Redesigning the kinetics of lysozyme amyloid aggregation by cephalosporin molecules

Abstract

In lysozyme amyloidosis, fibrillar aggregates of lysozyme are associated with severe renal, hepatic, and gastrointestinal manifestations, with no definite therapy. Current drugs are now being tested in amyloidosis clinical trials as aggregation inhibitors to mitigate disease progression. The tetracycline group among antimicrobials in use is in phase II of clinical trials, whereas some macrolides and cephalosporins have shown neuroprotection. In the present study, two cephalosporins, ceftazidime (CZD) and cefotaxime (CXM), and a glycopeptide, vancomycin (VNC), are evaluated for inhibition of amyloid aggregation of hen egg white lysozyme (HEWL) under two conditions (i) 4 M guanidine hydrochloride (GuHCl) at pH 6.5 and 37° C, (ii) At pH 1.5 and 65 °C. Fluorescence quench titration and molecular docking methods report that CZD, CXM, and VNC interact more strongly with the partially folded intermediates (PFI) in comparison to the protein’s natural state (N). However, only CZD and CXM proficiently inhibit the aggregation. Transmission electron microscopy, tinctorial assessments, and aggregation kinetics all support oligomer-level inhibition. Transition structures in CZD-HEWL and CXM-HEWL aggregation are shown by circular dichroism (CD). On the other hand, kinetic variables and soluble fraction assays point to a localized association of monomers. Intrinsic fluorescence (IF),1-Anilino 8-naphthalene sulphonic acid, and CD demonstrate structural and conformational modifications redesigning the PFI. GuHCl-induced unfolding and differential scanning fluorimetry suggested that the PFI monomers bound to CZD and CXM exhibited partial stability. Our results present two mechanisms that function in both solution conditions, creating a novel avenue for the screening of putative inhibitors for drug repurposing. We extend our proposed mechanisms in the designing of physical inhibitors of amyloid aggregation considering shorter time frames and foolproof methods.

Communicated by Ramaswamy H. Sarma

HIGHLIGHTS

• Drug repurposing has overcome failures in drug discovery and has reduced the overall time and cost of drug discovery and development.

• We examined the effect of screened antibiotics, ceftazidime (CZD), cefotaxime (CXM), and vancomycin (VNC) on lysozyme aggregation under two solution conditions.

• These antibiotics inhibit/modulate the aggregation reactions by strongly interacting with aggregation-prone intermediate and modulation of conformation and stability.

• Our study puts forward with caution two cephalosporins for aggregation inhibition studies.

Keywords:

• Amyloid aggregation
• fibril inhibition
• transmission electron microscopy
• conformational stability

Acknowledgments

The authors gratefully acknowledge Department of MM and ME&T, SPER Jamia Hamdard, New Delhi for laboratory facilities and financial assistance and SPER, Jamia Hamdard for TEM facility. B.A. gratefully acknowledges UGC-Faculty Recharge Program (UGC-FRP), UGC, Govt. of India for awarding UGC Assistant Professor position. SAM for ICMR-SRF position awarded by Indian Council of Medical Research (ICMR), Govt of India. Support of FIST (No. SR/FST/LS-I/2017/05[C]) grant to Department of Toxicology and PURSE grant (No. SR/PURSE Phase 2/39[C]) to Jamia Hamdard from Department of Science and Technology (DST), Ministry of Science and Technology, Govt. of India is acknowledged.

Author contributions

Shivani A. Muthu—Conceptualization; Methodology; Data curation; Formal analysis; Investigation; Visualization; Roles/Writing—original draft; Writing—review & editing. Afnaan Qureshi—Methodology, Data curation; Formal analysis; Investigation. Rahul Sharma—Methodology; Data curation; Formal analysis; Investigation. Ishita Bisaria—Methodology; Data curation; Formal analysis; Investigation. Suhel Parvez—Methodology; Data curation, Resources; Software. Sonam Grover—Methodology; Data curation. Basir Ahmad—Conceptualization; Funding acquisition; Project administration; Resources; Software; Supervision; Roles/Writing—original draft; Writing—review & editing.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.