Yixin Zhang

High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries

DNA encoding facilitates the construction and screening of large chemical libraries. Here, we describe general strategies for the stepwise coupling of coding DNA fragments to nascent organic molecules throughout individual reaction steps as well as the first implementation of high-throughput sequencing for the identification and relative quantification of the library members. The methodology was exemplified in the construction of a DNA-encoded chemical library containing 4,000 compounds and in the discovery of binders to streptavidin, matrix metalloproteinase 3, and polyclonal human IgG.

A Portable Albumin Binder from a DNA‐Encoded Chemical Library

Albumin represents the most abundant protein in human plasma, at a concentration of 45 mgmL . To keep physiological production rates to a minimum, albumin displays a long circulatory half-life in mammals thanks to its size above the renal filtration threshold and its unique ability to interact with the neonatal FcRn receptor. Fusions of biopharmaceuticals to albumin or to albumin-binding peptides have been devised to expose the body to adequate concentrations of the therapeutic agent for a sufficiently long period of time, thus improving efficacy and reducing the number of injections. In principle, small organic albumin-binding molecules could be used as functional analogues of albumin-binding peptides. However, although many small molecules are known to bind to albumin, the success in isolating small molecules as portable albumin-binding moieties has been limited, mainly because most albumin binders (for example, ibuprofen) lose binding affinity upon chemical modification. Myristoylation of insulin has been shown to significantly prolong the circulatory half-life, but this modification is not applicable to a broader set of molecules because of its negative effect on solubility. In another example, a 4,4diphenylcyclohexyl moiety has been connected through a phosphodiester bond to the metal chelator diethylenetriaminepentaacetic acid (DTPA) for magnetic resonance imaging (MRI) applications and to short peptides. These compounds display dissociation constants (Kd) from human serum albumin in the 100 mm range and are susceptible to hydrolysis in vivo. Thus, there is a considerable scientific and biotechnological interest in the identification of small portable binders that display a stable noncovalent interaction with serum albumin. Herein, we report the discovery and characterization of a class of 4-(p-iodophenyl)butyric acid derivatives from a DNA-encoded chemical library, which display a stable noncovalent binding interaction with both mouse serum albumin (MSA) and human serum albumin (HSA). One of these portable albumin-binding moieties was used to improve the performance of the contrast agents fluorescein and GdDTPA. HSA-binding molecules were selected from a DNAencoded chemical library consisting of 619 oligonucleotidecompound conjugates carrying a six-base-pair code for identification. After selection, the DNA sequences of the enriched compounds were amplified by PCR and decoded on oligonucleotide microarrays displaying the complementary sequences (Figure 1a), normalizing the signal intensities after selection against the intensities of compounds selected on empty resin (Figure 1b). Some of the identified binding molecules were excluded from further evaluation based on being promiscuous binders or because of the high standard deviations of the signal intensities on the microarrays (64, 313, 453, 454, 619). Several of the selected molecules (428, 533, 535, 539) displayed striking structural similarities. The basic structure featured a 4-phenylbutanoic acid moiety, with different hydrophobic substituents on the phenyl ring. To obtain further insights into structure–activity relationships, DNA-modified analogues containing propanoyl or pentanoyl skeletons, and/or carrying various substituents on the phenyl ring (Figure 1c; 536, 622–632), were characterized in a radioactivity-based chromatographic albumin-binding assay, which allowed a first classification of the potential binders (Retention: 428> 539> 624> 535> 533> 536> 326> others; see the Supporting Information). The absence of retention of compounds with propanoyl (625) and penta[*] S. Tr ssel, F. Buller, Dr. F. Bootz, Dr. Y. Zhang, L. Mannocci, Dr. J. Scheuermann, Prof. Dr. D. Neri Institut f r Pharmazeutische Wissenschaften Departement f r Chemie und Angewandte Biowissenschaften ETH Z rich Wolfgang-Pauli-Strasse 10, 8093 Z rich (Switzerland) Fax: (+41)44-633-1358 E-mail: neri@pharma.ethz.ch

DNA-encoded chemical libraries for the discovery of MMP-3 inhibitors.

Encoded self-assembling chemical (ESAC) libraries are characterized by the covalent display of chemical moieties at the extremity of self-assembling oligonucleotides carrying a unique DNA sequence for the identification of the corresponding chemical moiety. We have used ESAC library technology in a two-step selection procedure for the identification of novel inhibitors of stromelysin-1 (MMP-3), a matrix metalloproteinase involved in both physiological and pathological tissue remodeling processes, yielding novel inhibitors with micromolar potency.

Discovery of TNF Inhibitors from a DNA-Encoded Chemical Library based on Diels-Alder Cycloaddition

DNA-encoded chemical libraries are promising tools for the discovery of ligands toward protein targets of pharmaceutical relevance. DNA-encoded small molecules can be enriched in affinity-based selections and their unique DNA barcode allows the amplification and identification by high-throughput sequencing. We describe selection experiments using a DNA-encoded 4000-compound library generated by Diels-Alder cycloadditions. High-throughput sequencing enabled the identification and relative quantification of library members before and after selection. Sequence enrichment profiles corresponding to the bar-coded library members were validated by affinity measurements of single compounds. We were able to affinity mature trypsin inhibitors and identify a series of albumin binders for the conjugation of pharmaceuticals. Furthermore, we discovered a ligand for the antiapoptotic Bcl-xL protein and a class of tumor necrosis factor (TNF) binders that completely inhibited TNF-mediated killing of L-M fibroblasts in vitro.

Design and synthesis of a novel DNA-encoded chemical library using Diels-Alder cycloadditions.

DNA-encoded chemical libraries are increasingly being employed for the identification of binding molecules to protein targets of pharmaceutical relevance. Here, we describe the synthesis and characterization of a DNA-encoded chemical library, consisting of 4000 compounds generated by Diels-Alder cycloaddition reactions. The compounds were encoded with unique DNA fragments which were generated through a stepwise assembly process and serve as amplifiable bar codes for the identification and relative quantification of library members.

Discovery of Small‐Molecule Interleukin‐2 Inhibitors from a DNA‐Encoded Chemical Library

Libraries of chemical compounds individually coupled to encoding DNA tags (DNA-encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high-quality DNA-encoded chemical library comprising 30,000 drug-like compounds; this was screened in 170 different affinity capture experiments. High-throughput sequencing allowed the evaluation of 120u2005million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor-associated antigen carbonic anhydraseu2005IX (CAu2005IX) and the pro-inflammatory cytokine interleukin-2 (IL-2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL-2 was confirmed by molecular docking. Our findings suggest that DNA-encoded chemical libraries allow the facile identification of drug-like ligands principally to any protein of choice, including molecules capable of disrupting high-affinity protein-protein interactions.

Isolation of a Small‐Molecule Inhibitor of the Antiapoptotic Protein Bcl‐xL from a DNA‐Encoded Chemical Library

Bcl‐xL is an antiapoptotic member of the Bcl‐2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl‐xL from a DNA‐encoded chemical library using affinity‐capture selections and massively parallel high‐throughput sequencing of >30u2009000 sequence tags of library members. The most potent binder identified, compound 19/93 [(R)‐3‐(amido indomethacin)‐4‐(naphthalen‐1‐yl)butanoic acid], bound to Bcl‐xL with a dissociation constant (Kd) of 930u2005nM and was able to compete with a Bak‐derived BH3 peptide, an antagonist of Bcl‐xL function.

Automated screening for small organic ligands using DNA-encoded chemical libraries

DNA-encoded chemical libraries (DECLs) are collections of organic compounds that are individually linked to different oligonucleotides, serving as amplifiable identification barcodes. As all compounds in the library can be identified by their DNA tags, they can be mixed and used in affinity-capture experiments on target proteins of interest. In this protocol, we describe the screening process that allows the identification of the few binding molecules within the multiplicity of library members. First, the automated affinity selection process physically isolates binding library members. Second, the DNA codes of the isolated binders are PCR-amplified and subjected to high-throughput DNA sequencing. Third, the obtained sequencing data are evaluated using a C++ program and the results are displayed using MATLAB software. The resulting selection fingerprints facilitate the discrimination of binding from nonbinding library members. The described procedures allow the identification of small organic ligands to biological targets from a DECL within 10 d.