External ID: RMI-FANCM-MM2

Protocol: FP HTS. Screening took place at the University of Wisconsin Small Molecule Screening and Synthesis Facility. A master mix of RMI core complex and F-MM2 (30 microL per well) was plated in black 384 shallow well plates (ThermoFisher, Waltham, MA), using a BioTek "MicroFlo Select" reagent dispenser. Compounds were added using a Beckman FX liquid handler; 0.33 microL of 10 mM stock was added for a final compound concentration of 33 microM. MM2 and cMM2 were each added to 4 wells of master mix per plate to a final concentration of 10 muM to serve as controls. Following compound addition, plates were covered, centrifuged briefly, and incubated for 20 minutes at room temperature. FP measurements were taking using a Tecan "Safire 2" microplate reader. Instrument settings were as follows: top read, EX 470, EM 525/20, G-factor 0.89947. A suitable gain was calculated from the first plate of each day. Z' scores for were calculated for each plate; plates with Z' scores <0.5 were rerun prior to analysis.

Comment:

External ID: HSPH_Screening_CFS_002

Protocol: Preparation of assay plates and seeding cells:
Polyacrylamide based gel substrates were miniaturized in glass bottom 96-well plates. In the first method, each 96-well plate was treated with NaOH (6N in water) for 1hr followed by silane solution ((3-aminopropyl)trimethoxysilane, 10% in water) for an additional 1hr. Then, glass surfaces were treated with glutaraldehyde (0.25% in PBS) for 30min and further washed and dried. Acrylamide gels (5.5% acrylamide, 0.076% bisacrylamide, Young's modulus = 1.8kPa, thickness = 200 microm) were cast in each well using a custom-made gel caster. The gel surfaces were functionalized using sulfo-SANPAH (sulfosuccinimidyl-6-[4 -azido-2 -nitrophenylamino]hexanoate, 0.2mg/mL), coated with green beads (0.2microm sulfate microspheres, Invitrogen, 0.002% in water), coated with bovine collagen I (40microg/mL in PBS) and were stored at 4C (Fig. 1A) for more than 1 day. After washing off collagen solution, primary human airway smooth muscle cells were seeded in each well (10,000 cells/well). 1 day after seeding, media was replaced with serum-deprivation media and cells were kept in serum free media for 2 days.

Measurements of contractile forces using Fourier-transform traction microscopy:
On the day of screening, 96-well plate was mounted upon a motorized stage within a temperature controlled chamber and imaged using an inverted microscope (DMI 6000B, Leica Inc.). In each well, images were obtained in quick succession: one phase contrast image of cells and a fluorescent images of beads. The image set was obtained before plating cells (reference), immediately prior to adding drugs (baseline), and 1 hr after drug addition (treatment). By comparing fluorescent images obtained during baseline or treatment with the corresponding image from reference, we computed the cell-exerted displacement field using particle image velocimetry. From the displacement field, we computed the contractile force (per unit area) using Fourier-transform traction microscopy modified to the case of cell monolayers. This modified approach takes into consideration effects of finite gel thickness as well as force imbalances associated with the microscope field of view as we described previously. From each force map, we computed the root mean squared value to represent the averaged contractile force. Drug effects were quantified as the 'force response ratio' (FRR), namely, the contractile force before versus after drug addition.

Small molecule library and pooling:
ChemBridge Diverset was screened with pooling 8 compounds together.
Primary measurements of the screening are FRR (force response ratio). 1 means no change in average contractile force and less than 1 means the reduction of average contractile force after drug treatment.

Comment: Among the mixtures having FRR less than 1, 12 most effective mixtures were chosen as positives and deconvoluted to test individually. Finally, 9 single compounds were found effective.