Ute Scheffer

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.

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.

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.

Fragment based search for small molecule inhibitors of HIV-1 Tat-TAR.

Basic molecular building blocks such as benzene rings, amidines, guanidines, and amino groups have been combined in a systematic way to generate ligand candidates for HIV-1 TAR RNA. Ranking of the resulting compounds was achieved in a fluorimetric Tat-TAR competition assay. Although simple molecules such as phenylguanidine are inactive, few iteration steps led to a set of ligands with IC50 values ranging from 40 to 150 μM. 1,7-Diaminoisoquinoline 17 and 2,4,6-triaminoquinazoline 22 have been further characterized by NMR titrations with TAR RNA. Compound 22 is bound to TAR at two high affinity sites and shows slow exchange between the free ligand and the RNA complex. These results encourage investigations of dimeric ligands built from two copies of compound 22 or related heterocycles.

Cleavage of Phosphodiesters and of DNA by a Bis(guanidinium)naphthol Acting as a Metal-Free Anion Receptor

Phosphoric acid diesters form anions at neutral pH. As a result of charge repulsion they are notoriously resistant to hydrolysis. Nucleophilic attack, however, can be promoted by different types of electrophilic catalysts that bind to the anions and reduce their negative charge density. Although in most cases phosphodiester‐cleaving enzymes and synthetic catalysts rely on Lewis acidic metal ions, some exploit the guanidinium residues of arginine as metal‐free electrophiles. Here we report that a combination of two guanidines and a hydroxy group yields highly reactive receptor molecules that can attack a broad range of phosphodiester substrates by nucleophilic displacement at phosphorus in a single‐turnover mode. Some stable O‐phosphates were isolated and characterized further by NMR spectroscopy. The bis(guanidinium)naphthols also cleave plasmid DNA, presumably by a transphosphorylation mechanism.

Binding Sites of the Viral RNA Element TAR and of TAR Mutants for Various Peptide Ligands, Probed with LILBID: A New Laser Mass Spectrometry

A new laser-based mass spectrometry method, called laser induced liquid bead ion desorption (LILBID), was applied to investigate RNA:ligand interactions. As model system the HIV Tat:TAR transactivation complex and its binding behavior were analyzed. TARwt of HIV Type 1 and Type 2 and different artificial mutants were compared regarding their binding to Tat and different peptide ligands. Specific and nonspecific association to TAR was deduced, with the bulge being the well known specific binding site of TAR. In the case of triple arginine (RRR) as a nonspecific ligand, multiple electrostatic binding to TAR was found at higher concentration of RRR. This contrasted with the formation of only ternary complexes in competitive binding studies with TAR, Tat, and potential inhibitors. The fact that the stoichiometries of the complexes can be determined is a major advantage of MS methods over the widely applied fluorimetric methods. A quantitative evaluation of the spectra by a numeric model for ternary complex formation allows conclusions about the role and strength of the binding sites of the RNAs, the specificity and affinity of different ligands, the determination of apparent IC50 and KD values, and a comparison of the binding efficiencies of potential inhibitors.

Tripeptides from Synthetic Amino Acids Block the Tat–TAR Association and Slow Down HIV Spread in Cell Cultures

Non‐natural amino acids with aromatic or heteroaromatic side chains were incorporated into tripeptides of the general structure Arg‐X‐Arg and tested as ligands of the HIV RNA element TAR. Some of these compounds could compete efficiently with the association of TAR and Tat and downregulated a TAR‐controlled reporter gene in HeLa cells. Peptide 7, which contains a 2‐pyrimidinyl‐alkyl chain, also inhibited the spread of HIV‐1 in cell cultures. NMR studies of 7 bound to HIV‐2‐TAR gave evidence for contacts in the bulge region.

Detecting Specific Ligand Binding to Nucleic Acids: A Test for Ultrasoft Laser Mass Spectrometry

Results from a new laser based mass spectrometry method termed LILBID (laser induced liquid bead ion desorption) are presented, which demonstrate the potential for the soft mass analysis of nucleic acids and their specific complexes in solution. On-demand micro droplets, injected into vacuum, are irradiated by intense mid IR-laser pulses. A subsequent explosive phase transition leads to the emission of solvated biomolecular ions into vacuum, which are then mass analyzed by time of flight mass spectrometry. The method is very soft and allows the detection of specific noncovalent complexes. Furthermore it is very tolerant against various buffers and detergents and the charge states of the observed ions reflect qualitatively the charge state in solution. With a required volume of analyte solution in the micro litre range at micromolar concentration, the total consumption of analyte is in the picomolar region. Regarding a single droplet only it is even three orders of magnitude lower, underlining the very high sensitivity of this method. Here we present first examples of the LILBID mass analysis of single stranded DNA (up to a 77mer) and of specific DNA duplices, which show nearly no dissociation under ultrasoft desorption conditions. In case of model hairpin DNA, forming duplices, a sequence specific binding of Dervan-type polyamides into the minor groove of the DNA was observed, and in case of a Tat-TAR model complex strong indications for a specific binding of the Tat protein into the bulge of the wt-RNA was found. Mass spectra of the ribosome of thermos thermophilus give evidence of the applicability of LILBID-MS to analyze large RNA/protein complexes even in the MDa region.

A Bis(guanidinium)alcohol Attached to a Hairpin Polyamide: Synthesis, DNA Binding, and Plasmid Cleavage.

Bis(guanidinium)alcohols have been designed to react with phosphodiester substrates in a fast transphosphorylation step, a quasi‐intramolecular process taking place in contact ion pairs. Here the attachment of such compounds to Dervan‐type hairpin polyamides is described. The resulting conjugate 1 binds to AT‐rich DNA duplexes with affinity similar to that of the parent polyamide as shown by UV melting experiments and CD titrations. Conjugate 1 nicks plasmid DNA at concentrations ranging from micromolar to high nanomolar.