Novel semi-synthetic glycopeptide antibiotics active against methicillin-resistant staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE): doubly-modified water-soluble derivatives of chloroorienticin B

A series of N-alkylated and aminomethylated derivatives of chloroorienticin B, a vancomycin-related glycopeptide antibiotic, were synthesized. Doubly-modified derivatives having both hydrophobic and hydrophilic substituents exhibited potent antibacterial activity against MRSA and VRE along with considerable water-solubility.     

Design and synthesis of benzenesulfonanilides active against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Vancomycin is mainly used as an antibacterial agent of last resort, but recently vancomycin-resistant bacterial strains have been emerging. Although new antimicrobials have been developed in order to overcome drug-resistant bacteria, many are structurally complex beta-lactams or quinolones. In this study, we aimed to create new anti-drug-resistance antibacterials which can be synthesized in a few steps from inexpensive starting materials. Since sulfa drugs function as p-aminobenzoic acid mimics and inhibit dihydropteroate synthase (DHPS) in the folate pathway, we hypothesized that sulfa derivatives would act as folate metabolite-mimics and inhibit bacterial folate metabolism. Screening of our sulfonanilide libraries, including benzenesulfonanilide-type cyclooxygenase-1-selective inhibitors, led us to discover benzenesulfonanilides with potent anti-methicillin-resistant Staphylococcus aureus (MRSA)/vancomycin-resistant Enterococcus (VRE) activity, that is, N-3,5-bis(trifluoromethyl)phenyl-3,5-dichlorobenzenesulfonanilide (16b) [MIC=0.5microg/mL (MRSA), 1.0microg/mL (VRE)], and 3,5-bis(trifluoromethyl)-N-(3,5-dichlorophenyl)benzenesulfonanilide (16c) [MIC=0.5microg/mL (MRSA), 1.0microg/mL (VRE)]. These compounds are more active than vancomycin [MIC=2.0microg/mL (MRSA), 125microg/mL (VRE)], but do not possess an amino group, which is essential for DHPS inhibition by sulfa drugs. These results suggested that the mechanism of antibacterial action of compounds 16b and 16c is different from that of sulfa drugs. We also confirmed the activity of these compounds against clinical isolates of Gram-positive bacteria.     

Antimicrobial activity of mupirocin, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline against vancomycin-resistant enterococci (VRE) from clinical isolates in Korea (1998 and 2005)

It is a hot clinical issue whether newly approved antimicrobial agents such as daptomycin, linezolid, quinupristin/dalfopristin (synercid) and tigecycline are active enough to be used for infections caused by vancomycin resistant bacteria. We performed susceptibility tests for mupirocin, which is in widespread clinical use in Korea, and four new antimicrobials, daptomycin, linezolid, quinupristin/dalfopristin and tigecycline, against vancomycin-resistant Enterococcus faecalis and Enterococcus faecium isolated from Korean patients in 1998 and 2005 to evaluate and compare the in vitro activity of these antimicrobials. Among these agents, quinupristin/dalfopristin, which is rarely used in hospitals in Korea, showed relatively high resistance to several vancomycin-resistant enterococci (VRE) isolated in 2005. Likewise, daptomycin, linezolid and tigecycline have not yet been in clinical use in Korea. However, our results showed that most of the 2005 VRE isolates were already resistant to linezolid and daptomycin (highest minimum inhibitory concentration (MIC) value >100 microg/ml). Compared with the other four antimicrobial agents tested in this study, tigecycline generally showed the greatest activity against VRE. However, four strains of 2005 isolates exhibited resistance against tigecycline (MIC >12.5 microg/ml). Almost all VRE were resistant to mupirocin, whereas all E. faecium isolated in 1998 were inhibited at concentrations between 0.8 to approximately 1.6 microg/ml. In conclusion, resistances to these new antimicrobial agents were exhibited in most of VRE strains even though these new antibiotics have been rarely used in Korean hospitals.     

Identification of synthetic compounds active against VRE: the role of the lipidated aminoglucose and the structure of glycopeptide binding pocket

A modified vancomycin binding pocket (D-O-E ring) incorporating an alpha-hydroxy-beta-amino acid at the AA4 position is designed and synthesized. Some of these compounds display potent bioactivities against both sensitive- and resistant-strains (8 microg/ml against VREF). Both the lipidated aminoglucose and the structure of the 16-membered macrocycle are found to be important for the anti-VRE activities. The polyamine appendage at the C-terminal, on the other hand, improved the activity against vancomycin-sensitive strains.     

Isolation and characterization of Streptomyces sp KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics

The actinomycete strain KH-614 was antagonistic to vancomycin-resistant enterococci (VRE). Based on the diaminopimelic acid (DAP) type, morphological and physiological characteristics examined by scanning electron microscopy (SEM), KH-614 was confirmed as belonging to the genus Streptomyces. Based on the 16S rDNA nucleotide sequences, Streptomyces sp. KH-614 was found to have a relationship with Streptomyces lydicus. The production of antibiotic from this strain was most favorable when cultured in glucose, polypeptone, yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic was identified as a cyclo(L-leucyl-L-prolyl) by comparing it with the reported spectral data including MS and NMR. Cyclo(leu-pro) was found to be active against twelve VRE strains, including E. faecium (vanA, vanB), and E. faecalis (vanA, vanB), that had been isolated over a period three years (1998-2000). Cyclo(leu-pro) was especially effective against VRE strains such as E. faecalis (K-99-34), E. faecalis (K-00-184), E. faecalis (K-00-221), and the MIC values were 12.5 microg/ml. Moreover, cyclo(leu-pro) was effective against three leukemic cell lines at concentrations below 100 microg/ml. At 100 mg/ml cyclo(leu-pro), K562, HL60, and U937 leukemic cell lines showed growth inhibition of 95, 91, and 93%, respectively. In a normal cell line, MDBK, cyclo(leu-pro) exerted 24% growth inhibition at a concentration of 100 microg/ml, and showed no inhibitory activity at concentrations below 10 microg/ml. These results indicate that cyclo(leu-pro) is a potential anti-leukemic and anti-VRE agent.     

Structure-based design, synthesis and A-site rRNA co-crystal complexes of novel amphiphilic aminoglycoside antibiotics with new binding modes: a synergistic hydrophobic effect against resistant bacteria

Incorporation of an hydrophobic (phenethylamino)ethyl ether at C2″ of N1-(HABA)-3',4'-dideoxyparomomycin led to a novel analog with an excellent antibacterial profile against a host of resistant bacteria.     

Modelling the binding mode of macrocycles: Docking and conformational sampling

Drug discovery is increasingly tackling challenging protein binding sites regarding molecular recognition and druggability, including shallow and solvent-exposed protein-protein interaction interfaces. Macrocycles are emerging as promising chemotypes to modulate such sites. Despite their chemical complexity, macrocycles comprise important drugs and offer advantages compared to non-cyclic analogs, hence the recent impetus in the medicinal chemistry of macrocycles. Elaboration of macrocycles, or constituent fragments, can strongly benefit from knowledge of their binding mode to a target. When such information from X-ray crystallography is elusive, computational docking can provide working models. However, few studies have explored docking protocols for macrocycles, since conventional docking methods struggle with the conformational complexity of macrocycles, and also potentially with the shallower topology of their binding sites. Indeed, macrocycle binding mode prediction with the mainstream docking software GOLD has hardly been explored. Here, we present an in-depth study of macrocycle docking with GOLD and the ChemPLP scores. First, we summarize the thorough curation of a test set of 41 protein-macrocycle X-ray structures, raising the issue of lattice contacts with such systems. Rigid docking of the known bioactive conformers was successful (three top ranked poses) for 92.7% of the systems, in absence of crystallographic waters. Thus, without conformational search issues, scoring performed well. However, docking success dropped to 29.3% with the GOLD built-in conformational search. Yet, the success rate doubled to 58.5% when GOLD was supplied with extensive conformer ensembles docked rigidly. The reasons for failure, sampling or scoring, were analyzed, exemplified with particular cases. Overall, binding mode prediction of macrocycles remains challenging, but can be much improved with tailored protocols. The analysis of the interplay between conformational sampling and docking will be relevant to the prospective modelling of macrocycles in general.     

HINT predictive analysis of binding between retinol binding protein and hydrophobic ligands

The interaction between the retinol binding protein and four ligands was evaluated using HINT, a software based on experimental LogP values of individual atoms. A satisfactory correlation was found between the HINT scores and the experimental dissociation constants of three of the ligands, fenretinide, N-ethylretinamide and all-trans retinol, despite their hydrophobic nature. A prediction is made for the binding affinity of the fourth ligand, axerophtene, not yet determined in solution.     

Amphipathic benzoic acid derivatives: synthesis and binding in the hydrophobic tunnel of the zinc deacetylase LpxC

The first committed step in lipid A biosynthesis is catalyzed by uridine diphosphate-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC), a zinc-dependent deacetylase, and inhibitors of LpxC may be useful in the development of antibacterial agents targeting a broad spectrum of Gram-negative bacteria. Here, we report the design of amphipathic benzoic acid derivatives that bind in the hydrophobic tunnel in the active site of LpxC. The hydrophobic tunnel accounts for the specificity of LpxC toward substrates and substrate analogues bearing a 3-O-myristoyl substituent. Simple benzoic acid derivatives bearing an aliphatic 'tail' bind in the hydrophobic tunnel with micromolar affinity despite the lack of a glucosamine ring like that of the substrate. However, although these benzoic acid derivatives each contain a negatively charged carboxylate 'warhead' intended to coordinate to the active site zinc ion, the 2.25A resolution X-ray crystal structure of LpxC complexed with 3-(heptyloxy)benzoate reveals 'backward' binding in the hydrophobic tunnel, such that the benzoate moiety does not coordinate to zinc. Instead, it binds at the outer end of the hydrophobic tunnel. Interestingly, these ligands bind with affinities comparable to those measured for more complicated substrate analogue inhibitors containing glucosamine ring analogues and hydroxamate 'warheads' that coordinate to the active site zinc ion. We conclude that the intermolecular interactions in the hydrophobic tunnel dominate enzyme affinity in this series of benzoic acid derivatives.     

Clonal lineages, antibiotic resistance and virulence factors in vancomycin-resistant enterococci isolated from fecal samples of red foxes (Vulpes vulpes)

Fourteen vanA-containing enterococcal isolates were detected in seven of 52 fecal samples (13.5%) from free-ranging red foxes in Portugal. Nine of the vanA-containing isolates were Enterococcus faecium and five were E. durans. Both sequence types, ST262 and ST273, were identified among E. faecium isolates.     

The binding of anionic and nonionic surfactants to collagen through the hydrophobic effect

The adsorption of nonionic surfactants on hide powder previously treated with anionic surfactants has been studied. The adsorption of nonionic surfactants takes place through hydrophobic interactions. A mechanism has been proposed for this interaction, assuming that the nonionic surfactant has been fixed by means of secondary adsorption (hydrophobic interaction) after the primary adsorption of the anionic surfactant (ionic and hydrophobic interaction) which makes it possible.     

Design and synthesis of potent, orally active, inhibitors of carboxypeptidase U (TAFIa)

A series of 3-mercapto-propionic acid derivatives that function as reversible inhibitors of carboxypeptidase U have been prepared. We present a successful design strategy using cyclic, low basicity guanidine mimetics resulting in potent, selective and bioavailable inhibitors of carboxypeptidase U (TAFIa).     

Design and synthesis of conformationally restricted inhibitors of active thrombin activatable fibrinolysis inhibitor (TAFIa)

A series of 4,5,6,7-tetrahydro-1H-benzimidazole-5-carboxylic acid and 5,6,7,8-tetrahydroimidazo[1,2-a]pyridine-7-carboxylic acid derivatives designed as inhibitors of TAFIa has been prepared via a common hydrogenation–alkylation sequence starting from the appropriate benzimidazole and imidazopyridine system. We present a successful design strategy using a conformational restriction approach resulting in potent and selective inhibitors of TAFIa. The X-ray structure of compound 5 in complex with a H333Y/H335Q double mutant TAFI indicate that the conformational restriction is responsible for the observed potency increase.     

N-Benzyl-indolo carboxylic acids: Design and synthesis of potent and selective adipocyte fatty-acid binding protein (A-FABP) inhibitors

Small molecule inhibitors of adipocyte fatty-acid binding protein (A-FABP) have gained renewed interest following the recent publication of pharmacologically beneficial effects of such inhibitors. Despite the potential utility of selective A-FABP inhibitors within the fields of metabolic disease, inflammation and atherosclerosis, there are few examples of useful A-FABP inhibitors in the public domain. Herein, we describe the optimization of N-benzyl-tetrahydrocarbazole derivatives through the use of co-crystal structure guided medicinal chemistry efforts. This led to the identification of a potent and selective class of A-FABP inhibitors as illustrated by N-benzyl-hexahydrocyclohepta[b]indole 30.     

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.     

Structures and free-energy landscapes of the wild type and mutants of the Abeta(21-30) peptide are determined by an interplay between intrapeptide electrostatic and hydrophobic interactions

The initial events in protein aggregation involve fluctuations that populate monomer conformations, which lead to oligomerization and fibril assembly. The highly populated structures, driven by a balance between hydrophobic and electrostatic interactions in the protease-resistant wild-type Aβ_21-30 peptide and mutants E22Q (Dutch), D23N (Iowa), and K28N, are analyzed using molecular dynamics simulations. Intrapeptide electrostatic interactions were connected to calculated pK_a values that compare well with the experimental estimates. The pK_a values of the titratable residues show that E22 and D23 side chains form salt bridges only infrequently with the K28 side chain. Contacts between E22-K28 are more probable in “dried” salt bridges, whereas D23-K28 contacts are more probable in solvated salt bridges. The strength of the intrapeptide hydrophobic interactions increases as D23N < WT < E22Q < K28A. Free-energy profiles and disconnectivity representation of the energy landscapes show that the monomer structures partition into four distinct basins. The hydrophobic interactions cluster the Aβ_21-30 peptide into two basins, differentiated by the relative position of the DVG(23-25) and GSN(25-27) fragments about the G25 residue. The E22Q mutation increases the population with intact VGSN turn compared to the wild-type (WT) peptide. The increase in the population of the structures in the aggregation-prone Basin I in E22Q, which occurs solely due to the difference in charge states between the Dutch mutant and the WT, gives a structural explanation of the somewhat larger aggregation rate in the mutant. The D23N mutation dramatically reduces the intrapeptide interactions. The K28A mutation increases the intrapeptide hydrophobic interactions that promote population of structures in Basin I and Basin II whose structures are characterized by hydrophobic interaction between V24 and K28 side chains but with well-separated ends of the backbone atoms in the VGSN turn. The intrapeptide electrostatic interactions in the WT and E22Q peptides roughen the free-energy surface compared to the K28A peptide. The D23N mutation has a flat free-energy surface, corresponding to an increased population of random coil-like structures with weak hydrophobic and electrostatic interactions. We propose that mutations or sequences that enhance the probability of occupying Basin I would promote aggregation of Aβ peptides.     

Mechanistic insight of photo-induced aggregation of chicken egg white lysozyme: the interplay between hydrophobic interactions and formation of intermolecular disulfide bonds

Recently, it was reported that ultraviolet (UV) illumination could trigger the unfolding of proteins by disrupting the buried disulfide bonds. However, the consequence of such unfolding has not been adequately evaluated. Here, we report that unfolded chicken egg white lysozyme (CEWL) triggered by UV illumination can form uniform globular aggregates as confirmed by dynamic light scattering, atomic force microscopy, and transmission electron microscopy. The assembling process of such aggregates was also monitored by several other methods, such as circular dichroism, fluorescence spectroscopy, mass spectrometry based on chymotrypsin digestion, ANS-binding assay, Ellman essay, and SDS-PAGE. Our finding is that due to the dissociation of the native disulfide bonds by UV illumination, CEWL undergoes drastic conformational changes resulting in the exposure of some hydrophobic residues and free thiols. Subsequently, these partially unfolded molecules self-assemble into small granules driven by intermolecular hydrophobic interaction. With longer UV illumination or longer incubation time, these granules can further self-assemble into larger globular aggregates. The combined effects from both the hydrophobic interaction and the formation of intermolecular disulfide bonds dominate this process. Additionally, similar aggregation behavior can also be found in other three typical disulfide-bonded proteins, that is, α-lactalbumin, RNase A, and bovine serum albumin. Thus, we propose that such aggregation behavior might be a general mechanism for some disulfide-bonded proteins under UV irradiation.