Matthias Mentel

Of Two Make One: The Biosynthesis of Phenazines

Physicians of the 19th century were familiar with the conspicuous occurrence of “blue pus”, which they sometimes observed in patients with severe purulent wounds. Even older are reports of and folk remedies against “blue milk”, a coloration that sometimes developed in fresh milk after some days. Key insight into these phenomena was provided in 1859—exactly 150 years ago—when Mathurin-Joseph Fordos, at a session of the Societ d’ mulation pour les Sciences Pharmaceutiques, reported the isolation of the blue pigment “pyocyanine” (from the Greek words for “pus” and “blue”; pyocyanine is nowadays more commonly spelled as pyocyanin) from purulent wound dressings by chloroform extraction. Pyocyanin (5-N-methyl-1hydroxophenazinium betaine) was the first example of a phenazine natural product, a compound class that has grown to well over 100 members since this first report. Due to the improved understanding of their importance to the phenazinegenerating and also to commensal species, the phenazines have been reviewed several times in recent years. Here, we provide a historical perspective of the more than 100 years of research that led us to our current picture of the interesting biosynthesis of phenazine natural products. The details of Fordos’ pyocyanin isolation method, chloroform extraction followed by acidification and partition into an aqueous phase, were published one year later and are still in use today, but it took until 1882 for the French pharmacist Carle Gessard to show that the blue coloration in pus was due to the presence of a microorganism that he then termed Bacillus pyocyaneus. B. pyocyaneus is nowadays known as Pseudomonas aeruginosa, and the Latin term still reflects this strain’s capacity to secrete colored compounds in the modern name: “aerugo” is the Latin word for verdigris, the blue–green coating that develops on copper after long exposure to air. P. aeruginosa is an important human opportunistic pathogen responsible for a large number of nosocomial infections, and it is also the main course of low life expectancy in patients with cystic fibrosis due to chronic infections of the lungs. The production of pyocyanin is used both for identification in the clinic and as a reporter signal in P. aeruginosa research until today. The occurrence of blue milk, on the other hand, is probably due to an environmental strain of P. fluorescens, and it is not yet clear if this coloration also is a consequence of phenazine production. Gessard’s discovery of P. aeruginosa was resonated in many publications from the medical field, but it required more than 50 years before the correct chemical structure of pyocyanin was established. The chemical composition was first studied by Ledderhose, who derived a formula that was later corrected by McCombie and Scarborough and by Wrede and Strack. Wrede and Strack were also the first to discover a phenazine moiety in a breakdown product of pyocyanin, but their studies were hampered by the fact that they could only obtain a defined molecular weight when working in glacial acetic acid, under which circumstances they obtained a pyocyanin dimer. This dimer was questioned by the results of electrochemical studies by Elema and by Friedheim and Michaelis, before Hillemann finally derived the correct structure in 1938. In retrospect, it seems possible that the conditions employed by Wrede and Strack induced a 1:1 charge-transfer complex of reduced and oxidized pyocyanin, similar to the phenazine derivative chlororaphin, which is also produced by P. aeruginosa (Figure 1). Jensen and Holten later measured the dipole moment of pyocyanin and found that its zwitterionic mesomer is present in considerable amounts. In the course of these studies, it became clear that pyocyanin is a redox-active compound that changes its color depending on pH and oxidation state. This also explained the “chameleon phenomenon”, which describes a temporary color change of P. aeruginosa cultures on solid media after exposure to air by disturbance with a platinum needle. Since the first isolation by Fordos, more than 100 phenazine derivatives modified at all positions of the ring system and colored in all shades of

PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis.

Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine-1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives.

PIP3 Induces the Recycling of Receptor Tyrosine Kinases

EGFR is recycled to the cell surface in response to the phosphoinositide PIP3. Recycling Receptors The epidermal growth factor receptor (EGFR) promotes cellular proliferation. Activation of EGFRs by ligand binding typically leads to receptor internalization and then degradation of the receptor, thereby terminating signaling downstream of the receptor. Laketa et al. found that high concentrations of the phosphoinositide PIP3 (phosphatidylinositol 3,4,5-trisphosphate) triggered the internalization of EGFRs and their recycling to the cell surface. Because high concentrations of PIP3 can be generated both physiologically and pathophysiologically, this mechanism could prevent activated EGFRs from degradation, diverting them back to the surface to sustain the cell’s response to EGF. Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain–containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

Combinatorial Solid-Phase Natural Product Chemistry

One major challenge in combinatorial chemistry is to find biologically relevant starting points in the chemical universe around which compound libraries should be produced. Natural products represent such biologically validated starting points. Progress in Solid-Phase Organic Synthesis (SPOS) has enabled now the combination of natural product synthesis with combinatorial methods. Two strategies can be distinguished:(1) Attachment of a natural product core structure onto solid phase and subsequent modification of functional groups, or (2) total synthesis of the complete natural product scaffold on solid phase. A complementary approach identifies privileged structures among natural product scaffolds, which serve as inspiration for library synthesis. In this review the current status and the challenges of combinatorial natural product synthesis on solid phase are presented.

The Active Site of an Enzyme Can Host Both Enantiomers of a Racemic Ligand Simultaneously

Since Pasteur discovered the principle of chirality and its implications in interactions with biological systems, the effect of chirality in drugs has been the subject of intense investigation. The most common case is that only one enantiomer of a racemic mixture binds to a biological receptor while the other can be regarded as “isomeric ballast” (Figure 1a). There are also cases in which the second enantiomer shows different behavior, ranging from agonistic or antagonistic binding to the same receptor to interactions with other biological targets, which can lead to cooperative, side, or even counterproductive effects. Consequently, recent legal regulation requires that only single-enantiomer drugs may be marketed. 4] While the question of singleenantiomer drugs has been settled for the end of the drugdiscovery process, racemic mixtures are still preferentially used in primary screens, mainly because of by the considerable efforts necessary to produce enantiomerically pure

Combinatorial chemistry and the synthesis of compound libraries.

Solid Phase Organic Synthesis (SPOS) has become a powerful tool for the preparation of compound libraries used for screening efforts in Chemical Biology. While different types of screening libraries have become commercially available through several vendors, the elaboration of a hit compound in a screening campaign to a useful chemical probe still requires the preparation of a focused library. In this report, protocols are given which allow the synthesis of a focused compound library on solid phase by the diversification of a central scaffold using reliable functionalization reactions. Diversity elements can be introduced by attaching building blocks via amidation, esterification, reductive amination, and Suzuki cross-coupling.