Alexander Bujotzek

DNA-programmed spatial screening of carbohydrate–lectin interactions

A wide range of multivalent scaffolds was assembled by using only five different PNA oligomers and various DNA templates. The flexibility of the PNA–DNA duplexes could be increased by introducing nick-sites and partially unpaired regions, as confirmed by MD simulations. The self-organized glyco-assemblies were used in a spatial screening of accessible carbohydrate binding sites in the Erythrina cristagalli lectin (ECL). This systematic investigation revealed a distance dependence which is in agreement with the crystal structure analysis.

DNA-controlled bivalent presentation of ligands for the estrogen receptor.

The assembly of DNA complexes proceeds according to known rules. Thus, the mutual recognition of DNA conjugates can be used for the precise positioning of functional groups. For example, chromophores, metals, catalytic units, nanoparticles, fluorophores and even proteins have been arranged at well-defined distances by means of DNA hybridization. Until recently, the main interest was focused on issues within materials science as well as on the immobilization of biomolecules. We and others assumed that the ability to position functional units at defined distances could also be used to address biological problems. According to this, DNA may serve as a molecular ruler to determine the distance between binding pockets in biological receptors. Due to self-assembly of the DNA complex the rapid spatial screening of a receptor can be doen with minor synthetic effort. In this approach, the ligand of a biological receptor is covalently attached to an oligonucleotide (Figure 1a). The binding of two or more oligonucleotide–ligand conjugates to a template strand provides bior multivalent DNA–ligand conjugates. The distance between the two biologically active ligands can be readily adjusted by varying of the template strand. Herein we demonstrate, for the first time, the DNAcontrolled presentation of small molecules in the spatial screening of a protein receptor. We demonstrate the advantages conferred by DNA spacers by examining a well-studied nuclear receptor, the estrogen receptor, and by comparison with commonly applied oligoethyleneglycol spacers. The estrogen receptor (ER) is activated by the hormone estradiol and is involved in the regulation of gene expression. It is assumed that the formation of dimers is essential for the natural function of the receptor (Figure 1b). The dimerization constant is in the subnanomolar range, yet it must be considered that ligand binding can influence the dimerization equilibrium. The selective estrogen receptor modulators (SERMs) hexestrol, raloxifene, and 4-hydroxytamoxifene stabilize the receptor dimer and were thus deemed suitable for the spatial screening of the ER. The synthesis of the SERM–oligonucleotide conjugates was achieved by introducing the alkyne-modified uridine building block X during automated DNA synthesis (Scheme 1). The SERMs were equipped with azido functions to enable the covalent attachment to the oligonucleotides ODN-X by the Cu-catalyzed 1,3-dipolar cycloaddition. The resulting conjugates ODN-XR were obtained in 30–70% yield. The affinity of the oligonucleotide–SERM complexes to the estrogen receptor (ER-a) was assessed by means of the HitHunter assay. The conjugation of hexestrol (Hex) with an oligonucleotide diminished the affinity by several orders of magnitude (Figure 2). This result appears plausible because in the structure of the ER in complex with agonists such as hexestrol the binding pocket is nearly closed (Figure S33 in the Supporting Information). In contrast, the estrogen analogues raloxifene (Ral) and 4-hydroxytamoxifene (Tam) Figure 1. a) Bivalent presentation of estrogen receptor ligands (L) on ternary DNA complexes. b) Crystal structure of the ligand binding domain of the estrogen receptor (PDB ID: 1ERR) in complex with raloxifene (orange). The nitrogen atoms of raloxifene (blue) are 35 apart.

Nonsteroidal Bivalent Estrogen Ligands - An Application of the Bivalent Concept to the Estrogen Receptor

The estrogen receptor (ER) is a hormone-regulated transcription factor that binds, as a dimer, to estrogens and to specific DNA sequences. To explore at a fundamental level the geometric and topological features of bivalent-ligand binding to the ER dimer, dimeric ER crystal structures were used to rationally design nonsteroidal bivalent estrogen ligands. Guided by this structure-based ligand design, we prepared two series of bivalent ligands (agonists and antagonists) tethered by flexible spacers of varying lengths (7-47 Å) and evaluated their ER-binding affinities for the two ER subtypes and their biological activities in cell lines. Bivalent ligands based on the agonist diethylstilbestrol (DES) proved to be poor candidates, but bivalent ligands based on the antagonist hydroxytamoxifen (OHT) were well suited for intensive study. Binding affinities of the OHT-based bivalent ligands were related to spacer length in a distinctive fashion, reaching two maximum values at 14 and 29 Å in both ER subtypes. These results demonstrate that the bivalent concept can operate in determining ER-ligand binding affinity and suggest that two distinct modes operate for the binding of bivalent estrogen ligands to the ER dimers, an intermolecular as well as an intramolecular mode. Our insights, particularly the possibility of intramolecular bivalent binding on a single ER monomer, may provide an alternative strategy for preparing more selective and active ER antagonists for endocrine therapy of breast cancer.

Conformational analysis of bivalent estrogen receptor ligands: from intramolecular to intermolecular binding.

The estrogen receptor binding affinities of bivalent raloxifene ligands tethered by flexible spacers of different lengths have been evaluated in vitro. Two bivalent binding modes, intra‐ and intermolecular, were hypothesized to explain their different binding properties. The binding affinities of these bivalent ligands in an aqueous environment are influenced by their conformations, which can be determined by 2D NMR and UV spectral methods. Moreover, computer modeling and simulations were performed to explain the binding modes of these bivalent ligands and to estimate the conformational entropy difference between their unbound and bound states. It was found that bivalent ligands tethered by long spacers had weaker binding affinities because of the shielding of the binding moieties that results from their folded conformations; those tethered by short spacers had stronger affinities because they exposed their ligands to the receptor.

EFFICIENT SIMULATION OF LIGAND–RECEPTOR BINDING PROCESSES USING THE CONFORMATION DYNAMICS APPROACH

The understanding of biological ligand-receptor binding processes is relevant for a variety of research topics and assists the rational design of novel drug molecules. Computer simulation can help to advance this understanding, but, due to the high dimensionality of according systems, suffers from the severe computational cost. Based on the framework provided by conformation dynamics and transition state theory, a novel heuristic approach of simulating ligand-receptor binding processes is introduced, which is not dependent on calculating lengthy molecular dynamics trajectories. First, the relevant portion of conformational space is partitioned with meshless methods. Then, each region is sampled separately, using hybrid Monte Carlo. Finally, the dynamical binding process is reconstructed from the static overlaps between the partial densities obtained in the sampling step. The method characterizes the metastable steps of the binding process and can yield the corresponding transition probabilities.

Towards a rational spacer design for bivalent inhibition of estrogen receptor

Estrogen receptors are known drug targets that have been linked to several kinds of cancer. The structure of the estrogen receptor ligand binding domain is available and reveals a homodimeric layout. In order to improve the binding affinity of known estrogen receptor inhibitors, bivalent compounds have been developed that consist of two individual ligands linked by flexible tethers serving as spacers. So far, binding affinities of the bivalent compounds do not surpass their monovalent counterparts. In this article, we focus our attention on the molecular spacers that are used to connect the individual ligands to form bivalent compounds, and describe their thermodynamic contribution during the ligand binding process. We use computational methods to predict structural and entropic parameters of different spacer structures. We find that flexible spacers introduce a number of effects that may interfere with ligand binding and possibly can be connected to the low binding affinities that have been reported in binding assays. Based on these findings, we try to provide guidelines for the design of novel molecular spacers.

A Meshless Discretization Method for Markov State Models Applied to Explicit Water Peptide Folding Simulations

Markov State Models (MSMs) are widely used to represent molecular conformational changes as jump-like transitions between subsets of the conformational state space. However, the simulation of peptide folding in explicit water is usually said to be unsuitable for the MSM framework. In this article, we summarize the theoretical background of MSMs and indicate that explicit water simulations do not contradict these principles. The algorithmic framework of a meshless conformational space discretization is applied to an explicit water system and the sampling results are compared to a long-term molecular dynamics trajectory. The meshless discretization approach is based on spectral clustering of stochastic matrices (MSMs) and allows for a parallelization of MD simulations. In our example of Trialanine we were able to compute the same distribution of a long term simulation in less computing time.

ZIBgridfree: Efficient Conformational Analysis by Partition-of-Unity Coupling

Obtaining a sufficient sampling of conformational space is a common problem in molecular simulation. We present the implementation of an umbrella-like adaptive sampling approach based on function-based meshless discretization of conformational space that is compatible with state of the art molecular dynamics code and that integrates an eigenvector-based clustering approach for conformational analysis and the computation of inter-conformational transition rates. The approach is applied to three example systems, namely

Peptide–polymer ligands for a tandem WW-domain, an adaptive multivalent protein–protein interaction: lessons on the thermodynamic fitness of flexible ligands

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