Serum/glucocorticoid-regulated kinase 1 contributes to the proliferation of varicella-zoster virus and induction of cyclin B1 expression.

In this study, we investigated the role of serum/glucocorticoid-regulated kinase 1 (SGK1) in varicella-zoster virus (VZV) replication. VZV DNA replication and plaque formation were inhibited by SGK1 knockout and treatment with an SGK1 inhibitor. Furthermore, SGK1 inhibition suppressed the increase in cyclin B1 expression induced by VZV infection. These results suggest that VZV infection induces SGK1 activation, which is required for efficient viral proliferation through the expression of cyclin B1. This is the first study to report that SGK1 is involved in the VZV life cycle.

Structure activity relationship of N-1 substituted 1,5-naphthyrid-2-one analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-9).

Novel bacterial topoisomerase inhibitors (NBTIs) are the newest members of gyrase inhibitor broad-spectrum antibacterial agents, represented by the most advanced member, gepotidacin, a 4-amino-piperidine linked NBTI, which is undergoing phase III clinical trials for treatment of urinary tract infections (UTI). We have extensively reported studies on oxabicyclooctane linked NBTIs, including AM-8722. The present study summarizes structure activity relationship (SAR) of AM-8722 leading to identification of 7-fluoro-1-cyanomethyl-1,5-naphthyridin-2-one based NBTI (16, AM-8888) with improved potency and spectrum (MIC values of 0.016-4 µg/mL), with Pseudomonas aeruginosa being the least sensitive strain (MIC 4 µg/mL).

In Vitro Activities and Spectrum of the Novel Fluoroquinolone Lascufloxacin (KRP-AM1977).

Lascufloxacin exhibited a broad spectrum of activity against various clinical isolates. Furthermore, lascufloxacin showed the most potent activity against Gram-positive bacteria among the quinolones tested and incomplete cross-resistance against existing quinolone-resistant strains. Enzymatic analysis indicated that lascufloxacin had potent inhibitory activity against both wild-type and mutated target enzymes. These results suggest that lascufloxacin may be useful in treating infections caused by various pathogens, including quinolone-resistant strains.

Sulfonamide-based non-alkyne LpxC inhibitors as Gram-negative antibacterial agents.

We attempted to optimize sulfonamide-based non-alkyne LpxC inhibitors by focusing on improvements in enzyme inhibitory and antibacterial activity. It was discovered that inhibitors possessing 2-aryl benzofuran as a hydrophobe exhibited good activity. In particular, compound 21 displayed impressive antibacterial activity (E. coli MIC=0.063µg/mL, K. pneumoniae MIC=0.5µg/mL, and P. aeruginosa MIC=0.5µg/mL), and is a promising lead for further exploration as an antibacterial agent.

In Vitro and In Vivo Characterization of the Novel Oxabicyclooctane-Linked Bacterial Topoisomerase Inhibitor AM-8722, a Selective, Potent Inhibitor of Bacterial DNA Gyrase.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, and in vivo characterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli and displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergence in vitro at concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activity in vitro and in vivo.

LpxC Inhibitors: Design, Synthesis, and Biological Evaluation of Oxazolidinones as Gram-negative Antibacterial Agents.

Herein we report a scaffold-hopping approach to identify a new scaffold with a zinc binding headgroup. Structural information was used to give novel oxazolidinone-based LpxC inhibitors. In particular, the most potent compound, 23j, showed a low efflux ratio, nanomolar potencies against E. coli LpxC enzyme, and excellent antibacterial activity against E. coli and K. pneumoniae. Computational docking was used to predict the interaction between 23j and E. coli LpxC, suggesting that the interactions with C207 and C63 contribute to the strong activity. These results provide new insights into the design of next-generation LpxC inhibitors.

Structure activity relationship of pyridoxazinone substituted RHS analogs of oxabicyclooctane-linked 1,5-naphthyridinyl novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-6).

Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30 µM) profile.

Structure activity relationship of C-2 ether substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-5).

Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property.

Hydroxy tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of RHS moiety (Part-3).

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. (R)-Hydroxy-1,5-naphthyridinone left-hand side (LHS) oxabicyclooctane linked pyridoxazinone right-hand side (RHS) containing NBTIs showed a potent Gram-positive antibacterial profile. SAR around the RHS moiety, including substitutions around pyridooxazinone, pyridodioxane, and phenyl propenoids has been described. A fluoro substituted pyridoxazinone showed an MIC against Staphylococcus aureus of 0.5 µg/mL with reduced functional hERG activity (IC50 333 µM) and good in vivo efficacy [ED90 12 mg/kg, intravenous (iv) and 15 mg/kg, oral (p.o.)]. A pyridodioxane-containing NBTI showed a S. aureus MIC of 0.5 µg/mL, significantly improved hERG IC50 764 µM and strong efficacy of 11 mg/kg (iv) and 5 mg/kg (p.o.). A phenyl propenoid series of compounds showed potent antibacterial activity, but also showed potent hERG binding activity. Many of the compounds in the hydroxy-tricyclic series showed strong activity against Acinetobacter baumannii, but reduced activity against Escherichia coli and Pseudomonas aeruginosa. Bicyclic heterocycles appeared to be the best RHS moiety for the hydroxy-tricyclic oxabicyclooctane linked NBTIs.

Structure activity relationship of substituted 1,5-naphthyridine analogs of oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents (Part-4).

Bacterial resistance is rapidly growing, necessitating the need to discover new agents. Novel bacterial topoisomerase inhibitors (NBTIs) are new class of broad-spectrum antibacterial agents targeting bacterial DNA gyrase and topoisomerase IV. This class of inhibitors binds to an alternative binding site relative to fluoroquinolones and shows no cross-resistance to quinolones. NBTIs consist of three structural motifs. A structure activity relationship of the left hand motif 1,5-naphthyridine of oxabicyclooctane-linked NBTIs is described. Fifty five compounds were evaluated against a panel of key Gram-positive and Gram-negative strains of bacteria, as well as for hERG activity and five compounds were tested for in vivo efficacy in murine model of Staphylococcus aureus infection. These studies suggest that only a narrow range (activating and deactivating) of substitutions at C-2 and C-7 are tolerated for optimal antibacterial activity and spectrum. An alkoxy (methoxy) and CN at C-2, and a halogen and hydroxyl at C-7, appeared to be preferred in this series. Substitutions on the other three carbons generally have detrimental effect on the activity. No clear hERG activity SAR emerged from these substitutions.

Kibdelomycin is a bactericidal broad-spectrum aerobic antibacterial agent.

Bacterial resistance to antibiotics continues to grow and pose serious challenges, while the discovery rate for new antibiotics declines. Kibdelomycin is a recently discovered natural-product antibiotic that inhibits bacterial growth by inhibiting the bacterial DNA replication enzymes DNA gyrase and topoisomerase IV. It was reported to be a broad-spectrum aerobic Gram-positive agent with selective inhibition of the anaerobic bacterium Clostridium difficile. We have extended the profiling of kibdelomycin by using over 196 strains of Gram-positive and Gram-negative aerobic pathogens recovered from worldwide patient populations. We report the MIC50s, MIC90s, and bactericidal activities of kibdelomycin. We confirm the Gram-positive spectrum and report for the first time that kibdelomycin shows strong activity (MIC90, 0.125 µg/ml) against clinical strains of the Gram-negative nonfermenter Acinetobacter baumannii but only weak activity against Pseudomonas aeruginosa. We confirm that well-characterized resistant strains of Staphylococcus aureus and Streptococcus pneumoniae show no cross-resistance to kibdelomycin and quinolones and coumarin antibiotics. We also show that kibdelomycin is not subject to efflux in Pseudomonas, though it is in Escherichia coli, and it is generally affected by the outer membrane permeability entry barrier in the nonfermenters P. aeruginosa and A. baumannii, which may be addressable by structure-based chemical modification.

Tricyclic 1,5-naphthyridinone oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad-spectrum antibacterial agents-SAR of left-hand-side moiety (Part-2).

Novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. A series of novel oxabicyclooctane-linked NBTIs with new tricyclic-1,5-naphthyridinone left hand side moieties have been described. Compounds with a (R)-hydroxy-1,5-naphthyridinone moiety (7) showed potent antibacterial activity (e.g., Staphylococcus aureus MIC 0.25 µg/mL), acceptable Gram-positive and Gram-negative spectrum with rapidly bactericidal activity. The compound 7 showed intravenous and oral efficacy (ED50) at 3.2 and 27 mg/kg doses, respectively, in a murine model of bacteremia. Most importantly they showed significant attenuation of functional hERG activity (IC50 >170 µM). In general, lower logD attenuated hERG activity but also reduced Gram-negative activity. The co-crystal structure of a hydroxy-tricyclic NBTI bound to a DNA-gyrase complex exhibited a binding mode that show enantiomeric preference for R isomer and explains the activity and SAR. The discovery, synthesis, SAR and X-ray crystal structure of the left-hand-side tricyclic 1,5-naphthyridinone based oxabicyclooctane linked NBTIs are described.

Structures of kibdelomycin bound to Staphylococcus aureus GyrB and ParE showed a novel U-shaped binding mode.

Bacterial resistance to antibiotics continues to pose serious challenges as the discovery rate for new antibiotics fades. Kibdelomycin is one of the rare, novel, natural product antibiotics discovered recently that inhibits the bacterial DNA synthesis enzymes gyrase and topoisomerase IV. It is a broad-spectrum, Gram-positive antibiotic without cross-resistance to known gyrase inhibitors, including clinically effective quinolones. To understand its mechanism of action, binding mode, and lack of cross-resistance, we have co-crystallized kibdelomycin and novobiocin with the N-terminal domains of Staphylococcus aureus gyrase B (24 kDa) and topo IV (ParE, 24 and 43 kDa). Kibdelomycin shows a unique "dual-arm", U-shaped binding mode in both crystal structures. The pyrrolamide moiety in the lower part of kibdelomycin penetrates deeply into the ATP-binding site pocket, whereas the isopropyl-tetramic acid and sugar moiety of the upper part thoroughly engage in polar interactions with a surface patch of the protein. The isoproramic acid (1,3-dioxopyrrolidine) and a tetrahydropyran acetate group (Sugar A) make polar contact with a surface area consisting of helix α4 and the flexible loop connecting helices α3 and α4. The two arms are connected together by a rigid decalin linker that makes van del Waals contacts with the protein backbone. This "dual-arm", U-shaped, multicontact binding mode of kibdelomycin is unique and distinctively different from binding modes of other known gyrase inhibitors (e.g., coumarins and quinolones), which explains its lack of cross-resistance and low frequency of resistance. The crystal structures reported in this paper should enable design and discovery of analogues with better properties and antibacterial spectrum.

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors as broad spectrum antibacterial agents.

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC50 > 18 µM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase (IC50 = 1.02 µM) and topo IV (IC50 = 10.4 µM). AM8191 showed parenteral and oral efficacy (ED50) at less than 2.5 mg/kg doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker.

Contribution of topoisomerase IV mutation to quinolone resistance in Mycoplasma genitalium.

The mechanism of quinolone resistance in Mycoplasma genitalium remains poorly understood due to difficulties with in vitro culture, especially of clinical isolates. In this study, to confirm the association between mutations in topoisomerases and antimicrobial susceptibilities to quinolones, ciprofloxacin-resistant mutant strains were selected using the cultivable type strain ATCC 33530. Sequence analysis revealed that the mutant strains harbored mutations in topoisomerase IV: Gly81Cys in ParC, Pro261Thr in ParC, or Asn466Lys in ParE. The MICs of all quinolones tested against the mutant strains were 2- to 16-fold higher than those against the wild-type strain. No cross-resistance was observed with macrolides or tetracyclines. We determined the inhibitory activities of quinolones against DNA gyrase and topoisomerase IV in order to investigate the correlation between antimicrobial susceptibility and inhibitory activity against the target enzymes, considered the primary targets of quinolones. Furthermore, using enzymatic analysis, we confirmed that Gly81Cys in the ParC quinolone resistance-determining region (QRDR) contributed to quinolone resistance. This is the first study to isolate quinolone-resistant mutant strains of M. genitalium harboring substitutions in the parC or parE gene in vitro and to measure the inhibitory activities against the purified topoisomerases of M. genitalium.

Relationship between the expression of ompF and quinolone resistance in Escherichia coli.

The outer membrane porin protein, OmpF, is widely found in gram-negative bacteria. It is known that the decreased expression of OmpF causes resistance to multiple antibiotics, including quinolones. In order to characterize the influence of decreased OmpF expression on bacterial growth, the fitness of the ompF and gyrA mutant strain of Escherichia coli selected experimentally with quinolone was compared with that of the parent strain. The expression levels of ompF in clinical isolates and the mutant selected with quinolone were determined by real-time PCR. The bacterial growth of the experimentally selected mutants was also measured both in vitro and in a urinary tract infection model in mice. Decreased ompF phenotypes were frequently found in clinical isolates that exhibited alteration of topoisomerases. The mutant experimentally obtained by the resistance selection process with quinolone showed no loss of fitness either in vitro or in vivo. These results suggest that the decreased expression of ompF and gyrA mutation do not affect the survival of the bacteria, and in fact may be responsible for the spread of high-level resistance to quinolones.

Structural and functional studies of the HAMP domain of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli.

The HAMP domain plays an essential role in signal transduction not only in histidine kinase but also in a number of other signal-transducing receptor proteins. Here we expressed the EnvZ HAMP domain (Arg(180)-Thr(235)) with the R218K mutation (termed L(RK)) or with L(RK) connected with domain A (Arg(180)-Arg(289)) (termed LA(RK)) of EnvZ, an osmosensing transmembrane histidine kinase in Escherichia coli, by fusing it with protein S. The L(RK) and LA(RK) proteins were purified after removing protein S. The CD analysis of the isolated L protein revealed that it consists of a random structure or is unstructured. This suggests that the EnvZ HAMP domain by itself is unable to form a stable structure and that this structural fragility may be important for its role in signal transduction. Interestingly the substitution of Ala(193) in the EnvZ HAMP domain with valine or leucine in Tez1A1, a chimeric protein of Tar and EnvZ, caused a constitutive OmpC phenotype. The CD analysis of LA(RK)(A193L) revealed that this mutated HAMP domain possesses considerable secondary structures and that the thermostability of this entire LA(RK)(A193L) became substantially lower than that of LA(RK) or just domain A, indicating that the structure of the HAMP domain with the A193L mutation affects the stability of downstream domain A. This results in cooperative thermodenaturation of domain A with the mutated HAMP domain. These results are discussed in light of the recently solved NMR structure of the HAMP domain from a thermophilic bacterium (Hulko, M., Berndt, F., Gruber, M., Linder, J. U., Truffault, V., Schultz, A., Martin, J., Schultz, J. E., Lupas, A. N., and Coles, M. (2006) Cell 126, 929-940).

The effect of pharmacokinetic/pharmacodynamic (PK/PD) parameters of gatifloxacin on its bactericidal activity and resistance selectivity against clinical isolates of Streptococcus pneumoniae.

The impact of the pharmacokinetic/pharmacodynamic (PK/PD) parameters (the 24h area under the concentration-time curve [AUC24h]/minimum inhibitory concentration [MIC] and maximum concentration in serum [Cmax]/MIC ratio) after single oral dosing of gatifloxacin on its bactericidal activity and resistance selectivity against quinolone-susceptible clinical isolates of Streptococcus pneumoniae J-69 was investigated using an in vitro PK model. The MICs of gatifloxacin, levofloxacin, and ciprofloxacin were 0.25, 1, and 1 micro g/ml, respectively. When the range of AUC24h/MIC ratios was varied from 9.0 to 36 with a constant Cmax/MIC ratio of 3.4, the bactericidal activity was correlated with the AUC24h/MIC ratios. Eradication was observed at an AUC24h/MIC ratio of 36. On the other hand, the resistance selectivity was associated with the Cmax/MIC ratio. Mutant strains were selected at a Cmax/MIC ratio of 0.84, but not 1.7 with a constant AUC24h/MIC ratio of 9.0. These results suggested that an AUC24h/MIC ratio of > or =36 and a Cmax/MIC ratio of > or =1.7 might be possible benchmarks to show enough bacterial eradication and prevention of emergence of resistant strains to gatifloxacin, respectively. When the serum concentrations after clinical oral dosing of gatifloxacin (200 mg b.i.d.), levofloxacin (100 mg t.i.d.), and ciprofloxacin (200 mg t.i.d.) were simulated, the bactericidal activity of gatifloxacin was higher than those of levofloxacin and ciprofloxacin. Moreover, no resistant strain was obtained by the exposure to gatifloxacin and levofloxacin, whereas ciprofloxacin selected resistant strains. The clinically relevant oral dosage of gatifloxacin was anticipated to result in a high AUC24h/MIC90 ratio of 81 and a Cmax/MIC90 ratio of 4.4, suggesting that this agent is clinically effective in the treatment of pneumococcal infections.

Contribution of the 8-methoxy group to the activity of gatifloxacin against type II topoisomerases of Streptococcus pneumoniae.

The inhibitory activities (50% inhibitory concentrations [IC(50)s]) of gatifloxacin and other quinolones against both DNA gyrase and topoisomerase IV of the wild-type Streptococcus pneumoniae IID553 were determined. The IC(50)s of 10 compounds ranged from 4.28 to 582 microg/ml against DNA gyrase and from 1.90 to 35.2 microg/ml against topoisomerase IV. The inhibitory activity against DNA gyrase was more varied than that against topoisomerase IV among fluoroquinolones. The IC(50)s for DNA gyrase of the 8-methoxy quinolones gatifloxacin and AM-1147 were approximately seven times lower than those of their 8-H counterparts AM-1121 and ciprofloxacin, whereas the IC(50)s for topoisomerase IV were 1.5 times lower. Moreover, the IC(50) ratios (IC(50) for DNA gyrase/IC(50) for topoisomerase IV) of gatifloxacin, AM-1147, and moxifloxacin, which possess 8-methoxy groups, were almost the same. The 8-methoxy quinolones showed higher antibacterial activity and less mutant selectivity against IID553 than their 8-H counterparts. These results suggest that the 8-methoxy group enhances both target inhibition, especially for DNA gyrase, leading to potent antipneumococcal activity and dual inhibition against both DNA gyrase and topoisomerase IV in the bacterial cell.