications not only for the identification of enzyme inhibitors, but also for activators and allosteric ligands. [11,12] We carried out TSA with the StepOne ™ Real-Time PCR Systems. The protein (0.1-0.5 mg/mL final concentration) was equilibrated in the appropriate buffer (Na-Hepes 20 mM, NaCl 150 mM, pH 7.4 or Na-acetate 20 mM, NaCl 150 mM, pH 4.2 or Na-acetate 20 mM, NaCl 150 mM pH 5.2) with Sypro Orange 2.5X (Invitrogen Molecular Probes, lifetechnologies.com), both with and without ligands. The samples were distributed in PCR multi-strip (0.025 mL in each well), sealed with cap strips and...