Penicillin binding proteins (PBPs) are well validated antimicrobial targets, but the prevalence of β-lactamase driven resistance and, more rarely, target-based mutations, necessitates new classes of PBP-targeting drugs. Here we describe the discovery and optimisation of bicyclic peptide (Bicycle®) inhibitors of E. coli PBP3 (EcPBP3) using a proprietary phage display platform, and their conjugation to linear antimicrobial peptides to confer outer membrane permeation. These molecules exhibited high-affinity binding to E. coli PBP3 and a viable spectrum of killing activity against clinically relevant species of the Enterobacterales. X-ray crystallography was used to explore the mode of binding to PBP3, enabling increased target affinity and improvement of in vitro stability. These compounds bind to the transpeptidase active site cleft of PBP3 and represent, to our knowledge, a novel non-β-lactam chemical class of high affinity, non-covalent penicillin binding protein inhibitors. This work demonstrates an approach to rapidly find binders to antimicrobial targets, combined with an entry mechanism to provide access to the Gram negative cell.

Ten years ago, the O’Neill Review on Antimicrobial Resistance called for a major effort to discover and develop new antimicrobials. It outlined a strategy for achieving this through a combination of “push” and “pull” incentives and, importantly, measures to reduce the cost of antimicrobial development. The UK has played an important role in mobilising a global effort to implement this strategy. A lot of progress has been made, but the current situation does not match the expressed ambition. Recent developments in the science and technology capabilities of several middle-income countries and their pharmaceutical sectors, including in research and development, are creating new opportunities for implementing a strategy that could enable sustainable and affordable antimicrobial discovery, development and manufacturing. This would entail new kinds of international partnership based on the principle of shared commitment to contribute to a global public good but with differentiated responsibilities. The UK is well-placed to continue its catalytic role within international leadership, but a more systematic and implementable approach is required at national and cross-national levels.

Pseudomonas aeruginosa (PA) is a Gram negative opportunistic pathogen that is an important cause of nosicomial and antibiotic resistant infections.It can cause a myriad of infectious diseases including sepsis, pneumonia, skin and soft-tissue infections and osteomyelitis.Some strains are resistant to all available antibiotics.As a result, this organism is found near the top of WHO and CDC lists of bacteria that need new antibiotic development.In PA, the murI gene encodes the enzyme glutamate racemase (MurI).This enzyme catalyzes the interconversion of D-and L-glutamate.In bacteria, like PA it provides D-glutamate for cell wall biosynthesis.Since D-glutamate is an essential component of the bacterial cell wall peptidoglycan, glutamate racemase is an essential enzyme for PA growth and function.As a result, interfering in D-glutamate production by inhibiting glutamate racemase is potentially attractive as a target for antibacterial drug discovery.

Acinetobacter baumannii is a gram-negative bacterial pathogen that causes challenging nosocomial infections. β-lactam targeting of penicillin-binding protein (PBP)–mediated cell wall peptidoglycan (PG) formation is a well-established antimicrobial strategy. Exposure to carbapenems or zinc (Zn)-deprived growth conditions leads to a rod-to-sphere morphological transition in A. baumannii , an effect resembling that caused by deficiency in the RodA–PBP2 PG synthesis complex required for cell wall elongation. While it is recognized that carbapenems preferentially acylate PBP2 in A. baumannii and therefore block the transpeptidase function of the RodA–PBP2 system, the molecular details underpinning cell wall elongation inhibition upon Zn starvation remain undefined. Here, we report the X-ray crystal structure of A. baumannii PBP2, revealing an unexpected Zn coordination site in the transpeptidase domain required for protein stability. Mutations in the Zn-binding site of PBP2 cause a loss of bacterial rod shape and increase susceptibility to β-lactams, therefore providing a direct rationale for cell wall shape maintenance and Zn homeostasis in A. baumannii . Furthermore, the Zn-coordinating residues are conserved in various β- and γ-proteobacterial PBP2 orthologs, consistent with a widespread Zn-binding requirement for function that has been previously unknown. Due to the emergence of resistance to virtually all marketed antibiotic classes, alternative or complementary antimicrobial strategies need to be explored. These findings offer a perspective for dual inhibition of Zn-dependent PG synthases and metallo-β-lactamases by metal chelating agents, considered the most sought-after adjuvants to restore β-lactam potency against gram-negative bacteria.

An accumulation of evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesised around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to land plants at least as evolved as pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. We employed mass spectrometry and enzymology in a twofold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens . To drive the accumulation of peptidoglycan pathway intermediates, P.patens was cultured with the antibiotics phosphomycin, D-cycloserine and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the TCA-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from UDP-Glc N Ac through to the UDP-Mur N Ac-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram negative bacteria, including cyanobacteria, incorporate meso -diaminopimelate (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram positive bacteria. To establish the specificity of D,L-DAP incorporation into the P.patens precursors, we analysed the recombinant protein, UDP-Mur N Ac-tripeptide ligase ( MurE ), from both P.patens and the cyanobacterium Anabaena sp. strain PCC 7120. Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.

Abstract Despite their clinical importance, the mechanisms by which β-lactams inhibit penicillin-binding-proteins (PBPs) are not fully defined. X-ray crystallographic and mass spectrometric data studies of a Pseudomonas aeruginosa PBP3 active site variant (Tyr409Ala) reveal evidence for the in vitro acylation of P. aeruginosa PBP3 by reaction of nitrocefin with both Ser294 and Ser349. The results reveal a potentially novel binding mode for β-lactams with PBPs and will inform the design of new PBP inhibitors.

The opportunistic human pathogen Candida glabrata has become an increasingly important threat to human health, with infections globally characterized by high mortality rates and multidrug resistance. To face this threat, more efficient diagnostic and therapeutic approaches are required, underpinning research to help define the intraspecies epidemiology, genetic variability, and therefore, diagnostic and therapeutic target stability. Previous comparative genetics studies conducted on limited numbers of strains only revealed partial resolution of chromosomal settings. In this study, by combining short- and long-read genome sequencing, phenotypic characterization, and comparative genomics over a large set of strains, we detected strict relationships between large chromosomal rearrangements and phylogenetic clades, genes subjected to different selective pressures, and new sets of genes associated with resistance to antifungals. Overall, these results not only provide a fundamental contribution to our knowledge of C. glabrata evolution and epidemiology but may also lay the foundations for the future development of tailored therapeutic approaches. IMPORTANCE The human pathogen Candida glabrata has become a global threat to human health, with infections characterized by high mortality and multidrug resistance. We have obtained nine fully assembled genomes from clinical isolates through a combination of short- and long-read sequencing approaches. The quality and completeness of such genomes and their subsequent comparison to the broadest set of genomes so far allowed us to pinpoint chromosomal rearrangements in several genomes and detect phylogenetic clades that were not associated with geographic location or isolation source. We identified a new set of genes associated with resistance to antifungals coding for adhesin or adhesin-like proteins, suggesting C. glabrata resists antifungals by forming aggregates or adhering to the host tissue. These results, which provide a fundamental contribution to our knowledge of C. glabrata evolution and epidemiology, may initiate the development of precision medicine interventions for patients with suspected or proven invasive fungal infections.

The COVID-19 pandemic has refocused attention worldwide on the dangers of infectious diseases, in terms of both global health and the effects on the world economy. Even in high income countries, health systems have been found wanting in dealing with the new infectious agent. However, the even greater long-term danger of antimicrobial resistance in pathogenic bacteria and fungi is still under-appreciated, especially among the general public. Although antimicrobial drug development faces significant scientific challenges, the gravest challenge at the moment appears to be economic, where the lack of a viable market has led to a collapse in drug development pipelines. There is therefore a critical need for governments across the world to further incentivize the development of antimicrobials. Most incentive strategies over the past decade have focused on so-called “push” incentives that bridge the costs of antimicrobial research and development, but these have been insufficient for reviving the pipeline. In this Perspective, we analyze the current incentive strategies in place for antimicrobial drug development, and focus on “pull” incentives, which instead aim to improve revenue generation and thereby resolve the antimicrobial market failure challenge. We further analyze these incentives in a broader “One Health” context and stress the importance of developing and enforcing strict protocols to ensure appropriate manufacturing practices and responsible use. Our analysis reiterates the importance of international cooperation, coordination across antimicrobial research, and sustained funding in tackling this significant global challenge. A failure to invest wisely and continuously to incentivize antimicrobial pipelines will have catastrophic consequences for global health and wellbeing in the years to come.

Abstract Introduction The National Patient Safety Agency (NPSA) noted between September 2005 to June 2009, 259 incidents relating to suprapubic catheter (SPC) insertion were reported. 9 of these were bowel perforations. BAUS produced guidelines for SPC insertion which included recommending open or ultrasound guided insertion of SPCs in patients with previous lower abdominal surgery. The aim of this audit was to assess compliance with BAUS guidelines and complications following SPC insertion. Methods All patients who had a SPC inserted in theatre at this District General Hospital (DGH) between October 2012 to October 2019 were identified. Patient demographics, ASA grade, co-morbidities, previous abdominal surgery and complications were recorded. Results A total of 154 patients (59.1% male; 40.3% female) were identified. Mean age was 65 and mode ASA was 3. 21 (13.6%) of patients had previous lower abdominal surgery. Of these 2 (10%) had ultrasound guided insertion, 3 (14%) were open, 11 (52%) had cystoscopy guided insertion alone and for 5 (24%) the method was unknown. 4 (2.6%) of patients had a bowel injury following SPC insertion. Discussion At this DGH there was poor compliance with BAUS guidelines with a significant number of patients with lower abdominal surgery not having open or ultrasound guided insertion of SPC. 2.6% of patients had a bowel injury, however none of these had previous lower abdominal surgery. For these patients BAUS guidelines were adhered to, but bowel injury was not prevented. We therefore recommend the consideration of image guided insertion of SPCs in all patients where possible.

There is an increasingly urgent need for new antibiotics, yet there is a significant and persistent economic problem when it comes to developing such medicines. The problem stems from the perceived need for a “market” to drive commercial antibiotic development. In this article, we explore abandoning the market as a prerequisite for successful antibiotic research and development. Once one stops trying to fix a market model that has stopped functioning, one is free to carry out research and development (R&D) in ways that are more openly collaborative, a mechanism that has been demonstrably effective for the R&D underpinning the response to the COVID pandemic. New “open source” research models have great potential for the development of medicines for areas of public health where the traditional profit-driven model struggles to deliver. New financial initiatives, including major push/pull incentives, aimed at fixing the broken antibiotics market provide one possible means for funding an openly collaborative approach to drug development. We argue that now is therefore the time to evaluate, at scale, whether such methods can deliver new medicines through to patients, in a timely manner.

Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

The effectiveness of β-lactam antibiotics is increasingly compromised by β-lactamases. Boron-containing inhibitors are potent serine-β-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) β-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by β-lactamase-driven resistance.

SignificanceDuring infection, microbes must survive the hostile environmental conditions of the human host. When exposed to stresses, bacteria activate an intracellular response, known as the stringent response pathway, to ensure their survival. This study connects two fundamental pathways important for cellular growth in a Gram-positive pathogen: it demonstrates that enzymes responsible for cell wall biosynthesis are connected to the stringent response pathway via their ability to ameliorate errors in protein translation. Our study was performed onS. pneumoniae, where the MurM cell wall biosynthesis enzyme, a tRNA-dependent amino acyl transferase, is linked to penicillin resistance. We now demonstrate the importance of MurM in translation quality control and establish that it serves as a gatekeeper of the stringent response pathway.

Correction for ‘Metal complexes as a promising source for new antibiotics’ by Angelo Frei et al., Chem. Sci., 2020, 11, 2627–2639.

Branched Lipid II, required for the formation of indirectly crosslinked peptidoglycan, is generated by MurM, a protein essential for high-level penicillin resistance in the human pathogen Streptococcus pneumoniae. We have solved the X-ray crystal structure of Staphylococcus aureus FemX, an isofunctional homolog, and have used this as a template to generate a MurM homology model. Using this model, we perform molecular docking and molecular dynamics to examine the interaction of MurM with the phospholipid bilayer and the membrane-embedded Lipid II substrate. Our model suggests that MurM is associated with the major membrane phospholipid cardiolipin, and experimental evidence confirms that the activity of MurM is enhanced by this phospholipid and inhibited by its direct precursor phosphatidylglycerol. The spatial association of pneumococcal membrane phospholipids and their impact on MurM activity may therefore be critical to the final architecture of peptidoglycan and the expression of clinically relevant penicillin resistance in this pathogen.

Organic synthesis underpins the evolution of weak fragment hits into potent lead compounds. Deficiencies within current screening collections often result in the requirement of significant synthetic investment to enable multidirectional fragment growth, limiting the efficiency of the hit evolution process. Diversity-oriented synthesis (DOS)-derived fragment libraries are constructed in an efficient and modular fashion and thus are well-suited to address this challenge. To demonstrate the effective nature of such libraries within fragment-based drug discovery, we herein describe the screening of a 40-member DOS library against three functionally distinct biological targets using X-Ray crystallography. Firstly, we demonstrate the importance for diversity in aiding hit identification with four fragment binders resulting from these efforts. Moreover, we also exemplify the ability to readily access a library of analogues from cheap commercially available materials, which ultimately enabled the exploration of a minimum of four synthetic vectors from each molecule. In total, 10-14 analogues of each hit were rapidly accessed in three to six synthetic steps. Thus, we showcase how DOS-derived fragment libraries enable efficient hit derivatisation and can be utilised to remove the synthetic limitations encountered in early stage fragment-based drug discovery.

Correction for 'Metal complexes as a promising source for new antibiotics' by Angelo Frei et al., Chem. Sci., 2020, 11, 2627-2639.

Streptococcus pneumoniae serotype 1 is the predominant cause of invasive pneumococcal disease in sub-Saharan Africa, but the mechanism behind its increased invasiveness is not well understood. Here, we use mouse models of lung infection to identify virulence factors associated with severe bacteraemic pneumonia during serotype-1 (ST217) infection. We use BALB/c mice, which are highly resistant to pneumococcal pneumonia when infected with other serotypes. However, we observe 100% mortality and high levels of bacteraemia within 24 hours when BALB/c mice are intranasally infected with ST217. Serotype 1 produces large quantities of pneumolysin, which is rapidly released due to high levels of bacterial autolysis. This leads to substantial levels of cellular cytotoxicity and breakdown of tight junctions between cells, allowing a route for rapid bacterial dissemination from the respiratory tract into the blood. Thus, our results offer an explanation for the increased invasiveness of serotype 1.

The multi-drug resistance of the opportunistic pathogen Acinetobacter baumannii is of growing concern, with many clinical isolates proving to be resistant to last resort as well as front line antibiotic treatments. The use of bacteriophages is an attractive alternative to controlling and treating this emerging nosocomial pathogen. In this study, we have investigated bacteriophages collected from hospital wastewater in Thailand and we have explored their activity against clinical isolates of A. baumannii. Bacteriophage vB_AbaM_PhT2 showed 28% host range against 150 multidrug resistant (MDR) isolates and whole genome sequencing did not detect any known virulence factors or antibiotic resistance genes. Purified vB_AbaM_PhT2 samples had endotoxin levels below those recommended for preclinical trials and were not shown to be directly cytotoxic to human cell lines in vitro. The treatment of human brain and bladder cell lines grown in the presence of A. baumannii with this bacteriophage released significantly less lactate dehydrogenase compared to samples with no bacteriophage treatment, indicating that vB_AbaM_PhT2 can protect from A. baumannii induced cellular damage. Our results have also indicated that there is synergy between this bacteriophage and the end line antibiotic colistin. We therefore propose bacteriophage vB_AbaM_PhT2 as a good candidate for future research and for its potential development into a surface antimicrobial for use in hospitals.