Structure-Based Design of Covalent Siah Inhibitors

Transcription

1 Chemistry & Biology, Volume 20 Supplemental Information Structure-Based Design of Covalent Siah Inhibitors John L. Stebbins, Eugenio Santelli, Yongmei Feng, Surya K. De, Angela Purves, Khatereh Motamedchaboki, Bainan Wu, Ze ev A. Ronai, Robert C. Liddington, and Maurizio Pellecchia Inventory of Supplemental Information Table S1. PHYL Mimetics and IC50 Values relative to Their Ability to Displace Biotin-PHYL in the DELFIA Assay, Related to Table 1 Figure S1. Scheme for the Synthesis of Fmoc-Lys-(Acryl amide)-oh, Related to Table 1 Figure S2. Chemical Structure of BI-117C3, Related to Table 1 Supplemental Experimental Procedures Details relative to chemical library screening campaigns cited in the main text of the manuscript.

2 1) KLRPV(cpro)MVRPTVR IC 50 > 100 M 2) KLRPV(cbut)MVRPTVR IC 50 > 100 M 3) KLRPV(cpen)MVRPTVR IC 50 > 100 M 4) KLRPV(bzl)MVRPTVR IC 50 = 18 M 5) KLRPVQMVRPTVR IC 50 = 10 M 6) KLRPVIMVRPTVR IC 50 = 60 M 7) KLRPVTMVRPTVR IC 50 > 100 M 8) KLRPVVVVRPTVR IC 50 = 13 M 9) KLRPVAMVR(5OHP)TVR IC 50 > 100 M 10) KLRPVAMVR(pip)TVR IC 50 = 74 M 11) KLRPVAMVR(tBu)TVR IC 50 > 100 M 12) KLRPVAMVR(hyp)TVR IC 50 > 100 M 13) KLRPVE*M(Dab)*RPTVR IC 50 > 100 M 14) Ac-rvtprvmavprlk-CONH2 IC 50 > 100 M 15) Ac-rvtprvmqvprlk-CONH2 IC 50 > 100 M TABLE S1. PHYL mimetics and IC50 values relative to their ability to displace biotin-phyl in the DELFIA assay. Abbreviations: cpro= cyclopropyl; cpen = cyclopentyl; bzl = benzyl; Peptide 13 is cyclic via an intra-molecular peptide bond between the side chains of the amino-acids marked with an asterisk. The amino-acids in peptides 14 and 15 have a D stereochemistry at their C carbon. It relates to Table 1 in the manuscript.

3 Synthetic procedures. General. Unless otherwise indicated, all anhydrous solvents were commercially obtained and stored in Sure-seal bottles under nitrogen. All other reagents and solvents were purchased as the highest grade available and used without further purification. Thinlayer chromatography (TLC) analysis of reaction mixtures was performed using Merck silica gel 60 F254 TLC plates, and visualized using ultraviolet light. NMR spectra were recorded on Jeol 300 or 400 MHz instruments. Chemical shifts ( ) are reported in parts per million (ppm) referenced to 1 H (Me 4 Si at 0.00). Coupling constants (J) are reported in Hz throughout. Mass spectral data were acquired on Shimadzu LCMS-2010EV for low resolution, and on an Agilent ESI-TOF for either high or low resolution. Purity of all compounds was obtained in a HPLC Breeze from Waters Co. using an Atlantis T3 3µm 4.6x150 mm reverse phase column. The eluant was a linear gradient with a flow rate of 1 ml/min from 95% A and 5% B to 5% A and 95% B in 15 min followed by 5 min at 100% B (Solvent A: H 2 O with 0.1% TFA; Solvent B: acetonitrile with 0.1% TFA). The compounds were detected at =254 nm or 220 nm. Purity of key compounds was established by elemental analysis as performed on a Perkin Elmer series II Combustion analysis was performed by NuMega Resonance Labs, San Diego, CA, USA. Figure S1: Scheme for the synthesis of Fmoc-Lys-(Acryl amide)-oh. It relates to Table 1 in the manuscript.

4 Synthesis of (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-6- acrylamidohexanoic acid (Fmoc-Lys(Acryl amide)-oh): Fmoc-Lys-OH.HCl (500 mg, 1.35 mmol) was dissolved in dioxane (5 ml) and cooled at 0 O C. 10% Na 2 CO 3 in water (12 ml) was added to it. Acryl chloride (1.48 mmol) in dioxane (5 ml) was added to the reaction mixture dropwise at 0 O C, after completion of addition, the reaction mixture was stirred at room temperature for 1h. The reaction mixture was concentrated in vacuo. The crude mixture was re-dissolved in water (50 ml) followed by extraction with diethyl ether (3 x 50 ml) to remove non polar undesired compounds. The aqueous layer was acidified with 10% KHSO 4 and extracted with ethyl acetate. The ethyl acetate layer was dried (MgSO 4 ) and concentrated to give the desired compound as yellow solid (512 mg, 90%). This compound was used for the solid phase synthesis without further purification and it was almost 90% pure. 1 H NMR (400 MHz, DMSO-d 6 ) δ (m, 2 H), (m, 2 H), (m, 2 H), (m, 1 H), (m, 2 H), (m 3 H), (m, 1 H), (m, 1 H), (m, 1 H), 7.32 (t, J = 7.6 Hz, 2 H), 7.40 (t, J = 7.8 Hz, 2 H), 7.62 (d, J = 6 Hz, 1 H, NH), 7.71 (d, J = 7.6 Hz, 2 H), 7.88 (d, J = 7.6 Hz, 2 H), 8.06 (t, J = 5.6 Hz, 1 H, NH), (br s, 1 H, CO 2 H); HRMS calcd for C 24 H 27 N 2 O 5 (M+H) , found Similarly, Fmoc-Dap(acryl amide)-oh and Fmoc-Orn(acryl amide)-oh were prepared. (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-acrylamidopentanoic acid (Fmoc-Orn(acryl amide)-oh): 1 H NMR (400 MHz, DMSO-d 6 ) δ (m, 2 H), (m, 2 H), (m, 1 H), (m, 2 H), (m 3 H),

5 (m, 1 H), (m, 1 H), (m, 1 H), (m, 4 H), (m, 5 H), 7.87 (d, J = 7.6 Hz, 2 H), 8.08 (t, J = 5.6 Hz, 1 H, NH), (br s, 1 H, CO 2 H); HRMS calcd for C 23 H 25 N 2 O 5 (M+H) , found (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-acrylamidopropanoic acid (Fmoc-Dap(acryl amide)-oh). 1 H NMR (400 MHz, DMSO-d 6 ) δ (m, 2 H), (m, 1 H), (m 3 H), (m, 1 H), (m, 1 H), (m, 1 H), (m, 4 H), (m, 5 H), 7.89 (d, J = 7.6 Hz, 2 H), 8.10 (t, J = 5.6 Hz, 1 H, NH), (br s, 1 H, CO 2 H); HRMS calcd for C 21 H 21 N 2 O 5 (M+H) , found Solid Phase Peptide Synthesis. All linear peptides (acrylated and non acrylated) were assembled using standard Fmoc peptide synthesis protocol with the Rink amide resin on 433A Peptide Synthesizer (Applied Bio-Systems). For each coupling reaction, 10 equiv of Fmoc-amino acid, 0.45 M solution of HOBt/HBTU (9 equiv) in DMF, 2 M solution of DIEA in NMP were used. The coupling reaction was allowed to proceed for 9 min. Fmoc deprotection was performed by treating the resin-bound peptide with 20% piperidine in DMF for 10 min. The peptide was cleaved from the resin by treating the resin with a cleavage cocktail containing 94% TFA, 2 % water, 2% triisopropylsilane, 2% phenol for 3 h. All protecting groups also were removed during this cleavage reaction. TFA was removed under reduced pressure and the peptide was precipitated in diethyl ether, centrifuged, and washed with diethyl ether prior to drying in high vacuum. The crude peptide was purified

6 by preparative reverse phase HPLC. The final compound was characterized by NMR and MALDI-Mass. Figure S2: Chemical structure of BI-117C3: (S)-1-((S)-2-((S)-2-((S)-2-acetamido-6- aminohexanamido)-4-methylpentanamido)-5-guanidinopentanoyl)-n- ((6S,9S,12S,15S,18S)-15-(acrylamidomethyl)-1-amino-6-((S)-2-(((S)-1-(((S)-1-(((S)-1- amino-5-guanidino-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-3-(1h- indol-3-yl)-1-oxopropan-2-yl)carbamoyl)pyrrolidine-1-carbonyl)-1-imino-9-isopropyl- 19-methyl-12-(2-(methylthio)ethyl)-8,11,14,17-tetraoxo-2,7,10,13,16-pentaazaicosan-18- yl)pyrrolidine-2-carboxamide. It relates to Table 1 in the manuscript. Chemical library screens. Four different small molecule libraries totaling 16,009 compounds (240 TimTec; 14,009 Asinex; 1040 U.S. Drug Collection; 720 Microsource Natural Products) arranged in mixtures of 24 were screened at 50 M in the DELFIA assay format. We opted to use this assay platform given its excellent reproducibility (Figure S2). In addition, we also we also tested a L-hexa-peptide (TPI 1069) and D-deca-peptide positional scanning

7 libraries from the Torrey Pines Institute for Molecular Studies (TPI 1039). TPI 1069 consists of all the combinations of 19 natural amino acids, cysteine omitted, and TPI 1039 consists of all combinations 18 d-amino acids, cysteine and tryptophan omitted. All libraries used were peptide-like and arranged in an OXn format, where O represents one of the component in a defined position and X represents a mixture of all the components. The positional scanning libraries were prepared at the Torrey Pines Institute for Molecular Studies as described previously (1, 2). Hits were selected based on at least 50% inhibition of DELFIA signal obtained in control wells. IC 50 values were obtained upon titration of hit compounds. Upon deconvolutions and dose response titrations, no viable hits reproducibly displaced the biotin-qqertklrpvamvrptvrvqpql peptide. This is consistent with a parallel effort from the MLPCN SBMRI screening center that using a similar peptide displacement assay platforms (based on FPA and TR- TRET with a similar PHYL peptide) tested over 300,000 compounds from the NIH collection, resulting in no viable hits (see bioassays AIDs 1817, 1826, 2127 and 2141 for details). An additional fragment-based screening approach was also adopted consisting of obtaining a sample 13 C -Met-Siah2(DR), detect chemical shift perturbations induced by the PHYL peptide in 2D [ 13 C, 1 H ] HSQC spectra and in 1D 13 C-filtered 1D 1 H NMR spectra for such Siah2(DR) sample and subsequently use these NMR assays (1D and 2D) to screen a in house collection of rule of 3 compliant fragment like molecules (~500 compounds). Testing the library at 100 M concentration, with 13 C -Met-Siah2( R) at 10 M concentration revealed no viable hits.

8 (1) Pinilla, C.; Appel, J.R.; Blanc, P.; Houghten, R.A. Biotechniques 1992, 13, 901. (2) Pinilla, C.; Appel, J.R.; Houghten, R.A. Biochem. J. 1994, 301 ( Pt 3), 847.