Received: 26 May 2018 | Accepted: 7 August 2018 DOI: 10.1002/jcb.27576 RESEARCH ARTICLE Stabilizing proteins to prevent conformational changes required for amyloid fibril formation Mohammad Khursheed Siddiqi 1 | Parvez Alam 1,6 | Sadia Malik 1 | Nabeela Majid 1 | Sumit Kumar Chaturvedi 1 | Sudeepa Rajan 2 | Mohd Rehan Ajmal 1 | Mohsin Vahid Khan 1 | Vladimir N Uversky 3,4,5 | Rizwan Hasan Khan 1 1 Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India 2 National Institute of Immunology, Delhi, India 3 Protein Research Group, Institute for Biological Instrumentation of the Russian Academy of Sciences, Moscow, Russia 4 Department of Biological Sciences, Faculty of Sciences, King Abdulaziz University, Jeddah, Saudi Arabia 5 Department of Molecular Medicine, USF Health Byrd Alzheimer’s Research Institute, Morsani College of Medicine, University of South Florida, Tampa, Florida 6 Kususma School of Biological Sciences, Indian Institute of Technology, New Delhi, India Correspondence Rizwan Hasan Khan, PhD, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India. Email: rizwanhkhan1@gmail.com Abstract Amyloid fibrillation is associated with several human maladies, such as Alzheimer’s, Parkinson’s, Huntington’s diseases, prions, amyotrophic lateral sclerosis, and type 2 diabetes diseases. Gaining insights into the mechanism of amyloid fibril formation and exploring novel approaches to fibrillation inhibition are crucial for preventing amyloid diseases. Here, we hypothesized that ligands capable of stabilizing the native state of query proteins might prevent protein unfolding, which, in turn, may reduce the propensity of proteins to form amyloid fibrils. We demonstrated the efficient inhibition of amyloid formation of the human serum albumin (HSA) (up to 85%) and human insulin (up to 80%) by a nonsteroidal anti‐inflammatory drug, ibuprofen (IBFN). IBFN significantly increases the conformational stability of both HSA and insulin, as confirmed by differential scanning calorimetry (DSC). Moreover, increasing concentration of IBFN boosts its amyloid inhibitory propensity in a linear fashion by influencing the nucleation phase as assayed by thioflavin T fluorescence, transmission electron microscopy, and dynamic light scattering. Furthermore, circular dichroism analysis supported the DSC results, showing that IBFN binds to the native state of proteins and almost completely prevents their tendency to lose secondary and tertiary structures. Cell toxicity assay confirms that species formed in the presence of IBFN are less toxic to neuronal cells (SH‐SY5Y). These results demonstrate the feasibility of using a small molecule to stabilize the native state of proteins, thereby preventing the amyloidogenic conformational changes, which appear to be the common link in several human amyloid diseases. KEYWORDS amyloid, human insulin, protein stability, transmission electron microscopy (TEM), thioflavin T (ThT) assay 1 | INTRODUCTION Amyloid fibrils are pathological self‐assembly of various proteins with diverse sequences, structures, and functions into morphologically similar fibrillar struc- tures rich in cross‐β sheets, representing a molecular signature of multiple protein deposition diseases. 1-3 Amyloidosis may be localized or systemic depending J Cell Biochem. 2018;1-15. wileyonlinelibrary.com/journal/jcb © 2018 Wiley Periodicals, Inc. | 1