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Samu Melkko

Publications and source records attributed to Samu Melkko.

7 recordsLinked to original sources
PubMed2006

DNA-encoded chemical libraries.

The discovery and development of novel drugs for the multitude of targets originating from functional genomic research is a challenging task. While antibodies can nowadays be raised against virtually any given target using phage-display methodologies, a similar "selection/amplification" approach for the facile discovery of low-molecular weight compounds capable of specific binding to protein targets of choice has so far been lacking. The development of DNA-encoded chemical libraries, combined with suitable selection and high-throughput sequencing strategies, holds promises to fill this gap. Here, we review the latest developments in the field of DNA-encoded chemical libraries, commenting on the challenges and opportunities for the different experimental strategies in this rapidly evolving research area, which may gain importance for the future drug discovery process.

Animals↗
PubMed2006

On the magnitude of the chelate effect for the recognition of proteins by pharmacophores scaffolded by self-assembling oligonucleotides.

The simultaneous interaction of the binding moieties of a bidentate ligand on adjacent epitopes of a target protein represents an attractive avenue for the discovery of specific, high-affinity binders. We used short DNA fragments in heteroduplex format to scaffold pairs of binding molecules with defined spatial arrangements. Iminobiotin derivates were coupled either via bifunctional linkers or by using various oligonucleotides, thus allowing monovalent or bivalent binding to streptavidin. We determined the binding affinities of the synthesized constructs in solution. We also investigated the efficiency of recovery of superior bidentate ligands in affinity capture experiments, by using both radioactive counts and DNA microarrays as readouts. This analysis confirmed the suitability of the DNA heteroduplex as a scaffold for the identification of synergistic pairs of binding moieties, capable of a high-affinity interaction with protein targets by virtue of the chelate effect.

Base Sequence↗
PubMed2004

Encoded self-assembling chemical libraries.

The isolation of molecules capable of high-affinity and specific binding to biological targets is a central problem in chemistry, biology and pharmaceutical sciences. Here we describe the use of encoded self-assembling chemical (ESAC) libraries for the facile identification of molecules that bind macromolecular targets. ESAC technology uses libraries of organic molecules linked to individual oligonucleotides that mediate the self-assembly of the library and provide a code associated with each organic molecule. After panning ESAC libraries on the biomolecular target of interest, the 'binding code' of the selected compounds can be 'decoded' by a number of experimental techniques (e.g., hybridization on oligonucleotide microarrays). The potential of this technology was demonstrated by the affinity maturation (>40-fold) of binding molecules to human serum albumin and bovine carbonic anhydrase, leading to binders with dissociation constants in the nanomolar range.

Albumins↗
PubMed2002

An antibody-calmodulin fusion protein reveals a functional dependence between macromolecular isoelectric point and tumor targeting performance.

PURPOSE: Human monoclonal antibodies are promising agents for the development of improved anticancer therapeutics, because, unlike low-molecular-weight chemotherapeutic agents, they can selectively localize to solid tumors. In particular, the scFv(L19) antibody fragment, specific for the EDB domain of fibronectin, a marker of angiogenesis, has demonstrated an impressive tumor targeting performance in a variety of tumor-bearing animals and in patients with cancer. The purpose of this study was to develop a tumor pretargeting strategy, based on a novel anti-EDB fusion protein. METHODS AND MATERIALS: We have fused the scFv(L19) to calmodulin, a small acidic protein for which specific binding peptides with a dissociation constant in the picomolar range are available. The resulting fusion protein has been expressed in mammalian cells and purified to homogeneity, before being characterized by quantitative biodistribution analysis in mice bearing the F9 murine teratocarcinoma. RESULTS: Surprisingly, we have found that the fusion of scFv(L19) to calmodulin completely abrogated the tumor targeting ability of the antibody in vivo, although both scFv(L19) and calmodulin moieties within the fusion protein retained unaltered binding affinities toward their respective ligand. Furthermore, a systematic analysis of 13 derivatives of scFv(L19) recently produced in our laboratories showed that the 10 derivatives that retain the tumor targeting ability of the parental antibody have isoelectric points (pI) between 5.0 and 9.0, whereas scFv(L19)-calmodulin (pI = 4.49) and two other derivatives of scFv(L19) with pI >9.0 were unable to target tumors in vivo. CONCLUSIONS: Because the EDB domain of fibronectin is a component of the modified extracellular matrix, predominantly located at the abluminal side of tumor blood vessels, our data suggest that extreme pI values of antibody-based pharmaceuticals may inhibit protein extravasation, perhaps by virtue of electrostatic interactions with endothelial cells and/or components of the extracellular matrix.

Animals↗
PubMed2002

Two general methods for the isolation of enzyme activities by colony filter screening.

We describe two general methodologies, based on filter-sandwich assays, for isolating enzymatic activities from a large repertoire of protein variants expressed in the cytoplasm of E. coli cells. The enzymes are released by the freezing and thawing of bacterial colonies grown on a porous master filter and diffuse to a second "reaction" filter that closely contacts the master filter. Reaction substrates can be immobilized either on the filter or on the enzyme itself (which is then, in turn, captured on the reaction filter). The resulting products are detected with suitable affinity reagents. We used biotin ligase as a model enzyme to assess the performance of the two methodologies. Active enzymes were released by the bacteria, locally biotinylated the immobilized target substrate peptide, and allowed the sensitive and specific detection of individual catalytically active colonies.

Bacterial Proteins↗
PubMedDate not supplied

Selection of streptavidin binders from a DNA-encoded chemical library.

DNA-encoded libraries of small organic molecules facilitate the construction of large, encoded self-assembling chemical libraries for the identification of high-affinity binders to protein targets. We have constructed a library of 477 chemical compounds, coupled to 48mer-oligonucleotides, each containing a unique six-base sequence serving as "bar-code" for the identification of the chemical moiety. The functionality of the library was confirmed by selection and amplification of both high- and low-affinity binding molecules specific to streptavidin.

Animals↗