Rene Meier

Structure-activity studies on suramin analogues as inhibitors of NAD+-dependent histone deacetylases (sirtuins).

Suramin is a symmetric polyanionic naphthylurea originally used for the treatment of trypanosomiasis and onchocerciasis. Suramin and diverse analogues exhibit a broad range of biological actions in vitro and in vivo, including, among others, antiproliferative and antiviral activity. Suramin derivatives usually target purinergic binding sites. Class III histone deacetylases (sirtuins) are amidohydrolases that require nicotinamide adenine dinucleotide (NAD+) as a cofactor for their catalytic mechanism. Deacetylation of the target proteins leads to a change in conformation and alters the activity of the proteins in question. Suramin was reported to inhibit human sirtuin 1 (SIRT1). We tested a diverse set of suramin analogues to elucidate the inhibition of the NAD+‐dependent histone deacetylases SIRT1 and SIRT2 and discovered selective inhibitors of human sirtuins with potency in the two‐digit nanomolar range. In addition, the structural requirements for the binding of suramin derivatives to sirtuins were investigated by molecular docking. The recently published X‐ray crystal structure of human SIRT5 in complex with suramin and the human SIRT2 structure were used to analyze the interaction mode of the novel suramin derivatives.

Structure–Activity Studies on Splitomicin Derivatives as Sirtuin Inhibitors and Computational Prediction of Binding Mode

NAD (+)-dependent histone deacetylases (sirtuins) are enzymes that cleave acetyl groups from lysines in histones and other proteins. Potent selective sirtuin inhibitors are interesting tools for the investigation of the biological functions of those enzymes and may be future drugs for the treatment of cancer. Splitomicin was among the first two inhibitors that were discovered for yeast sirtuins but showed rather weak inhibition on human enzymes. We present detailed structure-activity relationships on splitomicin derivatives and their inhibition of recombinant Sirt2. To rationalize our experimental results, ligand docking followed by molecular mechanics Poisson-Boltzmann/surface area (MM-PBSA) calculations were carried out. These analyses suggested a molecular basis for the interaction of the beta-phenylsplitomicins with human Sirt2. Protein-based virtual screening resulted in the identification of a novel Sirt2 inhibitor chemotype. Selected inhibitors showed antiproliferative properties and tubulin hyperacetylation in MCF7 breast cancer cells and are promising candidates for further optimization as potential anticancer drugs.

Virtual screening and biological characterization of novel histone arginine methyltransferase PRMT1 inhibitors.

Lysine and arginine methyltransferases participate in the posttranslational modification of histones and regulate key cellular functions. Protein arginine methyltransferase 1 (PRMT1) has been identified as an essential component of mixed lineage leukemia (MLL) oncogenic complexes, revealing its potential as a novel therapeutic target in human cancer. The first potent arginine methyltransferase inhibitors were recently discovered by random‐ and target‐based screening approaches. Herein we report virtual and biological screening for novel inhibitors of PRMT1. Structure‐based virtual screening (VS) of the Chembridge database composed of 328 000 molecules was performed with a combination of ligand‐ and target‐based in silico approaches. Nine inhibitors were identified from the top‐scored docking solutions; these were experimentally tested using human PRMT1 and an antibody‐based assay with a time‐resolved fluorescence readout. Among several aromatic amines, an aliphatic amine and an amide were also found to be active in the micromolar range.

ParaDockS: a framework for molecular docking with population-based metaheuristics.

Molecular docking is a simulation technique that aims to predict the binding pose between a ligand and a receptor. The resulting multidimensional continuous optimization problem is practically unsolvable in an exact way. One possible approach is the combination of an optimization algorithm and an objective function that describes the interaction. The software ParaDockS is designed to hold different optimization algorithms and objective functions. At the current stage, an adapted particle-swarm optimizer (PSO) is implemented. Available objective functions are (i) the empirical objective function p-Score and (ii) an adapted version of the knowledge-based potential PMF04. We tested the docking accuracy in terms of reproducing known crystal structures from the PDBbind core set. For 73% of the test instances the native binding mode was found with an rmsd below 2 A. The virtual screening efficiency was tested with a subset of 13 targets and the respective ligands and decoys from the directory of useful decoys (DUD). ParaDockS with PMF04 shows a superior early enrichment. The here presented approach can be employed for molecular docking experiments and virtual screenings of large compound libraries in academia as well as in industrial research and development. The performance in terms of accuracy and enrichment is close to the results of commercial software solutions.

Thiobarbiturates as Sirtuin Inhibitors: Virtual Screening, Free‐Energy Calculations, and Biological Testing

NAD+‐dependent histone deacetylases (sirtuins) are enzymes that cleave acetyl groups from lysine residues in histones and other proteins. Potent selective sirtuin inhibitors are interesting tools for the investigation of the biological functions of these enzymes and may be future drugs for the treatment of cancer or neurodegenerative diseases. Herein we present the results from a protein‐based virtual screen of a commercial database with subsequent biological testing of the most promising compounds. The combination of docking and in vitro experimental testing resulted in the identification of novel sirtuin inhibitors with thiobarbiturate structure. To rationalize the experimental results, free‐energy calculations were carried out by molecular mechanics Poisson–Boltzmann/surface area (MM‐PBSA) calculations. A significant correlation between calculated binding free energies and measured Sirt2 inhibitory activities was observed. The analyses suggested a molecular basis for the interaction of the identified thiobarbiturate derivatives with human Sirt2. Based on the docking and MM‐PBSA calculations we synthesized and tested five further thiobarbiturates. The MM‐PBSA method correctly predicted the activity of the novel thiobarbiturates. The identified compounds will be used to further explore the therapeutic potential of sirtuin inhibitors.

Proteolytic activation of prochemerin by kallikrein 7 breaks an ionic linkage and results in C-terminal rearrangement

The excessive accumulation of adipose tissue in obesity is associated with multiple inflammatory dermatological diseases. Chemerin, a chemoattractant adipokine, dependent on proteolytical activation, is highly expressed in skin. Different proteases have been reported to activate prochemerin, but none is inherently expressed in human skin. In the present study, we identified a tissue-specific protease and investigated the underlying mechanism of activation at the molecular level. We characterized human KLK7 (kallikrein 7) as a prochemerin processing protease in vitro converting prochemerin into active chemerinF(156). The activating truncation by the protease might trigger a structural rearrangement leading to an increased affinity of chemerin to CMKLR1 (chemokine-like receptor 1). Molecular modelling and experimental data suggest an underlying ionic interaction in prochemerin C-terminal domains. These findings provide a general molecular basis for the necessity of C-terminal processing of prochemerin. Moreover, immunohistochemistry was used to investigate prochemerin, KLK7 and the recently identified KLK7 inhibitor vaspin expression in human skin biopsies, and distinct co-localization in psoriatic biopsies was observed. On the basis of these results, it is hypothesized that KLK7 activity may contribute to the development of psoriatic lesions as a consequence of excessive chemerin activation and impaired protease activity regulation by vaspin. Therefore this interaction represents an interesting target for psoriasis therapy and treatment of other obesity-related diseases.

Unwinding of the C‐Terminal Residues of Neuropeptide Y is critical for Y2 Receptor Binding and Activation

Despite recent breakthroughs in the structural characterization of G-protein-coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double-cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G-protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specific isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T(32) to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.

Engineered bacterial polyester hydrolases efficiently degrade polyethylene terephthalate due to relieved product inhibition.

Recent studies on the enzymatic degradation of synthetic polyesters have shown the potential of polyester hydrolases from thermophilic actinomycetes for modifying or degrading polyethylene terephthalate (PET). TfCut2 from Thermobifida fusca KW3 and LC‐cutinase (LCC) isolated from a compost metagenome are remarkably active polyester hydrolases with high sequence and structural similarity. Both enzymes exhibit an exposed active site in a substrate binding groove located at the protein surface. By exchanging selected amino acid residues of TfCut2 involved in substrate binding with those present in LCC, enzyme variants with increased PET hydrolytic activity at 65°C were obtained. The highest activity in hydrolyzing PET films and fibers were detected with the single variant G62A and the double variant G62A/I213S. Both variants caused a weight loss of PET films of more than 42% after 50 h of hydrolysis, corresponding to a 2.7‐fold increase compared to the wild type enzyme. Kinetic analysis based on the released PET hydrolysis products confirmed the superior hydrolytic activity of G62A with a fourfold higher hydrolysis rate constant and a 1.5‐fold lower substrate binding constant than those of the wild type enzyme. Mono‐(2‐hydroxyethyl) terephthalate is a strong inhibitor of TfCut2. A determination of the Rosetta binding energy suggested a reduced interaction of G62A with 2PET, a dimer of the PET monomer ethylene terephthalate. Indeed, G62A revealed a 5.5‐fold lower binding constant to the inhibitor than the wild type enzyme indicating that its increased PET hydrolysis activity is the result of a relieved product inhibition by mono‐(2‐hydroxyethyl) terephthalate. Biotechnol. Bioeng. 2016;113: 1658–1665.

Selective mode of action of guanidine-containing non-peptides at human NPFF receptors.

The binding pocket of both NPFF receptors was investigated, focusing on subtype-selective behavior. By use of four nonpeptidic compounds and the peptide mimetics RF9 and BIBP3226, agonistic and antagonistic properties were characterized. A set of Ala receptor mutants was generated. The binding pocket was narrowed down to the upper part of transmembrane helices V, VI, VII and the extracellular loop 2. Positions 5.27 and 6.59 have been shown to have a strong impact on receptor activation and were suggested to form an acidic, negatively charged binding pocket in both NPFF receptor subtypes. Additionally, position 7.35 was identified to play an important role in functional selectivity. According to docking experiments, the aryl group of AC-216 interacts with position 7.35 in the NPFF(1) but not in the NPFF(2) receptor. These results provide distinct insights into the receptor specific binding pockets, which is necessary for the development of drugs to address the NPFF system.

Influence of the Hinge Region and Its Adjacent Domains on Binding and Signaling Patterns of the Thyrotropin and Follitropin Receptor

Glycoprotein hormone receptors (GPHR) have a large extracellular domain (ECD) divided into the leucine rich repeat (LRR) domain for binding of the glycoprotein hormones and the hinge region (HinR), which connects the LRR domain with the transmembrane domain (TMD). Understanding of the activation mechanism of GPHRs is hindered by the unknown interaction of the ECD with the TMD and the structural changes upon ligand binding responsible for receptor activation. Recently, our group showed that the HinR of the thyrotropin receptor (TSHR) can be replaced by those of the follitropin (FSHR) and lutropin receptor (LHCGR) without effects on surface expression and hTSH signaling. However, differences in binding characteristics for bovine TSH at the various HinRs were obvious. To gain further insights into the interplay between LRR domain, HinR and TMD we generated chimeras between the TSHR and FSHR. Our results obtained by the determination of cell surface expression, ligand binding and G protein activation confirm the similar characteristics of GPHR HinRs but they also demonstrate an involvement of the HinR in ligand selectivity indicated by the observed promiscuity of some chimeras. While the TSHR HinR contributes to specific binding of TSH and its variants, no such contribution is observed for FSH and its analog TR4401 at the HinR of the FSHR. Furthermore, the charge distribution at the poorly characterized LRR domain/HinR transition affected ligand binding and signaling even though this area is not in direct contact with the ligand. In addition our results also demonstrate the importance of the TMD/HinR interface. Especially the combination of the TSHR HinR with the FSHR-TMD resulted in a loss of cell surface expression of the respective chimeras. In conclusion, the HinRs of GPHRs do not only share similar characteristics but also behave as ligand specific structural and functional entities.