Supporting Information. Binding-site compatible growing applied to the

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1 Supporting Information Binding-site compatible growing applied to the design of β 2 -adrenergic receptor ligands. Florent Chevillard, Helena Rimmer, Cecilia Betti, Els Pardon,, Steven Ballet, Niek van Hilten, Jan Steyaert,, Wibke E. Diederich, and Peter Kolb, Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, Marburg, Germany, Department of Pharmaceutical Chemistry & Center for Tumor Biology and Immunology, Philipps-University Marburg, Hans-Meerwein-Straße 3, Marburg, Germany, Research Group of Organic Chemistry, Departments of Chemistry and Bio-Engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium, VIB-VUB Center for Structural Biology, VIB, 1050 Brussels, Belgium, and Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium peter.kolb@uni-marburg.de To whom correspondence should be addressed Department of Pharmaceutical Chemistry, Philipps-University Marburg, Marbacher Weg 6, Marburg, Germany Department of Pharmaceutical Chemistry & Center for Tumor Biology and Immunology, Philipps-University Marburg, Hans-Meerwein-Straße 3, Marburg, Germany Research Group of Organic Chemistry, Departments of Chemistry and Bio-Engineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium VIB-VUB Center for Structural Biology, VIB, 1050 Brussels, Belgium Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussels, Belgium S1

2 Supplemental Information Web interface Workflow of the growing strategy Radioligand displacement assay PAINS filter results Similarity search results Compound 6-10 binding modes an discussion Page S2 S3 S3 S3 S3 S3 Methods Web interface The PINGUI toolbox is freely accessible at and contains four distinct modules. The first module Apply Organic Reactions applies 58 organic reactions to a molecule which has to be defined as SMILES. Only the compatible reactions are displayed under the Compatible reactions container. The second module Create your Virtual Library requires three inputs: a user-defined molecule as SMILES; one of the 58 reactions available in the list; and a chemical library to react with (currently available are chembridge: 7805 building blocks; molport: building blocks; or a user-provided library: up to building blocks). For the first two vendors, it is then possible to select a subset defined by its main reactive feature (carbonyls, alcohols, halides, amines, acids, or boronic acids). The third module Retrosynthesis deconstructs a user-defined molecule as SMILES based on the 58 organic reactions. This module can be used for retrosynthesis studies. The fourth module Filter your Library requires two inputs: a list of molecules as SMILES (file size limited to 2 MB: about fragment-sized molecules) and a chemical moiety (carbonyle, ketone, aldehyde, alcohol, halide, amine, acid, or boronic acid). The molecules will then be filtered and only the ones containing the selected chemical moiety will be retained. All chemical descriptors shown for each molecular entity were computed using the RDKit library. The partition coefficient LogP was predicted using Crippen s approach. 1 TPSA represents the topological polar surface area of the molecule. 2 S2

3 Workflow of the growing strategy Figure S1 shows a comprehensive scheme of the growing via merging (GVM) workflow. Results Radioligand displacement assay The IC 50 curves of the remaining compounds are reported in Figure S2. PAINS filter results The compounds were filtered using the PAINS filter 3 in order to get rid of compounds that have a high chance of generating artificial results during follow-up biological assays. This was done by employing a python script based on the rdkit library. 4 ne of the tested compounds were flagged with PAINS moieties. Similarity search results Every compound was compared to a set of 501 ligands of the β 2 AR extracted from the ChEMBL 5 (K i < 10µM) using ECFP4 fingerprints and a python script based on the rdkit library. All compounds are below a 0.4 Tanimoto coefficient (Table S1), suggesting that they are dissimilar to know actives of the β 2 AR. 6 Discussion and compound binding modes Compound 6 Compound 6 exists in two enantiomers. The S-enantiomer was predicted to make mostly apolar interactions in the SBP, with the trifluoro moiety interacting with Ile , Ile and Trp S3

4 next building-block Fragment to grow 58 organic reactions Fragment compatible with reaction? next reaction next fragment to grow Reaction compatible with the area to grow? more reaction in the database? Building Blocks Database BB compatible with the reaction? more BB in the database? Generate Surrogate Add to Surrogates dataset Generate corresponding products Docking: DOCK next docking pose more BB in the database? Docking: SEED Surrogate geometrically favorable? Top 500 docking poses Fragment overlap with virtual product docking pose? Surrogate overlap with virtual product docking pose? next docking pose Keep top 500 Visual inspection + final selection Figure S1: A schematic diagram illustrating all steps in the growing via merging workflow. S4

5 (a) Compound 6 (b) Compound 7 (c) Compound 8 (d) Compound 9 (e) Compound 10 (f) Compound 12 (g) Compound 13 Figure S2: IC 50 curves from radioligand displacement assay. The green curves correspond to the assay made with the active-locked conformation of the receptor (fused to Nb80), while the red curves correspond to the basal-locked conformation of the receptor (fused to Nb69). S5

6 Table S1: Tanimoto distance of every compound to the closest β 2 AR ligand of CheMBL using ECFP4 fingerprints. Compound Distance to the closest ligand (Tanimoto ECFP4) (Figure S3). The charged amine makes polar interactions with Asp and Asn Interestingly, the R-enantiomer keeps the overall binding mode except for the amine which flips, thus breaking the polar bond with Asp Thus, the R-enantiomer presents an unsatisfied hydrogen bond donor. Compound 6 was tested as racemic mixture and had a weaker affinity and LE (52.3 µm, LE = 0.23) compared to its initial core fragment compound 3 (K i = 33.5 µm, LE = 0.35). The docking predictions scored the S-enantiomer more favorable than the R-enantiomer, hence the affinity of one enantiomer could potentially be lower then the apparent K i of 52.3 µm. However, because of the low affinity, we decided not to attempt purification of the individual enantiomers. Compound 7 Compound 7 contains a chiral center, too, and was selected for synthesis because it contains a hydroxy moiety that was predicted to make a hydrogen bond with Tyr (Figure S3), albeit from the opposite side of the nitrogen compared to adrenaline. This motif was quite frequent among all the suggested products, so we wanted to explore its pharmacological relevance. The hydroxy moiety of the R-enantiomer was predicted to form a hydrogen bond with Tyr , while the furan makes hydrophobic contacts with Ile On the other hand, the S-enantiomer S6

7 (a) Compound 6 (b) Compound 7 (c) Compound 9 (d) Compound 10 Figure S3: (a-d) Prediction of the binding mode of compounds 6, 7, 9 and 10 within the β2 AR active conformation. S7

8 is predicted to lose all key polar interactions: the charged amine as well as the hydroxy group are too far from Tyr and Asp Thus, the S-enantiomer is left with two free hydrogen bond donors which is highly unfavorable. Compound 7 was tested as racemic mixture and had a lower affinity (95.4 µm, LE = 0.20) compared to its initial core fragment compound 3 (K i = 33.5 µm, LE = 0.35). As before, the docking score predicts the R-enantiomer to bind with a more favorable affinity than the S-enantiomer, suggesting that the affinity of the R-enantiomer could actually be lower than the one measured. Compound 9 Compound 9 was selected because the initial core fragment was predicted to flip its binding mode, therefore interacting in the SBP with Trp and Ile Compound 9 contains only hydrophobic moieties except for the charged amine. Compound 9 was tested and had an improved affinity (K i = 7.9 µm, LE = 0.27) compared to its initial core fragment compound 1 (K i = 33.8 µm, LE = 0.49). This slight improvement in affinity but decrease in LE for this product could be explained by a less unfavorable desolvation due to its hydrophobic nature. Compound 10 Compound 10 was selected due to the small size of its extension (only four additional heteroatoms) in comparison to the other products and also because it did not contain an aromatic moiety. Compound 10 showed an important fall in affinity (250-fold decrease in affinity) which can partially be explained by the lack of strong hydrophobic contacts with Trp or Ile Furthermore, the cyclic ether moiety introduces two free lone pairs that will induce a small desolvation penalty. References (1) Wildman, S. A.; Crippen, G. M. Prediction of physicochemical parameters by atomic contributions. J. Chem. Inf. Comput. Sci. 1999, 39, S8

9 (2) Ertl, P.; Rohde, B.; Selzer, P. Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties. J. Med. Chem. 2000, 43, (3) Baell, J. B.; Holloway, G. A. New Substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J. Med. Chem. 2010, 53, (4) Landrum, G. RDKit: Open-source chemoinformatics, ( accessed v 15, 2014). (5) Gaulton, A.; Bellis, L. J.; Bento, A. P.; Chambers, J.; Davies, M.; Hersey, A.; Light, Y.; McGlinchey, S.; Michalovich, D.; Al-Lazikani, B.; Overington, J. P. ChEMBL: a large-scale bioactivity database for drug discovery. Nucleic Acids Res. 2012, 40, D1100-D1107. (6) Chevillard, F.; Lagorce, D.; Reynès, C.; Villoutreix, B. O.; Vayer, P.; Miteva, M. In silico prediction of aqueous solubility : A multimodel protocol based on chemical similarity. Mol. Pharm. 2012, 9, S9

10 Product Specifications Product (S)-1-(4-Chlorophenyl)ethylamine,95+%,(sum of isomers), 95% e.e. General Product Data Version 0 CAS Molecular weight Molecular formula Linear formula Flash point ( C) 115 C8 H10 Cl N ClC6H4CH(CH3) NH2 Product Specifications Appearance (Color) Clear colorless to yellow Appearance (Form) Liquid Infrared spectrum Authentic Refractive index to (20 C, 589 nm) Specific optical rotation -22 to -25 (20 C, 589 nm) (c=2, methanol) GC >=95.0 % Enantiomeric excess >=95.0 % Acros Organics ENA23, zone 1, nr 1350, Janssen Pharmaceuticalaan 3a, B-2440 Geel, Belgium Tel / Fax / Internet: 1 Reagent Lane, Fair Lawn, NJ 07410,USA Fax

11 Product Specifications Product (S)-1-(4-Chlorophenyl)ethylamine,95+%,(sum of isomers), 95% e.e. General Product Data Version 0 CAS Molecular weight Molecular formula Linear formula Flash point ( C) 115 C8 H10 Cl N ClC6H4CH(CH3) NH2 Product Specifications Appearance (Color) Clear colorless to yellow Appearance (Form) Liquid Infrared spectrum Authentic Refractive index to (20 C, 589 nm) Specific optical rotation -22 to -25 (20 C, 589 nm) (c=2, methanol) GC >=95.0 % Enantiomeric excess >=95.0 % Acros Organics ENA23, zone 1, nr 1350, Janssen Pharmaceuticalaan 3a, B-2440 Geel, Belgium Tel / Fax / Internet: 1 Reagent Lane, Fair Lawn, NJ 07410,USA Fax

12 CERTIFICATE OF ANALYSIS Code. CC12296 Name N-(1-benzothien-2-ylmethyl)-N-methylamine hydrochloride Batch Last reviewed 03/03/2015 CAS Mol. Formula C10 H11 N S. Cl H Mol. Wt TEST SPECIFICATION RESULT Appearance White to off-white solid Off-white solid NMR Consistent with Structure Complies IR Consistent with Structure Complies GLC Purity HPLC Purity 97% 99.5% Melting Point Range C CHN C: N: C 55.92; H 5.65; N 6.49 Titration Optical Rotation: Water (kf): Comments This batch meets the current specification. Maybridge provides this Certificate of Analysis in good faith and to the best of its knowledge. It should not be substituted for the user s own raw material Quality Control. This report has been computer generated using reviewed and approved QC data and does not contain a signature. Maybridge, Thermo Fisher Scientific, Bakewell Road, Loughborough, LE11 5QY England. Telephone +44 (0) maybridge.sales@thermofisher.com

13 10/27/ :14 PM Chromatogram C:\ClarityVA\DataFiles\ORGC2\Data\CC 40246_10_27_ _32_18 AM.PRM Page 1 of 1 HPLC-2 Sample Info: Sample ID : CC Amount : 0 Sample : CC ISTD Amount : 0 Inj. Volume [µl] : 1 Dilution : 1 Column : Chromolith HR RP mm Detection : Mobile Phase : 1 to 97 % (ACN/H2O) Temperature : Flow Rate : 3 ml/min Pressure : te : [mau] 300 CC 40246_10_27_ _32_18 AM - PDA1-214nm- at- 4nm [%] Absorbance Components Time 0 [min] Result Table (Uncal - CC 40246_10_27_ _32_18 AM - PDA1-214nm-at-4nm) Reten. Time [min] Area [mau.s] Height [mau] Area [%] Height [%] Total

14 Powered by TCPDF ( CERTIFICATE OF ANALYSIS Catalog Number: Lot Number: Product Name: sc L Pyrimidin-2-ylbenzylamine CAS Number: Molecular Formula: C 11 H 11 N 3 Molecular Weight: Test Specification Result Appearance Off white solid Identification ( 1 H-NMR) Consistent with structure Complies Purity 95% This document was produced electronically and is valid without a signature. Santa Cruz Biotechnology, Inc fax Europe

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