2H96

Discovery of Potent, Highly Selective, and Orally Bioavailable Pyridine Carboxamide C-jun NH2-terminal Kinase Inhibitors

X-RAY DIFFRACTION

Crystallization

Crystalization Experiments
ID	Method	pH	Temperature	Details
1		6.2		PROTEIN WAS PREINCUBATED WITH THE JIP1 PEPTIDE AT A 5X MOLAR EXCESS. PROTEIN CONCENTRATION 9- 12.6 MG/ML. HANGING DROPS CONSISTED OF 2UL PROTEIN PLUS 2 UL WELL SOLUTION. WELL SOLUTION:2.8-3.1 M AMMONIUM SULFATE, 10- 14% GLYCEROL. FOR CO-CRYSTALLIZATION EXPERIMENT WITH THE COMPOUND, THE COMPOUND WAS DISSOLVED IN DMSO AT 100 MM CONCENTRATION. ALLOW TO INCUBATE FOR AT LEAST AN HOUR ON ICE. SOLUTION WAS SPUN FOR 5 MINUTES AT 2000G PRIOR TO SETTING UP FOR CRYSTALLIZATION, PH 6.2, VAPOR DIFFUSION, HANGING DROP, TEMPERATURE 277.0K, pH 6.20

Crystal Properties
Matthews coefficient	Solvent content
4.94	75.11

Crystal Data

Unit Cell
Length ( Å )	Angle ( ˚ )
a = 155.66	α = 90
b = 155.66	β = 90
c = 125.163	γ = 120

Symmetry
Space Group	P 32 2 1

Diffraction

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	110	CCD	ADSC QUANTUM 210		2004-10-18	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 17-ID		APS	17-ID

Data Collection

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	R Sym I (Observed)	Net I Over Average Sigma (I)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	3	20	89.1	0.094	0.094	22.5	7.2	34786	31447		1	54.6

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	R-Sym I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	3	3.11	99.5		0.754	0.754	2.2	6.8	3461

Refinement

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Cut-off Sigma (F)	Number Reflections (All)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (All)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	2G01	3	19.97	2	31348	28184	2801	79.8	0.2433	0.2433	0.225	0.278	RANDOM	59.4

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]
8.92	29.47		8.92		-17.83

RMS Deviations
Key	Refinement Restraint Deviation
c_dihedral_angle_d	22.9
c_angle_deg	1.4
c_improper_angle_d	0.87
c_bond_d	0.008

Non-Hydrogen Atoms Used in Refinement
Non-Hydrogen Atoms	Number
Protein Atoms	5939
Nucleic Acid Atoms
Solvent Atoms
Heterogen Atoms	82

Software

Software
Software Name	Purpose
ADSC	data collection
HKL-2000	data reduction
CNX	refinement
HKL-2000	data scaling
CNX	phasing

RCSB PDB Core Operations are funded by the National Science Foundation (DBI-1832184), the US Department of Energy (DE-SC0019749), and the National Cancer Institute, National Institute of Allergy and Infectious Diseases, and National Institute of General Medical Sciences of the National Institutes of Health under grant R01GM133198.