Jörg Rademann

New Tacrine–4-Oxo-4H-chromene Hybrids as Multifunctional Agents for the Treatment of Alzheimer’s Disease, with Cholinergic, Antioxidant, and β-Amyloid-Reducing Properties

By using fragments endowed with interesting and complementary properties for the treatment of Alzheimers disease (AD), a new family of tacrine-4-oxo-4H-chromene hybrids has been designed, synthesized, and evaluated biologically. The tacrine fragment was selected for its inhibition of cholinesterases, and the flavonoid scaffold derived from 4-oxo-4H -chromene was chosen for its radical capture and β-secretase 1 (BACE-1) inhibitory activities. At nano- and picomolar concentrations, the new tacrine-4-oxo-4H-chromene hybrids inhibit human acetyl- and butyrylcholinesterase (h-AChE and h-BuChE), being more potent than the parent inhibitor, tacrine. They are also potent inhibitors of human BACE-1, better than the parent flavonoid, apigenin. They show interesting antioxidant properties and could be able to penetrate into the CNS according to the in vitro PAMPA-BBB assay. Among the hybrids investigated, 6-hydroxy-4-oxo- N-{10-[(1,2,3,4-tetrahydroacridin-9-yl)amino]decyl}-4 H-chromene-2-carboxamide (19) shows potent combined inhibition of human BACE-1 and ChEs, as well as good antioxidant and CNS-permeable properties.

Lysosomal Pathology and Osteopetrosis upon Loss of H+-Driven Lysosomal Cl– Accumulation

Chloride Balancing Act The ionic composition of the cytosol and intracellular organelles must be regulated in the face of ongoing membrane traffic into and out of the cell. Now, two papers address the consequences of a change in the transport phenotype of an intracellular Cl− transport protein from a coupled exchanger to a passive Cl− conductor (see the Perspective by Smith and Schwappach). Novarino et al. (p. 1398, published online 29 April) investigated the consequence of a knock-in of the uncoupled ClC-5 transporter into mouse. The knock-out mouse of this endosomal kidney transporter has a severe endocytic phenotype believed to be due to a defect in vesicular acidification. The current study shows a similarly impaired endocytic phenotype for the uncoupled mutant, but the acidification of endosomes was unaffected. Weinert et al. (p. 1401, published online 29 April) used a similar strategy to investigate the consequence of the equivalent mutation in the lysosomal transporter ClC-7, which is highly expressed in the resorption lacuna of osteoclasts and whose knock-out in mice produces lysosomal storage disease and severe osteopetrosis. A similar (though less severe) phenotype was observed in the knock-in mice containing the uncoupled ClC-7, indicating that coupled transport plays a critical role in lysosomes. Chloride conductance and chloride-proton exchange have distinct effects on endolysosomal physiology in mice. During lysosomal acidification, proton-pump currents are thought to be shunted by a chloride ion (Cl–) channel, tentatively identified as ClC-7. Surprisingly, recent data suggest that ClC-7 instead mediates Cl–/proton (H+) exchange. We generated mice carrying a point mutation converting ClC-7 into an uncoupled (unc) Cl– conductor. Despite maintaining lysosomal conductance and normal lysosomal pH, these Clcn7unc/unc mice showed lysosomal storage disease like mice lacking ClC-7. However, their osteopetrosis was milder, and they lacked a coat color phenotype. Thus, only some roles of ClC-7 Cl–/H+ exchange can be taken over by a Cl– conductance. This conductance was even deleterious in Clcn7+/unc mice. Clcn7–/– and Clcn7unc/unc mice accumulated less Cl– in lysosomes than did wild-type mice. Thus, lowered lysosomal chloride may underlie their common phenotypes.

Specific inhibitors of the protein tyrosine phosphatase Shp2 identified by high-throughput docking

The protein tyrosine phosphatase Shp2 is a positive regulator of growth factor signaling. Gain-of-function mutations in several types of leukemia define Shp2 as a bona fide oncogene. We performed a high-throughput in silico screen for small-molecular-weight compounds that bind the catalytic site of Shp2. We have identified the phenylhydrazonopyrazolone sulfonate PHPS1 as a potent and cell-permeable inhibitor, which is specific for Shp2 over the closely related tyrosine phosphatases Shp1 and PTP1B. PHPS1 inhibits Shp2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocks Shp2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of the Erk1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibits activation of Erk1/2 by the leukemia-associated Shp2 mutant, Shp2-E76K, and blocks the anchorage-independent growth of a variety of human tumor cell lines. The PHPS compound class is therefore suitable for further development of therapeutics for the treatment of Shp2-dependent diseases.

Small-Molecule Scaffolds for CYP51 Inhibitors Identified by High-Throughput Screening and Defined by X-Ray Crystallography

ABSTRACT Sterol 14α-demethylase (CYP51), a major checkpoint in membrane sterol biosynthesis, is a key target for fungal antibiotic therapy. We sought small organic molecules for lead candidate CYP51 inhibitors. The changes in CYP51 spectral properties following ligand binding make CYP51 a convenient target for high-throughput screening technologies. These changes are characteristic of either substrate binding (type I) or inhibitor binding (type II) in the active site. We screened a library of 20,000 organic molecules against Mycobacterium tuberculosis CYP51 (CYP51Mt), examined the top type I and type II binding hits for their inhibitory effects on M. tuberculosis in broth culture, and analyzed them spectrally for their ability to discriminate between CYP51Mt and two reference M. tuberculosis CYP proteins, CYP130 and CYP125. We determined the binding mode for one of the top type II hits, α-ethyl-N-4-pyridinyl-benzeneacetamide (EPBA), by solving the X-ray structure of the CYP51Mt-EPBA complex to a resolution of 1.53 Å. EPBA binds coordinately to the heme iron in the CYP51Mt active site through a lone pair of nitrogen electrons and also through hydrogen bonds with residues H259 and Y76, which are invariable in the CYP51 family, and hydrophobic interactions in a phylum- and/or substrate-specific cavity of CYP51. We also identified a second compound with structural and binding properties similar to those of EPBA, 2-(benzo[d]-2,1,3-thiadiazole-4-sulfonyl)-2-amino-2-phenyl-N-(pyridinyl-4)-acetamide (BSPPA). The congruence between the geometries of EPBA and BSPPA and the CYP51 binding site singles out EPBA and BSPPA as lead candidate CYP51 inhibitors with optimization potential for efficient discrimination between host and pathogen enzymes.

A NEW METHOD FOR THE SOLID PHASE SYNTHESIS OF OLIGOSACCHARIDES

Abstract Trichloroacetimidates are employed as sugar donors in solid phase oligosaccharide synthesis as demonstrated for the glucose (1→6)-linkage. The use of a new thiol linker allows the direct attachment of the first sugar residue to the resin and a fast and convenient quantitative monitoring of all solid phase reactions. The synthesis of pentasaccharides 6 (n = 5) exhibits the efficiency of the method and the potential in combinatorial syntheses.

Endotoxin-like properties of a rhamnolipid exotoxin from Burkholderia (Pseudomonas) plantarii: immune cell stimulation and biophysical characterization

Abstract Here we report on the purification, structural characterization, and biological activity of a glycolipid, 2-O-α-L-rhamnopyranosyl-α-L-rhamnopyranosyl-α(R)-3-hydroxytetradecanoyl-(R)-3-hydroxytetradecanoate (RL-2,214) produced by Burkholderia (Pseudomonas) plantarii. RL-2,214 is structurally very similar to a rhamnolipid exotoxin from Pseudomonas aeruginosa and identical to the rhamnolipid of Burkholderia pseudomallei, the causative agent of melioidosis. Interestingly, RL-2,214 exhibits strong stimulatory activity on human mononuclear cells to produce tumor necrosis factor α, the overproduction of which is known to cause sepsis and the septic shock syndrome. Such a property has not been noted so far for rhamnolipid exotoxins, only for bacterial endotoxins (lipopolysaccharide, LPS). Consequently, we analyzed RL-2,214 with respect to its pathophysiological activities as a heat-stable extracellular toxin. Like LPS, the cell-stimulating activity of the rhamnolipid could be inhibited by incubation with polymyxin B. However, immune cell activation by RL-2,214 does nor occur via receptors that are involved in LPS (TLR4) or lipopeptide signaling (TLR2). Despite its completely different chemical structure, RL-2,214 exhibits a variety of endotoxin-related physicochemical characteristics, such as a cubic-inverted supramolecular structure. These data are in good agreement with our conformational concept of endotoxicity: intercalation of naturally originating virulence factors into the immune cell membrane leads to strong mechanical stress on integral proteins, eventually causing cell activation.

Discovery of Mycobacterium Tuberculosis Protein Tyrosine Phosphatase A (MptpA) Inhibitors Based on Natural Products and a Fragment-Based Approach

Protein phosphorylation and dephosphorylation reactions are at the heart of innumerable biological processes. Aberrant protein phosphorylation contributes to the development of many human diseases including cancer and diabetes. Due to this biological importance, protein kinases, which catalyse protein phosphorylation, and their antagonists, protein phosphatases (PPs), have moved into the focus of a rapidly growing number of medicinal-chemistry and chemicalbiology research programs. Several bacterial pathogens produce eukaryotic-like protein phosphatases that have been implicated in virulence. A particularly important case is Myobacterium tuberculosis, which is the causative agent of tuberculosis (TB) and a major cause of mortality around the world. M. tuberculosis has two functional phosphatases, MptpA and MptpB. These enzymes are secreted by growing mycobacterial cells. They are believed to mediate mycobacterial survival in host cells by dephosphorylating proteins that are involved in interferon-g signaling pathways. About one third of the world’s population is infected with M. tuberculosis, and there is an increasing spread of drug-resistant mycobacteria. Therefore, there is a growing need for the development of new therapeutic agents for the treatment of tuberculosis. In the light of this urgent demand, the Mptps have been proposed as new potential anti-TB drug targets. However, to date, inhibitors of these enzymes have not been described. Here we describe the discovery of MptpA inhibitors by two different and complementary approaches for the identification of initial hits in screening collections, namely natural-productinspired and fragment-based library development. We have previously forwarded the notion that biologically active natural products should be regarded as evolutionarily selected and biologically prevalidated starting points for inhibitor development. Based on this principle and the fact that MptpA is a tyrosine phosphatase, we have investigated whether natural products and their analogues that have already served as guiding structures for the discovery of new classes of phosphatase inhibitors could be employed for the identification of the first Mptp inhibitors. Initially the stevastelins (Scheme 1) were considered as possible starting points for the development of MptpA inhibitors.

Sensitized Detection of Inhibitory Fragments and Iterative Development of Non‐Peptidic Protease Inhibitors by Dynamic Ligation Screening

A potential anti‐SARS drug has been developed by dynamic ligation screening (DLS), by which nucleophilic fragments are directed to the proteins active site by reversible reaction with an aldehyde inhibitor. Their inhibitory effect is detected by competition with a fluorogenic enzyme substrate. With this concept, low‐affinity fragments binding specifically to the active site are quickly identified in a functional enzyme assay.WILEY-VCH

Design of chemical libraries with potentially bioactive molecules applying a maximum common substructure concept

Success in small molecule screening relies heavily on the preselection of compounds. Here, we present a strategy for the enrichment of chemical libraries with potentially bioactive compounds integrating the collected knowledge of medicinal chemistry. Employing a genetic algorithm, substructures typically occurring in bioactive compounds were identified using the World Drug Index. Availability of compounds containing the selected substructures was analysed in vendor libraries, and the substructure-specific sublibraries were assembled. Compounds containing reactive, undesired functional groups were omitted. Using a diversity filter for both physico-chemical properties and the substructure composition, the compounds of all the sublibraries were ranked. Accordingly, a screening collection of 16,671 compounds was selected. Diversity and chemical space coverage of the collection indicate that it is highly diverse and well-placed in the chemical space spanned by bioactive compounds. Furthermore, secondary assay-validated hits presented in this study show the practical relevance of our library design strategy.

Solid-Phase Supported Synthesis of the Branched Pentasaccharide Moiety That Occurs in Most Complex Type N-Glycan Chains

Repetitive glycosylation on a sulfanylalkyl-functionalized Merrifield resin leads to the branched, complex pentasaccharide 1 in 20% overall yield in ten steps when appropriately protected O-glycosyl trichloroacetimidates are used as glycosyl donors. A decisive factor here was the tuning of the reaction conditions for the solid-phase glycosylation and the conditions for selective removal of the protecting groups and for cleavage of the samples from the resin for characterization. The subsequent cleavage of the product was achieved with a thiophilic reagent that does not attack the O-glycosidic linkages.