2,2'-Bithiophene as sensor tag for ligand-protein binding assays based on Förster resonance energy transfer.

Ligand-protein binding assays based on intrinsic protein fluorescence are straightforward, inexpensive methods to study ligand-protein interactions. However, their applicability is limited to ligands that can interfere with protein emission. In this Note, we describe the applicability of 2,2'-bithiophene as a FRET-based sensor tag, that can be incorporated into high-affinity ligands to generate target-specific compounds able to quench protein fluorescence upon binding. The generated ligands were assessed in different assay designs. Considerations to account for possible sources of interference with the assay readout are addressed, besides interpretation of the obtained results.

Conformational selection in the flaviviral NS2B-NS3 protease.

The first x-ray structures of flaviviral proteases defined two conformational states, open and closed, depending on the relative position of NS2B with respect to NS3, a feature that affects the shape of the binding site. The degree of flexibility in the active site was limited to changes in the fold of NS2B rather than NS3 and an induced-fit mechanism was regarded as the main factor for ligand binding. A minor degree of conformational plasticity in NS3 is observed in the two protein chains in the asymmetric unit for the structure of Zika protease with a dipeptide boronate, synthesized in our group. We hypothesize that the NS3 fold has a crucial influence on the shape of the binding site and that a reevaluation of the induced-fit interpretation is warranted. A comparison of flaviviral protease structures identifies conformational dynamics of NS3 and their unexpected role in controlling the depth of the, otherwise shallow, active site. The structural changes of NS3 are mediated by conserved residues and reveal a subpocket, which we denote as subpocket B, extending beyond the catalytic aspartate 75 towards the allosteric binding site, providing a unique connection between the orthosteric and allosteric sites in the protease. The structural evidence supports a molecular recognition based primarily on conformational selection and population shift rather than induced-fit. Besides the implications on protease studies and drug development, this hypothesis provides an interpretation for the alternate binding modes with respect to the catalytic serine, which are observed for recently developed beta-lactam inhibitors incorporating benzyloxyphenylglycine.

Diversity-oriented synthesis of peptide-boronic acids by a versatile building-block approach.

A new strategy for the synthesis of peptide-boronic acids (PBAs) is presented. 20 Fmoc-protected natural amino acids with orthogonal side-chain protection were straightforwardly converted into their corresponding boron analogues in three simple steps. Subsequent immobilisation on commercially available 1-glycerol polystyrene resin and on-resin transformations yielded a diversity of sequences in high purity. The strategy eliminates various synthetic obstacles such as multi-step routes, low yields, and inseparable impurities. The described method comprises great potential to be implemented in automated combinatorial approaches by markedly facilitating the access to a variety of PBAs. The coupling of amino acids or other building blocks with α-aminoboronates allows the creation of hybrid molecules with significant potential in various scientific disciplines, such as medicinal chemistry, structural biology, and materials science.

Virtues of Volatility: A Facile Transesterification Approach to Boronic Acids.

Boronic acids are an increasingly important compound class for many applications, including C-C bond formation reactions, medicinal chemistry, and diagnostics. The deprotection of boronic ester intermediates is frequently a problematic and inefficient step in boronic acid syntheses. We describe an approach that highly facilitates this transformation by leveraging the volatility of methylboronic acid and its diol esters. The method is performed under mild conditions, provides high yields, and eliminates cumbersome and problematic purification steps.

Protein-dynamics of the putative HCV receptor CD81 large extracellular loop.

The human CD81 protein is a likely receptor for the binding of hepatitis C virus (HCV) to hepatocytes and therefore a possible target for novel anti-HCV drugs. The two published X-ray structures of the HCV binding region of CD81 (1G8Q and 1IV5) have, particularly in a substructure that is formed by two helices, a slightly different conformation. The abovementioned substructure is a candidate target region for virtual screening approaches. We present here a molecular dynamics study of the two X-ray structures. Our results indicate that the conformation of the two helical regions in one of the X-ray structures (1G8Q) is affected by crystallographic contacts and most likely does not represent the native state of the protein.

Protonation states of methionine aminopeptidase and their relevance for inhibitor binding and catalytic activity.

We have performed a computational study of different protomeric states of the methionine aminopeptidase active site using a combined quantum-mechanical/molecular mechanical simulation approach. The aim of this study was to clarify the native protonation state of the enzyme, which is needed for the development of novel irreversible inhibitors that can possibly be used as antiangiogenic and antibiotic drugs by virtual screening and other drug design methods. The results of the simulations indicated that two protonation states are possible without disturbing the overall geometry of the active site. We then verified experimentally the presence of the two protonation states by studying the substrate hydrolysis and inhibitor binding reactions at different pH values and come to the conclusion that one of the protomeric states is relevant for inhibitor binding, whereas the other is relevant for substrate hydrolysis. This result has implications for the development of other inhibitors of this class of enzymes and adds a new perspective to the pharmacological properties of the antiangiogenic drug fumagillin, which is an irreversible inhibitor of the human methionine aminopeptidase type II.

Understanding the selectivity of fumagillin for the methionine aminopeptidase type II.

The aim of this study is to explain the selectivity of the antiangiogenic drug fumagillin for the eukaryotic enzyme methionine aminopeptidase type II (MetAP-II, EC 3.4.11.18) over the structurally very similar MetAP-I. A homology model for the human MetAP-I is constructed and molecular dynamics simulations are performed on this model with and without a docked fumagillin molecule. These simulations are compared with analogous simulations that were performed on the experimentally determined structure of the human MetAP-II enzyme. We observe an increased flexibility of the active site histidine that is covalently modified by fumagillin in the MetAP-I enzyme. The MetAP-I active site residues, particularly the fumagillin-binding histidine, have a lower probability to be in a conformation that is prone to react with the drug than their MetAP-II counterparts. This result offers an explanation for the selectivity of fumagillin for the eukaryotic MetAP-II enzyme.

Inhibition of CYP 17, a new strategy for the treatment of prostate cancer.

Androgens are growth factors for approximately 80 percent of all prostate cancers. Suppressing androgen biosynthesis is therefore an important therapeutic strategy in order to inhibit tumor growth. Unfortunately, the drugs currently applied to lower androgen levels only affect testicular androgen production. Since androgens are also synthesized in the adrenal glands, tumor stimulation cannot be blocked completely. A new therapeutic target, CYP 17 (P450 17, 17alpha-hydroxylase-C17, C20 lyase), is likely to improve this situation. CYP 17 is a P450 enzyme and catalyzes the last step of androgen biosynthesis in both testes and adrenals. Inhibition of this enzyme will therefore result in a complete block of androgen production. This paper gives an overview of the current situation in this novel field of drug research and focuses on the development of steroidal and non-steroidal inhibitors of CYP 17.

Computational Methods Facilitate the Assignment of Protein Functions.

From genome data to a successful drug: Many companies are eager to make this journey in the future, though roadmaps are still scarce. We comment on a novel method that promises to make the trip from gene to drug shorter and more effective by predicting protein functions from their three-dimensional structure.