Beyond Amphiphilic Balance: Changing Subunit Stereochemistry Alters the Pore-Forming Activity of Nylon-3 Polymers.

Amphiphilic nylon-3 polymers have been reported to mimic the biological activities of natural antimicrobial peptides, with high potency against bacteria and minimal toxicity toward eukaryotic cells. Amphiphilic balance, determined by the proportions of hydrophilic and lipophilic subunits, is considered one of the most important features for achieving this activity profile for nylon-3 polymers and many other antimicrobial polymers. Insufficient hydrophobicity often correlates with weak activities against bacteria, whereas excessive hydrophobicity correlates with high toxicity toward eukaryotic cells.

Rice-induced secondary metabolite gene expression in Aspergillus nidulans.

Activation of silent biosynthetic gene clusters in fungi remains a challenge for discovery of new useful natural products. In this work, we identify a group of silent secondary metabolite gene clusters in Aspergillus nidulans that are induced by rice (Oryza sativa). Using reverse phase HPLC purification on extracts of rice, we identified the plant hormone gibberellic acid as one compound present in rice extracts that induced these silent genes.

Retention of Native Quaternary Structure in Racemic Melittin Crystals.

Racemic crystallography has been used to elucidate the secondary and tertiary structures of peptides and small proteins that are recalcitrant to conventional crystallization. It is unclear, however, whether racemic crystallography can capture native quaternary structure, which could be disrupted by heterochiral associations. We are exploring the use of racemic crystallography to characterize the self-assembly behavior of membrane-associated peptides, very few of which have been crystallized.

Quasiracemate Crystal Structures of Magainin 2 Derivatives Support the Functional Significance of the Phenylalanine Zipper Motif.

Quasiracemic crystallography has been used to explore the significance of homochiral and heterochiral associations in a set of host-defense peptide derivatives. The previously reported racemic crystal structure of a magainin 2 derivative displayed a homochiral antiparallel dimer association featuring a "phenylalanine zipper" notable for the dual roles of phenylalanines in mediating dimerization and formation of an exposed hydrophobic swath. This motif is seen as well in two new quasiracemate crystals that contain the d form of the magainin 2 derivative along with an l-peptide in which one Ala has been replaced by a β-amino acid residue.

Nylon-3 polymers active against drug-resistant Candida albicans biofilms.

Candida albicans is the most common fungal pathogen in humans, and most diseases produced by C. albicans are associated with biofilms. We previously developed nylon-3 polymers with potent activity against planktonic C.

Synthetic polymers active against Clostridium difficile vegetative cell growth and spore outgrowth.

Nylon-3 polymers (poly-β-peptides) have been investigated as synthetic mimics of host-defense peptides in recent years. These polymers are attractive because they are much easier to synthesize than are the peptides themselves, and the polymers resist proteolysis. Here we describe in vitro analysis of selected nylon-3 copolymers against Clostridium difficile, an important nosocomial pathogen that causes highly infectious diarrheal disease.

Ternary nylon-3 copolymers as host-defense peptide mimics: beyond hydrophobic and cationic subunits.

Host-defense peptides (HDPs) are produced by eukaryotes to defend against bacterial infection, and diverse synthetic polymers have recently been explored as mimics of these natural peptides. HDPs are rich in both hydrophobic and cationic amino acid residues, and most HDP-mimetic polymers have therefore contained binary combinations of hydrophobic and cationic subunits. However, HDP-mimetic polymers rarely duplicate the hydrophobic surface and cationic charge density found among HDPs ( Hu , K.

Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors.

RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target.

Structure-activity relationships among antifungal nylon-3 polymers: identification of materials active against drug-resistant strains of Candida albicans.

Fungal infections are a major challenge to human health that is heightened by pathogen resistance to current therapeutic agents. Previously, we were inspired by host-defense peptides to develop nylon-3 polymers (poly-β-peptides) that are toxic toward the fungal pathogen Candida albicans but exert little effect on mammalian cells. Based on subsequent analysis of structure-activity relationships among antifungal nylon-3 polymers, we have now identified readily prepared cationic homopolymers active against strains of C.

Tuning the biological activity profile of antibacterial polymers via subunit substitution pattern.

Binary nylon-3 copolymers containing cationic and hydrophobic subunits can mimic the biological properties of host-defense peptides, but relationships between composition and activity are not yet well understood for these materials. Hydrophobic subunits in previously studied examples have been limited mostly to cycloalkane-derived structures, with cyclohexyl proving to be particularly promising. The present study evaluates alternative hydrophobic subunits that are isomeric or nearly isomeric with the cyclohexyl example; each has four sp(3) carbons in the side chains.

Effects of Cyclic vs. Acyclic Hydrophobic Subunits on the Chemical Structure and Biological Properties of Nylon-3 Co-Polymers.

Nylon-3 co-polymers containing both hydrophobic and cationic subunits can mimic the activity profile of host-defense peptides, if subunit identity and proportion are carefully selected. These sequence- and stereo-random co-polymers inhibit bacterial growth at relatively low concentrations, apparently via disruption of bacterial membranes, but they are relatively non-disruptive toward eukaryotic cell membranes (low hemolytic activity). In all previous examples, the hydrophobic subunits have contained cycloalkyl groups that incorporate the backbone Cα-Cβ bond.

Evidence for phenylalanine zipper-mediated dimerization in the X-ray crystal structure of a magainin 2 analogue.

High-resolution structure elucidation has been challenging for the large group of host-defense peptides that form helices on or within membranes but do not manifest a strong folding propensity in aqueous solution. Here we report the crystal structure of an analogue of the widely studied host-defense peptide magainin 2. Magainin 2 (S8A, G13A, G18A) is a designed variant that displays enhanced antibacterial activity relative to the natural peptide.

Interplay among subunit identity, subunit proportion, chain length, and stereochemistry in the activity profile of sequence-random peptide mixtures.

Fmoc-based solid-phase synthesis methodology was used to prepare peptide mixtures containing one type of hydrophobic residue and one type of cationic residue. Each mixture was random in terms of sequence but highly controlled in terms of length. Analysis of the antibacterial and hemolytic properties of these mixtures revealed that selective antibacterial activity can be achieved with heterochiral binary mixtures but not homochiral binary mixture, if the proper amino acid residues are used.

West Nile virus methyltransferase domain interacts with protein kinase G.

Background: The flaviviral nonstructural protein 5 (NS5) is a phosphoprotein, though the precise identities and roles of many specific phosphorylations remain unknown. Protein kinase G (PKG), a cGMP-dependent protein kinase, has previously been shown to phosphorylate dengue virus NS5.

Methods: We used mass spectrometry to specifically identify NS5 phosphosites.

Nylon-3 polymers with selective antifungal activity.

Host-defense peptides inhibit bacterial growth but show little toxicity toward mammalian cells. A variety of synthetic polymers have been reported to mimic this antibacterial selectivity; however, achieving comparable selectivity for fungi is more difficult because these pathogens are eukaryotes. Here we report nylon-3 polymers based on a novel subunit that display potent antifungal activity (MIC = 3.

Functionally Diverse Nylon-3 Copolymers from Readily Accessible β-Lactams.

A new family of β-lactams is described that enables anionic ring-opening polymerization (AROP) to prepare nylon-3 materials bearing diverse appended functionality, including carboxylic acid, thiol, hydroxyl and secondary amine groups. Nylon-3 copolymers generated with the new β-lactams are shown to display distinctive self-assembly behavior and biological properties.

Phosphorylation of the Streptococcus pneumoniae cell wall biosynthesis enzyme MurC by a eukaryotic-like Ser/Thr kinase.

Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway.

Screen for agents that induce autolysis in Bacillus subtilis.

The growing prevalence of antibiotic-resistant infections underscores the need to discover new antibiotics and to use them with maximum effectiveness. In response to these needs, we describe a screening protocol for the discovery of autolysis-inducing agents that uses two Bacillus subtilis reporter strains, SH-536 and BAU-102. To screen chemical libraries, autolysis-inducing agents were first identified with a BAU-102-based screen and then subdivided with SH-536 into two major groups: those that induce autolysis by their direct action on the cell membrane and those that induce autolysis secondary to inhibition of cell wall synthesis.

C-terminal functionalization of nylon-3 polymers: effects of C-terminal groups on antibacterial and hemolytic activities.

Nylon-3 polymers contain β-amino-acid-derived subunits and can be viewed as higher homologues of poly(α-amino acids). This structural relationship raises the possibility that nylon-3 polymers offer a platform for development of new materials with a variety of biological activities, a prospect that has recently begun to receive experimental support. Nylon-3 homo- and copolymers can be prepared via anionic ring-opening polymerization of β-lactams, and use of an N-acyl-β-lactam as coinitiator in the polymerization reaction allows placement of a specific functional group, borne by the N-acyl-β-lactam, at the N-terminus of each polymer chain.

Improved small-molecule macroarray platform for the rapid synthesis and discovery of antibacterial chalcones.

Bacterial resistance to current antibiotics is a major global health threat. Consequently, there is an urgent need for the identification of new antibacterial agents. We are applying the small-molecule macroarray platform to rapidly synthesize and screen compounds for activity against methicillin-resistant Staphylococcus aureus (MRSA).

Screen for inducers of autolysis in Bacillus subtilis.

We describe a primary high-throughput screen that uses the reporter strain Bacillus subtilis BAU-102 to identify antibiotics that induce autolysis. The screen measures autolysis in terms of the incipient release of recombinant Escherichia coli beta-galactosidase (beta-Gal) from the periplasmic space of B. subtilis owing to a loss of integrity of the cell wall.

Structure-activity relationships among random nylon-3 copolymers that mimic antibacterial host-defense peptides.

Host-defense peptides are natural antibiotics produced by multicellular organisms to ward off bacterial infection. Since the discovery of these molecules in the 1980s, a great deal of effort has been devoted to elucidating their mechanisms of action and to developing analogues with improved properties for possible therapeutic use. The vast majority of this effort has focused on materials composed of a single type of molecule, most commonly a peptide with a specific sequence of alpha-amino acid residues.

Phosphorylation of the RitR DNA-binding domain by a Ser-Thr phosphokinase: implications for global gene regulation in the streptococci.

We report selective phosphorylation of the DNA-binding domain of the Streptococcus pneumoniae transcriptional regulator RitR. RitR is annotated as a two-component response regulator, but lacks a cognate His kinase as a neighbouring locus in the genome. In addition, Asn replaces Asp at the expected acceptor site.

Phosphorylation of yellow fever virus NS5 alters methyltransferase activity.

Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to characterize phosphorylated residues of yellow fever virus (YFV) NS5 expressed in mammalian cells. Multiple different phosphopeptides were detected.