Design of human ACE2 mimic miniprotein binders that interact with RBD of SARS-CoV-2 variants of concerns

Abstract

The world of medicine demands from the research community solutions to the emerging problem of SARS-CoV-2 variants and other such potential global pandemics. With advantages of specificity over small molecule drugs and designability over antibodies, miniprotein therapeutics offers a unique solution to the threats of rapidly emerging SARS-CoV-2 variants. Unfortunately, most of the promising miniprotein binders are de novo designed and it is not viable to generate molecules for each new variant. Therefore in this study, we demonstrate a method for design of miniprotein mimics from the interaction interphase of human angiotensin converting enzyme 2 (ACE2). ACE2 is the natural interacting partner for the SARS-CoV-2 spike receptor binding domain (RBD) and acts as a recognition molecule for viral entry into the host cells. Starting with ACE2 N-terminal triple helix interaction interphase, we generated more than 70 miniprotein sequences. Employing Rosetta folding and docking scores we selected 10 promising miniprotein candidates amongst which 3 were found to be soluble in lab studies. Further, using molecular mechanics (MM) calculations on molecular dynamics (MD) trajectories we test interaction of miniproteins with RBD from various variants of concern (VOC). Presently, we report two key findings; miniproteins in this study are generated using less than 10 lab testing experiments, yet when tested through in-vitro experiments, they show submicro to nanomolar affinities towards SARS-CoV-2 RBD. Also in simulation studies, when compared with previously developed therapeutics, our miniproteins display remarkable ability to mimic ACE2 interphase; making them an ideal solution to the ever evolving problem of VOCs.

Communicated by Ramaswamy H. Sarma

Keywords:

• SARS-CoV-2
• miniprotein
• therapeutics
• ACE2 mimics
• variant of concern
• protein design

Acknowledgment

We thank Dr. Ashwani Kumar for his help in executing molecular dynamics simulations. NKG acknowledges support from BRNS-JRF. Authors would like to acknowledge Dr. Kiran Kulkarni from CSIR-NCL and Central Instrumentation Facility, Savitribai Phule Pune University, Pune for access to MST.

Author contributions

RDM developed this idea and designed the project, NKG performed miniprotein design, expression and simulation studies. RDM and NKG wrote the manuscript and all authors read and reviewed the manuscript.

Disclosure statement

Authors would like to declare that results from this work is part of a provisional patent (202411001293) filed in India.