Michael W. Göbel

Nitrotetrazolate-2N-oxides and the Strategy of N-Oxide Introduction

The first anionic tetrazole-2N-oxide has been prepared by mild aqueous oxidation of easily prepared 5-nitrotetrazole with commercially available oxone in high yields. The result of protonating 5-nitrotetrazolate-2N-oxide has been identified as a hydroxytetrazole, and the nitrogen-rich salts including ammonium, hydroxylammonium, guanidinium, aminoguanidinum, diaminoguanidinium, and triaminoguanidinium have been prepared and characterized. When compared to the nitrogen-rich salts of nitrotetrazole, the nitrogen-rich salts of nitrotetrazole-2N-oxide show superior energetic performance as calculated by the EXPLO5 computer code, using heats of formation calculated using the CBS-4M level of quantum mechanical theory. The impact, friction, and electrical spark sensitivities of the nitrogen-rich nitrotetrazolate-2N-oxides were measured and cover the whole range from sensitive to insensitive energetic materials.

Molecular mechanism of inhibition of the human protein complex Hsp90-Cdc37, a kinome chaperone-cochaperone, by triterpene celastrol.

Inhibition of the ATPase activity of the kinome chaperone Hsp90 (kinome = set of all protein kinases in an organism) has long been known as molecular target for anticancer therapy. Cdc37, a cochaperone of Hsp90 in mammalian cells, targets protein kinases and is upregulated in various cancers. The protein–protein complex forms with a KD value of 1.2 mm and is considered to mediate carcinogenesis by stabilizing a variety of different oncogenic kinases in malignant cells. However, Cdc37 as well as Hsp90 can also act alone, at least in yeast. Low-molecular-weight molecules that interfere with Cdc37 or Hsp90 or disrupt the Hsp90–Cdc37 complex have recently been proposed as a new class of anticancer agents. 4] Gene-based expression studies have identified the triterpene celastrol, which represents a new class of non ATP-competitive inhibitors of Hsp90. Immunoprecipitation in a pancreatic cell line and docking experiments suggested that celastrol exerts its antiproliferative activity by binding to the N-terminal domain of Hsp90 (Hsp90N), thereby disrupting the complex between Hsp90N and Cdc37. In vivo, celastrol showed significant inhibition of tumor growth in nude mice with prostate or pancreatic cancer. 7] Herein, we describe in detail how celastrol disrupts the human Hsp90–Cdc37 complex. H,N-HSQC NMR experiments detect ligand binding to a target through chemical-shift perturbations (CSPs). We investigated the effects of celastrol on the complex of H,N-labeled Hsp90N (23 kDa) with unlabeled full-length Cdc37 (45 kDa) (Figure 1A). As a result of the increased transverse relaxation rates of the protein in complex (approx. 70 kDa), the HSQC spectrum of

A peptide template as an allosteric supramolecular catalyst for the cleavage of phosphate esters

The heptapeptide H-Iva-Api-Iva-ATANP-Iva-Api-Iva-NHCH3 (P1a), where Iva is (S)-isovaline, Api is 4-amino-4-carboxypiperidine, and ATANP is (S)-2-amino-3-[1-(1,4,7-triazacyclononane)]propanoic acid, has been synthesized. Its conformation in aqueous solution is essentially that of a 310-helix. By connecting three copies of P1a to a functionalized Tris(2-aminoethyl)amine (Tren) platform a new peptide template, [T(P1)3], was obtained. This molecule is able to bind up to four metal ions (CuII or ZnII): one in the Tren subsite and three in the azacyclononane subunits. The binding of the metals to the Tren platform induces a change from an open to a closed conformation in which the three short, helical peptides are aligned in a parallel manner with the azacyclonane units pointing inward within the pseudocavity they define. T(P1)3 shows a peculiar behavior in the transphosphorylation of phosphate esters; the tetrazinc complex is a catalyst of the cleavage of 2-hydroxypropyl-p-nitrophenyl phosphate (HPNP), whereas the free ligand is a catalyst of the cleavage of an oligomeric RNA sequence with selectivity for pyrimidine bases. In the case of HPNP, ZnII acts as a positive allosteric effector by enhancing the catalytic efficiency of the system. In the case of the polyanionic RNA substrate, ZnII switches off the activity, thus behaving as a negative allosteric regulator. It is suggested that the opposite behavior of the catalyst induced by ZnII is associated with the change of conformation of the Tren platform, and consequently of the relative spatial disposition of the three linked peptides, that occurs after binding of the metal ion.

Scaffold Hopping by “Fuzzy” Pharmacophores and its Application to RNA Targets

Ideally, a novel lead structure represents a different chemotype from known ligands of a given target macromolecule. Virtual screening techniques and molecular de novo design are able to perform “scaffold-hopping”, and can thus be used as idea generators for medicinal chemistry. The task is to find isofunctional bioactive molecules with different backbone architectures. Here, we present the successful application of our “fuzzy” pharmacophore method LIQUID (“ligand-based quantification of interaction distributions”) to finding novel RNA ligand scaffolds and new inhibitors of in vitro protein expression. LIQUID is akin to our previously introduced SQUID approach. Both techniques model pharmacophoric features as Gaussian densities. While SQUID operates with univariate distributions, LIQUID employs trivariate models. In a retrospective virtual-screening study, we first compared LIQUID and SQUID, which both work on 3D pharmacophores, with the corresponding 2D CATS approach. All three methods encode pharmacophoric information as a correlation vector; this allows for rapid alignment-free similarity searching (by pair-wise Euclidian distance) in large compound libraries. We assessed their ability to retrieve known ligands of six drug targets from a collection of drugs and lead compounds compiled from recent literature. Overall, the CATS 2D method outperformed the 3D methods in this study, and SQUID retrieved slightly more hits than LIQUID (Table 1). This outcome is not entirely surprising, as we employed only a single calculated conformation for each of the molecules, and owing to the fact that the trivariate pharmacophore points in the LIQUID models are more restrictive than their univariate counterparts in SQUID. Notably, within the error margins, all methods performed comparably and clearly demonstrated their ability to retrieve active species from a collection of drug-like compounds. As all targets employed for this preliminary retrospective assessment were proteins, we checked the scaffold-hopping abilities of SQUID and LIQUID by using an RNA target, namely the trans-activation response element (TAR) of human immunodeficiency virus (HIV) 1. AIDS is caused by infection with HIV, which belongs to the class of retroviruses. This viral class is able to transpose its genome and use the host’s replication machinery for proliferation. Regulatory viral units have been selected as targets for blocking viral replication. Specific interaction of TAR RNA with the Tat protein is essential for virus Table 1. Average enrichment of active species expressed as average enrichment factor <ef>=hitsfound/hitsexpected ( SD), yielded by retrospective virtual screening.

Molecular Structure of Hydrazoic Acid with Hydrogen-Bonded Tetramers in Nearly Planar Layers

Hydrazoic acid (HN(3))--potentially explosive, highly toxic, and very hygroscopic--is the simplest covalent azide and contains 97.7 wt % nitrogen. Although its molecular structure was established decades ago, its crystal structure has now been solved by X-ray diffraction for the first time. Molecules of HN(3) are connected to each other by hydrogen bonds in nearly planar layers parallel to (001) with stacking sequence A, B, ... The layer distance, at 2.950(1) Å, is shorter than that in 2H-graphite [3.355(2) Å]. The hydrogen bonds N-H···N are of great interest, since the azido group consists of three homonuclear atoms with identical electronegativity, but different formal charges. These hydrogen bonds are bifurcated into moderate ones with ≈2.0 Å and into weak ones with ≈2.6 Å. The moderate ones build up tetramers (HN(3))(4) in a nearly planar net of eight-membered rings. To the best of our knowledge, such a network of tetramers of a simple molecule is unique.

New Inhibitors of the Tat–TAR RNA Interaction Found with a “Fuzzy” Pharmacophore Model

TAR RNA is a potential target for AIDS therapy. Ligand‐based virtual screening was performed to retrieve novel scaffolds for RNA‐binding molecules capable of inhibiting the Tat–TAR interaction, which is essential for HIV replication. We used a “fuzzy” pharmacophore approach (SQUID) and an alignment‐free pharmacophore method (CATS3D) to carry out virtual screening of a vendor database of small molecules and to perform “scaffold‐hopping”. A small subset of 19 candidate molecules were experimentally tested for TAR RNA binding in a fluorescence resonance energy transfer (FRET) assay. Both methods retrieved molecules that exhibited activities comparable to those of the reference molecules acetylpromazine and chlorpromazine, with the best molecule showing ten times better binding behavior (IC50=46 μM). The hits had molecular scaffolds different from those of the reference molecules.

Enantioselective Synthesis of (+)-Estrone Exploiting a Hydrogen Bond-Promoted Diels−Alder Reaction

Starting from Dane’s diene and methylcyclopentenedione, (+)-estrone is synthesized along the Quinkert−Dane route in 24% total yield. The key step is an enantioselective Diels−Alder reaction promoted by an amidinium catalyst as efficiently as by a traditional Ti-TADDOLate Lewis acid.

Targeting the HIV Trans-Activation Responsive Region—Approaches Towards RNA-Binding Drugs

RNA±protein interactions are essential for many biological processes such as translation, RNA splicing, and transcription. For a long time, RNA was regarded mainly as a passive system for information delivery from DNA to proteins. Today it is known that the catalytically active part of the ribosome is its RNA component. A considerable number of antibiotics bind to the ribosomal RNA and thus block protein synthesis in microorganisms. This finding suggests that RNA in general is a potential target for small molecules that could alter its biological function. In the case of ribosomal RNA, however, the similarity between the human and procaryotic structures causes an intrinsic selectivity problem responsible for the well-known toxicity of aminoglycoside antibiotics. One strategy towards developing less toxic RNA-binding antiinfectives is to target sequences specific for bacterial or viral replication. The trans-activation responsive region (TAR) of HIV-1, for example, is essential for viral gene expression but absent in uninfected human cells. It was recognized early on that specific ligands for TAR have the potential to inhibit HIV-1 selectively. During the past decade, numerous methods of structure determination and techniques for drug discovery have been applied to TAR. Most recently, regulatory elements have been identified in the untranslated regions of many mRNAs. This discovery opens up fascinating opportunities for future drug development. Lessons learnt from well-characterized model systems such as TAR will be decisive for the successful treatment of these challenging problems.

The concept of template-based de novo design from drug-derived molecular fragments and its application to TAR RNA

Principles of fragment-based molecular design are presented and discussed in the context of de novo drug design. The underlying idea is to dissect known drug molecules in fragments by straightforward pseudo-retro-synthesis. The resulting building blocks are then used for automated assembly of new molecules. A particular question has been whether this approach is actually able to perform scaffold-hopping. A prospective case study illustrates the usefulness of fragment-based de novo design for finding new scaffolds. We were able to identify a novel ligand disrupting the interaction between the Tat peptide and TAR RNA, which is part of the human immunodeficiency virus (HIV-1) mRNA. Using a single template structure (acetylpromazine) as reference molecule and a topological pharmacophore descriptor (CATS), new chemotypes were automatically generated by our de novo design software Flux. Flux features an evolutionary algorithm for fragment-based compound assembly and optimization. Pharmacophore superimposition and docking into the target RNA suggest perfect matching between the template molecule and the designed compound. Chemical synthesis was straightforward, and bioactivity of the designed molecule was confirmed in a FRET assay. This study demonstrates the practicability of de novo design to generating RNA ligands containing novel molecular scaffolds.

Laser-Induced Liquid Bead Ion Desorption Mass Spectrometry: An Approach to Precisely Monitor the Oligomerization of the β-Amyloid Peptide

In the present work, the recently developed laser-induced liquid bead ion desorption mass spectrometry (LILBID MS) is applied as a novel technique to study Aβ oligomerization, thought to be crucial in Alzheimers disease (AD). The characterization of the earliest nucleation events of this peptide necessitates the application of several techniques to bridge the gap between small oligomers and large fibrils. We precisely monitored in time the transformation of monomeric Aβ (1-42) into oligomeric Aβ(n) (n < 20) and its dependence on concentration and agitation. The distribution shows signs of the hexamer being crucial in the assembly process. The intensity of the monomer decreases in time with a time constant of about 9 h. After a lag time of around 10 h, a phase transition occurred in which the total ion current of the oligomers goes to nearly zero. In this late stage of aggregation, protofibrils are formed and mass spectrometry is no longer sensitive. Here fluorescence correlation spectroscopy (FCS) and transmission electron microscopy (TEM) are complementary tools for detection and size characterization of these large species. We also utilized the oligomers of Aβ (1-42) as a model of the corresponding in vivo process to screen the efficacy and specificity of small molecule inhibitors of oligomerization. The LILBID results are in excellent agreement with condensed phase behavior determined in other studies. Our data identified LILBID MS as a powerful technique that will advance the understanding of peptide oligomerization in neurodegenerative diseases and represents a powerful tool for the identification of small oligomerization inhibitors.