Jörg Scheuermann

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.

Selection of Carbonic Anhydrase IX Inhibitors from One Million DNA-Encoded Compounds

DNA-encoded chemical libraries, i.e., collections of compounds individually coupled to distinctive DNA fragments serving as amplifiable identification barcodes, represent a new tool for the de novo discovery of small molecule ligands to target proteins of pharmaceutical interest. Here, we describe the design and synthesis of a novel DNA-encoded chemical library containing one million small molecules. The library was synthesized by combinatorial assembly of three sets of chemical building blocks using Diels-Alder cycloadditions and by the stepwise build-up of the DNA barcodes. Model selections were performed to test library performance and to develop a statistical method for the analysis of high-throughput sequencing data. A library selection against carbonic anhydrase IX revealed a new class of submicromolar bis(sulfonamide) inhibitors. One of these inhibitors was synthesized in the absence of the DNA-tag and showed accumulation in hypoxic tumor tissue sections in vitro and tumor targeting in vivo.

DNA-Encoded Chemical Libraries: Advancing beyond Conventional Small-Molecule Libraries

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

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

A Traceless Vascular‐Targeting Antibody–Drug Conjugate for Cancer Therapy

Monoclonal antibodies have demonstrated considerable utility in the clinical treatment of cancer, but unmodified immunoglobulins are rarely curative, especially when used as single agents. Thus, there is considerable interest in arming antibodies with bioactive payloads (e.g., drugs, radionuclides, cytokines), to improve their potency and selectivity, thus increasing activity at the tumor site while sparing normal tissues. Significant progress has beenmade in the past few years in the area of antibody–drug conjugates (ADCs) for the selective delivery of cytotoxic drugs to tumors. As a result of these investigations, new agents with pronounced clinical activities have been developed, including SGN-35 (an ADC directed against CD30-positive hematological malignancies) and trastuzumab-DM1 (which has shown activity in metastatic breast cancer). As conventional drug conjugation strategies yield heterogeneous ADC preparations, intense efforts are being devoted to the development of methods for site-selective modification of therapeutic antibodies, thus leading to products with improved performance and batch-tobatch reproducibility. Furthermore, comparative evaluations of intact immunoglobulins in IgG format and other recombinant antibody formats for ADC development have been conducted. It is generally assumed that ADCs may need to be internalized by the tumor cells for the active release of cytotoxic drugs. Once ADCs are internalized and the drug is released in the intracellular compartments, a cross-fire effect (corresponding to the migration to neighboring cells) may occur, as has been reported for the treatment of tumors consisting of a mixture of antigen-positive and antigennegative cells. However, monoclonal antibodies specific to tumor cell antigens often exhibit limited diffusion into the solid tumor mass by several mechanisms, including slow extravasation and antibody trapping by perivascular tumor cells (the so-called antigen barrier). In view of the fact that the formation of new blood vessels (angiogenesis) is a rare process in a healthy adult but a characteristic feature of virtually all types of aggressive cancers, it would be reasonable to develop vascular-targeting ADCs. Unlike the use of cell-specific markers, vascular targeting offers comprehensive tumor coverage, as the majority of cancers express splice isoforms of tenascin-C and of oncofetal fibronectin. In addition, vascular targeting helps address the issue of heterogeneity of antigen expression within the tumor mass (i.e., tumor cells which are positive or negative for the antigen). Despite the remarkable potency of cytotoxic compounds targeting the tumor vasculature and the strong dependence of growing neoplastic masses on florid angiogenesis, only limited efforts were directed in the past towards the investigation of ADCs that target tumor vascular antigens. We have recently shown that the antibody-based delivery of photosensitizers to tumor blood vessels and irradiation may induce complete and long-lasting cancer eradication, in a process that also involves the action of natural killer cells. Thus, there appears to be a strong rationale for the targeted delivery of cytotoxic agents to the tumor neovasculature for cancer therapy. Given that antibodies are large molecules compared to cytotoxic agents, potent drugs need to be used to generate ADCs that can be administered at reasonably low doses and that are compatible with industrial development activities at acceptable cost of goods. Herein, we aimed at generating a novel class of chemically defined vascular-targeting ADCs that release cytotoxic drugs with a mechanism that does not require antibody internalization. We reasoned that ADCs based on linkerless antibody modification with a potent thiolcontaining drug would allow the formation of homogeneous products by the formation of a mixed disulfide. These agents could release the cytotoxic payload in the extracellular space, when tumor cell death is initiated and releases high concentrations of reducing agents (e.g., cysteine, glutathione) to the surrounding environment. Provided that a sufficiently large amount of ADC can be delivered to the subendothelial extracellular matrix, the drug release process would be amplified as tumor cell death progresses. Dolastatins are a group of small peptides isolated from the Indian ocean hare Dolabella auricularia that bind to tubulin subunits and inhibit new microtubule assembly and depolymerize existing microtubules, thus blocking cell cycle [*] Dr. G. J. L. Bernardes, Dr. S. Tr!ssel, I. Hartmann, Dr. J. Scheuermann, Prof. D. Neri Department of Chemistry and Applied Biosciences Swiss Federal Institute of Technology (ETH Z!rich) Wolfgang-Pauli Str. 10, 8093 Z!rich (Switzerland) E-mail: dario.neri@pharma.ethz.ch Dr. G. Casi, Dr. K. Schwager Philochem AG, Libernstrasse 3, 8112 Otelfingen (Switzerland) [**] G.J.L.B. is an EMBO and Novartis Foundation Research Fellow. We thank Katrin Gutbrodt for her help in histology and immunofluorescence experiments, Dr. Kathrin Zuberb!hler and Nadine Pasche for their help during therapy experiments, and Dr. Eveline Traschel for treating tumor-bearing mice with IgG(F8). Financial contribution from ETH Z!rich, Swiss National Science Foundation, SwissBridge/Stammbach Stiftung, Kommission f!r Technologie und Innovation (KTI) and Philochem AG is gratefully acknowledged. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201106527. Angewandte Chemie

Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery.

Aldehyde drugs are gaining increasing research interest, considering that aldehyde dehydrogenases overexpression is characteristic of cancer stem cells. Here, we describe the traceless site-specific coupling of a novel potent drug, containing an aldehyde moiety, to recombinant antibodies, which were engineered to display a cysteine residue at their N-terminus, or a 1,2-aminothiol at their C-terminus. The resulting chemically defined antibody-drug conjugates represent the first example in which a thiazolidine linkage is used for the targeted delivery and release of cytotoxic agents.

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.

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 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor-associated antigen carbonic anhydrase IX (CA IX) 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.

Diagnostic and Therapeutic Applications of Recombinant Antibodies:Targeting the Extra-Domain B of Fibronectin, A Marker of Tumor Angiogenesis

Angiogenesis, the sprouting of new blood vessels from preexisting ones, is a phenomenon associated to several human pathologies, including different potentially blinding ocular disorders, rheumatoid arthritis, and cancer. Indeed, ongoing angiogenesis is a characteristic feature of the majority of aggressive solid tumors, and is also a pre-requisite for the progression towards the metastatic phenotype. One established marker of angiogenesis is represented by an isoform of the oncofoetal fibronectin (FN), containing an additional domain inserted by alternative splicing of the FN pre-mRNA and called extra-domain B (ED-B). This isoform has been found to be present almost exclusively in the modified extra-cellular matrix surrounding newly-formed blood vessels in tumors (and other animal models of ocular pathologies), being completely absent from the normal vasculature in adult organs. This article reviews the recombinant antibodies raised against ED-B and the different methodologies used for the generation of these antibodies. Moreover, new diagnostic and therapeutic applications based on the delivery of bioactive molecules to tumor blood vessels by means of ED-B targeting will be discussed.