One-pot stapling of interchain disulfides of antibodies using an isobutylene motif.

Monoclonal antibodies have emerged as an important class of therapeutics in oncological and autoimmune diseases due to their several attractive properties, such as high binding affinity and specificity. However, it has recently become clear that antibodies recovered from serum show a significantly decreased potency owing to various reasons, including deamidation, oxidation, fragment antigen binding (Fab) exchange, and disulfide shuffling. Fab exchange and disulfide shuffling result because of the instability of disulfides in serum. Herein, we reported a 'one-pot' stapling strategy using isobutylene motifs to stabilise the interchain disulfides of antibodies. This general method was applied to a Fab fragment of the anti-HER2 antibody. The stapled Fab was completely stable in the presence of biological thiols. The approach was further applied to two different full-length IgGs, trastuzumab and rituximab, under mild and biocompatible conditions. The binding affinity of the antibody was enhanced, relative to its native form, after being stapled. The stapled structure maintained its effector functions and behaved similarly to its native form in vivo. This work provides a straightforward and scalable method for the stabilisation of antibodies in various formats

A Biodegradable Polycationic Paint that Kills Bacteria <i>in Vitro</i> and <i>in Vivo</i>

Bacterial colonization and subsequent formation of biofilms onto surfaces of medical devices and implants is a major source of nosocomial infections. Most antibacterial coatings to combat infections are either metal-based or nondegradable-polymer-based and hence limited by their nondegradability and unpredictable toxicity. Moreover, to combat infections effectively, the coatings are required to display simultaneous antibacterial and antibiofilm activity. Herein we report biocompatible and biodegradable coatings based on organo-soluble quaternary chitin polymers which were immobilized noncovalently onto surfaces as bactericidal paint. The polycationic paint was found to be active against both drug-sensitive and -resistant bacteria such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), vancomycin-resistant <i>Enterococcus faecium</i> (VRE), and β-lactam-resistant <i>Klebsiella pneumoniae</i>. The cationic polymers were shown to interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thereby causing leakage of intracellular constituents and cell death upon contact. Importantly, surfaces coated with the polymers inhibited formation of biofilms against both Gram-positive <i>S. aureus</i> and Gram-negative <i>E. coli</i>, two of the most clinically important bacteria that form biofilms. Surfaces coated with the polymers displayed negligible toxicity against human erythrocytes and embryo kidney cells. Notably, the polymers were shown to be susceptible toward lysozyme. Furthermore, subcutaneous implantation of polymer discs in rats led to 15–20% degradation in 4 weeks thereby displaying their biodegradability. In a murine model of subcutaneous infection, polymer-coated medical-grade catheter reduced MRSA burden by 3.7 log compared to that of noncoated catheter. Furthermore, no biofilm development was observed on the coated catheters under <i>in vivo</i> conditions. The polycationic materials thus developed herein represent a novel class of safe and effective coating agents for the prevention of device-associated infections

Gene structure and expression of the aspen cytosolic copper/zinc-superoxide dismutase (PtSodCc1)

Genomic and cDNA clones, corresponding to an ozone-induced cytosolic copper–zinc superoxide dismutase, were isolated from quaking aspen (Populus tremuloides Michx.). The cytosolic superoxide dismutase (SOD) appears to be part of a multi-gene family in aspen and is interrupted by five introns in the coding region. Northern blot analysis with a gene-specific probe revealed an increase in the expression of this gene in response to ozone in the leaves of an ozone-tolerant aspen clone, compared with an ozone-sensitive clone. Cytosolic SOD transcript expression levels in leaves were also found to increase significantly within 6hof mechanical wounding, after which the level of the transcript decreases. Under normal growing conditions, immature male and female aspen floral bud tissues contained the highest levels of the cytosolic SOD gene transcript, whereas transcript levels wer

Cleavable Cationic Antibacterial Amphiphiles: Synthesis, Mechanism of Action, and Cytotoxicities

The development of novel antimicrobial agents having high selectivity toward bacterial cells over mammalian cells is urgently required to curb the widespread emergence of infectious diseases caused by pathogenic bacteria. Toward this end, we have developed a set of cationic dimeric amphiphiles (bearing cleavable amide linkages between the headgroup and the hydrocarbon tail with different methylene spacers) that showed high antibacterial activity against human pathogenic bacteria (<i>Escherichia coli</i> and <i>Staphylococcus aureus</i>) and low cytotoxicity. The Minimum Inhibitory Concentrations (MIC) were found to be very low for the dimeric amphiphiles and were lower or comparable to the monomeric counterpart. In the case of dimeric amphiphiles, MIC was found to decrease with the increase in the spacer chain length (<i>n</i> = 2 to 6) and again to increase at higher spacer length (<i>n</i> > 6). It was found that the compound with six methylene spacers was the most active among all of the amphiphiles (MICs = 10–13 μM). By fluorescence spectroscopy, fluorescence microscopy, and field-emission scanning electron microscopy (FESEM), it was revealed that these cationic amphiphiles interact with the negatively charged bacterial cell membrane and disrupt the membrane integrity, thus killing the bacteria. All of the cationic amphiphiles showed low hemolytic activity (HC₅₀) and high selectivity against both gram-positive and gram-negative bacteria. The most active amphiphile (<i>n</i> = 6) had a 10–13-fold higher HC₅₀ than did the MIC. Also, this amphiphile did not show any cytotoxicity against mammalian cells (HeLa cells) even at a concentration above the MIC (20 μM). The critical micellar concentration (CMC) values of gemini surfactants were found to be very low (CMC = 0.30–0.11 mM) and were 10–27 times smaller than the corresponding monomeric analogue (CMC = 2.9 mM). Chemical hydrolysis and thermogravimetric analysis (TGA) proved that these amphiphiles are quite stable under both acidic and thermal conditions. Collectively, these properties make the newly synthesized amphiphiles potentially superior disinfectants and antiseptics for various biomedical and biotechnological applications

Small Molecular Antibacterial Peptoid Mimics: The Simpler the Better!

The emergence of multidrug resistant bacteria compounded by the depleting arsenal of antibiotics has accelerated efforts toward development of antibiotics with novel mechanisms of action. In this report, we present a series of small molecular antibacterial peptoid mimics which exhibit high in vitro potency against a variety of Gram-positive and Gram-negative bacteria, including drug-resistant species such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium. The highlight of these compounds is their superior activity against the major nosocomial pathogen Pseudomonas aeruginosa. Nontoxic toward mammalian cells, these rapidly bactericidal compounds primarily act by permeabilization and depolarization of bacterial membrane. Synthetically simple and selectively antibacterial, these compounds can be developed into a newer class of therapeutic agents against multidrug resistant bacterial species

Broad Spectrum Antibacterial and Antifungal Polymeric Paint Materials: Synthesis, Structure–Activity Relationship, and Membrane-Active Mode of Action

Microbial attachment and subsequent colonization onto surfaces lead to the spread of deadly community-acquired and hospital-acquired (nosocomial) infections. Noncovalent immobilization of water insoluble and organo-soluble cationic polymers onto a surface is a facile approach to prevent microbial contamination. In the present study, we described the synthesis of water insoluble and organo-soluble polymeric materials and demonstrated their structure–activity relationship against various human pathogenic bacteria including drug-resistant strains such as methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), vancomycin-resistant enterococci (VRE), and beta lactam-resistant <i>Klebsiella pneumoniae</i> as well as pathogenic fungi such as <i>Candida</i> spp. and <i>Cryptococcus</i> spp. The polymer coated surfaces completely inactivated both bacteria and fungi upon contact (5 log reduction with respect to control). Linear polymers were more active and found to have a higher killing rate than the branched polymers. The polymer coated surfaces also exhibited significant activity in various complex mammalian fluids such as serum, plasma, and blood and showed negligible hemolysis at an amount much higher than minimum inhibitory amounts (MIAs). These polymers were found to have excellent compatibility with other medically relevant polymers (polylactic acid, PLA) and commercial paint. The cationic hydrophobic polymer coatings disrupted the lipid membrane of both bacteria and fungi and thus showed a membrane-active mode of action. Further, bacteria did not develop resistance against these membrane-active polymers in sharp contrast to conventional antibiotics and lipopeptides, thus the polymers hold great promise to be used as coating materials for developing permanent antimicrobial paint