Conformationally restricted short peptides inhibit human islet amyloid polypeptide (hIAPP) fibrillization†

Type 2 Diabetes Mellitus (T2DM) is one of the most prevalent endocrine disorders underlining the importance of developingmolecular therapies to mitigate T2DM. It is characterized by a significant decrease in b-cell mass, insulin resistance and presence of amyloid plaques in which human islet amyloid polypeptide (hIAPP) is the major protein component. hIAPP is a 37-residue polypeptide co-secreted with insulin in b-cells of islets of Langerhans. A large amount of evidence favors its wide role in glucose metabolism. hIAPP is known to form amyloid fibrils with cross-beta structure, and amyloid deposits as the product of aggregation, but the process proceeds through oligomerization. It has been suggested that hIAPP oligomers of pore-like morphology are formed by association of helical monomers which then perform membrane fragmentation by pore formation. Thus, these prefibrillar oligomers are considered to be toxic and are implicated in b-cell dysfunction and death. Hence, the impairment of oligomerization of helices by using designed small molecule inhibitors such as short peptides is a therapeutically relevant strategy for the prevention of T2DM. In this report, we show that two pentapeptides related to one of the core fibrillization regions of hIAPP inhibit fibril formation of hIAPP. Crystal structure analysis revealed an anion receptor ‘nest’ motif in these inhibitors, which based on computational studies was shown to interact with helical monomers of hIAPP. We also propose a model for fibrillization inhibition by these peptides. Among the core fibrillization motifs/fragments of hIAPP, hIAPP(22–27), i.e. NFGAIL, has been shown to form amyloid fibrils similar to those formed by the full-length polypeptide. Based on the motif hIAPP(22–27), we designed several peptides as possible inhibitors of hIAPP fibrillization by strategically incorporating a non-natural amino acid a,b-dehydrophenylalanine (DF). DF is an analogue of phenylalanine with a double bond between C and C atoms and its presence induces b-turn in short peptides and helical secondary structures in longer peptides. Also, peptides containing DF resist enzymatic proteolysis, an added advantage for inhibitor design. NFGAIL contains two b-favoring residues, F and I, and their replacement with the helicogenic residue DF, individually or together, was a preferred choice for inhibitor design. I is an important residue; I P mutation in full length hIAPP resulted in a hIAPP fibrillization inhibitor. Designed peptides (Table S1, ESI†) were synthesized using solid phase methods, purified on reverse phase HPLC and their identity confirmed by mass spectroscopy (ESI†). Fibrillization was quantified by the enhancement of thioflavin T (ThT) fluorescence upon its binding to fibrils. The % fibrillization inhibition activities are presented in Table S1 (ESI†). I DF mutation in the fibrillizing motif resulted in pentaand hexapeptides, FGADFL and NFGADFL, respectively. Neither of the two peptides showed b-sheet conformation and fibrillization property. ThT assay revealed (Table S1, ESI†) that FGADFL inhibited hIAPP fibrillization much more efficiently (75 8%) than NFGADFL (7 5%). Therefore, we focussed further studies on FGADFL. The fibrillization kinetics of hIAPP in the presence of the pentapeptide was studied. The exponential increase in ThT intensity, considered as a hallmark of fibril formation, was suppressed greatly when hIAPP was incubated with FGADFL in 1 : 5molar ratio (Fig. 1a) suggesting that the peptide probably curtailed fibrillization at the stage of pre-fibrillar intermediates. Transmission electron microscopy (TEM) studies also confirmed that FGADFL significantly decreased hIAPP fibril formation (Fig. 1b and c). To explore the structure–function relationship, we determined the 3D structure of F–G–A–DF–L through X-ray crystallography a International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India. E-mail: [email protected]; Fax: +91-11-26742316; Tel: +91-11-26741358 b Department of Physics, Indian Institute of Science, Bangalore 560012, India. E-mail: [email protected]; Tel: +91-80-22932312 † Electronic supplementary information (ESI) available: Experimental procedures, list of all the synthesized peptides and their % hIAPP fibrillization inhibition, MTT cytotoxicity assay, crystallization, details of X-ray structure determination, in silico docking of FGADFI with hIAPP, CD studies, Tables S1–S4, and Fig. S1–S6. CCDC 822015 and 904790. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c3cc38982k ‡ These authors contributed equally to this work. Received 16th December 2012, Accepted 11th February 2013