Martin Empting

Triazole bridge: disulfide-bond replacement by ruthenium-catalyzed formation of 1,5-disubstituted 1,2,3-triazoles.

About one fourth of the peptidic macromolecular structures deposited in the protein data base (PDB) contain at least one disulfide bridge. In nature, disulfide bonds are formed in a milieu where oxidizing conditions prevail, for example, on the cell surface or in the extracellular matrix. Many proteins benefit from disulfide contributions to their conformational stability. In particular, the defined tertiary folding of oligopeptides smaller than 30 residues essentially relies on macrocyclization through the cystine motif because of the restricted number of noncovalent intramolecular interactions available. Moreover, formation of the disulfide pattern results in structural rigidity of the peptidic framework, as for example, in the family of cystine knot miniproteins, leading to conformationally constrained scaffolds with extraordinary thermal stability and resistance against proteolytic degradation. Hence, the discovery and development of disulfidebridged peptides suitable for diagnostic and therapeutic applications remains a field of intense research. The in-vitro generation of disulfide bonds in peptides is usually achieved post-synthetically and mediated by DMSO, air oxygen, or other oxidizing agents. Although this reaction step can be achieved under relatively mild conditions in solution, it remains one of the most demanding obstacles towards high-yield peptide synthesis, especially for disulfiderich species in which the controlled regiospecific formation of several disulfide bonds is not trivial to control. In addition, to suppress unwanted intermolecular reactions of the thiol groups of individual peptides, oxidative folding usually has to be conducted in highly diluted solutions. In spite of the use of gluthathione-based redox buffers, polymer-supported oxidation systems, macrocyclization on the solid support and/or orthogonal protecting groups, control over the topology of the disulfide bridges formed is still a challenge. 5] In view of these difficulties and to improve the redox stability of bridged peptides, several routes towards synthetic disulfide surrogates have been developed. Straightforward approaches usually employ thioether, olefin, or alkane-based isosters. However, cystathione bridges require multiple synthetic steps and careful choice of orthogonal protection, and dicarba bridges give cis/trans isomers during ring-closing metathesis (RCM). Only an additional purification step or the subsequent palladium-catalyzed hydrogenation of the unsaturated species to the corresponding alkane leads to a construct with defined configuration. In 2004, Meldal et al. described the utility of copper(I)catalyzed azide–alkyne cycloaddition (CuAAC) for a triazole-based disulfide replacement. Owing to the compelling characteristics of this prototypic “click” reaction, it has been extensively applied in peptide chemistry exploiting the almost perfect orthogonality to side-chain reactivities. The introduction of 1,4-disubstituted 1,2,3-triazoles into peptides has also been used to mimic and rigidify conformations of the amide backbone. Moreover, a variety of examples of CuAAC-based macrocyclizations of peptides in solution and on solid supports has been reported. Using the same azideand alkyne-functionalized buidling blocks, 1,5-disubstituted 1,2,3-triazoles can be generated in the ruthenium(II)-catalyzed variant (RuAAC) of the CuAAC. This reaction expands the range of peptidomimetic structures selectively accessible from the same precursor and having different biological activities governed by the architecture of the incorporated triazole. To our knowledge, 1,5-disubstitiuted 1,2,3-triazoles have not been taken into consideration as disulfide mimics to date. Herein, we report the facile introduction of 1,4and 1,5disubstituted 1,2,3-triazoles into a monocyclic variant of the sunflower trypsin inhibitor-I (SFTI-1[1,14], 1; Figure 1) and show that the macrocyclic peptidomimeticum 2 with the “1,5” substitution pattern retains nearly full biological activity in contrast to the “1,4” variants 3 and 4. The choice of 1 as the model peptide for the investigation of triazole-based disulfide replacements had several reasons. SFTI-1 is a small, though very potent, inhibitor of trypsin. Therefore, the influence of different modes of macrocyclization on the bioactivity of the corresponding synthetic variant can be routinely examined by serine protease inhibition assays. [*] M. Empting, Dr. O. Avrutina, Dr. R. Meusinger, S. Fabritz, M. Reinwarth, Prof. Dr. H. Kolmar Clemens-Sch pf-Institut f r Organische Chemie und Biochemie Technische Universit t Darmstadt Petersenstrasse 22, 64287 Darmstadt (Germany) Fax: (+49)6151-16-5399 E-mail: kolmar@biochemie-tud.de Homepage: http://www.chemie.tu-darmstadt.de/kolmar

Novel Strategies for the Treatment of Pseudomonas aeruginosa Infections

Infections with Pseudomonas aeruginosa have become a concerning threat in hospital-acquired infections and for cystic fibrosis patients. The major problem leading to high mortality lies in the appearance of drug-resistant strains. Therefore, a vast number of approaches to develop novel anti-infectives is currently pursued. These diverse strategies span from killing (new antibiotics) to disarming (antivirulence) the pathogen. Particular emphasis lies on the development of compounds that inhibit biofilms formed in chronic infections to restore susceptibility toward antibiotics. Numerous promising results are summarized in this perspective. Antibiotics with a novel mode of action will be needed to avoid cross resistance against currently used therapeutic agents. Importantly, antivirulence drugs are expected to yield a significantly reduced rate of resistance development. Most developments are still far from the application. It can however be expected that combination therapies, also containing antivirulence agents, will pave the way toward novel treatment options against P. aeruginosa.

Towards click bioconjugations on cube-octameric silsesquioxane scaffolds

Cube-octameric silsesquioxane (POSS) based conjugation scaffolds for copper catalysed azide-alkyne [3+2] cycloaddition are reported. The synthetic route to octaazido and octaalkyno functionalised POSS templates without cage rearrangements is described. A set of click couplings is conducted including the first effective conjugation with a fully unprotected functional peptide towards a POSS assembled peptide octamer.

Structural insights and biomedical potential of IgNAR scaffolds from sharks

In addition to antibodies with the classical composition of heavy and light chains, the adaptive immune repertoire of sharks also includes a heavy-chain only isotype, where antigen binding is mediated exclusively by a small and highly stable domain, referred to as vNAR. In recent years, due to their high affinity and specificity combined with their small size, high physicochemical stability and low-cost of production, vNAR fragments have evolved as promising target-binding scaffolds that can be tailor-made for applications in medicine and biotechnology. This review highlights the structural features of vNAR molecules, addresses aspects of their generation using immunization or in vitro high throughput screening methods and provides examples of therapeutic, diagnostic and other biotechnological applications.

Braces for the peptide backbone: insights into structure-activity relationships of protease inhibitor mimics with locked amide conformations.

The architecture of protein macromolecules fundamentallydepends on the sequential arrangement of peptide backbonebonds in defined conformations. Among the three torsionangles(f,y,andw)presentateachaminoacid,itistheamidebond (w) which is intrinsically hindered as a result of itspartial double-bond character and it is thus more or lessrestricted to either a trans or a cis conformation (Figure 1).

Shark Attack: high affinity binding proteins derived from shark vNAR domains by stepwise in vitro affinity maturation.

A novel method for stepwise in vitro affinity maturation of antigen-specific shark vNAR domains is described that exclusively relies on semi-synthetic repertoires derived from non-immunized sharks. Target-specific molecules were selected from a CDR3-randomized bamboo shark (Chiloscyllium plagiosum) vNAR library using yeast surface display as platform technology. Various antigen-binding vNAR domains were easily isolated by screening against several therapeutically relevant antigens, including the epithelial cell adhesion molecule (EpCAM), the Ephrin type-A receptor 2 (EphA2), and the human serine protease HTRA1. Affinity maturation was demonstrated for EpCAM and HTRA1 by diversifying CDR1 of target-enriched populations which allowed for the rapid selection of nanomolar binders. EpCAM-specific vNAR molecules were produced as soluble proteins and more extensively characterized via thermal shift assays and biolayer interferometry. Essentially, we demonstrate that high-affinity binders can be generated in vitro without largely compromising the desirable high thermostability of the vNAR scaffold.

Design and synthesis of a library of lead-like 2,4-bisheterocyclic substituted thiophenes as selective Dyrk/Clk inhibitors.

The Dyrk family of protein kinases is implicated in the pathogenesis of several diseases, including cancer and neurodegeneration. Pharmacological inhibitors were mainly described for Dyrk1A so far, but in fewer cases for Dyrk1B, Dyrk2 or other isoforms. Herein, we report the development and optimization of 2,4-bisheterocyclic substituted thiophenes as a novel class of Dyrk inhibitors. The optimized hit compounds displayed favorable pharmacokinetic properties and high ligand efficiencies, and inhibited Dyrk1B in intact cells. In a larger selectivity screen, only Clk1 and Clk4 were identified as additional targets of compound 48, but no other kinases frequently reported as off-targets. Interestingly, Dyrk1A is implicated in the regulation of alternative splicing, a function shared with Clk1/Clk4; thus, some of the dual inhibitors might be useful as efficient splicing modulators. A further compound (29) inhibited Dyrk1A and 1B with an IC50 of 130 nM, showing a moderate selectivity over Dyrk2. Since penetration of the central nervous system (CNS) seems possible based on the physicochemical properties, this compound might serve as a lead for the development of potential therapeutic agents against glioblastoma. Furthermore, an inhibitor selective for Dyrk2 (24) was also identified, which might be are suitable as a pharmacological tool to dissect Dyrk2 isoform–mediated functions.

Single-domain antibodies for biomedical applications

Abstract Single-domain antibodies are the smallest antigen-binding units of antibodies, consisting either only of one variable domain or one engineered constant domain that solely facilitates target binding. This class of antibody derivatives comprises naturally occurring variable domains derived from camelids and sharks as well as engineered human variable or constant antibody domains of the heavy or light chain. Because of their high affinity and specificity as well as stability, small size and benefit of multiple re-formatting opportunities, those molecules emerged as promising candidates for biomedical applications and some of these entities have already proven to be successful in clinical development.

Potent inhibitors of human matriptase-1 based on the scaffold of sunflower trypsin inhibitor

Sunflower trypsin inhibitor‐1 (SFTI‐1), a bicyclic tetradecapeptide, has become a versatile tool as a scaffold for the development of the inhibitors of therapeutically relevant serine proteases, among them matriptase and kallikreins. Herein, we report the rational design of potent monocyclic and bicyclic inhibitors of human matriptase‐1. We found that the presence of positive charge and lack of bulky residues at the peptide N‐terminus is required for the maintenance of inhibitory activity. Replacement of the N‐terminal glycine residue by lysine allowed for the chemical conjugation with a fluorophor via the ε‐amino group without significant loss of inhibitory activity. Head‐to‐tail and side‐chain‐to‐tail cyclization resulted in potent inhibitors with comparable activities against matriptase‐1. The most potent synthetic bicyclic inhibitor found in this study (Ki = 2.6 nM at pH 7.6) is a truncated version of SFTI‐1 (cyclo‐KRCTKSIPPRCH) lacking a C‐terminal proline and aspartate residue. It combines an internal disulfide bond with a peptide macrocycle that is formed through side‐chain‐to‐tail cyclization of the ε‐amino group of an N‐terminal lysine and a C‐terminal proline. Copyright

Application of Dual Inhibition Concept within Looped Autoregulatory Systems toward Antivirulence Agents against Pseudomonas aeruginosa Infections

Pseudomonas aeruginosa quorum-sensing (QS) is a sophisticated network of genome-wide regulation triggered in response to population density. A major component is the self-inducing pseudomonas quinolone signal (PQS) QS system that regulates the production of several nonvital virulence- and biofilm-related determinants. Hence, QS circuitry is an attractive target for antivirulence agents with lowered resistance development potential and a good model to study the concept of polypharmacology in autoloop-regulated systems per se. Based on the finding that a combination of PqsR antagonist and PqsD inhibitor synergistically lowers pyocyanin, we have developed a dual-inhibitor compound of low molecular weight and high solubility that targets PQS transcriptional regulator (PqsR) and PqsD, a key enzyme in the biosynthesis of PQS-QS signal molecules (HHQ and PQS). In vitro, this compound markedly reduced virulence factor production and biofilm formation accompanied by a diminished content of extracellular DNA (eDNA). Additionally, coadministration with ciprofloxacin increased susceptibility of PA14 to antibiotic treatment under biofilm conditions. Finally, disruption of pathogenicity mechanisms was also assessed in vivo, with significantly increased survival of challenged larvae in a Galleria mellonella infection model. Favorable physicochemical properties and effects on virulence/biofilm establish a promising starting point for further optimization. In particular, the ability to address two targets of the PQS autoinduction cycle at the same time with a single compound holds great promise in achieving enhanced synergistic cellular effects while potentially lowering rates of resistance development.