Table 1.

qHTS protocol

Step	Parameter	Value	Description
1	Reagent	2 μl	Dispense HEK293-ACE2 cells at 2000 cells/well in media
2	Time	16 – 24 hr	Incubate at standard cell culture conditions
3a	Reagent	23 nl	Dispense library compounds to columns 5–48
3b	Reagent	23 nl	Dispense DMSO control to columns 1–4
4	Time	1 hr	Incubate at standard cell culture conditions
5a	Reagent	2 μl	Dispense bald PP in columns 1–2
5b	Reagent	2 μl	Dispense SARS-CoV-2-S PP in columns 3–48
6	Centrifugation	45 min	Spinoculation at 453 xg
7	Time	48 hr	Incubate at standard cell culture conditions
8	Aspiration	−4 μl	Supernatant removal via centrifugation
9	Reagent	4 μl	Dispense luciferase detection reagent
10	Time	5 min	Incubate at room temperature
11	Detector		Luminescence read

Step	Protocol details
1	Dispense with Multidrop. Growth media: DMEM 10% FBS, 1 mg/ml puromycin
2	Overnight incubation at 37 oC, 5% CO2
3	Dispense via pintool transfer. Compounds in DMSO.
4	Incubate at 37 oC, 5% CO2
5	Dispense with Multidrop.
6	Spinoculation at 1500 rpm (453 xg) at room temperature.
7	Incubate at 37 oC, 5% CO2
8	Blue Washer (BlueCat Bio) gentle spin setting.
9	Dispense Bright-Glo Luciferase detection reagent (Promega) with BioRapr
10	Incubate at room temperature.
11	PHERAStar plate reader (BMG Labtech) luminescence setting.