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: MTASE-p

Protocol: PROTOCOL TABLE (as described by Inglese J, Shamu CE and Guy RK. 2007)
SEQUENCE No. (e.g., 1, 2, 3, etc.); PARAMETER (e.g., Cells, Incubation, Reagent, etc.); VALUE; DESCRIPTION.
1; Reagent; 2 uL; SMMTase Enzyme (2x) in reaction buffer, columns 1- 48.
2; Reagent; 1 uL; SAM (4x) in reaction buffer, columns 1-48.
3; Controls; 23 nL; DMSO in column 4; sinefungin in DMSO (0 uM - 40 uM) in 7-point 1:2 dilution series (n = 2) in column 2.
4; Compounds; 23 nL; Columns 5-48.
5; Reagent; 1 uL; Substrate (4x) in reaction buffer, columns 2-48.
6; Time; 20-30 min; Incubation.
7; Reagent; 1 uL; MTase-Glo reagent (5X), columns 1-48.
8; Time; 30 min; Incubation.
9; Reagent; 5 uL; MTase-Glo Detection reagent, columns 1-48.
10; Time; 30 min; Incubation, luminescence evolution.
11; Detection; Luminescence; ViewLux uHTS Microplate Imager (PerkinElmer).

NOTES (numbers refer to Sequence numbers above)
1, 2, 5. White Medium Binding Greiner 1536-well plates (Cat #789175-F, Greiner Bio-One, Monroe, NC); Reaction buffer: 50 mM Tris, pH 8.0, 3 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, and 0.1 mg/mL BSA. Evaporation was prevented by covering assay plates with metal lids containing holes to allow gas diffusion.
1. Six SMMTase were profiled: HNMT, GNMT, PNMT, COMT, NNMT, GAMT
2. The corresponding SAM cofactor concentration was used for each SMMTase: HNMT - 9.3 uM SAM; GNMT - 12.1 uM SAM; PNMT - 3.6 uM SAM; COMT - 14.5 uM SAM; NNMT 11.9 uM SAM; GAMT - 5.6 uM SAM.
3, 4. Pintool transfer
5. The corresponding substrate was used for each enzyme: HNMT - 5.3 uM Histamine; GNMT - >500 uM Glycine; PNMT - 16.2 uM Norephinephrine; COMT - 13.7 uM Norephinephrine; NNMT - 3.8 uM Nicotinamide; GAMT - 2.5 uM Guanidinoacetate.6, 8, 10. Room temperature
6. Final reaction conditions: 10 nM COMT, 5 uM SAM, 15 uM norepinephrine, 50 mM Tris, pH 8.0, 3 mM MgCl2, 1 mM EDTA, 50 mM NaCl, 1 mM DTT, and 0.1 mg/mL BSA; refer to tables 2 and S1 for specific timing.
8. Conversion of SAH to ADP; MTase Glo Kit (Promega, Madison, WI).
10. Conversion of ADP to ATP and detection by UltraGlo luciferase; MTase Glo Kit (Promega, Madison, WI).
11. Settings: 20 s exposure, 1X binning, high gain, medium speed.

REFERENCES:
Inglese J, Shamu CE and Guy RK, Reporting data from high throughput screening of small molecule libraries, Nature Chemical Biology, 2007, 3(8): 438-441. doi.org/10.1038/nchembio0807-438.

Comment: Disclaimer:
Although all reasonable efforts have been made to ensure the accuracy and reliability of the data, caution should be exercised when interpreting the results as artifacts are possible from nonspecific effects such as assay signal interference. The curve fitting and activity calls presented here are based on the NCATS analysis methods.

Compound Ranking:
1. Compounds are assayed at single point activity (40 uM).
2. Active / Inactive compound calling is based on the results of the enzyme assay panel. ACTIVE compounds have PUBCHEM_ACTIVITY_SCORE = 50 and are compounds that have <= -50% max response in ANY ONE of the six enzymes. INACTIVE compounds have PUBCHEM_ACTIVITY_SCORE = 0 and are those that have >= -30% max response in ALL SIX enzymes. The remaining compounds are INCONCLUSIVE and have PUBCHEM_ACITIVITY_SCORE = 30.