Since the emergence of SARS-CoV-2, the public health domain has been impacted on a global scale. The deployment of vaccines against SARS-CoV-2 has been an important step in lowering morbidity and mortality, however there remains a continuing need for effective antivirals for the treatment of acute viral infections (1). Anti-coronavirus drug discovery programs commonly depend on assays which are expensive and restricted to high level biosafety conditions (2). This study aimed to combine the development of a new in-vitro assay, with the unique bioresources available in Western Australia, to form an inexpensive, streamlined compound screening pipeline. This pipeline utilizes HCoV-OC43 as a low biosafety surrogate for pathogenic human coronaviruses, as well as a combination of acoustically-focused compound dispensing (to accurately pre-dispense nanomolar levels of test compounds), resazurin reduction assays and qRT-PCR.
In-vitro, cell based resazurin reduction assays were optimised using remdesivir for the assessment of anti-HCoV-OC43 activity. Following optimisation, this method was applied to the screening of 246, natural compounds from the University of Western Australia’s collection for validation. Initial screening identified six compounds as potential leads, which were then subjected to dose response analysis using qRT-PCR. Following validation this process was applied to 384 Western Australian plant extracts. Two lead extracts were identified and subjected to a bioassay guided fractionation process using the resazurin reduction method. From this two lead compounds were isolated, and dose response analysis conducted by qRT-PCR.
From the initial validation screening of 246 natural compounds, five compounds with a relative viral inhibition >60% and a relative cell viability >70% were identified. Dose response analysis identified two compounds with an IC50 value less than 10 µM. The application of this method to 384 plant extracts led to the identification of two lead extracts, from which four pure compounds were isolated, two of which displayed IC50 values of less than 5 µM.
This study validated a compound screening pipeline which utilises the resazurin reduction assay for the rapid an inexpensive identification of novel compounds and extracts which display anti-HCoV-OC43 activity. This combined with the unique bioresources of Western Australia make it a powerful antiviral screening tool.