Meldal Group - Evolutionary Chemical Biology

Molecular Understanding of Cellular Behavior

Chemical control of adhesion, proliferation, signaling and phenotype

Cells may be controlled in terms of their adhesion and spreading by molecular interaction with membrane and surface receptors. Phenotypes can be modified by small molecules derived from molecular libraries.

At CECB, we have developed screening technologies for selection of molecules that interact with receptors or directly with the cell membrane through "on bead" selection technologies using fluorescence as a readout.

Using this approach, GPCR agonists have been identified. Moreover, super-adhesion molecules have been developed through iterative rounds of synthesis and screening of D-amino acid libraries (Figure 1).

CECB intends to develop "on bead" screening for phenotypic changes of stem cells induced by peptidomimetic small molecules prepared in the form of split mix libraries. By combining cell adhesion with libraries of small molecule that modulate cell behavior and proliferation, compounds for use in cell development may be derived at high velocity.

MPM-encoding technology and the development of stable on-bead fluorescence assays are essential to these studies.

"Click"-Chemistries: CuAAC and INAIC Reactions

The CuAAC-click reaction, which we was introduced in 2001, has become the Royalty amongst click reactions.

CECB uses CuAAC for click assembly of molecular LEGO using functional alkyne and azide modified peptides and proteins.

For example the triazole analogue of the tachyplesin 1 was prepared in this manner and was shown to be a selective functional dimer (see figure) lysing only bacterial membranes.

CECB is working on development of a range of intramolecular multicomponent click reactions.

These are the so called intramolecular N-acyl iminium cascade (INAIC)-reactions, which proceed quantitatively in synthesis of combinatorial libraries and convert easily assembled peptides into chiral small-molecule scaffolds.

The products have great potential as ligands for receptors and can tap into the great diversity of amino acid derivatives currently available.

The INIAC reaction is initiated by intramolecular nucleophilic attack of an amide bond on a proximate aldehyde. The formed hydroxylactam looses water and forms highly reactive N-acyl iminium ions.

These collapse in yet another intramolecular reaction with side-chain C-, N-, S- or O-nucleophiles.

Combinatorial chemistry has developed out of the observation of natural selection and combichem presenting its power in key biological functions, including replication and immune response.

CECB has a great interest in applying both computational design, combinatorial technology and combinatorial selection methods to build and screen molecular libraries.

In 1988, we introduced the parallel synthesis block that in various forms has since become standard laboratory equipment in CombiChem laboratories worldwide. In 1992, we described a multicolumn library synthesizer for split mix synthesis that greatly facilitated the preparation of molecular libraries.

Combinatorial methods were gradually discovered 1984 - 1991 culminating with the simultaneous presentation of the solid phase split mix approach by Lam and Furka in 1991. This event created an outburst of combinatorial chemistry groups around the world. The power of combinatorial technology in discovery of new active substances was immediately realized. However, reliable analysis of structures from single beads has remained a real difficulty in this approach.

Center for Evolutionary Chemical Biology has developed a range of enabling and facilitating methods in the field of combinatorial chemistry, both with respect to screening and structure elucidation. The screens include on-bead fluorescence resonance energy transfer (FRET) assays for protease activity and inhibition, on bead cellular assays and solid phase binding assays.

At CECB structural analysis from single beads is performed on the center's ICR-MS (Figure 1) and microTOF LC-MS instruments. Rapid structure determination and determination of structure activity relationship may alternatively be performed by our 3D-MPM-encoding.

Optical 3D-MPM-Encoding of Beads

CECB has a great interest in selection and combinatorial technology for the building and screening of molecular libraries.  One of the most important technologies developed at CECB is a microsized 3D-equivalent to the all-important barcode we all use in our everyday life.

This so-called Micro-Particle Matrix or MPM-encoding facilitate structure elucidation in a manner that is not comprimized by structural class or complexity. The decoding principles are completely detached from the target structures attached to the beads.

The encoded macrobeads with a diameter of ~500 micron are polymerized with a novel detergent optimized for homogeneous formation of the desired macrobeads with even but random distribution of fluorescently labeled micro-particles.

Decoding is performed through imaging of all beads in each library conversion step and in the hit identification step (Figure 1).

Description of Figure 1

The three orthogonal (perpendicular) projections of an MPM-encoded bead which can be transformed into 3D coordinates used for the reading of the code (left), and the simple decoder instrument (right), which not only can read the three images but also reads a fluorescence signal as a result of the interaction of a fluorescently labelled protein with a resin bound ligand and automatically relate this to the ligand structure.

This technology may significantly facilitate future combinatorial chemistry approaches.

Green Material from Natural Fiber and Water Purification Membranes

CECB employs controled thin-layer polymer technology to produce aquaporin- containing polymer membranes for water purification. Nanotechnology and self-assembly processes are used to control the membrane structure and function.  The project is a collaboration between CECB, AquaZ and the Danish Foundation for Advanced Technology.

CECB also utilizes long experience in polymer chemistry in an attempt to convert waste streams of agriculture into new fiber-based materials with special properties for packaging, construction and home utilities. The resulting biodegradable plastics are produced from 100% non-fossils.

The polymers are dense and non porous and can be moulded into various shapes. Chemical surface modification of the fibers by environment-friendly gas-phase reactions subsequently ensures covalent anchoring of the fibers in the polymer leading to extraordinary strong materials at low cost.

Transition Metal Catalysts by Combinatorial Chemistry

Artificial enzymes have a huge potential for becoming our next generation of nanotools in medicine as well as in the processing of raw materials for the food and beverage industry.

These enzyme-like catalysts may be derived through folding and coordination of peptides containing phosphines or carbenes around a central transition metal.

Imagine a compound that binds to a target protein and processes this protein selectively in the presence of all other proteins of the cell.

With such artificial enzymes it may become feasible to trigger the activation of the various starch processing enzymes in the brewing process or cure disease with artificial proteases that cleave essential proteins in severe patogens such as the HIV envelope or the plaque forming 1-42 APP peptide of Alzheimer.

Peptide Synthesis and Technology

The PEG-based resins PEGA, SPOCC and POEPOP are versatile polymers which are suitable for both aqueous and organic chemistry and are ideally suited for peptide and peptidomimetic assembly.

The extreme swelling and the solvating power of these resins make difficult peptide synthesis easy. We favor active- ester couplings and introduced the O-Dhbt esters as auto-monitoring agents in solid- phase peptide synthesis.

This allowed direct, real-time monitoring of the kinetic behavior of the solid-phase peptide couplings in solid supports. We introduced multicolumn peptide synthesis for simultaneous synthesis of hundreds of peptides and used this technology in the development of the combinatorial multicolumn peptide synthesizer that we employ for the assembly of large split-mix molecular libraries.

This technology has enabled synthesis of a large variety of glycopeptide and phosphopeptide. We combine solid-phase peptide synthesis with enzyme reactions and organic chemistry.

The merger of organic chemistry with solid-phase peptide synthesis is particularly interesting because structurally, molecular libraries may bebefit from both the great variety of the peptide world and the multitude of functionality derived by organic synthesis.

PEG-Hydrogels and Biocompatible Polymers

Over the years the group of professor Meldal has developed a large range of polymers for synthesis and biomolecular screening.

The polymers are typically prepared from PEG- or PEI-macromonomers, which are used in either radical or ring-opening polymerization reactions.

PEGA resins are widely used for on-resin biochemical assays, due to the stealth-like spectroscopic properties and beneficial interaction with proteins and cells.

These special properties have allowed a range of sensitive, on-bead bioassays based on fluorescence to be developed e.g. the Abz/YNO2 FRET assay or the cells- on-beads GPCR-assay.

One of the many beneficial properties of PEG-based resins is the extraordinary high swelling potential of the resin in aqueous and apolar solvents alike, allowing first solid-phase organic synthesis and then screening of compound libraries with enzymes in aqueous buffers.

Enzymes transverse throughout the polymer network and are protected by favorable interactions with the PEG-matrix.

Protein Function and "Click" Chemistry

At CECB we are interested in the subtleties of molecular recognition in the transmission of biological function. An example of this is our work with Tachyplesin I analogs, which were irreversibly locked by click chemistry replacement of disulfide bonds.

This allowed us to measure the formation of dimers by 1H-NMR and suggested a mechanism for antibacterial selectivity.

Dimerization and oligomerization of G-protein coupled receptors are involved in regulation of the signal transduction.

In order to be able to study receptor signaling we are attempting to express proteins and protein fragments with genetic code reprograming to introduce alkynes and azides genetically and site-specifically in the protein.

The aim is to be able to click-activate protein function and to establish protein-protein dimerization irreversibly. Internalization of receptors and dimerization dependent signaling is investigated.

The interface in receptor dimerization allows the azide and alkyne to be incorporated at proximate residues which are in direct contact during dimerization.

Protease Specificity and Function

CECB has developed a range of technologies to monitor and determine protease specificity and inhibition.

Protease research serves to investigate the physiological significance of the proteases and to determine the when, the where and the why of proteolytic activity.

A unique combinatorial chemistry toolbox for the investigation of enzymes has been developed at the Center for Evolutionary Chemical Biology and is employed during investigation of the mamalian proteases.

This toolbox is employed to investigate the specificity of proteases in a variety of live cells and tissue. Genes encoding for proteases involved in plant germination, arthritis and in programmed cell death are cloned and expressed.

CECB has established a platform with bioinformatics, modelling, molecular biology, protein expression, FRET, enzymology, combinatorial chemistry, synthesis, high-throughput screening, in vivo substrates and microscopy.

These tools are used to identify the genes, express the proteases, analyze the protease activity profile, locate and study the proteases in vivo in a time- and tissue-specific manner and identify the natural target for the protease.

Artificially induced arthritis may be monitored using specific in vivo probes that can be visualized through intact tissue. Fluorescence increases selectively at the site of inflammation.

Substrate specificity of a target protease over that of related proteases is determined through screening of combinatorial libraries.

The most selective substrates are converted into in vivo probes for live monitoring of disease progression.