Michael J. Dawson

Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclature

This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.

Alternatives to antibiotics—a pipeline portfolio review

Antibiotics have saved countless lives and enabled the development of modern medicine over the past 70 years. However, it is clear that the success of antibiotics might only have been temporary and we now expect a long-term and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. A broader approach to address bacterial infection is needed. In this Review, we discuss alternatives to antibiotics, which we defined as non-compound approaches (products other than classic antibacterial agents) that target bacteria or any approaches that target the host. The most advanced approaches are antibodies, probiotics, and vaccines in phase 2 and phase 3 trials. This first wave of alternatives to antibiotics will probably best serve as adjunctive or preventive therapies, which suggests that conventional antibiotics are still needed. Funding of more than £1·5 billion is needed over 10 years to test and develop these alternatives to antibiotics. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches in phase 2 trials, which would be a catalyst for active engagement and investment by the pharmaceutical and biotechnology industry. Only a sustained, concerted, and coordinated international effort will provide the solutions needed for the future.

Treatment of health-care-associated infections caused by Gram-negative bacteria: a consensus statement

This consensus statement presents the conclusions of a group of academic and industrial experts who met in London in September, 2006, to consider the issues associated with the treatment of hospital infections caused by Gram-negative bacteria. The group discussed the severe clinical problems arising from the emergence of antibiotic resistance in these bacteria and the lack of new antibacterial agents to challenge the threat. The discovery of new drugs active against hospital-acquired Gram-negative bacteria is essential to prevent a future medical and social catastrophe. An important strategy to promote drug discovery will be the development of focused cooperations between academic institutions and small pharmaceutical companies.

Discovery research: the scientific challenge of finding new antibiotics

The dwindling supply of new antibiotics largely reflects regulatory and commercial challenges, but also a failure of discovery. In the 1990s the pharmaceutical industry abandoned its classical ways of seeking antibiotics and instead adopted a strategy that combined genomics with high-throughput screening of existing compound libraries. Too much emphasis was placed on identifying targets and molecules that bound to them, and too little emphasis was placed on the ability of these molecules to permeate bacteria, evade efflux and avoid mutational resistance; moreover, the compound libraries were systematically biased against antibiotics. The sorry result is that no antibiotic found by this strategy has yet entered clinical use and many major pharmaceutical companies have abandoned antibiotic discovery. Although a raft of start-up companies-variously financed by venture capital, charity or public money--are now finding new antibiotic compounds (some of them very promising in vitro or in early trials), their development through Phase III depends on financial commitments from large pharmaceutical companies, where the discouraging regulatory environment and the poor likely return on investment remain paramount issues.

Directed evolution of an amine oxidase for the preparative deracemisation of cyclic secondary amines.

Enantiomerically pure primary and secondary amines are widely used as chiral auxiliaries and resolving agents and are also valuable intermediates for the synthesis of pharmaceuticals and agrochemicals. Although enantiomerically pure amines are traditionally prepared by classical resolution of the corresponding racemate, alternative approaches have been developed based upon i) asymmetric reduction of imines, ii) hydroamination of alkenes and iii) lipase-catalysed kinetic resolution of racemic amines. However, secondary amines, many of which have pronounced biological activity, are poor substrates for lipases compared to the corresponding primary amines, with only a few documented examples in the literature. Hence the use of lipase resolution does not offer a general route to this class of chiral molecule. Moreover, to date it has generally not been possible to achieve the in situ racemisation of amines to effect a dynamic kinetic resolution process due to the relatively harsh conditions required to racemise amines. Against this backdrop, we sought to extend our chemo-enzymatic deracemisation method to encompass chiral secondary amines. Based upon our earlier work with a-amino acids, we recently reported the deracemisation of a-methylbenzyl amine (a-MBA, 1) in a one-pot procedure by the combined use of an enantioselective amine oxidase and ammonia borane as the reducing agent (Scheme 1). In order to identify an enzyme with appropriate activity and enantioselectivity towards a-methylbenzylamine, the amine oxidase from Aspergillus niger (MAO-N) was subjected to directed evolution, with (S)-1 as the probe substrate, by random mutagenesis and selection employing a high-throughput agarplate-based colorimetric screen. This approach led to the identification of an important amino acid substitution (Asn336Ser) that resulted in a variant enzyme possessing significantly enhanced activity (ca. 50-fold) and greater enantioselectivity towards 1 than the wild-type enzyme. Subsequently, we showed that this variant was also characterised by broad substrate specificity, being able to oxidize a wide range of chiral primary amines with high enantioselectivity. However, although this variant showed some activity towards chiral secondary amines (relative activity of 1-methyltetrahydroisoquinoline (MTQ, 2) 15 % of a-MBA), the rates of oxidation were too low to permit efficient preparative deracemisation reactions. Our goal therefore was to evolve a “secondary amine oxidase” for preparative-scale deracemisation reactions to complement the existing “primary amine oxidase”. The MAO-N gene used as the starting point for further directed evolution contained four amino acid substitutions compared to the wild-type. In addition to the Asn336Ser mutation, which is important for catalytic activity/enantioselectivity, mutations were Arg259Lys and Arg260Lys (improved expression) and Met348Lys (improved activity). This gene was subjected to random mutagenesis, by using the E. coli XL1-Red mutator strain (mutation frequency ca. 1–2 base changes per gene), followed by transformation and screening of the library (ca. 20 000 clones) against (R/S)-2 as the substrate, as previously described. A number of clones (ca. 10) showed greater activity than the parent, with one in particular appearing to be significantly more active. Purification of this variant amine oxidase showed that it possessed a kcat value about 5.5-fold higher than the parent towards (S)-2 (Table 1) and also a higher KM

An efficient process for production of n-acetylneuraminic acid using n-acetylneuraminic acid aldolase

N-acetyl-D-neuraminic acid (Neu5Ac) aldolase (EC 4.1.3.3) has bee reported for synthesis of Neu5Ac,1-5 but there are no reports of processes which do not have significant drawbacks for large-scale operation. Here, Neu5Ac aldolase from an overexpressing recombinant strain of Escherichia coli has been used to develop an immobilized enzyme process for production of Neu5Ac. The enzyme was immobilized onto Eupergit-C and could be reused many times in the reaction. Base-catalyzed epimerization of N-acetyl-D-glucosamine (GlcNAc) yielded GlcNAc/N-acetyl-D-mannosamine (ManNAc) mixtures (c 4:1) which could be used directly in the aldolase reaction; however, inhibition of the enzyme by GlcNAc limited the concentration of ManNAc which could be used in the reaction by this approach. This necessitated the addition of a large molar excess of pyruvate (five- to seven-fold) to drive the equilibrium over to Neu5Ac; nevertheless, a method has been developed to remove the excess pyruvate effectively by complexation with bisulfite, thus allowing Neu5Ac to be recovered by absorption onto an anion-exchange resin. In a second approach, a method has been developed to enrich GlcNAc/ManNAc mixtures for ManNAc. ManNAc can be used at high concentrations in the reaction, thus obviating the need to use a large molar excess of pyruvate. Neu5Ac can be isolated from such reaction mixtures by a simple crystallization. This work shows the importance of integrated process solutions for the effective scale-up of biotransformation reactions.

The urgent need for new antibacterial agents

I find it continually amazing that society as a whole does not recognize the consequences of rising antimicrobial resistance as the threat it most certainly is. This is not for a lack of sustained activity by those who share these concerns. Far from it. Since 1997 there have been a plethora of enquiries, reports and recommendations—many from important bodies in both Europe and North America, yet little meaningful action has materialized. Some might consider this to be rather negative and an overstatement, yet can they point out a concrete outcome to all this activity? I like to think that the UK has led the way in raising concerns that antibiotic use, especially overuse (in animals as well as man) will hasten the day when these essential agents will lose their efficacy. The Swann Committee first brought this to our attention in 1969, and in 1998 a House of Lords report starkly stated that antimicrobial resistance was a ‘major threat to public health’. Most recently, the Infectious Diseases Society of America (IDSA) and the European Union, among others, have voiced their concerns. In 2009 the WHO called antibiotic resistance one of the three greatest threats to human health, and in 2011 the focus of World Health Day was ‘Combating Antibiotic Resistance’. However, antimicrobial resistance moves on in an inexorable fashion and the prospects for new agents are as bleak as ever. Perhaps it is us, the health professionals, who are at fault, either in the nature of our message, or in approaching the wrong groups who cannot influence outcomes? The BSAC has changed tack in its report on ‘The Urgent Need’, as outlined in the articles accompanying this one. – 9 Rather than restate the concerns surrounding antimicrobial resistance, its surveillance and how it might be contained (or more accurately, how its progress might be slowed), the BSAC adopted a different approach, focusing on the barriers to discovery and development of new technologies that might combat resistance (including new antimicrobial agents) and how these might be overcome. The Working Party of the BSAC examined three areas, namely research, regulation and economics. While recognizing that these areas are not distinct and there is much important overlap, the Working Party was challenged to suggest a practical framework for action. Critically there was an awareness on the part of the Working Party that the BSAC cannot undertake this immense task on its own, and co-operation with others is key. I do not wish to précis the report here, but rather make some personal comments on what I consider to be a few important areas. In research there is a major concern that international expertise in natural product discovery is being rapidly lost—how long has it been since such an antibacterial compound has been marketed? Overoptimism in genomics and highthroughput screening as the answer to the discovery of new agents in the 1990s would appear to have put back the cause by at least a decade. Research into how to influence the public’s perceptions of the risks confronting them (hence the political response) is also needed. Most certainly the regulatory issues relating to the licensing of new antimicrobials are extremely important. The bureaucrats are risk averse, yet do not take account of the risks to society of their inaction. This would change if political concerns were more loudly voiced. It is my personal opinion that it is changes in the economic field that are most likely to yield results. We were not the first to expound the economic arguments. Everything the Working Party heard from industry makes me believe that the marketplace must change. A course of antibiotics costs a few pounds or dollars and can save lives. In hospital practice we shudder if the costs rise into the hundreds. The angiogenesis inhibitor bevacizumab (trade name Avastin) is one of the most expensive widely marketed drugs. In 2008 sales generated nearly US

Dissecting Structural and Functional Diversity of the Lantibiotic Mersacidin

2.7 billion for Genentech, yet it has only modest effects on patient survival in a number of cancers. This is not to say it should not be used, but rather that there should be a rebalancing of risks and, more importantly, benefits. I would suggest that antimicrobials (other than a few antifungals) should be at a higher premium. Antimicrobial development must allow pharmaceutical companies realistic returns on their investment. This is crucial if society is to obtain new agents. So what actions should the BSAC undertake? The Working Party has suggested a number of short-, mediumand long-term activities. These, realistically, revolve around communication, in its broadest sense, with clinicians and academics, but possibly more importantly, with opinion formers in the UK and further afield. Such a programme of work, which will not be cheap, should include other parties and could usefully include the participation of the pharmaceutical industry.

Organization of the genes encoding the biosynthesis of actagardine and engineering of a variant generation system

Summary Mersacidin is a tetracyclic lantibiotic with antibacterial activity against Gram-positive pathogens. To probe the specificity of the biosynthetic pathway of mersacidin and obtain analogs with improved antibacterial activity, an efficient system for generating variants of this lantibiotic was developed. A saturation mutagenesis library of the residues of mersacidin not involved in cycle formation was constructed and used to validate this system. Mersacidin analogs were obtained in good yield in approximately 35% of the cases, producing a collection of 82 new compounds. This system was also used for the production of deletion and insertion mutants of mersacidin. The outcome of these studies suggests that this system can be extended to produce mersacidin variants with multiple changes that will allow a full investigation of the potential use of modified mersacidins as therapeutic agents.

Characterization of the Chemoenzymatic Synthesis of N‐Acetyl‐d‐neuraminic Acid (Neu5Ac)

The biosynthetic pathway of the type B lantibiotic actagardine (formerly gardimycin), produced by Actinoplanes garbadinensis ATCC31049, has been cloned, sequenced and annotated. The gene cluster contains the gene garA that encodes the actagardine prepropeptide, a modification gene garM, involved in the dehydration and cyclization of the prepeptide, several putative transporter and regulatory genes as well as a novel luciferase‐like monooxygenase gene designated garO. Expression of these genes in Streptomyces lividans resulted in the production of ala(0)‐actagardine while deletion of the garA gene from A. garbadinensis generated a strain incapable of producing actagardine. Actagardine production was successfully restored however, by the delivery of the plasmid pAGvarX. This plasmid contains an engineered cassette of the actagardine encoding gene garA and offers an alternative route to generating extensive libraries of actagardine variants. Using this plasmid, an alanine scanning library has been constructed and the mutants analysed. Further modifications include the removal of the novel garO gene from A. garbadinensis. Deletion of this gene resulted in the production of deoxy variants of actagardine, demonstrating that the formation of the sulfoxide group is enzyme catalysed and not a spontaneous chemical modification as previously believed.