Michael Zahn

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug-Resistant Gram-Negative Pathogens

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a K(D) of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic E. coli strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable in vitro and in vivo properties including a promising safety margin.

Rational Design of Oncocin Derivatives with Superior Protease Stabilities and Antibacterial Activities Based on the High-Resolution Structure of the Oncocin-DnaK Complex.

Despite the success story of antibiotics, which began nearly a hundred years ago, bacterial infections are still a major cause of death worldwide. The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global problem of growing importance, with an estimated annual death toll of 50 000 in the EU and 60 000 in the USA. More recently, MDR bacteria have also caused severe community-acquired infections, indicating that we will soon face bacterial strains with the ability to overcome existing antibiotic treatments, and which will therefore represent a severe global threat. Agents responsible for important resistance mechanisms in Gram-negative bacteria include extended spectrum b-lactamases (ESBLs) in Enterobacteriaceae (e.g. , E. coli, K. pneumoniae, and Enterobacter cloacae) or broad-range b-lactamases (e.g. , KPC in Klebsiella pneumoniae or metallo-b-lactamases in Pseudomonas aeruginosa). MDR Acinetobacter baumannii, associated with invasive infections such as pneumonia, meningitis, and bacteremia, has been found to be responsible for outbreaks in intensive care units, including “panresistant” A. baumannii clones susceptible only to polymyxin. To provide effective future treatment options, novel antimicrobial drug classes with novel modes of action are urgently needed. Inducible, gene-encoded antimicrobial peptides (AMPs) represent such a promising alternative, having been selected and optimized by evolution over millions of years. Although AMPs that kill bacteria by lytic effects on the membrane are often toxic to human cells at higher doses, the class of small prolinerich AMPs (PR-AMPs) expressed in mammals and insects has attracted considerable interest. These peptides specifically target intracellular components in Gram-negative bacteria with no indication of any resulting toxic side effects. Despite their favorable antibacterial spectrum against Enterobacteriaceae and nonfermenting species (e.g. , A. baumannii and P. aeruginosa), there are multiple obstacles to be overcome in their further development for therapeutic consideration. We have recently used rational design to optimize the 19-residue-long PR-AMP oncocin (VDKPPYLPRPRPPRRIYNR-NH2) as a potential means of countering the five human pathogens discussed. Substitution of Arg15 and Arg19 by ornithine drastically improved the half-life in mouse serum. Mechanistically, oncocin freely penetrates the bacterial membrane and distributes homogenously within E. coli cells. Here we report its further optimization, based on a positional Ala-scan to deduce residues critical to its antibacterial activity and the crystal structure of an oncocin-DnaK (ligand–target) complex. The new lead compounds were highly resistant against serum (t1=2 8 h) and E. coli proteases (t1=2 >10 h). The mode of action assumed for oncocin and all other PRAMPs has not been worked out in detail, but most likely consists of at least three steps: passive penetration of the bacterial outer membrane, active transport from the periplasmic space into the cytoplasm, and inhibition of DnaK and maybe other targets. 12] A lead optimization strategy therefore has to consider all aspects and cannot focus only on the target binding. We first identified residues crucial for the antibacterial activity of oncocin by determining the minimal inhibitory concentrations (MICs) and inhibition zones for all 19 peptides resulting from a positional Ala-scan (see Figure S1 in the Supporting Information). Substitution of residues 1, 2, 4, 5, 10, and 12–19 reduced the antibacterial activity slightly, whereas substitutions at positions 3, 6–9, and 11 abolished it almost completely. Although the target of oncocin has not yet been identified, the high sequence homology to other insect-derived PR-AMPs, especially pyrrhocoricin, suggests that it might be the bacterial chaperone DnaK. Full-length DnaK was therefore expressed in E. coli and purified in order to study oncocin binding by fluorescence polarization. The binding constants for oncocin and its analogue O2, with a 5(6)-carboxyfluorescein unit at the N terminus, were 1.0 0.2 mm and 4.0 1.0 mm, respectively, whereas all-d oncocin did not bind (Table 1, Figures S2 and S3). These values correspond to binding constants reported for other DnaK-binding sequences, ranging from 0.1 to 10 mm. Cocrystallization of oncocin O2 with the substrate binding domain of DnaK (residues 389 to 607), demonstrated that oncocin residues 4 to 10 (PPYLPR) bound to the peptide binding site of DnaK, whereas the remaining terminal residues of the peptide were flexible (Figures 1 and S4–S8). Interestingly, this sequence stretch matched the residues identified by the Ala-scan as crucial for antibacterial activity. The antibacterial activity of oncocin thus mostly depends on its DnaK binding site, although the activity is also abolished by shortening the sequence, either from the N or the C terminus. Such inactivation, which can occur through the action of proteases either in the bacteria or in blood, should be minimized for systemic applications. Because the peptide O2 is rel[a] D. Knappe, M. Zahn, Prof. Dr. N. Str ter, Prof. Dr. R. Hoffmann Institut f r Bioanalytische Chemie Biotechnologisch-Biomedizinisches Zentrum Fakult t f r Chemie und Mineralogie, Universit t Leipzig Deutscher Platz 5, 04103 Leipzig (Germany) Fax: (+ 49) 341-97-31339 E-mail : hoffmann@chemie.uni-leipzig.de [b] U. Sauer, Dr. G. Schiffer AiCuris GmbH & Co KG Friedrich-Ebert-Strasse 475, Building 302, 42117 Wuppertal (Germany) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201000792.

Structural Studies on the Forward and Reverse Binding Modes of Peptides to the Chaperone DnaK

Hsp70 chaperones have been implicated in assisting protein folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. For these functions, the chaperones need to exhibit a significant promiscuity in binding to different sequences of hydrophobic peptide stretches. To characterize the structural basis of sequence specificity and flexibility of the Escherichia coli Hsp70 chaperone DnaK, we have analyzed crystal structures of the substrate binding domain of the protein in complex with artificially designed peptides as well as small proline-rich antimicrobial peptides. The latter peptides from mammals and insects were identified to target DnaK after cell penetration. Interestingly, the complex crystal structures reveal two different peptide binding modes. The peptides can bind either in a forward or in a reverse direction to the conventional substrate binding cleft of DnaK in an extended conformation. Superposition of the two binding modes shows a remarkable similarity in the side chain orientations and hydrogen bonding pattern despite the reversed peptide orientation. The DnaK chaperone has evolved to bind peptides in both orientations in the substrate binding cleft with comparable energy without rearrangements of the protein. Optimal hydrophobic interactions with binding pockets -2 to 0 appear to be the main determinant for the orientation and sequence position of peptide binding.

Structural Basis of the Stereospecificity of Bacterial B12-dependent 2-Hydroxyisobutyryl-CoA Mutase

Background: Bacterial B12-dependent 2-hydroxyisobutyryl-CoA mutase specifically catalyzes the isomerization of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. Results: The crystal structure of 2-hydroxyisobutyryl-CoA mutase shows decisive differences in the active site when compared with the well studied methylmalonyl-CoA mutase. Conclusion: Specificity toward (S)-3-hydroxybutyryl-CoA strongly depends on the active site amino acid AspA117. Significance: This is the first structural characterization of a B12-dependent mutase with α2β2 organization isomerizing 2-hydroxyisobutyryl-CoA. Bacterial coenzyme B12-dependent 2-hydroxyisobutyryl-CoA mutase (HCM) is a radical enzyme catalyzing the stereospecific interconversion of (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA. It consists of two subunits, HcmA and HcmB. To characterize the determinants of substrate specificity, we have analyzed the crystal structure of HCM from Aquincola tertiaricarbonis in complex with coenzyme B12 and the substrates (S)-3-hydroxybutyryl- and 2-hydroxyisobutyryl-CoA in alternative binding. When compared with the well studied structure of bacterial and mitochondrial B12-dependent methylmalonyl-CoA mutase (MCM), HCM has a highly conserved domain architecture. However, inspection of the substrate binding site identified amino acid residues not present in MCM, namely HcmA IleA90 and AspA117. AspA117 determines the orientation of the hydroxyl group of the acyl-CoA esters by H-bond formation, thus determining stereospecificity of catalysis. Accordingly, HcmA D117A and D117V mutations resulted in significantly increased activity toward (R)-3-hydroxybutyryl-CoA. Besides interconversion of hydroxylated acyl-CoA esters, wild-type HCM as well as HcmA I90V and I90A mutant enzymes could also isomerize pivalyl- and isovaleryl-CoA, albeit at >10 times lower rates than the favorite substrate (S)-3-hydroxybutyryl-CoA. The nonconservative mutation HcmA D117V, however, resulted in an enzyme showing high activity toward pivalyl-CoA. Structural requirements for binding and isomerization of highly branched acyl-CoA substrates such as 2-hydroxyisobutyryl- and pivalyl-CoA, possessing tertiary and quaternary carbon atoms, respectively, are discussed.

Structural Identification of DnaK Binding Sites within Bovine and Sheep Bactenecin Bac7

Bacterial resistance against common antibiotics is an increasing health problem. New pharmaceuticals for the treatment of infections caused by resistant pathogens are needed. Small proline-rich antimicrobial peptides (PrAMPs) from insects are known to bind intracellularly to the conventional substrate binding cleft of the E. coli Hsp70 chaperone DnaK. Furthermore, bactenecins from mammals, members of the cathelicidin family, also contain potential DnaK binding sites. Crystal structures of bovine and sheep Bac7 in complex with the DnaK substrate binding domain show that the peptides bind in the forward binding mode with a leucine positioned in the central hydrophobic pocket. In most structures, proline and arginine residues preceding leucine occupy the hydrophobic DnaK binding sites -1 and -2. Within bovine Bac7, four potential DnaK binding sites were identified.

Fluorine-Containing 6,7-Dialkoxybiaryl-Based Inhibitors for Phosphodiesterase 10 A: Synthesis and in vitro Evaluation of Inhibitory Potency, Selectivity, and Metabolism

Based on the potent phosphodiesterase 10 A (PDE10A) inhibitor PQ‐10, we synthesized 32 derivatives to determine relationships between their molecular structure and binding properties. Their roles as potential positron emission tomography (PET) ligands were evaluated, as well as their inhibitory potency toward PDE10A and other PDEs, and their metabolic stability was determined in vitro. According to our findings, halo‐alkyl substituents at position 2 of the quinazoline moiety and/or halo‐alkyloxy substituents at positions 6 or 7 affect not only the compounds′ affinity, but also their selectivity toward PDE10A. As a result of substituting the methoxy group for a monofluoroethoxy or difluoroethoxy group at position 6 of the quinazoline ring, the selectivity for PDE10A over PDE3A increased. The same result was obtained by 6,7‐difluoride substitution on the quinoxaline moiety. Finally, fluorinated compounds (R)‐7‐(fluoromethoxy)‐6‐methoxy‐4‐(3‐(quinoxaline‐2‐yloxy)pyrrolidine‐1‐yl)quinazoline (16 a), 19 a–d, (R)‐tert‐butyl‐3‐(6‐fluoroquinoxalin‐2‐yloxy)pyrrolidine‐1‐carboxylate (29), and 35 (IC50 PDE10A 11–65 nM) showed the highest inhibitory potential. Further, fluoroethoxy substitution at position 7 of the quinazoline ring improved metabolic stability over that of the lead structure PQ‐10.

Crystal structure of a supercharged variant of the human enteropeptidase light chain

The highly specific serine protease human enteropeptidase light chain cleaves the Asp4Lys recognition sequence and represents an interesting enzyme for biotechnological applications. The human enzyme shows 10 times faster kinetics compared to other animal sources but low solubility under low salt conditions, which hampers protein production and crystallization. Therefore, a supercharged variant (N6D/G21D/G22D/N142D/K210E/C112S) with increased solubility was used for crystallization. The structure (resolution, 1.9 Å) displays a typical α/β trypsin‐like serine protease‐fold. The mutations introduced for protein supercharging generate larger clusters of negative potential on both sites of the active cleft but do not affect the structural integrity of the protein. Proteins 2012.