Multidrug-resistant Gram-negative bacteria are a particular problem in both hospital and community settings and treatment is rendered more difficult by low intrinsic permeability to antibacterial compounds and by the presence of multidrug efflux pumps. The small number of molecular targets against which antibacterial compounds are directed is thought to have contributed to the emergence of multidrug-resistance but, despite much effort, only two new classes of antibiotics have reached the clinic in the past forty years. Connecting phenotype with mechanism in cell-based screening programmes has proved a significant challenge, but scientists at McMaster University have used high copy suppression to determine the cellular targets of new antibacterial leads identified by high-throughput screening. In this approach, the abundance of an essential target at high copy is believed to exceed the amount of available compound, leading to a suppression of a growth-inhibitory phenotype. The team used an array of E. coli clones over-expressing essential genes to screen for those that were able to suppress the activity of high-throughput screening actives. As well as discovering new chemical genetic interactions for some known antibiotics, the team identified MAC13243, a new antibacterial compound that interferes with the bacterial lipoprotein targeting pathway. MAC13243 inhibits the function of LolA, a key component of this pathway, which is present in Gram-negative (but not Gram-positive) bacteria, and is the first compound that has been shown to act via this mechanism.

A small array of tetrahydotriazines was then prepared to explore structure-activity relationships around MAC13243. Key findings were that a Cl- or Br-substituent in the para-position of the thiobenzyl moiety is needed for good activity, and that potency is maintained if the dimethoxy-phenethyl group is truncated to a methyl group (Compound 19). Both MAC13243 and Compound 19 showed good activity against a number of Gram-negative bacteria, including clinical isolates, but had no impact on Gram-positive organisms, confirming their proposed mechanism of action. The effectiveness of MAC13243 was not impaired by over-expression of acrB, which encodes the most pervasive multidrug efflux system, suggesting that the compound may represent an excellent starting point for the development of new treatments for infections caused by multidrug-resistant Gram-negative organisms.

The study is published in the journal Nature Chemical Biology.

Since the late 1980s, when nitric oxide (NO) was first shown to play a physiological role in mammals, this small molecule has been found to be a key mediator of an extraordinary variety of biological processes including blood pressure regulation, learning and memory, penile erection, digestion and the fighting of infection and cancer. Researchers at NYU School of Medicine have shown that bacteria also use NO to protect against oxidative stress and a new study by the same team shows that a broad spectrum of antibacterial drugs kill bacteria (at least in part) by inducing oxidative stress and that this effect is opposed by NO.

As well as opposing the antibacterial effects of oxidative stress, NO also reduced the effectiveness of antibiotics by chemical modifications that resulted in detoxification. Antibiotics were found to be more potent when the NO-mediated bacterial defence was eliminated, suggesting that co-administration of an inhibitor of NO-synthase could increase the effectiveness of existing antibiotics and might make antibiotic-resistant bacteria such as MRSA and anthrax more sensitive to available drugs during acute infection.

The study was published on September 11^th in the journal Science.

Brain infections such as bacterial meningitis and encephalitis can cause death or serious disability and are difficult to treat with conventional antibiotics because of poor CNS-penetration and bacterial drug-resistance. Small cationic antimicrobial peptides form an important part of the host defence system and have potential as therapeutic agents. Unlike conventional antibiotics, antimicrobial peptides appear to be bacteriocidal rather than bacteriostatic, require a short contact time to induce killing, and do not easily induce resistance. Most small antimicrobial peptides form α-helices or β-sheet-like structures that can insert into, and subsequently disrupt, negatively charged bacterial cell surfaces.

Scientists at Singapore’s Institute of Bioengineering and Nanotechnology have described novel antimicrobial peptide nanoparticles that are able to readily cross the blood brain barrier. The nanoparticles are self-assembled from an amphiphilic oligopeptide containing a motif that promotes cell penetration. The nanoparticles were effective against a range of bacteria, yeasts and fungi in vitro and were also more effective against Staphylococcus aureus infection in mice than their unassembled peptide counterparts. Importantly, the nanoparticles were also able to cross the blood brain barrier and suppress bacterial growth in rabbits with Staphylococcus aureus meningitis. The study showed that antimicrobial nanoparticles are effective against bacterial brain infections in animals and do not damage red blood cells, liver or kidneys at the tested doses. The next challenge will be to develop nanoparticles that can be used in people.

The study is published in the journal Nature Nanotechnology.

Although the ubiquitination pathway in eukaryotes was characterised in the early 1980s, it has only recently been recognised that bacteria also tag proteins to determine their fate. Ubiquitination controls a variety of cellular processes, but one of its major roles is to label proteins for degradation by the proteasome. In Mycobacterium tuberculosis (Mtb), the small ubiquitin-like protein that targets other proteins for proteasomal degradation is called prokaryotic ubiquitin-like protein (Pup) and, because proteasome function is essential for the Mtb virulence, interruption of the Pup pathway could potentially be a target for anti-tuberculosis drugs. Writing in Nature Structural & Molecular Biology, researchers at the Institute of Molecular Biology & Biophysics of ETH Zurich have now described an enzyme called Pup deaminase (Dop) that is involved in the Pupylation of proteins in Mtb. Dop modifies Pup by deaminating the C-terminal glutamine to glutamate and the Pup ligase, proteasome accessory factor A (PafA), then couples the modified Pup to the ε-amino group of lysine residues in the target proteins. This formation of an isopeptide bond requires hydrolysis of ATP to ADP, suggesting that the C-terminal glutamate of the modified Pup is activated for attachment via phosphorylation.

Since the Pupylation pathway differs from the ubiquitination pathway, drugs targeting the bacterial Pup pathway may offer the potential for safe and effective treatments for Mtb.

Recent research has shown that gene expression can be regulated at the level of mRNA by riboswitches. A riboswitch is an aptamer region on an mRNA molecule that can specifically bind a small effector molecule, causing changes in the structure of the expression platform and so regulating the activity of the mRNA. Riboswitches most usually switch off the ability of mRNA to carry out protein synthesis but can also switch it on. A variety of riboswitch classes have been identified, with most of the examples being discovered in bacteria including E. coli and streptococcus as well as bacteria causing anthrax, gonorrhoea, meningitis and dysentry.

Researchers at the University of Rochester Medical Center have now solved the crystal structure of the smallest known riboswitch, the preQ₁ riboswitch. The preQ₁ riboswitch controls the ability of bacteria to produce queuosine (Q), a molecule which enables accurate gene expression by overcoming an inbuilt defect in the mRNA-ribosome-tRNA system known as tRNA wobble, and which is essential for the survival of many important pathological bacteria. The preQ₁ riboswitch ‘senses’ the level of preQ₁, a precursor to Q. If too much preQ₁ is present, genes responsible for producing preQ₁, or for its transport, are shut down. The preQ₁ precursor, known as preQ₀, has the same effect in reducing production of Q. One gene that is regulated by the preQ₁ riboswitch is that which codes for the enzyme queF, which converts preQ₀ into preQ₁.

The structure of the preQ₁ riboswitch from Thermoanaerobacter tengcongensis complexed with preQ₀ shows preQ₀ bound in a buried pocket. The structure also reveals how the first base of the mRNA ribosome binding site binds to a loop of the riboswitch, and how the loop end of the preQ₁ riboswitch aptamer domain binds to preQ₀. Binding of the preQ₁ aptamer loop to the first base in the ribosome binding site was found to be mediated by a standard G to C base pairing interaction. The preQ₁ aptamer (34 nucleotides) is about 2.5-fold shorter than functionally related riboswitches that recognize similar metabolites.

An understanding of how bacterial species sequester their ribosome binding sites using divergent preQ₁ riboswitch aptamers could lead to the design of a new class of antibiotics. There is evidence that some existing antibiotics act – in part at least – by targeting riboswitches and, since riboswitches have not yet been found in human cells, the hope is that antibiotics acting on riboswitches will have a low propensity for side effects. The study is published in the Journal of Biological Chemistry.

Whereas mammalian fatty acid synthase (FASI) is a multidomain, multifunctional homodimeric protein which carries out all of the enzymatic steps needed for de novo synthesis of long chain fatty acids, bacterial fatty acid synthesis is carried out by a number of discrete enzymes, collectively known as FASII. This difference between FASI and FASII has led to the identification of FASII as a target for antibiotic therapy. The hypothesis is strengthened by the activities of the antiseptic, triclosan, the anti-Mycobacterium tuberculosis agent, isoniazid, and the antifungal antibiotic, cerulenin, which are believed to act primarily by inhibiting steps in the FASII pathway. The natural products, platensimycin and platensin, have also been shown to exhibit broad spectrum Gram-positive antibacterial activity and to inhibit fatty acid biosynthesis.

A recently published letter in the journal Nature, however, suggests that inhibition of FASII as an approach to antimicrobial therapy may be fundamentally flawed. The new study showed that major Gram-positive pathogens, such as streptococci, enterococci and staphylococci, were able to grow in the presence of FASII pathway inhibitors, cerulenin and triclosan, if supplied with exogenous fatty acids at levels that would be present in human serum. Using Streptococcus agalactiae – an opportunistic pathogen that can cause serious meningitis in newborns – as a model, the authors demonstrated that the unsaturated fatty acids, linoleic acid and oleic acid, but not the saturated fatty acids, palmitic acid and stearic acid, were able to overcome the inhibitory effect of cerulenin treatment.

The authors also demonstrated that when S agalactiae is grown in the presence of serum, there is an overall decrease in FASII gene expression. In further experiments, deletion mutants were used to demonstrate that FASII enzymes are dispensable in vivo during S agalactiae infection. Growth of all deletion mutants was severely restricted in standard Todd Hewitt (TH) medium, but all grew comparably with wild type strains in serum or in the presence of added oleic acid or linoleic acid. The mutant strains were also as virulent as wild type strains in animal models, even if the animals were treated with the hypolipidemic agent, fenofibrate. The authors believe that drugs targeting FASII would be ineffective in natural infections unless the bacteria had a requirement for specific fatty acids not present in serum.

Bacterial resistance to available antibiotics is becoming an increasing problem; methicillin-resistant Staphylococcus aureus (MRSA), which is broadly resistant to penicillins and cephalosporins, is a particular problem in hospital settings.

A recent report in the journal Science describes new compounds effective against MRSA. The compounds target FtsZ, a bacterial homologue of mammalian β-tubulin, which is essential for bacterial cell division. One of the compounds, PC190723, has been shown to have potent in vitro bactericidal activity against staphylococci, including MRSA, and also to cure mice infected with a lethal dose of MRSA.

The binding site for PC190723 has been mapped to a region of FtsZ that is analogous to the paclitaxel -binding site of tubulin. The activity of PC190723 further validates FtsZ as an antibacterial target, and provides the basis for optimisation to provide new treatments for MRSA.

Quorum sensing is used by bacteria to coordinate gene expression according to local population densities. The bacteria secrete signalling molecules and have receptors that can specifically recognize signalling molecules released by other bacteria of the same or different species. When the concentration of the signalling molecule reaches a certain concentration (i.e. many bacteria in the location), a response is triggered.

In 2006, researchers at UT Southwestern Medical Center described how blocking a newly discovered receptor in a strain of E. Coli could prevent infection. When contaminated food containing a virulent strain of E. Coli is eaten, the bacteria cause no damage until they encounter signalling molecules produced by native gut flora together with the human hormones, adrenaline and noradrenaline. These molecular signals prompt the virulent E.Coli bacteria to release enterotoxins which, in extreme cases, can be fatal.

In a recent report in the journal Science, Dr Sperandio’s group now describe the activity of a small molecule, LED209, which doesn’t inhibit bacterial growth but which markedly inhibits the virulence of several bacterial strains, both in vitro and in infected animals.

Many bacterial pathogens rely on signalling pathways using the same “adrenergic-type” receptor to promote the expression of virulence factors, so inhibition of this pathway may offer a strategy for the development of new broad-spectrum antimicrobial drugs. It is also possible that antagonists of this signalling pathway may not give rise to the widespread resistance seen with traditional antimicrobial agents.

There has been interest in the DNA polymerase sliding clamp as an antibacterial target for the last 15 years. Sliding clamp proteins, found in all organisms, encircle DNA (and slide along it!) and tether polymerases to enable rapid and processive DNA replication. The proteins are known as proliferating cell nuclear antigen (PCNA) in eukaryotes and as the β-clamp in prokaryotes. In PCNA the clamp is composed of three subunits of two domains each, whilst the bacterial β-clamp is assembled from two subunits of three domains. Although the overall structures of the eukaryotic and prokaryotic clamps are similar, there is no detectable sequence homology.

In a paper to be published in the August 12^th edition of PNAS, the authors disclose a small molecule inhibitor of the E.coli β-clamp, RU7, which differentially inhibits polymerases II, III and IV.

RU7 selectively inhibits Pol III in β-dependent replication assays, with no activity in the eukaryotic PCNA system. The compound, which has modest potency, was identified by screening for compounds able to displace a Pol III peptide from the β-clamp. The authors have also determined the co-crystal structure of RU7 bound to the clamp (pdb identifier 3d1g), paving the way for structure-based design.

PDB:3D1G