Christopher T. Walsh

Antibiotics for Emerging Pathogens

Toward New Scaffolds Most existing antibiotics are derived from a small number of core molecular structures or scaffolds. As more and more pathogens emerge that are resistant to existing antibiotics, Fischbach and Walsh (p. 1089) review why renewed efforts must be made to find not only new antibiotics but new scaffolds. Approaches in the areas of natural products, synthesis, and target-based discovery are all yielding promising antibiotics candidates. The battle against resistance should also involve researching narrow-spectrum antibiotics and using combination therapies to extend the usefulness of drugs with high intrinsic resistance rates. Antibiotic-resistant strains of pathogenic bacteria are increasingly prevalent in hospitals and the community. New antibiotics are needed to combat these bacterial pathogens, but progress in developing them has been slow. Historically, most antibiotics have come from a small set of molecular scaffolds whose functional lifetimes have been extended by generations of synthetic tailoring. The emergence of multidrug resistance among the latest generation of pathogens suggests that the discovery of new scaffolds should be a priority. Promising approaches to scaffold discovery are emerging; they include mining underexplored microbial niches for natural products, designing screens that avoid rediscovering old scaffolds, and repurposing libraries of synthetic molecules for use as antibiotics.

Molecular mechanisms that confer antibacterial drug resistance

Antibiotics — compounds that are literally ‘against life’ — are typically antibacterial drugs, interfering with some structure or process that is essential to bacterial growth or survival without harm to the eukaryotic host harbouring the infecting bacteria. We live in an era when antibiotic resistance has spread at an alarming rate and when dire predictions concerning the lack of effective antibacterial drugs occur with increasing frequency. In this context it is apposite to ask a few simple questions about these life-saving molecules. What are antibiotics? Where do they come from? How do they work? Why do they stop being effective? How do we find new antibiotics? And can we slow down the development of antibiotic-resistant superbugs?

Lessons from natural molecules

Natural products have inspired chemists and physicians for millennia. Their rich structural diversity and complexity has prompted synthetic chemists to produce them in the laboratory, often with therapeutic applications in mind, and many drugs used today are natural products or natural-product derivatives. Recent years have seen considerable advances in our understanding of natural-product biosynthesis. Coupled with improvements in approaches for natural-product isolation, characterization and synthesis, these could be opening the door to a new era in the investigation of natural products in academia and industry.

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.

Genetics and Assembly Line Enzymology of Siderophore Biosynthesis in Bacteria

SUMMARY The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.

Where will new antibiotics come from

There is a constant need for new antibacterial drugs owing to the inevitable development of resistance that follows the introduction of antibiotics to the clinic. When a new class of antibiotic is introduced, it is effective at first, but will eventually select for survival of the small fraction of bacterial populations that have an intrinsic or acquired resistance mechanism. Pathogens that are resistant to multiple drugs emerge around the globe, so how robust are antibiotic discovery processes?

New antibiotics from bacterial natural products.

For the past five decades, the need for new antibiotics has been met largely by semisynthetic tailoring of natural product scaffolds discovered in the middle of the 20th century. More recently, however, advances in technology have sparked a resurgence in the discovery of natural product antibiotics from bacterial sources. In particular, efforts have refocused on finding new antibiotics from old sources (for example, streptomycetes) and new sources (for example, other actinomycetes, cyanobacteria and uncultured bacteria). This has resulted in several newly discovered antibiotics with unique scaffolds and/or novel mechanisms of action, with the potential to form a basis for new antibiotic classes addressing bacterial targets that are currently underexploited.

Suicide substrates: mechanism-based enzyme inactivators

Abstract This review article defines suicide substrates as a class of irreversible inactivators of specific target enzymes where the target enzyme participates in its own destruction by catalytic unmasking of a latent functional group at some stage in the catalytic cycle of the enzyme. Criteria for evaluation of suicide substrates are presented. Then, examples of the various types of latent functional groups and their likely enzymic routes of activation are presented for both natural and synthetic compounds. These examples include gabaculine, secobarbital, allopurinol, clavulanate and penicillin sulfones, tranylcypramine, rhizobitoxin, fluoroalanine, 5-fluorodeoxyuridylate and mitomycin.

Identification of a Mycobacterium tuberculosis gene cluster encoding the biosynthetic enzymes for assembly of the virulence-conferring siderophore mycobactin.

BACKGROUND Many pathogenic bacteria secrete iron-chelating siderophores as virulence factors in the iron-limiting environments of their vertebrate hosts to compete for ferric iron. Mycobacterium tuberculosis mycobactins are mixed polyketide/nonribosomal peptides that contain a hydroxyaryloxazoline cap and two N-hydroxyamides that together create a high-affinity site for ferric ion. The mycobactin structure is analogous to that of the yersiniabactin and vibriobactin siderophores from the bacteria that cause plague and cholera, respectively. RESULTS A ten-gene cluster spanning 24 kilobases of the M. tuberculosis genome, designated mbtA-J, contains the core components necessary for mycobactin biogenesis. The gene products MbtB, MbtE and MbtF are proposed to be peptide synthetases, MbtC and MbtD polyketide synthases, MbtI an isochorismate synthase that provides a salicylate activated by MbtA, and MbtG a required hydroxylase. An aryl carrier protein (ArCP) domain is encoded in mbtB, and is probably the site of siderophore chain initiation. Overproduction and purification of the mbtB ArCP domain and MbtA in Escherichia coli allowed validation of the mycobactin initiation hypothesis, as sequential action of PptT (a phosphopantetheinyl transferase) and MbtA (a salicyl-AMP ligase) resulted in the mbtB ArCP domain being activated as salicyl-S-ArCP. CONCLUSIONS Mycobactins are produced in M. tuberculosis using a polyketide synthase/nonribosomal peptide synthetase strategy. The mycobactin gene cluster has organizational homologies to the yersiniabactin and enterobactin synthetase genes. Enzymatic targets for inhibitor design and therapeutic intervention are suggested by the similar ferric-ion ligation strategies used in the siderophores from Mycobacteria, Yersinia and E. coli pathogens.

Bacterial resistance to vancomycin: five genes and one missing hydrogen bond tell the story

A plasmid-borne transposon encodes enzymes and regulator proteins that confer resistance of enterococcal bacteria to the antibiotic vancomycin. Purification and characterization of individual proteins encoded by this operon has helped to elucidate the molecular basis of vancomycin resistance. This new understanding provides opportunities for intervention to reverse resistance.