Targeted inhibition of amyloidogenesis using a non-toxic, serum stable strategically designed cyclic peptide with therapeutic implications
Introduction
Protein amyloidogenesis plays a crucial role in the pathophysiology of several human diseases, including neurodegenerative disorders such as Alzheimer's and Parkinson's as well as metabolic disorders like Diabetes [[1], [2], [3]]. The association of amyloidogenesis with the devastating pathological implications has led to an increase in developing inhibitors against this aggregation. Scientific advancements using in vitro, in vivo, and in silico strategies have added to our gradual understanding of the system [[4], [5], [6], [7]]. However, success in implementing such approaches has been limited, in part, due to the complexity of the aggregation process and also in part because of the fact that the mechanisms and targets of the inhibitors are often poorly defined.
Over the years, several amyloidogenic model proteins have been identified, including insulin, amyloid β, α-synuclein, etc. that underly some of the most devastating disorders (such as Type-II Diabetes, Alzheimer's and Parkinson's Disease). The ease of availability of insulin over other amyloidogenic proteins has attracted several research groups to use it as a model protein for understanding amyloidogenesis [[8], [9], [10], [11], [12], [13]]. Insulin, apart from being an excellent amyloid model, is widely known for its significant pharmaceutical importance. However, owing to its innate aggregation propensity, insulin forms amyloid intermediates during its commercial production, formulation, transportation, and storage [14]. Furthermore, insulin has been shown to form amyloid deposits at the site of administration [[15], [16], [17], [18], [19], [20], [21], [22]], mostly due to a membrane-induced amyloidogenic attribute. Such deposits have often been referred to as the “insulin ball,” [15,19] which not only complicates the diagnosis of systemic amyloidosis in diabetes but also reduces insulin bioavailability and hence raises treatment costs [15,17]. In fact, biomembranes have been found to play crucial roles in modulating the overall aggregation propensity of several known amyloidogenic proteins at the lipid interface, thus adding further complexities to the system [[23], [24], [25], [26], [27], [28], [29], [30]]. Though the adoption of next-generation needle-free drug delivery can be a solution to this problem, it requires liposome-based drug delivery formulations [[31], [32], [33], [34], [35], [36], [37]].
Alternatively, the inhibition of the nucleating intermediates has been gaining interest as a particularly attractive therapeutic target in insulin and other amyloidogenic pathogenesis. Several researchers have been focusing on the development of biocompatible alternatives that would specifically inhibit the target species modulating systemic amyloidogenesis. However, their mechanism of action and the underlying side effects has remained elusive owing to the limited knowledge of the amyloidogenic intermediates. In this context, small peptides and peptidomimetics are much-valued aggregation inhibitors. Specifically, small peptides are effective in delaying fibrillation either by preventing the unfolding of native protein conformations or by interacting with the partially folded intermediates through covalent or non-covalent interactions [38,39]. In our previous study, we have reported a small peptide, KR7 (KPWWPRR-NH2), designed from an antimicrobial peptide, indolicidin, to be particularly potential in targeting insulin fibrillation in vitro [40]. In the present study, we have systematically modified the original peptide to enhance its therapeutic potential. We have introduced Cys residues either before or after the “PWWP motif” resulting in two linear peptides, KCR7 (KCPWWPRR-NH2) and KR7C (KPWWPCRR-NH2) along with a cyclic peptide KR7CC (KCPWWPCRR-NH2) with a disulfide bond between the two Cys residues (Figs. 1A and S1AB, Table S1). Our studies including various low-resolution spectroscopic tools in conjunction with high-resolution microscopy enabled us to gain mechanistic insights into the functional interface of inhibiting bovine insulin (BI) aggregation. Furthermore, high-resolution nuclear magnetic resonance (NMR) spectroscopy was employed to characterize the epitope of inhibitory action at atomic resolution.
In accordance with the previous reports [[41], [42], [43]] on cyclic peptides, KR7CC displayed superior activity in inhibiting the self-assembly of aggregation-prone sequences possibly due to its rigid conformation. In comparison to linear peptides, rigid cyclic peptides are known to help in reducing the randomness, thereby diminishing the entropy of the peptides upon binding with the targets [42]. The structural rigidity imparts a specific orientation, which facilitates enhanced binding towards target molecules. It has also been reported that cyclic peptides have better penetrating capability into the cell membranes [43]. Our results show that KR7CC not only has higher activity in comparison to other peptides against insulin fibrillation but also prevents the fibril formation and assists in arresting the non-toxic intermediates. Biophysical studies indicated a KR7CC-mediated reduction in the membrane-disruptive activity of insulin amyloids. The present study confirmed KR7CC to be a serum stable, non-toxic therapeutic alternative that effectively checks the insulin-imparted cytotoxicity in vitro as well as in pancreatic islet cells.
Section snippets
Results and discussion
The KR7-series of peptides modulate amyloid aggregation kinetics in vitro, resulting in effective inhibition.
The kinetics of amyloid fibril formation follows a nucleation dependent pathway, initiating from non-native monomers to meta-stable oligomeric species followed by protofibril formation and finally, the mature fibrils [12]. An in-depth understanding of the amyloidogenic pathways and toxic intermediate nucleating steps enables effective screening of inhibitors for a targeted therapeutic
Conclusion
Among a series of second-generation small peptides designed from the previously reported KR7 peptide comprising of a PWWP motif, our in vitro studies conclude that KR7CC is an effective inhibitor against amyloidogenesis of insulin. KR7CC effectively arrests all the early oligomeric intermediates, inducing the off-pathway aggregates. Furthermore, KR7CC is a nontoxic, serum stable peptide and is a good candidate for targeted therapeutic intervention against insulin amyloidogenesis in solution as
Materials and methods
Chemicals. Bovine insulin (BI) from bovine pancreas was purchased from Sigma Aldrich Co. (St. Louis, USA). The KR7, KCR7 and KR7C. KR7CC peptides were obtained from GL Biochem (Sanghai, China). HPLC and mass spectral (electrospray ionization-MS) analyses confirmed that the KR7-series peptides were > 95% pure. 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and Cholesterol were bought from Avanti Polar Lipids Inc. (AL, USA). The highest analytical grade solvents and reagents were used directly
Author contributions
AB designed the research; RP, BG, ZB, KGV, SR and AB performed the experiments; RP, ZB, ZG, VS, AKM, BNR and AB analyzed the results; RP, DB, ZG, VS, AKM and AB wrote the manuscript; all authors reviewed the manuscript; and AB arranged funding for this work.
Declaration of competing interest
None.
Acknowledgment
This work was partly supported by Council of Scientific and Industrial Research (02(0292)/17/EMR-II to AB) and partly by Department of Biotechnology (BT/PR29978/MED/30/2037/2018 to AB) Govt. of India. The work was also partly supported by VEGA 2/0145/17, MVTS COST 083/14 action BM1405 (to ZB) and by the Slovak Research and Development Agency under contracts nos. APVV-18-0284 (to ZG). RP thanks UGC, Govt. of India for JRF. The Central Institute Facility (CIF) of Bose Institute is greatly
Abbreviations
BI
Bovine insulin; NMR
Nuclear magnetic resonance; ThT
Thioflavin T; CD
Circular dichroism; AFM
Atomic force microscopy; MTT
3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide; NOESY
Nuclear Overhauser Effect Spectroscopy; STD
Saturation Transfer Difference; LUV
Large
Unilamellar Vesicles; DMF
Dimethylformamide; TFA
Trifluoroacetic acid; 6-CF
6-Carboxyfluorescein; FBS
Fetal bovine serum; DOPC,1,2-Dioleoyl-sn-glycero-3-phosphocholine; DLS
Dynamic light scattering
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