Fig 1.
SIRPα and TSP1 interaction with CD47.
The SIRPα-CD47 interface is defined by the co-crystal structure (PDB 2JJT) and is represented on the extracellular domain surfaces of CD47 (residues 20–136; gray) and SIRPα (residues 31–149; orange). The TSP1 interaction with CD47 is not well-defined but depends on a heparan-sulfate proteoglycan modification (HSPG; green) at CD47 Ser64 (magenta) and can be functionally minimized to the TSP1 peptide known as 7N3 (1120FIRVVMYEGKK1130, light blue) shown on the surface of the C-terminal CD47-interacting domain of TSP1 (E3CaG1; PDB 1UX6; residues 834–1170; blue). As a molecular size reference, the clinical stage protein-protein interaction (PPI) inhibitor navitoclax (974.6 Da) is shown (PDB 4LVT). PPI inhibitors are generally larger small molecules, and navitoclax is roughly 2x the mass of a typical small molecule drug. The N- and C-termini of the constructs are indicated.
Table 1.
Optimized assay conditions for 1536-well plate format.
Table 2.
TR-FRET assay protocol.
Table 3.
ALPHAScreen assay protocol.
Fig 2.
Validation of recombinant construct binding affinity by SPR.
SIRPα-biotin was immobilized on a SAV-chip followed by injection of CD47-CD4-6His at varying concentrations. (A) The association and dissociation responses are shown as an overlay for each concentration tested. (B) The maximum response of the association phase was plotted against concentration and fitted to a steady-state binding curve to approximate the Kd of the interaction pair.
Fig 3.
TR-FRET based assays (upper panel) and ALPHAScreen assay (lower panel). CD47 and SIRPα represented as their x-ray crystal structures from PDB 2JJS, chain D and chain B, respectively.
Fig 4.
Optimization of CisBio TR-FRET assay.
(A) Initial assay feasibility performed in 384-well plate format using CisBio TR-FRET reagents. CD47 and SIRPα were tested at 2 concentrations (20 nM and 100 nM), and the FRET ratio of the complete reaction was compared to controls lacking either CD47 or SIRPα to calculate the S/B (n = 16). (B) CD47 or (C) SIRPα concentrations were titrated while the other was fixed as indicated in (A) in 384-well plates to find their optimal assay concentrations as judged by S/B (n = 4). (D) Assay performance in 384-well or 1536-well formats compared using optimized concentrations derived from panels B and C. Assay Z’ indicated for each plate type (n = 32).
Table 4.
Optimized assay performance in 1536 well plate format.
Fig 5.
Validation of assay specificity and performance.
(A) Assay specificity was validated using SIRPα-cold titrated to inhibit the CD47-binding activity of SIRPα-biotin in the screening assays. Hill slope and IC50 are indicated (n = 4). (B) Assay performance using a positive control small molecule (biotin) was assessed in all three assays (n = 1). Normalized activity calculated with neutral control (+CD47, +SIRPα, no inhibitor) as 0% and low control (-CD47 +SIRPα, no inhibitor) as -100% activity. Assay activity with tested agents was then compared and normalized to these controls.
Fig 6.
High and low control signal performance in pilot screening.
Results for the (A) CisBio, (B) LANCE, and (C) AlphaScreen assays shown. Individual signal data points from neutral control (+CD47, +SIRPα, no inhibitor) and low control (+CD47 -SIRPα, no inhibitor) plotted by well for each plate in the 10-plate pilot screening run (n = 32 points per plate). Dotted lines represent + and– 20% of average signal for each high and low control. The Z’ factor was calculated from the high and low controls on each plate and averaged across all plates.
Fig 7.
Control inhibitor performance in pilot screening.
Results for the (A) CisBio, (B) LANCE, and (C) AlphaScreen assays shown. SIRPα-cold was titrated as a control inhibitor in each assay (n = 2). Error bars represent the standard deviation of two replicates. The average IC50 and minimum significant ratio (MSR) was calculated over the 10-plate run for each screening assay. MSR is defined as the smallest ratio between the potencies of two compounds that is statistically significant and is calculated as MSR = 102√2s, where s is an estimate of the standard deviation of a log potency for one compound.
Fig 8.
qHTS activity plots for LOPAC compounds.
Results shown using (A) CisBio, (B) LANCE, and (C) AlphaScreen assays. Concentration response curves fit to data of active compounds using a 4-parameter logistic equation are included with solid lines. Black lines indicate activity only within the indicated assay. Blue lines indicate activity in both CisBio and AlphaScreen assays. Red lines indicate activity in all three assays.
Table 5.
Classification of active compounds from LOPAC screening.
Fig 9.
LOPAC active compounds from the CisBio TR-FRET assay.
Normalized activity signal for the donor and acceptor channels and calculated ratio signal for each active compound reported for the 7-point concentration response generated using qHTS. Normalized activity was calculated with neutral control (+CD47 +SIRPα, no inhibitor) as 0% and low control (+CD47 -SIRPα, no inhibitor) as -100% activity. Assay activity with tested compounds was then compared and normalized within this range. (A) Compounds that interfered with donor fluorescence indicated by activity in the donor channel. (B) Reactive compound active in a variety of other unrelated screening assays.
Fig 10.
LOPAC active compounds from the LANCE TR-FRET assay.
Normalized activity signal for the donor and acceptor channels and calculated ratio signal for each active compound reported for the 7-point concentration response generated using qHTS. Normalized activity was calculated with neutral control (+CD47 +SIRPα, no inhibitor) as 0% and low control (+CD47 -SIRPα, no inhibitor) as -100% activity. Assay activity with tested compounds was then compared and normalized within this range. (A) Compounds that interfered with donor fluorescence indicated by activity in the donor channel. (B) Reactive compound active in a variety of other unrelated screening assays (Pubchem).
Fig 11.
LOPAC active compounds from the AlphaScreen assay.
Normalized activity signal for the specific AlphaScreen assay (Alpha) and the non-specific counter screen assay (counter) for each active compound reported for the 7-point concentration response generated using qHTS. Normalized activity in the AlphaScreen assay was calculated with neutral control (CD47 + SIRPα, no inhibitor) as 0% and negative control (CD47—SIRPα, no inhibitor) as -100% activity. Normalized activity in the counter screen assay was calculated with neutral control (+TruHits reagent, no inhibitor) as 0% and negative control (-TruHits reagent, no inhibitor) as -100% activity. Assay activity with tested compounds was then compared and normalized within this range. (A) Compounds that interfered with the assay as indicated by significant activity in the counter screen assay. (B) Reactive compounds identified as active in a variety of other unrelated screening assays.
Fig 12.
6-Hydroxy-DL-Dopamine follow-up testing comparison to primary screening.
Results shown for the (A) CisBio, (B) LANCE, and (C) AlphaScreen assays. Follow-up testing was conducted using freshly plated compound from powder stocks in an 11-point concentration response (n = 1).
Fig 13.
Comparison of 6-Hydroxy-DL-DOPA AlphaScreen.
Activity shown using the CD47-SIRPα PPI assay (left panel) and the counter screen assay run under different reagent addition sequences (right panel).
Fig 14.
qHTS screen of the NCATS Genesis library (94,965 compounds) using the LANCE TR-FRET assay.
(A) Z’ factor calculated from the intraplate controls. (B) Normalized activity of all compounds tested in a 6-point concentration response. Rendered curves represent all compounds displaying an activity with negative curve class. The curves rendered in red have a maximum activity ≤-25%.
Fig 15.
Large library screening workflow and results.
Left panel: schematic of screening process showing input library size, assay utilization, advanced compounds (green arrow), and non-advanced compounds (red arrow). Right panel: activity profiles for compounds selected for follow-up testing from the primary screen. Activities are shown for each assay performed and the compound profiles are segregated by level of assay advancement. (A) No activity on TR-FRET follow up. (B) Unacceptable counter screen activity. (C) Confirmed active with AlphaScreen.
Fig 16.
Venn diagram of LOPAC active compounds in all screening assays.
Grey denotes compounds triaged in counter screening. Black denotes remaining active compounds.