A handbook for DNA encoded chemistry theory and applications for exploring chemical space and drug discovery

A Handbook for DNA-Encoded Chemistry A Handbook for DNA-Encoded Chemistry Theory and Applications for Exploring Chemical Space and Drug Discovery Edited by Robert A Goodnow, Jr AstraZeneca Waltham, MA, USA GoodChem Consulting, LLC Gillette, NJ, USA Copyright © 2014 by John Wiley & Sons, Inc All rights reserved Published by John Wiley & Sons, Inc., Hoboken, New Jersey Published simultaneously in Canada No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose No warranty may be created or extended by sales representatives or written sales materials The advice and strategies contained herein may not be suitable for your situation You should consult with a professional where appropriate Neither the publisher nor author shall be liable for any loss of profit or any other commercial damages, including but not limited to special, incidental, consequential, or other damages For general information on our other products and services or for technical support, please contact our Customer Care Department within the United States at (800) 762-2974, outside the United States at (317) 572-3993 or fax (317) 572-4002 Wiley also publishes its books in a variety of electronic formats Some content that appears in print may not be available in electronic formats For more information about Wiley products, visit our web site at www.wiley.com Library of Congress Cataloging-in-Publication Data: A handbook for DNA-encoded chemistry : theory and applications for exploring chemical space and drug discovery / edited by Robert A Goodnow, Jr    p ; cm   Includes bibliographical references and index   ISBN-13: 978-1-118-48768-6 (cloth) I.  Goodnow, Robert A., Jr., editor of compilation  [DNLM: 1. Combinatorial Chemistry Techniques–methods. 2. DNA–chemical synthesis.  3.  Drug Discovery–methods.  4.  Gene Library.  5.  Small Molecule Libraries–chemical synthesis QV 744] RS420  615.1′9–dc23 2013042727 Printed in the United States of America 10 9 8 7 6 5 4 3 2 1 Contents Prefacevii Acknowledgmentsix Introductory Comments xi Contributorsxxiii Just enough knowledge… Agnieszka Kowalczyk A brief history of the development of combinatorial chemistry and the emerging need for DNA-encoded chemistry19 Robert A Goodnow, Jr A brief history of DNA-encoded chemistry 45 4 DNA-Compatible Chemistry 67 5 Foundations of a DNA-encoded library (DEL) 99 Anthony D Keefe Kin-Chun Luk and Alexander Lee Satz Alexander Lee Satz 6 EXERCISES IN THE SYNTHESIS OF DNA-ENCODED LIBRARIES 123 Steffen P Creaser and Raksha A Acharya the dna tag: A Chemical gene designed for DNA-encoded libraries Andrew W Fraley Analytical challenges for DNA-encoded library systems George L Perkins and G John Langley Informatics: Functionality and architecture for DNA-encoded library production and screening John A Feinberg and Zhengwei Peng 153 171 201 v viContents 10 Theoretical considerations of the application of DNA-encoded libraries to drug discovery Charles Wartchow 11 Begin with the End in Mind: The hit-to-lead process 213 231 John Proudfoot 12 Enumeration and Visualization of Large Combinatorial Chemical Libraries Sung-Sau So 13 Screening Large Compound Collections 247 281 Stephen P Hale 14 Reported applications of DNA-encoded library chemistry Johannes Ottl 15 Dual-Pharmacophore DNA-Encoded Chemical Libraries Jörg Scheuermann and Dario Neri 16 Hit Identification and Hit Follow-up 319 349 357 Yixin Zhang 17 Using DNA to Program Chemical Synthesis, Discover New Reactions, and Detect Ligand Binding Lynn M McGregor and David R Liu 377 18 the changing feasibility and economics of chemical diversity exploration with DNA-encoded combinatorial approaches Robert A Goodnow, Jr 19 Keeping the promise? An outlook on DNA chemical library technology Samu Melkko and Johannes Ottl 417 427 Index435 Preface The concept for this book came about after the rejection of an invitation to write a book about combinatorial chemistry Although a highly interesting field of chemistry, the initial invitation was declined upon the assumption that excellent books already exist in sufficient numbers on various subjects of combinatorial chemistry However, upon further reflection, the editor realized that a new chapter in the story of combinatorial chemistry had begun with the emergence and development of DNA-encoded chemistry methods Despite the existence of publications about the concept and practice of DNAencoded chemistry since 1992 by Brenner and Lerner and DNA-directed chemistry roughly a decade later, the editor found no single, authoritative summary of the theories, practice, and results of DNA-encoded chemistry Therefore, it seemed a worthy endeavor to recruit experts in the field and create a handbook summarizing theories, methods, and results for this exciting, new field It is hoped that this handbook will provide a good understanding of the practice of DNA-encoded and DNA-directed chemistry and that such chemistry methods will be more widely embraced and developed by a large community of scientists Readers may notice some overlap and/or repetition among various chapters The editor has tended to allow such commonality as a means not only to highlight the multiple points of view and interpretation on this new technology as it has been applied to organic chemistry and drug discovery, but also as a means to indicate those results which have been received with particular interest by those skilled in the art vii Target protein A B Target protein A B Target protein A B C Figure 3.6.  Alternative architectures of ESAC libraries are shown The figure was kindly provided by Dr Jörg Scheuermann Figure 6.1.  Ethanol precipitation of DNA By adding two volumetric equivalents of ethanol followed by 5 M NaCl to a solution of DNA, the DNA will precipitate from solution and can later be separated as a pellet by centrifugation Figure 6.2.  Split-and-pool By splitting the growing library into a 96-well plate at the beginning of each ligation and synthesis cycle, a unique DNA tag is paired with a unique building block and 96 new encoded molecules can be generated When the products of each cycle are collectively pooled into a single reservoir and split once more, the number of components in each well is multiplied by 96n (n = number of plates used per cycle) AOP-head piece (AOP-HP) TGACTCCC 3′ Primer (P) + 5′ Cycle tag (T1) 5′ (p)XXXXXXXAG 3′ ACXXXXXXX(p) Cycle tag (T2) + (p)AAATCGATGTG 3′ + 3′GGTTTAGCTAC (p) 5′ ACTGAG(p)5′ (p)XXXXXXXGT TCXXXXXXX(p) Cycle tag (T3) + (p)XXXXXXXGA CAXXXXXXX(p) Cycle tag (T4) + (p)XXXXXXXTT 3′ CTXXXXXXX(p) 5′ Closing primer (CP) + 5′ (p)XXXXXXXXXX 3′ 3′ AAXXXXXXXXXXNNNNNNNNNNNNAGTCTGTTCGAAGTGGACG(p) 5′ Figure 6.4.  Example of a DNA tagging strategy [7] Each segment will enzymatically ligate to the growing DEL structure via 2-base-pair “sticky ends” at the 3′-ends The 5′-ends contain a free phosphate group (p) The X regions in the encoding tags (T1, T2, T3, and T4) represent undefined oligonucleotides that will encode the building blocks used in each synthesis cycle The closing primer (CP) contains a long single-stranded segment, which includes a degenerate region (N), that will later be filled in by Klenow reaction at the completion of the DEL Figure 6.5.  Loading a polyacrylamide gel Performing gel electrophoresis on samples taken from representative wells after each ligation cycle allows you to follow the progression of the lengthening DEL and also look for problematic ligations Figure 6.9.  Essential tools for DEL synthesis A collection of well-maintained single and ­ ultichannel pipettes will be used throughout the DEL production Single pipettes will be m needed for preparing building block solutions and reagents, while the multichannel pipettes will drive the split-and-pool plate syntheses Table 6.10.  P ligation P (1) Ligation H2N H2N AOP-HP (1 component) Step P ligation Add Volume Mols Equiv Total volume 1.1 AOP-HP (50 μM in H2O) 50mL 50àmol 50mL 1.2 10ì ligation buffer a 20 mL – – 70 mL 1.3 T4 DNA ligase 2 mL – – 72 mL 1.4 H2O 64 mL – – 136 mL 1.5 DNA P (1.0 mM in H2O) 64 mL 64 µmol 1.28 200 mL 1.6 Stand overnight at room temperature 1.7 Analyze by gel electrophoresis Step Ethanol crash Add Volume Total volume 2.1 5 M NaCl(aq) 20 mL 220 mL 2.2 Cold EtOH 440 mL 660 mL 2.3 −78°C for ≥30 or −20°C for 1 h 2.4 Centrifuge and discard solvent 2.5 Lyophilize DNA pellet 2.6 Dissolve in approx 50 mL of H2O to make 1.0 mM solution 2.7 Confirm conc via OD measurement Table 6.10 outlines a step-by-step approach to attaching the P to the AOP-HP described in Table 6.4 a  500 mM Tris pH 7.5, 500 mM NaCl, 100 mM MgCl2, 100 mM DTT, 20 mM ATP Table 6.11.  Cycle (4) 96 ligations (6) 96 FMOC-AAs (10) 10% piperidine P H2N O H2N R1 P T1 N H (1 plate = 96 components) Step Split Split 6 mL of AOP-HP P (1.0 mM) into 96 wells Step Cycle 1— tag (T1) ligation Add Volume Mols Equiv Total volume (per well) 4.1 AOP-HP P (1.0 mM in H2O) 62.5 μL 62.5 nmol 62.5 μL 87.5 μL 4.2 10× ligation buffer 25 μL – – 4.3 T4 DNA ligase 2.5 μL – – 90 μL 4.4 H2O 35 μL – – 125 μL 4.5 DNA tags (T1) (1.0 mM in H2O) 125 μL 125 nmol 250 μL 4.6 Stand overnight at room temperature 4.7 Analyze by gel electrophoresis Step Ethanol crash Add 5.1 5 M NaCl(aq) 25 μL 275 μL 5.2 Cold EtOH 550 μL 825 μL 5.3 Volume Total volume (per well) −78°C for ≥30 or −20°C for 1 h 5.4 Centrifuge and discard solvent Step Cycle 1—building block addition Add Volume Mols Equiv Total volume (per well) 6.1 Sodium borate buffer (150 mM, pH 9.4) 62.5 μL 62.5 nmol 62.5 μL 6.2 Fmoc-AAs (200 mM in DMF) 12.5 μL 2.5 µmol 40 75 μL 6.3 DMT-MM (200 mM in H2O) 12.5 μL 2.5 µmol 40 87.5 μL 40 90 μL 6.4 6.5 Agitate for 2 h at 4°C Fmoc-AAs (200 mM in DMF) 12.5 μL 2.5 µmol (Continued) Table 6.11. (cont'd) Add Volume Mols Equiv Total volume (per well) 6.6 DMT-MM (200 mM in H2O) 12.5 μL 2.5 µmol 40 92.5 μL 6.7 Agitate overnight at 4°C 6.8 Analyze by LC/MS Step Pool Pool all 96 wells into a single vessel Step Ethanol crash Add Volume Total volume (per well) 8.1 5 M NaCl(aq) 0.89 mL 9.77 mL 8.2 Cold EtOH 19.5 mL 29.3 mL 8.3 −78°C for ≥30 or −20°C for 1 h 8.4 Centrifuge and discard solvent 8.5 Dissolve in H2O to make 1.0 mM solution Step Purify Purify by reverse-phase HPLC 9.1 Lyophilize pooled fractions Step Remove Fmoc 10 Add Volume Total volume 10.1 10% piperidine in H2O 36 mL 36 mL 10.2 Agitate for 1 h at room temperature 10.3 Analyze by LC/MS Step Ethanol crash 11 Add Volume 11.1 5 M NaCl(aq) 3.6 mL 39.6 mL 11.2 Cold EtOH 80 mL 119.6 mL 11.3 −78°C for ≥30 or −20°C for 1 h 11.4 Centrifuge and discard solvent 11.5 Lyophilize DNA pellet 11.6 Dissolve in H2O to make 1.0 mM solution 11.7 Confirm conc via OD measurement Total volume Table 6.11 outlines a step-by-step approach to performing the first cycle of a four-cycle DEL using a single 96-well plate Table 6.12.  Cycle O H2N R1 (13) 96 ligations (15) Cyanuric chloride/96 amines P T1 N H R3 R2 O H N N N N P R1 N Cl T1 T2 N H (1 plate = 9,216 components) Step Split 12 Split 5.76 mL of cycle product (1.0 mM) into 96 wells Step Cycle 2—tag (T2) ligation 13 Add Volume Mols Equiv Total volume (per well) 13.1 Cycle product (1.0 mM in H2O) 60 μL 60 nmol 60 μL 13.2 10 × ligation buffer 24 μL – – 84 μL 13.3 T4 DNA ligase 2.4 μL – – 86.4 μL 13.4 H2O 33.6 μL – – 120 μL 13.5 DNA tags (T2) (1.0 mM in H2O) 110 μL 110 nmol 1.8 230 μL 13.6 Stand overnight at room temperature 13.7 Analyze by gel electrophoresis Step Ethanol crash 14 Add Volume Total volume (per well) 14.1 5 M NaCl(aq) 23 μL 253 μL 14.2 Cold EtOH 506 μL 759 μL 14.3 −78°C for ≥30 or −20°C for 1 h 14.4 Centrifuge and discard solvent Step Cycle 2—building block addition 15 Add Volume Mols Equiv Total volume (per well) 15.1 Sodium borate buffer (150 mM, pH 9.4) 60 μL 60 nmol 60 μL 15.2 Cyanuric chloride (200 mM in ACN) 3 μL 0.6 µmol 10 63 μL 15.3 Agitate for 1 h 15.4 Cool plate to 4°C (Continued) Table 6.12. (cont'd) 15 Add Volume Mols Equiv Total volume (per well) 15.5 Amines (200 mM in DMA or ACN:H2O (1:1)) 15 μL 3 µmol 50 78 μL 15.6 Agitate for 40 h at 4°C 15.7 Analyze by LC/MS Step Pool 16 Pool all 96 wells into a single vessel Step Ethanol crash 17 Add Volume Total volume 17.1 5 M NaCl(aq) 0.75 mL 8.24 mL 17.2 Cold EtOH 16.5 mL 24.7 mL 17.3 −78°C for ≥30 or −20°C for 1 h 17.4 Centrifuge and discard solvent 17.5 Dissolve in approx 5.76 mL H2O to make 1.0 mM stock 17.6 Confirm conc via OD measurement Table 6.12 outlines a step-by-step approach to performing the second cycle of a four-cycle DEL using a single 96-well plate Table 6.13.  Cycle R3 N R2 N N O H N N R1 P (19) 96 ligations (21) 96 diamines (inc.60 Nvoc-diamines) T1 T2 N H (24) 365 nm R3 CL R2 N N H2N R5 N N Step O H N N R4 P T1 T2 T3 N H R1 (1 plate = 884,736 components) Split 18 Split 5.76 mL of Cycle product (1.0 mM) into 96 wells Step Cycle 3—tag (T3) ligation 19 Add Volume Mols Equiv Total volume (per well) 19.1 Cycle product (1.0 mM in H2O) 60 μL 60 nmol 60 μL 19.2 10 × ligation buffer 24 μL – – 84 μL (Continued ) Table 6.13. (cont'd) 19 Add Volume Mols Equiv Total volume (per well) 19.3 T4 DNA ligase 2.4 μL – – 86.4 μL 19.4 H2O 33.6 μL – – 120 μL 19.5 DNA tags (T3) (1.0 mM in H2O) 180 μL 180 nmol 300 μL 19.6 Stand overnight at room temperature 19.7 Analyze by gel electrophoresis Step Ethanol crash 20 Add Volume Total volume (per well) 20.1 5 M NaCl(aq) 30 μL 330 μL 20.2 Cold EtOH 660 μL 990 μL 20.3 −78°C for ≥30 or −20°C for 1 h 20.4 Centrifuge and discard solvent Step Cycle 3—building block addition 21 Add Volume Mols Equiv Total volume (per well) 21.1 Sodium borate buffer (150 mM, pH 9.4) 60 μL 60 nmol 60 μL 21.2 Nvoc-diamines (200 mM in DMA or ACN:H2O (1:1)) 15 μL 3 µmol 50 75 μL 21.3 Agitate for h at 80°C 21.4 Analyze by LC/MS Step Pool 22 Pool all 96 wells into a single vessel Step Ethanol crash 23 Add Volume Total volume 23.1 5 M NaCl(aq) 0.72 mL 7.92 mL 23.2 Cold EtOH 15.8 mL 23.7 mL 23.3 −78°C for ≥30 or −20°C for h 23.4 Centrifuge and discard solvent Step Nvoc deprotection 24 Dissolve in 3.6 mL AcOH aq buffer (100 mM, pH 4.5) 24.1 Transfer to flat-bottomed crystallizing dish 24.2 Cool to 4°C and irradiate at 365 nm for 16 h 24.3 Transfer to a centrifuge tube (Continued) Table 6.13. (cont'd) Step Ethanol crash 25 Add Volume Total volume 25.1 5 M NaCl(aq) 0.36 mL 3.96 mL 25.2 Cold EtOH 7.9 mL 11.86 mL 25.3 −78°C for ≥30 or −20°C for 1 h 25.4 Centrifuge and discard solvent 25.5 Dissolve in H2O Step Purify 26 Purify by reverse-phase HPLC 26.1 Lyophilize pooled fractions 26.2 Dissolve in H2O to make 1.0 mM stock 26.3 Confirm conc via OD measurement Table 6.13 outlines a step-by-step approach to performing the third cycle of a four-cycle DEL using a single 96-well plate Table 6.14.  Cycle R3 N R2 N H2N R5 N N O H N N R1 (28) 96 ligations P T1 T2 T3 (30) 96 sulfonyl chlorides N H R3 R4 R2 N R5 O H N N N N P T1 T2 T3 T4 N H R1 N O NH S O R6 R4 (1 DEL = 84,934,656 components) Step Split 27 Split 2.5 mL of Cycle product (1.0 mM) into 96 wells Step Cycle 4—tag (T4) ligation 28 Add Volume Mols Equiv Total volume (per well) 28.1 Cycle product (1.0 mM in H2O) 26 μL 26 nmol 26 μL 28.2 10 × ligation buffer 10.4 μL – – 36.4 μL (Continued ) Table 6.14. (cont'd) 28 Add Volume Mols Equiv Total volume (per well) 28.3 T4 DNA ligase 1.04 μL – – 37.4 μL 28.4 H2O 14.6 μL – – 52.0 μL 28.5 DNA tag (T4) (1.0 mM in H2O) 60 μL 60 nmol 2.3 112 μL 28.6 Stand overnight at room temperature 28.7 Analyze by gel electrophoresis Step Ethanol crash 29 Add Volume Total volume (per well) 29.1 5 M NaCl(aq) 11.2 μL 123 μL 29.2 Cold EtOH 246 μL 369 μL 29.3 −78°C for ≥30 or −20°C for 1 h 29.4 Centrifuge and discard solvent Step Cycle 4—building block addition 30 Add Volume Mols Equiv Total volume (per well) 30.1 Sodium borate buffer (150 mM, pH 9.4) 26 μL 26 nmol 26 μL 30.2 Sulfonyl chlorides (500 mM in ACN) 2.1 μL 1.05 µmol 40 28.1 μL 30.3 Agitate for 16 h at room temperature 30.4 Analyze by LC/MS Step Pool 31 Pool all 96 wells into a single vessel Step Ethanol crash 32 Add Volume Total volume 32.1 32.2 5 M NaCl(aq) 270 μL 2.97 mL Cold EtOH 5.93 mL 8.90 mL 32.3 −78°C for ≥30 or −20°C for 1 h 32.4 Centrifuge and discard solvent Dissolve DNA pellet in H2O Step Purify 33 Purify by reverse-phase HPLC 33.1 Dissolve in H2O to make 1.0 mM solution 33.2 Confirm conc via OD measurement Table 6.14 outlines a step-by-step approach to performing the last cycle of a four-cycle DEL using a single 96-well plate Phase partitioning Proximity=enhanced linkage Solution phase Stationary phase Proximity Figure 13.1.  Identification of target-associated ( ) compounds from libraries of encoded compounds ( ) can be achieved by a physical phase separation or signal generation linked to a target-proximity-dependent event Load Bind Wash Elute Figure 13.2.  Affinity-based library screening applying a chromatographic phase-separation ­process is defined by the adsorption of target-binding library compounds ( ) to the solid-phase ( ) target ( ) in the bind step, separation of weakly target-associated compounds in the wash step, and recovery of target-associated compounds in the elute step Covalent Non-covalent Figure 13.8.  Direct linkage of the target ( ) to the solid-phase matrix can be through the covalent coupling to an activated resin bead (blue sphere) or through noncovalent interactions with the plastic surface blue well of a polystyrene plate Encoding region Library DNA PCR product Constant primers Unique identifier Sequencing adapters Figure 13.17.  PCR-based amplification of library DNA with an appropriately designed primer set enables the creation of a product compatible with commercially available DNA-sequencing techniques 70 Enrichment 60 20 10 0 56 50 100 150 200 Sequence abundance 1500 1550 Figure 16.2.  Analysis of high-throughput sequencing results Plot of enrichment factor versus sequence abundance for library members after selection for binding to Src kinase [25] ... A Handbook for DNA- Encoded Chemistry A Handbook for DNA- Encoded Chemistry Theory and Applications for Exploring Chemical Space and Drug Discovery Edited by Robert A Goodnow, Jr AstraZeneca Waltham,... Luk and Alexander Lee Satz Alexander Lee Satz 6 EXERCISES IN THE SYNTHESIS OF DNA- ENCODED LIBRARIES 123 Steffen P Creaser and Raksha A Acharya the dna tag: A Chemical gene designed for DNA- encoded. .. www.wiley.com Library of Congress Cataloging-in-Publication Data: A handbook for DNA- encoded chemistry : theory and applications for exploring chemical space and drug discovery / edited by Robert A Goodnow,