Roger G. Linington

Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene clusters.

Although biosynthetic gene clusters (BGCs) have been discovered for hundreds of bacterial metabolites, our knowledge of their diversity remains limited. Here, we used a novel algorithm to systematically identify BGCs in the extensive extant microbial sequencing data. Network analysis of the predicted BGCs revealed large gene cluster families, the vast majority uncharacterized. We experimentally characterized the most prominent family, consisting of two subfamilies of hundreds of BGCs distributed throughout the Proteobacteria; their products are aryl polyenes, lipids with an aryl head group conjugated to a polyene tail. We identified a distant relationship to a third subfamily of aryl polyene BGCs, and together the three subfamilies represent the largest known family of biosynthetic gene clusters, with more than 1,000 members. Although these clusters are widely divergent in sequence, their small molecule products are remarkably conserved, indicating for the first time the important roles these compounds play in Gram-negative cell biology.

On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds

Backbone N-methylation is common among peptide natural products and has a significant impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was determined by backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N-methyl variants were corroborated by computational studies on a 1024-member virtual library of N-methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (MW = 755) with three N-methyl groups, showed an oral bioavailability of 28% in rat.

Molecular Networking as a Dereplication Strategy

A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases. This approach can accommodate different ionization platforms, enabling cross correlations of MS/MS data from ambient ionization, direct infusion, and LC-based methods. Molecular networking not only dereplicates known molecules from complex mixtures, it also captures related analogues, a challenge for many other dereplication strategies. To illustrate its utility as a dereplication tool, we apply mass spectrometry-based molecular networking to a diverse array of marine and terrestrial microbial samples, illustrating the dereplication of 58 molecules including analogues.

Living in the matrix: assembly and control of Vibrio cholerae biofilms

Nearly all bacteria form biofilms as a strategy for survival and persistence. Biofilms are associated with biotic and abiotic surfaces and are composed of aggregates of cells that are encased by a self-produced or acquired extracellular matrix. Vibrio cholerae has been studied as a model organism for understanding biofilm formation in environmental pathogens, as it spends much of its life cycle outside of the human host in the aquatic environment. Given the important role of biofilm formation in the V. cholerae life cycle, the molecular mechanisms underlying this process and the signals that trigger biofilm assembly or dispersal have been areas of intense investigation over the past 20 years. In this Review, we discuss V. cholerae surface attachment, various matrix components and the regulatory networks controlling biofilm formation.

Antimalarial peptides from marine cyanobacteria: isolation and structural elucidation of gallinamide A.

As part of a continuing program to identify novel treatments for neglected parasitic diseases, the Panama International Cooperative Biodiversity Group (ICBG) program has been investigating the antimalarial potential of secondary metabolites from Panamanian marine cyanobacteria. From over 60 strains of cyanobacteria evaluated in our biological screens, the organic extract of a Schizothrix species from a tropical reef near Piedras Gallinas (Caribbean coast of Panama) showed potent initial antimalarial activity against the W2 chloroquine-resistant strain of Plasmodium falciparum. Bioassay-guided fractionation followed by 2D NMR analysis afforded the planar structure of a new and highly functionalized linear peptide, gallinamide A. Subsequent degradation and derivatization methods were used to determine the absolute configuration at most stereogenic centers in this unusual new metabolite.

Highlights of marine invertebrate-derived biosynthetic products: Their biomedical potential and possible production by microbial associants

Coral reefs are among the most productive marine ecosystems and are the source of a large group of structurally unique biosynthetic products. Annual reviews of marine natural products continue to illustrate that the most prolific source of bioactive compounds consist of coral reef invertebrates-sponges, ascidians, mollusks, and bryozoans. This account examines recent milestone developments pertaining to compounds from invertebrates designated as therapeutic leads for biomedical discovery. The focus is on the secondary metabolites, their inspirational structural scaffolds and the possible role of micro-organism associants in their biosynthesis. Also important are the increasing concerns regarding the collection of reef invertebrates for the discovery process. The case examples considered here will be useful to insure that future research to unearth bioactive invertebrate-derived compounds will be carried out in a sustainable and environmentally conscious fashion. Our account begins with some observations pertaining to the natural history of these organisms. Many still believe that a serious obstacle to the ultimate development of a marine natural product isolated from coral reef invertebrates is the problem of compound supply. Recent achievements through total synthesis can now be drawn on to forcefully cast this myth aside. The tools of semisynthesis of complex natural products or insights from SAR efforts to simplify an active pharmacophore are at hand and demand discussion. Equally exciting is the prospect that invertebrate-associated micro-organisms may represent the next frontier to accelerate the development of high priority therapeutic candidates. Currently in the United States there are two FDA approved marine-derived therapeutic drugs and two others that are often cited as being marine-inspired. This record will be examined first followed by an analysis of a dozen of our favorite examples of coral reef invertebrate natural products having therapeutic potential. The record of using complex scaffolds of marine invertebrate products as the starting point for development will be reviewed by considering eight case examples. The potential promise of developing invertebrate-derived micro-organisms as the starting point for further exploration of therapeutically relevant structures is considered. Also significant is the circumstance that there are some 14 sponge-derived compounds that are available to facilitate fundamental biological investigations.

Interpretation of Tandem Mass Spectra Obtained from Cyclic Nonribosomal Peptides

Natural and non-natural cyclic peptides are a crucial component in drug discovery programs because of their considerable pharmaceutical properties. Cyclosporin, microcystins, and nodularins are all notable pharmacologically important cyclic peptides. Because these biologically active peptides are often biosynthesized nonribosomally, they often contain nonstandard amino acids, thus increasing the complexity of the resulting tandem mass spectrometry data. In addition, because of the cyclic nature, the fragmentation patterns of many of these peptides showed much higher complexity when compared to related counterparts. Therefore, at the present time it is still difficult to annotate cyclic peptides MS/MS spectra. In this current work, an annotation program was developed for the annotation and characterization of tandem mass spectra obtained from cyclic peptides. This program, which we call MS-CPA is available as a web tool (http://lol.ucsd.edu/ms-cpa_v1/Input.py). Using this program, we have successfully annotated the sequence of representative cyclic peptides, such as seglitide, tyrothricin, desmethoxymajusculamide C, dudawalamide A, and cyclomarins, in a rapid manner and also were able to provide the first-pass structure evidence of a newly discovered natural product based on predicted sequence. This compound is not available in sufficient quantities for structural elucidation by other means such as NMR. In addition to the development of this cyclic annotation program, it was observed that some cyclic peptides fragmented in unexpected ways resulting in the scrambling of sequences. In summary, MS-CPA not only provides a platform for rapid confirmation and annotation of tandem mass spectrometry data obtained with cyclic peptides but also enables quantitative analysis of the ion intensities. This program facilitates cyclic peptide analysis, sequencing, and also acts as a useful tool to investigate the uncommon fragmentation phenomena of cyclic peptides and aids the characterization of newly discovered cyclic peptides encountered in drug discovery programs.

Almiramides A−C: Discovery and Development of a New Class of Leishmaniasis Lead Compounds

Leishmaniasis is a debilitating disease caused by protozoan parasites of the genus Leishmania, which affects an estimated 12 million people worldwide. The discovery of new lead compounds for leishmaniasis is therefore a pressing concern for global health programs. The organic extract of a Panamanian collection of the marine cyanobacterium Lyngbya majuscula showed strong in vitro activity in two complementary screens against the tropical parasite Leishmania donovani, the causative agent of visceral leishmaniasis. Chromatographic separation of this complex mixture led to the isolation of the highly N-methylated linear lipopeptides, almiramides A-C (1-3). Comparison with the biological activities of a number of related metabolites and semisynthetic derivatives revealed key features required for activity and afforded one new compound (11) with superior in vitro activity. Subsequent synthesis of a library of simplified analogues led to the discovery of several compounds with improved therapeutic indices to the natural products.

Chemical inhibitors of the type three secretion system: disarming bacterial pathogens

ABSTRACT The recent and dramatic rise of antibiotic resistance among bacterial pathogens underlies the fear that standard treatments for infectious disease will soon be largely ineffective. Resistance has evolved against nearly every clinically used antibiotic, and in the near future, we may be hard-pressed to treat bacterial infections previously conquered by “magic bullet” drugs. While traditional antibiotics kill or slow bacterial growth, an important emerging strategy to combat pathogens seeks to block the ability of bacteria to harm the host by inhibiting bacterial virulence factors. One such virulence factor, the type three secretion system (T3SS), is found in over two dozen Gram-negative pathogens and functions by injecting effector proteins directly into the cytosol of host cells. Without T3SSs, many pathogenic bacteria are unable to cause disease, making the T3SS an attractive target for novel antimicrobial drugs. Interdisciplinary efforts between chemists and microbiologists have yielded several T3SS inhibitors, including the relatively well-studied salicylidene acylhydrazides. This review highlights the discovery and characterization of T3SS inhibitors in the primary literature over the past 10 years and discusses the future of these drugs as both research tools and a new class of therapeutic agents.

Development of Antibiotic Activity Profile Screening for the Classification and Discovery of Natural Product Antibiotics

Despite recognition of the looming antibiotic crisis by healthcare professionals, the number of new antibiotics reaching the clinic continues to decline sharply. This study aimed to establish an antibiotic profiling strategy using a panel of clinically relevant bacterial strains to create unique biological fingerprints for all major classes of antibiotics. Antibiotic mode of action profile (BioMAP) screening has been shown to effectively cluster antibiotics by structural class based on these fingerprints. Using this approach, we have accurately predicted the presence of known antibiotics in natural product extracts and have discovered a naphthoquinone-based antibiotic from our marine natural product library that possesses a unique carbon skeleton. We have demonstrated that bioactivity fingerprinting is a successful strategy for profiling antibiotic lead compounds and that BioMAP can be applied to the discovery of new natural product antibiotics leads.