Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development.

Historically, targeting protein-protein interactions with small molecules was not thought possible because the corresponding interfaces were considered mostly flat and featureless and therefore 'undruggable'. Instead, such interactions were targeted with larger molecules, such as peptides and antibodies. However, the past decade has seen encouraging breakthroughs through the refinement of existing techniques and the development of new ones, together with the identification and exploitation of unexpected aspects of protein-protein interaction surfaces. In this Review, we describe some of the latest techniques to discover modulators of protein-protein interactions and how current drug discovery approaches have been adapted to successfully target these interfaces.

Molecular docking is a computational method for predicting the placement of ligands in the binding sites of their receptor(s). In this review, we discuss the methodological developments that occurred in the docking field in 2012 and 2013, with a particular focus on the more difficult aspects of this computational discipline. The main challenges and therefore focal points for developments in docking, covered in this review, are receptor flexibility, solvation, scoring, and virtual screening. We specifically deal with such aspects of molecular docking and its applications as selection criteria for constructing receptor ensembles, target dependence of scoring functions, integration of higher-level theory into scoring, implicit and explicit handling of solvation in the binding process, and comparison and evaluation of docking and scoring methods.

The neuraminidase inhibitors zanamivir and oseltamivir are marketed for the treatment and prophylaxis of influenza and have been stockpiled by many countries for use in a pandemic. Although recent surveillance has identified a striking increase in the frequency of oseltamivir-resistant seasonal influenza A (H1N1) viruses in Europe, the United States, Oceania, and South Africa, to date there have been no reports of significant zanamivir resistance among influenza A (H1N1) viruses or any other human influenza viruses. We investigated the frequency of oseltamivir and zanamivir resistance in circulating seasonal influenza A (H1N1) viruses in Australasia and Southeast Asia. Analysis of 391 influenza A (H1N1) viruses isolated between 2006 and early 2008 from Australasia and Southeast Asia revealed nine viruses (2.3%) that demonstrated markedly reduced zanamivir susceptibility and contained a previously undescribed Gln136Lys (Q136K) neuraminidase mutation. The mutation had no effect on oseltamivir susceptibility but caused approximately a 300-fold and a 70-fold reduction in zanamivir and peramivir susceptibility, respectively. The role of the Q136K mutation in conferring zanamivir resistance was confirmed using reverse genetics. Interestingly, the mutation was not detected in the primary clinical specimens from which these mutant isolates were grown, suggesting that the resistant viruses either occurred in very low proportions in the primary clinical specimens or arose during MDCK cell culture passage. Compared to susceptible influenza A (H1N1) viruses, the Q136K mutant strains displayed greater viral fitness than the wild-type virus in MDCK cells but equivalent infectivity and transmissibility in a ferret model.

Intracellular nucleotide binding and oligomerization domain (NOD) receptors recognize antigens including bacterial peptidoglycans and initiate immune responses by triggering the production of pro-inflammatory cytokines through activating NF-kB and MAP kinases. Receptor interacting protein kinase 2 (RIPK2) is critical for NOD-mediated NF-kB activation and cytokine production. Here we develop and characterize a selective RIPK2 kinase inhibitor, WEHI-345, which delays RIPK2 ubiquitylation and NF-kB activation downstream of NOD engagement. Despite only delaying NF-kB activation on NOD stimulation, WEHI-345 prevents cytokine production in vitro and in vivo and ameliorates experimental autoimmune encephalomyelitis in mice. Our study highlights the importance of the kinase activity of RIPK2 for proper immune responses and demonstrates the therapeutic potential of inhibiting RIPK2 in NOD-driven inflammatory diseases.

To identify mutations that can arise in highly pathogenic A(H5N1) viruses under neuraminidase inhibitor selective pressure, two antigenically different strains were serially passaged with increasing levels of either oseltamivir or zanamivir. Under oseltamivir pressure, both A(H5N1) viruses developed a H274Y neuraminidase mutation, although in one strain the mutation occurred in combination with an I222M neuraminidase mutation. The H274Y neuraminidase mutation reduced oseltamivir susceptibility significantly (900-to 2,500-fold compared to the wild type). However the dual H274Y/I222M neuraminidase mutation had an even greater impact on resistance, with oseltamivir susceptibility reduced significantly further (8,000-fold compared to the wild type). A similar affect on oseltamivir susceptibility was observed when the dual H274Y/I222M mutations were introduced, by reverse genetics, into a recombinant seasonal human A(H1N1) virus and also when an alternative I222 substitution (I222V) was generated in combination with H274Y in A(H5N1) and A(H1N1) viruses. These viruses remained fully susceptible to zanamivir but demonstrated reduced susceptibility to peramivir. Following passage of the A(H5N1) viruses in the presence of zanamivir, the strains developed a D198G neuraminidase mutation, which reduced susceptibility to both zanamivir and oseltamivir, and also an E119G neuraminidase mutation, which demonstrated significantly reduced zanamivir susceptibility (1,400-fold compared to the wild type). Mutations in hemagglutinin residues implicated in receptor binding were also detected in many of the resistant strains. This study identified the mutations that can arise in A(H5N1) under either oseltamivir or zanamivir selective pressure and the potential for dual neuraminidase mutations to result in dramatically reduced drug susceptibility.

The TRIM family of proteins is distinguished by its tripartite motif (TRIM). Typically, TRIM proteins contain a RING finger domain, one or two B-box domains, a coiled-coil domain and the more variable C-terminal domains. TRIM16 does not have a RING domain but does harbour two B-box domains. Here we showed that TRIM16 homodimerized through its coiled-coil domain and heterodimerized with other TRIM family members; TRIM24, Promyelocytic leukaemia (PML) protein and Midline-1 (MID1). Although, TRIM16 has no classic RING domain, three-dimensional modelling of TRIM16 suggested that its B-box domains adopts RING-like folds leading to the hypothesis that TRIM16 acts as an ubiquitin ligase. Consistent with this hypothesis, we demonstrated that TRIM16, devoid of a classical RING domain had auto-polyubiquitination activity and acted as an E3 ubiquitin ligase in vivo and in vitro assays. Thus via its unique structure, TRIM16 possesses both heterodimerization function with other TRIM proteins and also has E3 ubiquitin ligase activity.

Along with influenza vaccines, the world is currently almost completely dependent on two licensed drugs for the treatment or prevention of seasonal (influenza A and B viruses) and pandemic influenza (influenza A viruses). These drugs - oseltamivir (Tamiflu) and zanamivir (Relenza) - are classified as neuraminidase inhibitors (NAIs) because they act by inhibiting one of the key surface proteins of the influenza virus, the neuraminidase, which in turn reduces the ability of the virus to infect other respiratory cells. Our dependence on these drugs has arisen because of high levels of resistance with seasonal influenza viruses to the older class of anti-influenza drugs, the adamantanes (amantadine and rimantadine), combined with the lack of activity of these drugs against influenza B viruses. Recently, however, significant levels of oseltamivir-resistant influenza A(H1) seasonal influenza viruses have also been encountered, which has been associated with a single amino acid change in the viral neuraminidase (H274Y). Oseltamivir is the most widely used and stockpiled NAI and, while these A(H1) viruses are still sensitive to zanamivir, it highlights the ease with which the influenza virus can mutate and reassort to circumvent available drugs. Fortunately, the current pandemic A(H1N1) 2009 virus, which is circulating globally, remains largely sensitive to both NAIs, although a small number of oseltamivir-resistant viruses have been isolated from patients to date, again with the H274Y mutation. Clearly there is a need to use the NAI drugs prudently to ensure they remain an effective defence against future seasonal and pandemic influenza viruses, along with careful monitoring of levels of resistance in the circulating viruses combined with the further development of new anti-influenza drugs.