Case Study of High-Throughput Drug Screening and Remote Data Collection for SARS-CoV-2 Main Protease by Using Serial Femtosecond X-ray Crystallography

Ömür Güven ,Chun Hong Yoon,Brandon Hayes,Christopher Kupitz,Zhen Su,Gozde Usta,Nurettin Tokay,Raymond G Sierra,J Austin Johnson,Gihan Ketawala,Sabine Botha,Esra Ayan,Frédéric Poitevin ,Serdar Durdağı ,Fatma Betül Ertem ,Cengizhan Büyükdağ ,Oktay Göcenler ,A Batyuk ,Mehmet Gül ,Barış Çakilkaya ,Büşra Yüksel ,Hasan Demi̇rci̇

doi:10.3390/cryst11121579

Abstract

Since early 2020, COVID-19 has grown to affect the lives of billions globally. A worldwide investigation has been ongoing for characterizing the virus and also for finding an effective drug and developing vaccines. As time has been of the essence, a crucial part of this research has been drug repurposing; therefore, confirmation of in silico drug screening studies have been carried out for this purpose. Here we demonstrated the possibility of screening a variety of drugs efficiently by leveraging a high data collection rate of 120 images/second with the new low-noise, high dynamic range ePix10k2M Pixel Array Detector installed at the Macromolecular Femtosecond Crystallography (MFX) instrument at the Linac Coherent Light Source (LCLS). The X-ray Free-Electron Laser (XFEL) is used for remote high-throughput data collection for drug repurposing of the main protease (Mpro) of SARS-CoV-2 at ambient temperature with mitigated X-ray radiation damage. We obtained multiple structures soaked with nine drug candidate molecules in two crystal forms. Although our drug binding attempts failed, we successfully established a high-throughput Serial Femtosecond X-ray crystallographic (SFX) data collection protocol.

Highlights

Emerging infectious diseases (EID) rapidly spread in a population [1]
severe acute respiratory syndrome (SARS)-CoV-2, like other coronaviruses, generates numerous polyproteins that must be processed into functional, unique proteins, such as nonstructural proteins required for viral replication [7]
The Mpro03 datasets were collected in 5 h 20 min as it was an early dataset; the Mpro19 samples took 36 min in a total of three runs, as the process was optimized during the first two days of our MFX beamtime (Table 1)

Summary

Introduction

Emerging infectious diseases (EID) rapidly spread in a population [1]. Acquired immune deficiency syndrome (AIDS) [2], Lyme disease [3], H1N1 influenza [4], severe acute respiratory syndrome (SARS) [5], Zika, and Middle East respiratory syndrome (MERS) are among the recent outbreaks. For the SARS-CoV-2 lifecycle, 3CLpro is necessary This main protease 3CLpro (referred to as Mpro hereafter) is responsible for functional protein maturation and can be an attractive primary antiviral target [9]. Besides functional importance, targeting proteases, such as Mpro, is a common approach for combating viral infections because those proteins are highly conserved between species [10]. Mpro is enzymatically active in the dimeric form, and its Cys-His catalytic dyad provides sequence-specific cleavage after Gln residues of polyproteins [10]. This conserved protein has no homologs or similar cleavage sites for proteases found in the human proteome, making it an ideal potential drug repurposing and screening target. Drugs targeting Mpro are expected to have less or no side effects and toxicity while still acting as a broad-spectrum antiviral agent [14,15]

Methods

Results

Discussion

Conclusion