John C. Vederas

Drug Discovery and Natural Products: End of an Era or an Endless Frontier?

Toward Drug Development The vast majority of pharmaceuticals on the market are based on natural product molecules originally derived from living organisms. When it comes to newly approved drugs, however, the situation looks rather different. Difficulties with high-throughput screening and laboratory synthesis of natural products have led drug companies to focus on libraries of synthetic compounds, despite their providing a much lower “hit rate.” Li and Vederas (p. 161) review methodologies that facilitate the screening, analysis, and synthesis of natural product molecules and their derivatives. Given these advances and the vast numbers of organisms and environments that remain to be explored for potential drug candidates, the current lull in newly approved drugs based on natural products will likely be temporary. Historically, the majority of new drugs have been generated from natural products (secondary metabolites) and from compounds derived from natural products. During the past 15 years, pharmaceutical industry research into natural products has declined, in part because of an emphasis on high-throughput screening of synthetic libraries. Currently there is substantial decline in new drug approvals and impending loss of patent protection for important medicines. However, untapped biological resources, “smart screening” methods, robotic separation with structural analysis, metabolic engineering, and synthetic biology offer exciting technologies for new natural product drug discovery. Advances in rapid genetic sequencing, coupled with manipulation of biosynthetic pathways, may provide a vast resource for the future discovery of pharmaceutical agents.

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.

Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile

The last decade has seen numerous outbreaks of Clostridium difficile-associated disease (CDAD), which presented significant challenges for healthcare facilities worldwide. We have identified and purified thuricin CD, a two-component antimicrobial that shows activity against C. difficile in the nanomolar range. Thuricin CD is produced by Bacillus thuringiensis DPC 6431, a bacterial strain isolated from a human fecal sample, and it consists of two distinct peptides, Trn-α and Trn-β, that act synergistically to kill a wide range of clinical C. difficile isolates, including ribotypes commonly associated with CDAD (e.g., ribotype 027). However, this bacteriocin thuricin CD has little impact on most other genera, including many gastrointestinal commensals. Complete amino acid sequencing using infusion tandem mass spectrometry indicated that each peptide is posttranslationally modified at its respective 21st, 25th, and 28th residues. Solution NMR studies on [13C,15N] Trn-α and [13C,15N]Trn-β were used to characterize these modifications. Analysis of multidimensional NOESY data shows that specific cysteines are linked to the α-carbons of the modified residues, forming three sulfur to α-carbon bridges. Complete sequencing of the thuricin CD gene cluster revealed genes capable of encoding two S′-adenosylmethionine proteins that are characteristically associated with unusual posttranslational modifications. Thuricin CD is a two-component antimicrobial peptide system with sulfur to α-carbon linkages, and it may have potential as a targeted therapy in the treatment of CDAD while also reducing collateral impact on the commensal flora.

Complete Reconstitution of a Highly Reducing Iterative Polyketide Synthase

Dissecting Megaenzyme Mechanisms Filamentous fungi contain a class of multidomain enzymes, the highly-reducing iterative polyketide synthases (HR-IPKSs), which produce important natural products such as the cholesterol-lowering drug lovastatin. To produce their complex products, these megasynthases use multiple catalytic domains repeatedly in different combinations, but mechanistic details remain unclear. Ma et al. (p. 589) now report in vitro reconstitution of the complete catalytic function of lovastatin nonaketide synthase (LovB), the megasynthase that works together with a partner enzyme LovC to complete nearly 40 chemical steps required to construct the core of lovastatin. Analyses of the dependency of enzyme function on cofactors and on the partner enzyme elucidate the programming rules for this system. Reconstitution of catalytic function provides insight into how multifunctional enzymes synthesize important natural products. Highly reducing iterative polyketide synthases are large, multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of the lovastatin nonaketide synthase (LovB) from an engineered strain of Saccharomyces cerevisiae, as well as complete reconstitution of its catalytic function in the presence and absence of cofactors (the reduced form of nicotinamide adenine dinucleotide phosphate and S-adenosylmethionine) and its partner enzyme, the enoyl reductase LovC. Our results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding of the functions and structures of this family of enzymes.

Two-peptide bacteriocins produced by lactic acid bacteria

Bacteriocins from lactic acid bacteria are ribosomally produced peptides (usually 30-60 amino acids) that display potent antimicrobial activity against certain other Gram-positive organisms. They function by disruption of the membrane of their targets, mediated in at least some cases by interaction of the peptide with a chiral receptor molecule (e.g., lipid II or sugar PTS proteins). Some bacteriocins are unmodified (except for disulfide bridges), whereas others (i.e. lantibiotics) possess extensive post-translational modifications which include multiple monosulfide (lanthionine) bridges and dehydro amino acids as well as possible keto amide residues at the N-terminus. Most known bacteriocins are biologically active as single peptides. However, there is a growing class of two peptide systems, both unmodified and lantibiotic, which are fully active only when both partners are present (usually 1:1). In some cases, neither peptide has activity by itself, whereas in others, the activity of one is enhanced by the other. This review discusses the classification, structure, production, regulation, biological activity, and potential applications of such two-peptide bacteriocins.

Isolation and characterization of carnocyclin a, a novel circular bacteriocin produced by Carnobacterium maltaromaticum UAL307.

ABSTRACT Carnobacterium maltaromaticum UAL307, isolated from fresh pork, exhibits potent activity against a number of gram-positive organisms, including numerous Listeria species. Three bacteriocins were isolated from culture supernatant, and using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Edman sequencing, two of these bacteriocins were identified as piscicolin 126 and carnobacteriocin BM1, both of which have previously been described. The remaining bacteriocin, with a molecular mass of 5,862 Da, could not be sequenced by traditional methods, suggesting that the peptide was either cyclic or N-terminally blocked. This bacteriocin showed remarkable stability over a wide temperature and pH range and was unaffected by a variety of proteases. After digestion with trypsin and α-chymotrypsin, the peptide was de novo sequenced by tandem mass spectrometry and a linear sequence deduced, consisting of 60 amino acids. Based on this sequence, the molecular mass was predicted to be 5,880 Da, 18 units higher than the observed molecular mass, which suggested that the peptide has a cyclic structure. Identification of the genetic sequence revealed that this peptide is circular, formed by a covalent linkage between the N and C termini following cleavage of a 4-residue peptide leader sequence. The results of structural studies suggest that the peptide is highly structured in aqueous conditions. This bacteriocin, named carnocyclin A, is the first reported example of a circular bacteriocin produced by Carnobacterium spp.

Lipopeptides from Bacillus and Paenibacillus spp.: A Gold Mine of Antibiotic Candidates

The emergence of multidrug‐resistant bacteria has placed a strain on health care systems and highlighted the need for new classes of antibiotics. Bacterial lipopeptides are secondary metabolites, generally produced by nonribosomal peptide synthetases that often exhibit broad‐spectrum antimicrobial activity. Only two new structural types of antibiotics have entered the market in the last 40 years, linezolid and the bacterial lipopeptide daptomycin. A wide variety of bacteria produce lipopeptides, however Bacillus and Paenibacillus spp. in particular have yielded several potent antimicrobial lipopeptides. Many of the lipopeptides produced by these bacteria have been known for decades and represent a potential gold mine of antibiotic candidates. This list includes the polymyxins, octapeptins, polypeptins, iturins, surfactins, fengycins, fusaricidins, and tridecaptins, as well as some novel examples, including the kurstakins. These lipopeptides have a wide variety of activities, ranging from antibacterial and antifungal, to anticancer and antiviral. This review presents a reasonably comprehensive list of each class of lipopeptide and their known homologues. Emphasis has been placed on their antimicrobial activities, as well other potential applications for this interesting class of substances.

Loss of Apelin Exacerbates Myocardial Infarction Adverse Remodeling and Ischemia-reperfusion Injury: Therapeutic Potential of Synthetic Apelin Analogues

Background Coronary artery disease leading to myocardial ischemia is the most common cause of heart failure. Apelin (APLN), the endogenous peptide ligand of the APJ receptor, has emerged as a novel regulator of the cardiovascular system. Methods and Results Here we show a critical role of APLN in myocardial infarction (MI) and ischemia‐reperfusion (IR) injury in patients and animal models. Myocardial APLN levels were reduced in patients with ischemic heart failure. Loss of APLN increased MI‐related mortality, infarct size, and inflammation with drastic reductions in prosurvival pathways resulting in greater systolic dysfunction and heart failure. APLN deficiency decreased vascular sprouting, impaired sprouting of human endothelial progenitor cells, and compromised in vivo myocardial angiogenesis. Lack of APLN enhanced susceptibility to ischemic injury and compromised functional recovery following ex vivo and in vivo IR injury. We designed and synthesized two novel APLN analogues resistant to angiotensin converting enzyme 2 cleavage and identified one analogue, which mimicked the function of APLN, to be markedly protective against ex vivo and in vivo myocardial IR injury linked to greater activation of survival pathways and promotion of angiogenesis. Conclusions APLN is a critical regulator of the myocardial response to infarction and ischemia and pharmacologically targeting this pathway is feasible and represents a new class of potential therapeutic agents.

Characteristics and genetic determinant of a hydrophobic peptide bacteriocin, carnobacteriocin A, produced by Carnobacterium piscicola LV17A.

Carnobacteriocin A is a hydrophobic nonlantibiotic bacteriocin that is detected early in the growth cycle of Carnobacterium piscicola LV17A and encoded by a 49 MDa plasmid. The bacteriocin was purified using hydrophobic interaction and gel filtration chromatography, and reversed-phase HPLC. Three different active peaks (A1, A2 and A3) were detected, but the purified samples had identical N-terminal amino acid sequences for the first 15 amino acids as determined by Edman degradation analysis. Only a 2.4 kb fragment of the EcoRI digest of the plasmid pCP49 hybridized with a 23-mer oligonucleotide probe derived from amino acids 5 to 13 of the amino acid sequence. The structural gene for carnobacteriocin A is located 600 base pairs into the 2.4 kb EcoRI fragment, but no other genetic information was detected on this unit. The structural gene includes an 18 amino acid N-terminal extension of the bacteriocin, ending with Gly-Gly residues in the -2, -1 positions with respect to the cleavage site. The bacteriocin consists of 53 amino acids that differ markedly from the majority of hydrophobic peptide bacteriocins characterized to date. Based on the amino acid sequence derived from the nucleotide sequence a molecular mass of 5052.85 Da was calculated. Mass spectrometric analysis showed that the molecular mass of the major component (A3) was 2 Da lower, thereby indicating the presence of a disulphide bridge between Cys 22 and Cys 51. Carnobacteriocin A2 has a similar structure except that Met 52 is oxidized to a sulphoxide, whereas A1 appears to be a mixture of peptides derived proteolytically from A3 or A2.

Aspects of the biosynthesis of non-aromatic fungal polyketides by iterative polyketide synthases.

Lovastatin biosynthesis in Aspergillus terreus involves two unusual type I multifunctional polyketide syntheses (PKSs). Lovastatin nonaketide synthase (LNKS), the product of the lovB gene, is an iterative PKS that interacts with LovC, a putative enoyl reductase, to catalyze the 35 separate reactions in the biosynthesis of dihydromonacolin L, a lovastatin precursor. LNKS also displays Diels-Alderase activity in vitro. Lovastatin diketide synthase (LDKS) made by lovF, in contrast, acts non-iteratively like the bacterial modular PKSs to make (2R)-2–methylbutyric acid. Then, like LNKS, LDKS interacts closely with another protein, the LovD transesterase enzyme that catalyzes attachment of the 2–methylbutyric acid to monacolin J in the final step of the lovastatin pathway. Key features of the genes for these four enzymes and others, plus the regulatory and self-resistance factors involved in lovastatin production, are also described.