Supplemental Online Material - Science

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www.sciencemag.org/cgi/content/full/310/5750/1022/DC1 Supporting Online Material for Small-Molecule Inhibition of TNF-α Molly M. He, Annemarie Stroustrup Smith, Johan D. Oslob, William M. Flanagan, Andrew C. Braisted, Adrian Whitty, Mark T. Cancilla, Jun Wang, Alexey A. Lugovskoy, Josh C. Yoburn, Amy D. Fung, Graham Farrington, John Eldredge, Eric S. Day, Leslie A. Cruz, Teresa G. Cachero, Stephan Miller, Jessica E. Friedman, Ingrid C. Choong, Brian C. Cunningham* *To whom correspondence should be addressed. E-mail: [email protected] Published 11 November 2005, Science 310, 1022 (2005) DOI: 10.1126/science.1116304 This PDF file includes: Materials and Methods Figs. S1 to S6 Tables S1 to S3

Transcript of Supplemental Online Material - Science

Page 1: Supplemental Online Material - Science

www.sciencemag.org/cgi/content/full/310/5750/1022/DC1

Supporting Online Material for

Small-Molecule Inhibition of TNF-α Molly M. He, Annemarie Stroustrup Smith, Johan D. Oslob, William M. Flanagan,

Andrew C. Braisted, Adrian Whitty, Mark T. Cancilla, Jun Wang, Alexey A. Lugovskoy, Josh C. Yoburn, Amy D. Fung, Graham Farrington, John Eldredge, Eric S. Day, Leslie A. Cruz, Teresa G. Cachero, Stephan Miller, Jessica E. Friedman, Ingrid C. Choong,

Brian C. Cunningham*

*To whom correspondence should be addressed. E-mail: [email protected]

Published 11 November 2005, Science 310, 1022 (2005) DOI: 10.1126/science.1116304

This PDF file includes:

Materials and Methods Figs. S1 to S6 Tables S1 to S3

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Supplemental Online Material Materials and Methods Preparation of reagents: Compound synthesis. ESI mass spectra were obtained on a HP 1100MSD mass spectrometer. 1H NMR spectra were recorded on a Bruker 400 MHz spectrometer with chemical shifts reported in units of parts per million (ppm). High resolution mass spectra (HRMS) were determined on an Applied Biosystems Qstar Pulsar-i. Flash column chromatography was performed using EM Science silica gel 60 (230-400 mesh). Solvents for chromatography are listed as volume/volume ratios. Analytical thin layer chromatograms (TLCs) were run using glass thin layer plates coated with silica gel as supplied by E. Merck and were visualized by viewing under 254 nm UV or by exposure to a potassium permanganate solution in ethanol. Analytical HPLC was conducted on an Agilent 1100 LC/MS system using a C-18 Synergi Hydro-RP 4.6 mm x 150 mm (Phenomenex) at a flow rate of 1.5 ml per minute. The mobile phase consisted of water and acetonitrile each containing 0.1% trifluoroacetic acid by volume. Two methods were employed to assess the purity of compounds. In Method I, the gradient began at 5% acetonitrile and ramped linearly to 100% over 25 minutes. In Method II, the mobile phase was kept isocratic at 35% acetonitrile for 24 minutes, then ramped up to 100% over one minute. UV absorbance was monitored at 220 nm and 254 nm. Preparative reverse phase high pressure liquid chromatography (RP-HPLC) was carried out on a Gilson HPLC fitted with a Waters Nova-Pak C-18 (25 x 100 mm) column eluting at 25 ml/min employing a gradient of acetonitrile:water (each containing 0.1% TFA) from 10% to 100% acetonitrile over 20 min and holding at 100% acetonitrile for 3 min.

F3C

Br

+

NH

N

F3C

O

OOHC

NH

HN

N

N

N

F3C

O

O

N

F3C

CHO

CuI, Cs2CO3

DMF, 164oC+

1) 4, DCE, NaBH(OAc)32) Prep. RP-HPLC

1

5

6

2

3

SPD00000304

DMF, POCl3, DCE

0oC to reflux4

3) Aq. NaHCO3/DCM4) HCl/Et2O, DCM

x 2 HCl

77% 89%

16%

1-(3-Trifluoromethyl-phenyl)-1H-indole (3). A mixture of indole (1, 2.35 g, 20.0 mmol), 3-bromobenzotrifluoride (2, 12.0 ml, 80.0 mmol), copper(I) iodide (3.81 g, 20.0 mmol), and cesium carbonate (9.20 g, 28.0 mmol) in 5.0 ml DMF was stirred at 164°C under nitrogen until LC/MS indicated complete conversion (ca. 2 h). After cooling to room temperature, the mixture was diluted with dichloromethane, filtered through a plug

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of celite, and washed twice with water. The organic phase was dried (Na2SO4) and concentrated. Purification of the crude residue by flash column chromatography (0-1.25% ethyl acetate in hexane) yielded 4.02 g (77%) of a solid. 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1 H), 7.70 (m, 2 H), 7.66 – 7.59 (m, 2 H), 7.54 (d, 1 H, J = 8.2 Hz), 7.34 (d, 1 H, J = 3.3 Hz), 7.25 (t, 1 H, J = 7.3 Hz), 7.19 (t, 1 H, J = 7.6 Hz), 6.72 (d, 1 H, J = 3.2 Hz). ES (+) MS m/e = 262 (M + H)+. 1-(3-Trifluoromethyl-phenyl)-1H-indole-3-carbaldehyde (4). To DMF (1.24 ml, 16.1 mmol) at 0 °C under nitrogen was added dropwise phosphorous oxychloride (1.42 ml, 15.3 mmol). After 15 min, a solution of 3 (2.00 g, 7.66 mmol) in dichloroethane (15.0 ml) was added and the resulting mixture was heated to reflux. After 2 h, the reaction mixture was cooled to room temperature, diluted with dichloromethane, and washed with 1 M aqueous NaOH (x2) followed by brine. The organic layer was dried (Na2SO4) and concentrated. Purification of the crude residue by flash column chromatography (0-30% ethyl acetate in hexane) yielded 1.97 g (89%) of a solid, Rf 0.31 (20% ethyl acetate in hexane). 1H NMR (400 MHz, CDCl3) δ 10.16 (s, 1 H), 8.42 (d, 1 H, J = 5.9 Hz), 7.97 (s, 1 H), 7.83 (s, 1 H), 7.77 (m, 3 H), 7.50 – 7.41 (m, 3 H). ES (+) MS m/e = 290 (M + H)+. 6,7-Dimethyl-3-{[methyl-(2-{methyl-[1-(3-trifluoromethyl-phenyl)-1H-indol-3-ylmethyl]-amino}-ethyl)-amino]-methyl}-chromen-4-one bis trifluoroacetate (“compound 1” x 2 TFA). To a solution of 4 (283 mg, 0.977 mmol), 3-formyl-6,7-dimethyl chromone(1) (5, 198 mg, 0.977 mmol), and N-N’-dimethylethylenediamine (6, 105 µL, 0.977 mmol) in 1,2-dichloroethane (2.50 ml) was added sodium triacetoxyborohydride (618 mg, 2.93 mmol). The resulting mixture was stirred at ambient temperature until LC/MS indicated complete consumption of 4. The mixture was then diluted with dichloromethane, washed with water, and concentrated. The crude residue was purified by RP-HPLC. The fractions containing pure compound were combined and concentrated. The residue was lyophilized under high-vacuum to yield 144 mg (19 %) of a white powder. ES (+) MS m/e = 548 (M + H)+.

6,7-Dimethyl-3-{[methyl-(2-{methyl-[1-(3-trifluoromethyl-phenyl)-1H-indol-3-ylmethyl]-amino}-ethyl)-amino]-methyl}-chromen-4-one bis hydrochloride (“compound 1” x 2 HCl). The bis-TFA salt of “SPD00000304” was taken up in chloroform and washed with aqueous saturated NaHCO3. The organic layer was dried (Na2SO4) and concentrated. The residue was dissolved in dichloromethane/ether (2.0 ml) and treated dropwise with 1.0 M HCl in ether (1.0 ml). The resulting precipitate was collected, washed with ether, and dried under high-vacuum to yield 98 mg (85 %) of a white solid. 1H NMR (400 MHz, CD3OD) δ 8.34 (s, 1 H), 7.92 – 7.86 (m, 3 H), 7.82 – 7.77 (m, 3 H), 7.66 (s, 1 H), 7.54 (d, 1 H, J = 8.3 Hz), 7.42 (s, 1 H), 7.27 (t, 1 H, J = 7.9 Hz), 7.13 (t, 1 H, J = 7.7 Hz), 4.73 (s, 2 H), 3.86 (br s, 2 H), 3.79 (br s, 2 H), 3.41 (br s, 2 H), 3.01 (s, 3 H), 2.63 (s, 3 H), 2.42 (s, 3 H), 2.28 (s, 3 H). ES (+) MS m/e = 548 (M + H)+. HRMS (TOF): calcd for C32H33F3N3O2 (M + H)+, 548.2519; found, 548.2546. Cloning, expression, and purification of human TNF-α. The TNF-α gene was PCR amplified from ATCC #65947 extracted plasmid and cloned into E. coli vector pRSET B (Invitrogen) using Nde I and Xho I restriction sites. Sequence verified RSET TNF-α

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plasmid was used to transform the expression strain BL21 Star DE3 cells (Invitrogen). From fresh colonies two 1.5 liter cultures of 2YT/ampicillin (100 µg/ml) were cultured at 37 ˚C and 200 rpm in 4.0 liter dented bottom shake flasks, induced with 0.4 mM IPTG at an optical density of 0.8 (λ = 550 nm) and shifted to a room temperature incubator for overnight expression. The cells were pelleted by centrifugation, frozen overnight at –20 ˚C, then resuspended in 100 ml of ice cold 25 mM ammonium acetate pH 6.0, 1 mM DTT, and 1 mM EDTA. The chilled solution was run through a microfluidizer twice (model 110S Microfluidics Corp, Newton Massachusetts), centrifuged at 9000 rpm for 15 minutes, and ultrafiltered (0.2 µm) to remove insoluble material. This solution was loaded onto tandem 5 ml HiTrap S HP columns, washed with Buffer A (0.2 M ammonium acetate pH 6.0, 1 mM DTT), and fractions collected over a 0-100% gradient with Buffer B (1 M ammonium acetate pH 6.0, 1 mM DTT). Fractions containing TNF-α protein as determined by SDS-PAGE were pooled and dialyzed overnight at 4 ˚C in a 3000 dalton mwco dialysis bag against 4 liters of 10 mM Tris pH 7.5, 10 mM NaCl, and 1 mM DTT. The protein solution was clarified by centrifugation at 13,500 rpm for 10 minutes and ultrafiltration (0.22 µm), loaded onto tandem HiTrap Q HP columns (Amersham), washed with Buffer A (10 mM Tris pH 7.5, 10 mM NaCl, 1 mM DTT), and fractions collected over a 0-100% gradient with Buffer B (10 mM Tris pH 7.5, 0.5 M NaCl, 1 mM DTT). Fractions that contained the TNF-α protein were pooled and dialyzed against 4 liters of 10 mM Tris pH 7.5, 50 mM NaCl, 1 mM β-ME and 1 mM cysteamine at 4 ˚C overnight to complete native disulfide bond formation. β-ME and cysteamine were removed by dialysis against 4 liters of 10 mM Tris 7.5, 50 mM NaCl overnight at 4 ˚C. The solution was ultrafiltered (0.2 µm) and TNF-α concentration calculated using a 20,460 M-1 cm-1 OD280 extinction coefficient. Aliquots were frozen in ethanol dry ice bath and stored at –80 ˚C. Cloning, expression, and purification of human TNF-R1 ECD. The extracellular domain of the human TNF-R1 gene (coding for N-terminal RDSV and for C-terminal QIEN sequences) was PCR amplified from a placenta cDNA library (Clontech) and cloned into E. coli vector pRSET (Invitrogen) using Nde I and Xho I restriction sites. Sequence verified RSET TNF-R1 plasmid was used to transform BL21 Star DE3 cells (Invitrogen). From fresh colonies two 1.5 liter cultures of 2YT/ampicillin (100 µg/ml) were cultured at 37 ˚C and 200 rpm in 4.0 liter dented bottom shake flasks, induced with 0.4 mM IPTG after reaching an optical density of 0.8 (λ = 550 nm) and shifted to a room temperature incubator for overnight expression. The cells were pelleted by centrifugation, frozen overnight at –20 ˚C, resuspended in 100 ml of ice cold 20mM Tris 7.5 with 5mM EDTA, and passed through a microfluidizer four times. Inclusion bodies were then pelleted twice by centrifugation at 4000g for 15minutes at 4˚C and resuspended. Inclusion bodies were dissolved in 40 ml denaturing solution (6M guanidine-HCl, 100mM Tris 8.5, 4mM PMSF) for 40 minutes at room temperature, then 20 mM DTT was added for 30 minutes, and then 30 mM oxidized glutathione added for 30 minutes. This solution was slowly diluted 10-fold with 50mM Tris 10.7, 6mM cysteine, and 4mM PMSF, and incubated overnight with gentle stirring at 4˚C. This solution was centrifuged at 20,000g for 20 minutes at 4˚C to remove insoluble material, dialyzed twice against 4 liters of 50mM Tris 7.5 at 4˚C, and dialyzed twice again with 4 liters of 50mM MES pH 6.2 with 25mM NaCl at 4˚C. Insoluble material was removed

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by centrifugation at 17,000g for 60 minutes at 4˚C and subsequent passage through a 0.2 µm filter. The solution was loaded onto an Uno-S column (Pharmacia) at 10 ml per minute, and fractionated over a 10-500 mM NaCl gradient buffered with 25mM MES pH6.2. Fractions containing TNF-R1 identified by SDS PAGE were pooled and concentrated by ultrafiltration, and sterilized with 0.2 µm filter. TNF-R1 concentration calculated from OD280 using a 13,530 M-1 cm-1 extinction coefficient. Aliquots were frozen in ethanol dry ice bath and stored at –80 ˚C. TNF-R1 HRP conjugate: TNF-R1 in 50 mM sodium carbonate pH 9.0 with 150 mM NaCl was conjugated by exposure to 1.5 molar equivalents of peroxidase using EZ-Link Plus Activated Peroxidase kit (Pierce, cat.# 31489), purified by gel filtration over a NAP-5 column (Pierce) equilibrated with PBS 0.1% bsa, brought to 50% glycerol and stored at –20o C. Sparsely biotinylated TNF-α: Sparsely biotinylated TNF-α designed to possess no more than one biotin molecule per TNF-α trimer was produced as follows. 10 molar equivalents of TNF-α (calculated from monomer) was reacted with 1 molar equivalent of Sulfo-NHS-LC-biotin (Pierce) in 50 mM sodium carbonate pH 9.0 with 150 mM NaCl for 60 minutes at room temperature, quenched with 10 mM ethanolamine, purified by gel filtration over a NAP-5 column (Pierce) equilibrated with PBS 0.1% bsa, brought to 50% glycerol, and then stored at –20o C. Sparsely biotinylated 3H- TNF-α: Sparsely biotinylated 3H-TNF-α was produced as follows. TNF-α was biotinylated as described before except that immediately following the biotinylation reaction the protein was further reacted with 3 molar equivalents of N-succinimidyl (2,3-3H) propionate (TRK556 Amersham cat. #TRK556) in 100 mM borate pH 8.5 for 60 minutes. The reaction was then quenched with 10 mM ethanolamine, purified by gel filtration over a Nap-5 column (Pierce) equilibrated with PBS 0.1% bsa, and stored at 4oC. TNF-α T7C-fluoroscein conjugate. A T7C mutation was introduced into TNF-α by standard protocols and TNF-α T7C protein produced as described for the wild-type protein. The protein was deblocked by TCEP under conditions that leave the TNF-α native disulfide intact, and the protein purified over a PBS equilibrated NAP-5 column. Freshly deblocked TNF-α T7C protein was reacted with 2.5 molar equivalents of 5-iodoacetamido-fluorescein (per TNF monomer) (Molecular Probes, catalog #I-3) for 2 hours in the dark at room temperature. The conjugated protein was separated from uncoupled fluorophore by separation over a PBS equilibrated NAP-5 column and aliquots stored at –20oC. Crystallography of TNF-α dimer: compound complex. Purified 1mM human TNF-α protein was pre-incubated with equimolar compound for 30 minutes and the mixture screened for crystals by hanging drop vapor diffusion using screening kits from Hampton Research (Irvine, CA). Crystals formed over night at room temperature from a mixture of 2 µl TNF-α: compound complex and 2 µl mother liquor (2.5 M Sodium Formate at pH 7.5). Full size crystals were obtained after 5 days.

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Diffraction data were collected on beam line 7.0.1 at Stanford Synchrotron Radiation Laboratory and was processed and scaled using MOSFLM and SCALA (2). The structure was solved by molecular replacement using AMORE (2) with a monomer of the TNF-α structure (PDB ID 1TNF.pdb, (3)) as a search model. REFMAC (2) was used for refinement and O (4) was used for manual model rebuilding. The model of small molecule was created using PyMol (DeLano Scentific; http://www.pymol.org), and waters were picked with ARP/warp (2). Positive difference density was observed in the dimer:dimer interface on top of the modeled 307 molecules. The density is symmetric along the NCS axis between the two dimers. Modeling of missing protein chains or compound in a compact conformation did not yield satisfactory R factors and density maps. Another co-structure with different crystal packing interactions was recently obtained with no positive difference density observed on top of the compound molecules at the dimer:dimer interface. Therefore, this density is likely a crystal packing effect that is insignificant in solution. Cell assays. HeLa cells (American Type Culture Collection) was cultured in DMEM supplemented with 10% FCS and grown to near confluency. The media was exchanged with pre-warmed DMEM containing serial additions of TNF-α or IL-1β and incubated for 15 minutes prior to measuring cell lysate total IKB-α (Bio-Rad Bioplex assay kit). This prewarmed DMEM was modified from standard by adjusting to pH 6.75 with HCl (to improve compound solubility) and by having 0.1% dmso (to control for compound carrier effects). Under these conditions analysis indicated that good partial IKβ-α depletion responses, representing about 80% of the maximum obtainable, occurred at about 0.8 ng/ml TNF-α or .08 ng/ml IL-1β. In subsequent experiments serial dilutions of compound were preincubated with these fixed concentrations of cytokine for 30 minutes and the mixture added to cells and total IKB-α was measured as described above. TNF-α receptor binding ELISA assay. Microtiter plates (Nunc 96F Maxisorp) were coated overnight with 100 µl of 5 µg/ml NeutrAvidin in 50 mM sodium carbonate pH 9.0 at 4°C. In morning the coat solution was removed, the wells blocked with 275 µL PBS Superblock (Pierce) for 60 minutes, and washed with PBS/0.01% Tween 20. Wells were incubated with 100 µl 7.5 nM sparsely biotinylated human TNF-α in PBS Superblock for 60 minutes with shaking and washed as before. Serial dilutions of test compound in 100 µl PBS Superblock pre-mixed with 0.5 nM TNFR-HRP (1% dmso) were added to the wells and the microtiter plates incubated with shaking for 60 minutes. The plates were washed as before, developed with TMB peroxidase substrate (Pierce) for 10 minutes, quenched with 100 µl 1M phosphoric acid, and TNF-R1 binding measured by absorbance at λ450. TNF-α detrimerization surface proximity assay. Biotin-3H-TNF-α was immobilized to the wells of a streptavidin microtiter flash plate by the addition of 100 µl of 25 nM biotin-3H-TNF-α in PBS Superblock for one hour at room temperature. The flash plate was then washed with 275 µl per well of PBS Superblock three times to remove unbound label, and 100 µl per well of PBS Superblock is added to the plate. Separately, test compound was serially diluted in DMSO in a conical bottom polypropylene microtiter

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plate (Costar) to give a set of concentrations 100-fold higher than the final test concentrations. Droplets of 1.5ul size were transferred to another polypropylene microtiter plate then resuspended into 150 µl of PBS Superblock to give final compound concentrations of 500 µM, 167 µM, 56 µM, 19 µM, 6.2 µM, 0.7 µM, and 0.2 µM, each in PBS Superblock 1% dmso. The flash plates were then counted with a Wallac Trilux 1450 Microbeta liquid scintillation counter to quantify immobilized biotin-TNF-3H. Next the buffer was removed from the flash plates and 100 µl of each of the freshly prepared compound dilutions added. The compound solutions were allowed to incubate for 60 minutes and the tritium counts read a second time. Next, the plate was washed again with 275 µL of PBS Superblock three times, and the tritium counts read a third time. TNF-α detrimerization assay by ELISA. Microtiter plates (96 well Nunc Maxisorp) were coated overnight with 100 µl of 5 µg/ml NeutrAvidin in 50 mM sodium carbonate pH 9.0 at 4°C. In morning the coat solution was removed, the wells blocked with 275 µL PBS Superblock (Pierce) for 60 minutes, and then washed with PBS/0.01% Tween 20. Wells were then incubated with 100 µl 7.5 nM sparsely biotinylated human TNF-α in PBS Superblock for 60 minutes with shaking and then washed as before. Serial dilutions of test compound in 100 µl PBS Superblock (1% dmso) were then added to the wells and the microtiter plates incubated with shaking for 60 minutes. The plates were then washed and incubated with 100 µl 0.5 nM TNFR-HRP for 60 minutes to stain remaining binding competent TNF-α trimer. Finally the plates were washed again, developed with TMB peroxidase substrate (Pierce) for 10 minutes, quenched with 100 µl 1M phosphoric acid, and TNF-R1 binding measured by absorbance at λ450. Mass spectrometry analysis of TNF-α oligomeric state. TNF-α was buffer exchanged by 2 cycles of dilution and ultrafiltration using 10 mM ammonium acetate pH7.0 and Biomax 10,000 dalton mwco ultrafilters (Millipore Corp.). The protein was then brought to a final concentration of 10 µM with 100 µM test compound (same buffer, final DMSO below 1%), and equilibrated at room temperature for 15 min prior to non-covalent mass spectrometry analysis. A QSTAR Pulsar-i hybrid quadrupole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) equipped with a Protana (MDS Proteomics Inc.) nanoelectrospray source was used for all noncovalent experiments. Borosilicate glass (New Objective, Woburn, MA) PicoTips were filled with 10 uL of appropriate TNFα test solution and used as nanoelectrospary emitters. Identical instrument operating conditions were used on all samples in the positive ion mode as follows: nanospray voltage, 1250 V; declustering potential, 40 V; focusing potential, 350 V. Data was acquired for five min from m/z 1000 – 7000. Each spectrum was combined from the average of the complete data set and deconvoluted with Bayesian Protein Reconstruct software. All data was collected and processed using Analyst QS system software (Applied Biosystems/MDS Sciex, Toronto, Canada). H/D exchange measurements. A 10uM TNF-α solution was prepared in 50 mM NH4OAc pH6.5 about 60 minutes prior to its scheduled addition to D20. Compound was pre-dissolved at 33.3 µM in D20 with 50 mM NH4OAc for 10 minutes with shaking. The TNF-α solution was then diluted 10-fold with compound solution (to give 1 µM TNF-α

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and 30 µM compound) and aliquots were quenched at various time points by addition of ice cold 100 mM DCl to lower the pH to 2.5 and frozen immediately on dry ice. Hydrogen-deuterium exchange was quantified by electrospray mass spectrometry obtained on a ThermoFinnigan LCQ Classic ion-trap mass spectrometer (San Jose, CA). The samples were loaded on a Protein µ-Trap (MichromBioresources, Auburn, CA) running chilled 0.29% formic acid (pH 2.5) as solvent A and 0.05% TFA, 05% acetic acid in acetonitrile as solvent B. Ballistic two-minute gradients were run to rapidly desalt the samples and allow for minimal back exchange. All necessary components were chilled with ice baths. Final reported HD exchange values were adjusted for back-exchange that occurred during the LCMS analysis. The theoretical number of exchangeable protons for the TNF-α trimer, dimer, and monomer forms were calculated from the solvent accessible surface areas of the backbone atoms of these different states (5) as determined from the native TNF-α trimer coordinates (3). Amide protons were considered exchangeable if respective nitrogens had a SASA of at least 8.7 Å2 (calculated area buried by a hydrogen atom). Structure analysis and calculations were performed using MOLMOL program(6). All the calculations omit the first 6 N-terminal residues that are disordered in X-ray crystal structures. TNF-α subunit dissociation rate measurements using fluorescence homoquenching assay. Microtiter plates (Nunc Black 96F Maxisorp) were blocked overnight at room temperature with 275 µL PBS Superblock. The blocking solution was then removed, 30 µl of 5X TNF-α T7C-AF added to each well, the plate briefly shaken and fluorescence emission measured at λ533 after excitation at λ490 (λ515 cutoff filter). Separately, droplets (2.1 µl) of 100X compound solution in DMSO were added to a conical bottom polypropylene microtiter plate (Costar), and resuspended in 168 µl of 1.25X unlabeled TNF-α solution in PBS Superblock by vigorous shaking for 5 minutes. Aliqouts (120 µl) of these compound solutions were added to the TNF-α T7C-AF distributed in black microtiter plates, the mixture shaken for 3 seconds, and an appropriate time course of fluorescence measurements taken. For long time courses (24 hours) plates were sealed with clear plastic film to prevent evaporation. The final concentration of critical components in the wells measured were 100 nM TNF-α T7C-AF, 20 µM unlabeled TNF-α, 30 µM test compound, and 1% DMSO. Plots of fluorescence versus time were fit to equation below after fixing starting fluorescence (m1), and starting concentration of TNF-α T7C-AF (m4) to known values. total fluorescence = m1*((3*m4*exp(-m5*m0))+(2*(m2/m1)*((m5*m4)/(-1/3*m5))*(exp(-m5*m0)-exp(-2/3*m5*m0)))+((m3/m1)*((3*m4)-(3*m4*exp(-m5*m0))-(((2*m5*m4)/(-1/3*m5))*(exp(-m5*m0)-exp(-2/3*m5*m0))))))-(m6*m0); m0 = time (seconds) m1 = fluorescence per nM labeled TNF-α subunit in quenched triply-labeled trimer. m2 = fluorescence per nM labeled TNF-α subunit, in partially unquenched doubly-labeled trimer. m3 = fluorescence per nM of labeled TNF-α subunit, in unquenched singly-labeled trimer. m4 = initial concentration of triply-labeled TNF-α trimer (nM). m5 = rate constant (sec-1) for exchange of TNF-α subunit from the TNF-α trimer.

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m6 = loss of fluorescence per unit time due to NSB and photobleaching. Sedimentation equilibrium analysis. 190 µl of sample (45 µM TNF-α with and without 67.5 µM compound, in 50 mM MOPS pH 6.75 with 1.0 % DMSO) and 260 µl buffer only (50 mM MOPS pH 6.75 with 1.0 % DMSO) were added to experimental and reference cells of a 2 channel cell assembly, respectively. Cells were run at 20,000 rpm and 25oC in an AN-50-Ti rotor using a Beckman Coulter Optima XL-1 analytical ultracentrifuge, and optical density radial scans from 6.6 to 7.25 cm collected at wavelengths of 293 and 310 nm after 48, 72, and 96 hours. Plot overlays of all three time points verified the samples had reached equilibrium. Single species curve fitting was performed using XL-A/XL-1 Data Analysis software version 60-4 using a partial specific volume value of 0.74. Offsets were determined by curve fitting to extended asymptotes rather than those obtained by high g-force runs due to inaccuracies that resulted from the adsorption of compound. Gel filtration-dynamic light scattering analysis of oligomeric state of TNF-α. TNF-α at 3, 9, and 30 µM was incubated overnight with and without 30 µM added compound in the sample buffer (50 mM MOPS pH 7.0, and 1% DMSO). 50 µl samples were subsequently chromatographed at a flow rate of 0.6 ml/min on a Toso Haas SWXL 3000 gel filtration column pre-equilibrated with 30 µM compound in the same sample buffer. Prior to each run the column was pre-equilibrated with 2 column volumes and stable UV absorbance baseline verified. Eluents were monitored in line using a UV detector at 280 nm, a refractive index detector (Waters), and a Precison Detector PD2000 light scattering instrument. Molecular weight determinations were calculated with Precision Detector software. Measurment of TNF-α intrinsic fluorescence. All samples were brought to 10 mM citrate pH 6.5, and 1% DMSO and equilibrated for 4 hours prior to making measurements. At that time fluorescence readings were made with an Aminco-Bowman Series 2 luminescence spectrometer (Rochester, NY) by exciting TNF-α at 290 nm and measuring the emission peaks at 322 nm (for TNF-α and SP307 mixtures) or 355 nm (for TNF-α guanidine mixtures). Compound inner filter effects were corrected using absorbance measurements of compound alone solutions at 290 and 322 or 355 nm (7). Notes on the origin of the TNF-α inhibitor SPD00000304. A combinatorial fragment assembly-based strategy (8, 9) was used to identify small molecule inhibitors of TNF-α. A dimethyl amine library containing 285 fragments was prepared from commercially available aldehydes and screened at 3 mM in the TNF-α receptor binding ELISA assay (quadruplicate controls gave %CV values of 1.4-5.6 and z’ values of 0.82-0.94). A subset of 15 fragments that exhibited 16-63 % inhibition in a rescreen of the best hits was chosen to generate a combinatorial library of linked fragments that were connected through common chemical linkage groups to a set of flexible linkers. This library consisted of 120 pools that linked all possible fragment pairs together with four N,N'-dimethyl-alkyl-diamine linkers. Each pool was screened by ELISA with the individual compound members present at 25 µM (quadruplicate controls gave %CV values of 4.0-

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4.3 and z’ values of 0.87-0.88). Inhibition curves were generated for individual compounds from 5 pools that exhibited 45-76 % inhibition. The most potent of these compounds was resynthesized to high purity (SPD00000035) and yielded an IC50 of 21 µM. An analog of this compound SPD00000304 is the focus of this report. Structures of both compounds are shown in Figure S6.

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Supporting Figure Legends Fig. S1. Orientation of TNF-α dimer: compound complexes found within one crystallographic asymmetric unit. (A) (B) Two TNF-α dimer: compound complexes are found face to face but with a substantial offset along the 2-fold non-crystallographic symmetry axis within each crystallographic asymmetric unit. (C) Comparison of the two dimers in the asymmetric unit demonstrates both adopt similar folds with a 0.2 Å r.m.s.d. for the main chain atoms. Fig. S2. TNF-α subunit dissociation rates in the presence and absence of 1% DMSO. The decrease in fluorescence homoquenching of 100 nM T7C-AF TNF-α after dilution in a 200-fold excess of unlabeled TNF-α was used to track subunit disassociation from TNF-α trimer. Curve fitting of the time course of the observed fluorescence increase under buffer only conditions (filled triangles) gave a calculated rate of 0.000058 sec-1 per monomer dissociation event. Curve fitting of the time course of the observed fluorescence increase under buffer with 1.0% DMSO (filled circles) gave a calculated rate of 0.000091 sec-1 per monomer dissociation event. Open triangles and circles show respective fluorescence in the absence of added unlabeled TNF-α. Error bars show standard deviation from the average of triplicate sample values. Fig. S3. Time course for inactivation of TNF-α receptor binding functionality as measured by ELISA. Singly biotinylated but unlabeled TNF-α immobilized on a strepavidin coated microtiter plate was exposed to a titration of compound for different durations. The microtiter plates were washed and functional TNF-α trimer measured by ability to bind TNF-R1 peroxidase conjugate. The graph shows losses of the relative binding competent TNF-α (y-axis) resulting from 5, 10, 30, 60, and 120 minutes of compound exposure for curves with open circles to progressively darker filled circles, respectively. Fig. S4. Analysis of the oligomeric state of high concentration TNF-α: compound complex by analytical ultracentrifugation. Sedimentation equilibrium runs of a mixture of 45 µM TNF-α and 67.5 µM compound were performed and migration tracked by optical density measurements at wavelengths of 293 nm and 310 nm. (A) Curve fits of equilibria obtained at 293 nm, which measures optical absorbance from both TNF-α and compound, give a single species mass of 50,540 daltons. (B) Curve fits of equilibria obtained at 310 nm, which measures optical absorbance from compound alone, give a single species mass of 57,200 daltons. Actual mass for TNF-α trimer is 52,452 daltons. Fig. S5. Intrinsic tryptophan fluorescence measurements. (A) Compound concentration dependent quenching was measured of the intrinsic fluorescence of 0.5 µM TNF-α). (B) The 300-400 nm emission spectra of 5 µM TNF-α either alone (thick trace), or with 100 µM compound added (thin trace). The observed compound induced fluorescence quench is immediately reversed by a 10-fold dilution of the sample (dashed trace). (C) The effect of 30 µM compound on 3 µM tryptophan or on the intrinsic fluorescence of 0.5 µM TNFα with or without 4 M guanidine added. The results indicate that the quenching of

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TNF-α fluorescence requires properly folded TNFα, and is not simply a non-specific effect of the compound on tryptophan. (D) Figures show the position of the tryptophans in the TNF trimer. The orientation W28 and W114 are shown in red and yellow, respectively. The compound as found in the TNF dimer complex structure is superimposed on the trimer and shown in green for reference. Fig. S6. Chemical structure of the parent compound SPD00000035 (left), and the analog described in the report SPD00000304 (right) are shown.

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Supplemental Figure 1

A

B

C

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Supplemental Figure 2

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Supplemental Figure 3

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Supplemental Figure 4

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Supplemental Figure 5

300 320 340 360 380 400

1

2

3

4

5

6

Wavelength (nm) TNF

TNF + Gdn

TNF + SP30

7

TNF + Gdn

+ SP30

7Trp

Trp + S

P307

SP307

12345678

0 25 50 75 100 125

1234567

[Compound] (uM)

A B C

D

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Supplemental Figure 6

N N

O

O

N

F

FF

N N

O

O

N

F

FF

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Table S1. Data collection and refinement statistics. Crystals were rhombahedral, space group was R3, cell dimension was a = b = 165.3, c = 63.7 with two TNF-α dimer: compound complexes per asymmetric unit. aNumbers in parentheses refer to the highest resolution shell. bRmerge = [ΣhΣi|Ii(h) - <I(h) > > |/ΣhΣiIi(h)] x 100, where <I(h)> is the mean of the I(h) observation of reflection. cRcryst = Σh||Fo|-|Fc||/Σh|Fo||, where Fo can Fc are the observed and calculated structure factor amplitudes. dRfree (%) is the same as Rcryst, but for 5% of the data randomly omitted from refinement. The coordinates and structure factors of the TNF-α : compound complex have been deposited in the Protein Data Bank (accession code 2AZ5). Data Collection resolution (Å) 20 - 2.1 (2.2 - 2.1) wave length (Å) 1.0 # unique reflections 37559 multiplicity 2.3 (2.3) completeness (%) 99.3 (99.3)

Rsymb (%) 6.7 (31.7)

Refinement

Rcrystc (%) 22.0

Rfreed (%) 27.9

number of protein atoms 4194 number of waters 226 average protein B-value (Å2) 37.2 average solvent B-value (Å2) 40.7 average ligand B-value (Å2) 33.8 RMS Deviation from Ideal bond lengths (Å) 0.007 bond angles (deg) 1.396 Ramachandran Plot (%) most favored 90.2 additionally allowed 9.6 generally allowed 0.2 not allowed 0

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Table S2. Sequence identity of compound contact residues on TNF-α dimer with other trimeric TNF superfamily members. Top row shows residue numbers for compound contact residues on chain A of the TNF-α dimer. Likewise, the next row shows residue numbers for compound contact residues on chain. Two of the residues (Q61 and G122) contact chain A but not chain B and are marked with dashes. Sequence alignment is shown for other TNF superfamily members (10) and those sharing TNF-α sequence identity are shaded.

chain A 57 59 60 61 119 120 121 122 151 chain B 57' 59' 60' - 119' 120' 121' - 151'

TNFalpha L Y S Q Y L G G Y APRIL L Y S Q Y S A G F LIGHT Y Y S K F L G G Y Apo2/TRAIL Y Y S Q Y Q G G F TNFbeta F Y S Q Y H G A F CD40L Y Y A Q H L G G S FasL F Y S K Y L G A F EDA-A1 F Y S Q Y T A G F EDA-A2 F Y S Q Y T A G F BAFF F Y G Q Y S A G F OPGL Y Y A N N V G G Y RANKL Y Y A N N V G G Y TWEAK Y Y C Q Q V S G Y OX40L L S L K Y L N V - VEGI - - - P Y L G A F LT beta Y Y C L G F G G F 4-1BBL Y F F Q F Q G R V CD27L M H I Q V S Q R F CD30L F I C Q S Q F L V

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Table S3. Analysis of the oligomeric state of high concentration TNF-α: compound complex by tandem gel filtration - dynamic light scattering. In the first row TNF-α (30 µM) chromatographed in the absence of compound is shown to give a mass of 54,460 daltons by dynamic light scattering measurements versus the expected mass for the TNF-α trimer of 52,250 (average of plus and minus N-terminal methionine forms). In the subsequent rows TNF-α at 3, 9, and 30 µM are identically analyzed but in the presence of 30 µM compound, and give the shown dynamic light scattering mass determinations.

TNF-α (µM) compound (µM) measured mass (daltons)

30 0 54,460

3 30 52,230

9 30 54,190

30 30 52,700

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Supporting References 1. A. Nohara, T. Umetani, Y. Sanno, Tetrahedron 30, 3553 (1974). 2. Acta Crystallogr D Biol Crystallogr 50, 760 (Sep 1, 1994). 3. M. J. Eck, S. R. Sprang, J Biol Chem 264, 17595 (1989). 4. T. A. Jones, J. Y. Zou, S. W. Cowan, Kjeldgaard, Acta Crystallogr A 47, 110

(1991). 5. M. Connolly, J. Appl. Cryst. 16, 548 (1983). 6. R. Koradi, M. Billeter, K. Wuthrich, J Mol Graph 14, 51 (1996). 7. J. R. Lakowicz, in Principles of Fluorescence Spectroscopy. (Kluwer

Academic/Plenum Publishers, New York, N. Y., 1999) pp. 52-54. 8. US Patents 6,344,330 and 6,344,334. 9. D. J. Maly, I. C. Choong, J. A. Ellman, Proc Natl Acad Sci U S A 97, 2419 (Mar 14,

2000). 10. R. M. Locksley, N. Killeen, M. J. Lenardo, Cell 104, 487 (2001).

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