Julian G. Hurdle

Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections.

Persistent infections involving slow-growing or non-growing bacteria are hard to treat with antibiotics that target biosynthetic processes in growing cells. Consequently, there is a need for antimicrobials that can treat infections containing dormant bacteria. In this Review, we discuss the emerging concept that disrupting the bacterial membrane bilayer or proteins that are integral to membrane function (including membrane potential and energy metabolism) in dormant bacteria is a strategy for treating persistent infections. The clinical applicability of these approaches is exemplified by the efficacy of lipoglycopeptides that damage bacterial membranes and of the diarylquinoline TMC207, which inhibits membrane-bound ATP synthase. Despite some drawbacks, membrane-active agents form an important new means of eradicating recalcitrant, non-growing bacteria.

Prospects for Aminoacyl-tRNA Synthetase Inhibitors as New Antimicrobial Agents

The dramatic rise in the prevalence of antibiotic resistance among bacteria currently poses a serious threat to public health worldwide. Of particular concern are infections caused by methicillin-resistant Staphylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Enterococcus (62), and Mycobacterium tuberculosis (60), since many of these organisms are resistant to several classes of established antibiotics (60, 62). This situation is driving the search for novel antibacterial agents that inhibit targets that are essential to bacteria and that are not affected by mechanisms of resistance to current chemotherapeutic agents (18, 19). In this regard, the aminoacyl-tRNA synthetase (AaRS) enzymes have been a focus of recent research for antibacterial drug discovery. These enzymes play crucial roles in protein biosynthesis by catalyzing the synthesis of aminoacyl-tRNAs (aa-rRNA). Once these enzymes are inhibited, protein biosynthesis is halted, which in turn results in the attenuation of bacterial growth under both in vitro and infectious conditions (80). Consequently, these enzymes are interesting antibacterial drug targets. An important example of the clinical application of an AaRS inhibitor is provided by the antibiotic mupirocin (marketed as Bactroban), which selectively inactivates bacterial isoleucyl-tRNA synthetase (IleRS). This product is currently the worlds most widely used topical antibiotic for the control of MRSA (12). In recent years, reports of compounds (both natural and synthetic) which inhibit AaRS have increased. While many of these new inhibitors also affect the counterpart human enzymes, there has been some success in identifying molecules that are specific for bacterial AaRS enzymes and that also exhibit antibacterial activities against several species in experimental infections (37, 54, 71, 75). However, mupirocin is currently the only clinically available AaRS inhibitor and therefore acts as the paradigm for the prospective clinical development and deployment of future AaRS inhibitors. Several suggestions for the prioritization of targets in the current process of antibiotic discovery and development have been made (63). The design of antibiotics which minimize the potential for the subsequent emergence of resistance is paramount (23, 32, 33, 63, 78). An opportunity to minimize drug-resistant organisms occurs when resistance emergence is accompanied by substantial reductions in the biological fitness of bacteria (3, 27, 59). Consequently, it is perceived that unfit drug-resistant mutants would be unable to survive upon withdrawal of an antibacterial drug and that policies involving antibiotic cycling would provide a means of eliminating drug-resistant mutants from natural populations (3, 27, 51, 58, 59). However, drug targets for which this approach may be appropriate have not yet been clearly identified, since in most cases compensatory evolution restores the fitness of drug-resistant mutants without the concomitant loss of resistance (2, 3). Nevertheless, recent novel findings in our laboratory suggest that AaRS could represent therapeutic targets where the loss of fitness associated with resistance could be compatible with antimicrobial restriction policies to eliminate resistance (46, 48). Another opportunity to search for new drugs with reduced potential to select drug-resistant variants concerns the identification of novel agents with the capacity for multiple-target inhibition (23, 63, 76). Simultaneous inhibition of more than one molecular target renders the emergence of resistance less likely because mutations are required in all targets to confer resistance to the drug (23). The β-lactam antibiotics provide an excellent example to support this contention (76), since they are multisite inhibitors which target bacterial penicillin binding proteins (PBPs). Resistance to β-lactam antibiotics rarely results from target site modification and is more commonly due to expression of β-lactamases, efflux mechanisms, or β-lactam-resistant PBPs acquired by horizontal gene transfer (76). Approaches that have been considered or that are currently under investigation include dual or multitargeting of DNA topoisomerase IV and DNA gyrase (78); cell wall biosynthetic ATP-dependent amino acid ligases (33); MurA and AroA (32); DNA gyrase and dihydropteroate synthetase (1); and DNA gyrase, DNA topoisomerase IV, and rRNA (43). Due to the existence of homologous sequences in phylogenetically related synthetases (Table ​(Table1),1), there is an important opportunity to develop single molecules which simultaneously inhibit multiple AaRS enzymes. These molecules could prove essential in limiting the emergence of both chromosomally and horizontally acquired forms of resistance to AaRS inhibitors. TABLE 1. Structural classification of aminoacyl-tRNA synthetase (49) In this minireview, we examine the prospects for the development of several recently reported AaRS inhibitors as chemo-therapeutic agents. The chemistry of these inhibitors will not be described in detail, as comprehensive reviews of these compounds from chemical perspectives have already been published (54, 71). Consequently, we examine the modes of action and potential mechanisms of clinical resistance to these compounds. We also discuss ways in which novel strategies aimed at reducing the emergence of resistance to these compounds can be exploited.

Analysis of Mupirocin Resistance and Fitness in Staphylococcus aureus by Molecular Genetic and Structural Modeling Techniques

ABSTRACT Chromosomal resistance to mupirocin in clinical isolates of Staphylococcus aureus arises from V588F or V631F mutations in isoleucyl-tRNA synthetase (IRS). Whether these are the only IRS mutations that confer mupirocin resistance or simply those that survive in the clinic is unknown. Mupirocin-resistant mutants of S. aureus 8325-4 were therefore generated to examine their ileS genotypes and the in vitro and in vivo fitness costs associated with them before and after compensatory evolution. Most spontaneous first-step mupirocin-resistant mutants carried V588F or V631F mutations in IRS, but a new mutation (G593V) was also identified. Second-step mutants carried combinations of previously identified IRS mutations (e.g., V588F/V631F and G593V/V631F), but additional combinations also occurred involving novel mutations (R816C, H67Q, and F563L). First-step mupirocin-resistant mutants were not associated with substantial fitness costs, a finding that is consistent with the occurrence of V588F or V631F mutations in the IRS of clinical strains. Second-step mutants were unfit, but fitness could be restored by subculture in the absence of mupirocin. In most cases, this was the result of compensatory mutations that also suppressed mupirocin resistance (e.g., A196V, E190K, and E195K), despite retention of the original mutations conferring resistance. Structural explanations for mupirocin resistance and loss of fitness were obtained by molecular modeling of mutated IRS enzymes, which provided data on mupirocin binding and interaction with the isoleucyl-AMP reactive intermediate.

A microbiological assessment of novel nitrofuranylamides as anti-tuberculosis agents

OBJECTIVES Nitrofuranylamides (NFAs) are nitroaromatic compounds that have recently been discovered and have potent anti-tuberculosis (TB) activity. A foundational study was performed to evaluate whether this class of agents possesses microbiological properties suitable for future antimycobacterial therapy. METHODS Five representative compounds of the NFA series were evaluated by standard microbiological assays to determine MICs, MBCs, activity against anaerobic non-replicating persistent Mycobacterium tuberculosis, post-antibiotic effects (PAEs), antibiotic synergy and the basis for resistance. RESULTS The antimicrobial activity of these compounds was restricted to bacteria of the M. tuberculosis complex, and all compounds were highly active against drug-susceptible and -resistant strains of M. tuberculosis, with MICs 0.0004-0.05 mg/L. Moreover, no antagonism was observed with front-line anti-TB drugs. Activity was also retained against dormant bacilli in two in vitro low-oxygen models for M. tuberculosis persistence. A long PAE was observed, which was comparable to that of rifampicin, but superior to isoniazid and ethambutol. Spontaneous NFA-resistant mutants arose at a frequency of 10(-5)-10(-7), comparable to that for isoniazid (10(-5)-10(-6)). Some of these mutants exhibited cross-resistance to one or both of the nitroimidazoles PA-824 and OPC-67683. Cross-resistance was associated with inactivation of the reduced F(420)-deazaflavin cofactor pathway and not with inactivation of the Rv3547, the nitroreductase for PA-824 and OPC-67683. CONCLUSIONS Based on these studies, NFAs have many useful antimycobacterial properties applicable to TB chemotherapy and probably possess a unique mode of action that results in good activity against active and dormant M. tuberculosis. Therefore, the further development of lead compounds in this series is warranted.

The structure-activity relationship of urea derivatives as anti-tuberculosis agents

The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure-activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolase enzymes. The inhibitors also showed potent inhibition of humans soluble epoxide hydrolase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolase inhibition towards the M. tuberculosis enzymes.

Structural studies of pterin-based inhibitors of dihydropteroate synthase.

Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.

Synthesis, Structure–Activity Relationship Studies, and Antibacterial Evaluation of 4-Chromanones and Chalcones, as Well as Olympicin A and Derivatives

On the basis of recently reported abyssinone II and olympicin A, a series of chemically modified flavonoid phytochemicals were synthesized and evaluated against Mycobacterium tuberculosis and a panel of Gram-positive and -negative bacterial pathogens. Some of the synthesized compounds exhibited good antibacterial activities against Gram-positive pathogens including methicillin resistant Staphylococcus aureus with minimum inhibitory concentration as low as 0.39 μg/mL. SAR analysis revealed that the 2-hydrophobic substituent and the 4-hydrogen bond donor/acceptor of the 4-chromanone scaffold together with the hydroxy groups at 5- and 7-positions enhanced antibacterial activities; the 2′,4′-dihydroxylated A ring and the lipophilic substituted B ring of chalcone derivatives were pharmacophoric elements for antibacterial activities. Mode of action studies performed on selected compounds revealed that they dissipated the bacterial membrane potential, resulting in the inhibition of macromolecular biosynthesis; further studies showed that selected compounds inhibited DNA topoisomerase IV, suggesting complex mechanisms of actions for compounds in this series.

N-Substituted 3-Acetyltetramic Acid Derivatives as Antibacterial Agents

In order to expand the structure-activity relationship of tetramic acid molecules with structural similarity to the antibiotic reutericyclin, 22 compounds were synthesized and tested against a panel of clinically relevant bacteria. Key structural changes on the tetramic acid core affected antibacterial activity. Various compounds in the N-alkyl 3-acetyltetramic acid series exhibited good activity against Gram-positive bacterial pathogens including Bacillus anthracis, Propionibacterium acnes, Enterococcus faecalis, and both Methicillin-sensitive and -resistant Staphylococcus aureus.

Novel acyl phosphate mimics that target PlsY, an essential acyltransferase in gram-positive bacteria.

PlsY is a recently discovered acyltransferase that executes an essential step in membrane phospholipid biosynthesis in Gram‐ positive bacteria. By using a bioisosteric replacement approach to generate substrate‐based inhibitors of PlsY as potential novel antibacterial agents, a series of stabilized acyl phosphate mimetics, including acyl phosphonates, acyl α,α‐difluoromethyl phosphonates, acyl phosphoramides, reverse amide phosphonates, acyl sulfamates, and acyl sulfamides were designed and synthesized. Several acyl phosphonates, phosphoramides, and sulfamates were identified as inhibitors of PlsY from Streptococcus pneumoniae and Bacillus anthracis. As anticipated, these inhibitors were competitive inhibitors with respect to the acyl phosphate substrate. Antimicrobial testing showed the inhibitors to have generally weak activity against Gram‐positive bacteria with the exception of some acyl phosphonates, reverse amide phosphonates, and acyl sulfamates, which had potent activity against multiple strains of B. anthracis.

Mode of Action and Bactericidal Properties of Surotomycin against Growing and Nongrowing Clostridium difficile

ABSTRACT Surotomycin (CB-183,315), a cyclic lipopeptide, is in phase 3 clinical development for the treatment of Clostridium difficile infection. We report here the further characterization of the in vitro mode of action of surotomycin, including its activity against growing and nongrowing C. difficile. This was assessed through time-kill kinetics, allowing a determination of the effects on the membrane potential and permeability and macromolecular synthesis in C. difficile. Against representative strains of C. difficile, surotomycin displayed concentration-dependent killing of both logarithmic-phase and stationary-phase cultures at a concentration that was ≤16× the MIC. Exposure resulted in the inhibition of macromolecular synthesis (in DNA, RNA, proteins, and cell wall). At bactericidal concentrations, surotomycin dissipated the membrane potential of C. difficile without changes to the permeability of propidium iodide. These observations are consistent with surotomycin acting as a membrane-active antibiotic, exhibiting rapid bactericidal activities against growing and nongrowing C. difficile.