Christoph E. Dumelin

In vivo targeting of tumor-associated carbonic anhydrases using acetazolamide derivatives.

We describe the synthesis and characterization of two acetazolamide derivatives containing either a charged fluorophore or an albumin-binding moiety, which restrict binding to carbonic anhydrase IX and XII present on tumor cells. In vivo studies showed the preferentially targeting of tumor cells by the fluorescent acetazolamide derivative and the ability of the albumin-binding acetazolamide derivative to cause tumor retardation in a SK-RC-52 xenograft model of cancer.

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

Label‐free determination of protein–ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry

We performed a systematic comparison of three label‐free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI‐MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (KD). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et al., Chem. Rev. 2008, 108: 946–1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the KD value determined for ethoxzolamide by nESI‐MS was 5 ± 1 nM and the KD value for sulfanilamide was 145.7 ± 10.0 µM. The agreement of the determined KD values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4‐carboxybenzenesulfonamide (variation by ∼ one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the KD values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations. Copyright

New Strategy for the Extension of the Serum Half-Life of Antibody Fragments

Antibody fragments can recognize their cognate antigen with high affinity and can be produced at high yields, but generally display rapid blood clearance profiles. For pharmaceutical applications, the serum half-life of antibody fragments is often extended by chemical modification with polymers or by genetic fusion to albumin or albumin-binding polypeptides. Here, we report that the site-specific chemical modification of a C-terminal cysteine residue in scFv antibody fragments with a small organic molecule capable of high-affinity binding to serum albumin substantially extends serum half-life in rodents. The strategy was implemented using the antibody fragment F8, specific to the alternatively spliced EDA domain of fibronectin, a tumor-associated antigen. The unmodified and chemically modified scFv-F8 antibody fragments were studied by biodistribution analysis in tumor-bearing mice, exhibiting a dramatic increase in tumor uptake for the albumin-binding antibody derivative. The data presented in this paper indicate that the chemical modification of the antibody fragment with the 2-(3-maleimidopropanamido)-6-(4-(4-iodophenyl)butanamido)hexanoate albumin-binding moiety may represent a general strategy for the extension of the serum half-life of antibody fragments and for the improvement of their in vivo targeting performance.

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.

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 >30 000 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 930 nM and was able to compete with a Bak‐derived BH3 peptide, an antagonist of Bcl‐xL function.

DNA‐Encoded Chemical Libraries

Conventional libraries of chemical compounds, which are individually screened for binding to pharmaceutical targets, are typically limited by screening costs and logistics to an upper size of few hundred thousand compounds. The use of DNA-encoded chemical libraries promises to revolutionize the way chemical libraries are screened, allowing the synthesis and selection of libraries of unprecedented size. This article reviews the most recent strategies for the construction and selection of DNA-encoded chemical libraries.