A lipoglycopeptide antibiotic for Gram-positive biofilm-related infections.

Drug-resistant Gram-positive bacterial infections are still a substantial burden on the public health system, with two bacteria (Staphylococcus aureus and Streptococcus pneumoniae) accounting for over 1.5 million drug-resistant infections in the United States alone in 2017. In 2019, 250,000 deaths were attributed to these pathogens globally. We have developed a preclinical glycopeptide antibiotic, MCC5145, that has excellent potency (MIC90 ≤ 0.06 µg/ml) against hundreds of isolates of methicillin-resistant S. aureus (MRSA) and other Gram-positive bacteria, with a greater than 1000-fold margin over mammalian cell cytotoxicity values. The antibiotic has therapeutic in vivo efficacy when dosed subcutaneously in multiple murine models of established bacterial infections, including thigh infection with MRSA and blood septicemia with S. pneumoniae, as well as when dosed orally in an antibiotic-induced Clostridioides difficile infection model. MCC5145 exhibited reduced nephrotoxicity at microbiologically active doses in mice compared to vancomycin. MCC5145 also showed improved activity against biofilms compared to vancomycin, both in vitro and in vivo, and a low propensity to select for drug resistance. Characterization of drug action using a transposon library bioinformatic platform showed a mechanistic distinction from other glycopeptide antibiotics.

A comparison study of superovulation strategies for C57BL/6J and B6D2F1 mice in CRISPR-Cas9 mediated genome editing.

Reproductive techniques such as superovulation and in vitro fertilisation (IVF) have been widely used in generating genetically modified animals. The current gold standard for superovulation in mice is using coherent treatments of equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). An alternative method using inhibin antiserum (IAS) instead of eCG has been recently reported. Here, we evaluate different superovulation strategies in C57BL/6J and B6D2F1 mice. Firstly, we found that using 5-week-old C57BL/6J and 4-week-old B6D2F1 donors could achieve better superovulation outcomes. Then, we compared eCG-hCG, IAS-hCG and eCG-IAS-hCG with different dosages in both mouse strains. Significantly increased numbers of oocytes were obtained by using IAS-hCG and eCG-IAS-hCG methods. However, low fertilisation rates (36.3-38.8%) were observed when natural mating was applied. We then confirmed that IVF could dramatically ameliorate the fertilisation rates up to 89.1%. Finally, we performed CRISPR-Cas9 mediated genome editing targeting Scn11a and Kcnh1 loci, and successfully obtained mutant pups using eCG-hCG and IAS-hCG induced zygotes, which were fertilised by either natural mating or IVF. Our results showed that IAS is a promising superovulation reagent, and the efficiency of genome editing is unlikely to be affected by using IAS-induced zygotes.

Construction of a full-length antibody phage display vector.

Antibody phage display technology plays an important role in the development of monoclonal antibodies, humanization, and affinity evolution of antibodies. Thus far, antibody phage display mainly focuses on the display of antibody variable region or antigen-binding fragments. In this study, we constructed a new phage display system that can display full-length IgG antibodies on M13 phage. The phage display vector contains open reading frames (ORFs) encoding full-length the heavy and light chains of the antibody. NcoI/XhoI restriction enzyme sites were used to clone the variable region of the heavy chain into the heavy chain ORF, and SalI/NotI sites were used to clone the light chain variable region. SnaBI and SbfI restriction enzyme sites were designed between the cloning sites of heavy and light chains, respectively, to increase the cloning efficiency. The full-length antibodies of nivolumab against programmed death factor 1, trastuzumab against human epidermal growth factor 2, diL2K against the cluster of differentiation 3 epsilon, and adalimumab against tumor necrosis factor- alpha were displayed on phage with the vector. Phage-displayed antibodies showed their original antigen-binding activity. An amber codon shifted the vector to express IgG in non-suppressed Escherichia coli. The heavy and light chains of the E. coli-expressed antibodies could be detected through western blotting, and the antigen-binding activity was confirmed using an enzyme-linked immunosorbent assay. Biopanning was carried out with a model phage display antibody library, and the results showed that the novel phage system could be used for antibody library construction and highly efficient antibody screening. The reported system is the first full-length antibody phage display system.

An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria.

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.

Genome-Wide Off-Target Analysis in CRISPR-Cas9 Modified Mice and Their Offspring.

The emergence of the CRISPR-Cas9 system has triggered a technical revolution in mammalian genome editing. Compared to traditional gene-targeting strategies, CRISPR-Cas9 technology offers a more efficient and cost-effective approach for generating genetically modified animal models. However, off-target cleavage in CRISPR-mediated genome editing is a major concern in the analysis of phenotypes as well as the selection of therapeutic targets. Here, we analyzed whole-genome sequencing (WGS) data from two knock-out (KO) mouse strains generated by using the CRISPR-Cas9 system targeting the Mmd and Paqr8 loci. A total of nine individuals were sequenced including two parents, four F1 offspring and three uninjected control mice. Using GATK and bcftools software, we identified two off-target events in the founder mice. The two CRISPR-Cas9-induced off-target events were predictable using Cas-OFFinder and were not passed on to the offspring that we investigated. In addition, our results indicated that the number of CRISPR-Cas9-induced mutations was not statistically distinguishable from the background de novo mutations (DNMs).

A novel murine antibody and an open sandwich immunoassay for the detection of clenbuterol.

Clenbuterol (CLEN) is a sympathomimetic amine used as a decongestant and bronchodilator while treating breathing disorders. It is also used in food-producing animals as it improves the rate of red meat production. However, it is prohibited in many countries nowadays due to human health and safety concerns. Unfortunately, the illegal use of CLEN is still rampant. Thus, monitoring it in food and livestock is important. Here, we report a novel murine antibody and an open sandwich enzyme linked immunosorbent assay (OS-ELISA) to detect CLEN based on antigen-antibody reactions. The genes of antibody variable regions in mice immunized with CLEN conjugated with bovine serum albumin were cloned into a phagemid (pDong1/Fab) to construct a phage-display antibody library, from which a novel antibody, A12, was selected. Then, an OS-ELISA was developed to detect CLEN using separated variable regions of the A12 antibody. The limit of detection of the assay was found to be 8 ng/mL, which was useful for monitoring CLEN usage.

Structure-Function Studies of Polymyxin B Lipononapeptides.

The emerging threat of infections caused by highly drug-resistant bacteria has prompted a resurgence in the use of the lipodecapeptide antibiotics polymyxin B and colistin as last resort therapies. Given the emergence of resistance to these drugs, there has also been a renewed interest in the development of next generation polymyxins with improved therapeutic indices and spectra of action. We report structure-activity studies of 36 polymyxin lipononapeptides structurally characterised by an exocyclic FA-Thr²-Dab³ lipodipeptide motif instead of the native FA-Dab¹-Thr²-Dab³ tripeptide motif found in polymyxin B, removing one of the positively charged residues believed to contribute to nephrotoxicity. The compounds were prepared by solid phase synthesis using an on-resin cyclisation approach, varying the fatty acid and the residues at position 2 (P2), P3 and P4, then assessing antimicrobial potency against a panel of Gram-negative bacteria, including polymyxin-resistant strains. Pairwise comparison of N-acyl nonapeptide and decapeptide analogues possessing different fatty acids demonstrated that antimicrobial potency is strongly influenced by the N-terminal L-Dab-1 residue, contingent upon the fatty acid. This study highlights that antimicrobial potency may be retained upon truncation of the N-terminal L-Dab-1 residue of the native exocyclic lipotripeptide motif found in polymyxin B. The strategy may aid in the design of next generation polymyxins.

Structure, Function, and Biosynthetic Origin of Octapeptin Antibiotics Active against Extensively Drug-Resistant Gram-Negative Bacteria.

Resistance to the last-resort antibiotic colistin is now widespread and new therapeutics are urgently required. We report the first in toto chemical synthesis and pre-clinical evaluation of octapeptins, a class of lipopeptides structurally related to colistin. The octapeptin biosynthetic cluster consisted of three non-ribosomal peptide synthetases (OctA, OctB, and OctC) that produced an amphiphilic antibiotic, octapeptin C4, which was shown to bind to and depolarize membranes. While active against multi-drug resistant (MDR) strains in vitro, octapeptin C4 displayed poor in vivo efficacy, most likely due to high plasma protein binding. Nuclear magnetic resonance solution structures, empirical structure-activity and structure-toxicity models were used to design synthetic octapeptins active against MDR and extensively drug-resistant (XDR) bacteria. The scaffold was then subtly altered to reduce plasma protein binding, while maintaining activity against MDR and XDR bacteria. In vivo efficacy was demonstrated in a murine bacteremia model with a colistin-resistant P. aeruginosa clinical isolate.

Protein-inspired antibiotics active against vancomycin- and daptomycin-resistant bacteria.

The public health threat posed by a looming 'post-antibiotic' era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature's control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the 'vancapticins', possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to 'revitalise' antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.

Cationic Acrylate Oligomers Comprising Amino Acid Mimic Moieties Demonstrate Improved Antibacterial Killing Efficiency.

Cu(0)-mediated polymerization was employed to synthesize a library of structurally varied cationic polymers and their application as antibacterial peptide mimics was assessed. Eight platform polymers were first synthesized with low degrees of polymerization (DP) using (2-Boc-amino)ethyl acrylate as the monomer and either ethyl α-bromoisobutyrate or dodecyl 2-bromoisobutyrate as the initiator (thus providing hydrocarbon chain termini of C2 or C12, respectively). A two-step modification strategy was then employed to generate the final sixteen-member polymer library. Specifically, an initial deprotection was employed to reveal the primary amine cationic polymers, followed by guanylation. The biocidal activity of these cationic polymers was assessed against various strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae. Polymers having a short segment of guanidine units and a C12 hydrophobic terminus were shown to provide the broadest antimicrobial activity against the panel of isolates studied, with MIC values approaching those for Gram-positive targeting antibacterial peptides: daptomycin and vancomycin. The C12-terminated guanidine functional polymers were assayed against human red blood cells, and a concomitant increase in haemolysis was observed with decreasing DP. Cytotoxicity was tested against HEK293 and HepG2 cells, with the lowest DP C12-terminated polymer exhibiting minimal toxicity over the concentrations examined, except at the highest concentration. Membrane disruption was identified as the most probable mechanism of bacteria cell killing, as elucidated by membrane permeability testing against E. coli.

Synthesis of octapeptin C4 and biological profiling against NDM-1 and polymyxin-resistant bacteria.

The first synthesis of octapeptin C4 was achieved using a combination of solid phase synthesis and off-resin cyclisation. Octapeptin C4 displayed antibiotic activity against multi-drug resistant, NDM-1 and polymyxin-resistant Gram-negative bacteria, with moderate activity against Staphylococcus aureus. The linear analogue of octapeptin C4 was also prepared, which showed reduced activity.

Plasma Protein Binding Structure-Activity Relationships Related to the N-Terminus of Daptomycin.

Daptomycin is a lipopeptide antibiotic that is highly bound to plasma proteins. To date, the plasma components and structure-activity relationships responsible for the plasma protein binding profile of daptomycin remain uncharacterized. In the present study we have employed a surface plasmon resonance assay together with molecular docking techniques to investigate the plasma protein binding structure-activity relationships related to the N-terminal fatty acyl of daptomycin. Three compounds were investigated: (1) native daptomycin, which displays an N-terminal n-decanoyl fatty acid side chain, and two analogues with modifications to the N-terminal fatty acyl chain; (2) des-acyl daptomycin; and (3) acetyl-daptomycin. The surface plasmon resonance (SPR) data showed that the binding profile of native daptomycin was in the rank order human serum albumin (HSA) ≫ α-1-antitrypsin > low-density lipoprotein ≥ hemoglobin > sex hormone binding globulin > α-1-acid-glycoprotein (AGP) > hemopexin > fibrinogen > α-2-macroglobulin > ß2-microglobulin > high-density lipoprotein > fibronectin > haptoglobulin > transferrin > immunoglobulin G. Notably, binding to fatty acid free HSA was greater than binding to nondelipidated HSA. SPR and ultrafiltration studies also indicated that physiological concentrations of calcium increase binding of daptomycin and acetyl-daptomycin to HSA and AGP. A molecular model of the daptomycin-human serum albumin A complex is presented that illustrates the pivotal role of the N-terminal fatty acyl chain of daptomycin for binding to drug site 1 of HSA. In proof-of-concept, the capacity of physiological cocktails of the identified plasma proteins to inhibit the antibacterial activity of daptomycin was assessed with in vitro microbiological assays. We show that HSA, α-1-antitrypsin, low-density lipoprotein, sex hormone binding globulin, α-1-acid-glycoprotein, and hemopexin are responsible for the majority of the sequestering activity in human plasma. The findings are relevant to medicinal chemistry programs focused on the development of next-generation daptomycin lipopeptides. Tailored modifications to the N-terminal fatty acyl domain of the daptomycin molecule should yield novel daptomycin lipopeptides with more ideal plasma protein binding profiles to increase the levels of active (free) drug in plasma and improved in vivo activity.

Truncated Latrunculins as Actin Inhibitors Targeting Plasmodium falciparum Motility and Host Cell Invasion.

Polymerization of the cytosolic protein actin is critical to cell movement and host cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesized and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with that of latrunculin B against P. falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.

An "Unlikely" Pair: The Antimicrobial Synergy of Polymyxin B in Combination with the Cystic Fibrosis Transmembrane Conductance Regulator Drugs KALYDECO and ORKAMBI.

Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC ≥ 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 mg/L) + lumacaftor (8 mg/L) displayed synergistic killing activity against polymyxin-resistant P. aeruginosa isolates as demonstrated by a 100-fold decrease in the bacterial count (CFU/mL) even after 24 h. The combinations also displayed excellent antibacterial activity against P. aeruginosa under CF relevant conditions in a sputum medium assay. The combination of lumacaftor (alone) with polymyxin B showed additivity against P. aeruginosa. The potential antimicrobial mode of action of the combinations against P. aeruginosa was investigated using different methods. Treatment with the combinations induced cytosolic GFP release from P. aeruginosa cells and showed permeabilizing activity in the nitrocefin assay, indicating damage to both the outer and inner Gram-negative cell membranes. Moreover, scanning and transmission electron micrographs revealed that the combinations produce outer membrane damage to P. aeruginosa cells that is distinct from the effect of each compound per se. Ivacaftor was also shown to be a weak inhibitor of the bacterial DNA gyrase and topoisomerase IV with no effect on either human type I or type IIα topoisomerases. Lumacaftor displayed the ability to increase the cellular production of damaging reactive oxygen species. In summary, the combination of polymyxin B with KALYDECO or ORKAMBI exhibited synergistic activity against highly polymyxin-resistant P. aeruginosa CF isolates and can be potentially useful for otherwise untreatable CF lung infections.

Programmed Death-1 Ligand 2-Mediated Regulation of the PD-L1 to PD-1 Axis Is Essential for Establishing CD4(+) T Cell Immunity.

Many pathogens, including Plasmodium spp., exploit the interaction of programmed death-1 (PD-1) with PD-1-ligand-1 (PD-L1) to "deactivate" T cell functions, but the role of PD-L2 remains unclear. We studied malarial infections to understand the contribution of PD-L2 to immunity. Here we have shown that higher PD-L2 expression on blood dendritic cells, from Plasmodium falciparum-infected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for establishing effective CD4(+) T cell immunity against malaria, because it not only inhibited PD-L1 to PD-1 activity but also increased CD3 and inducible co-stimulator (ICOS) expression on T cells. Importantly, administration of soluble multimeric PD-L2 to mice with lethal malaria was sufficient to dramatically improve immunity and survival. These studies show immuno-regulation by PD-L2, which has the potential to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due to PD-1-mediated signaling.

Antibacterial Low Molecular Weight Cationic Polymers: Dissecting the Contribution of Hydrophobicity, Chain Length and Charge to Activity.

The balance of cationicity and hydrophobicity can profoundly affect the performance of antimicrobial polymers. To this end a library of 24 cationic polymers with uniquely low degrees of polymerization was synthesized via Cu(0)-mediated polymerization, using three different cationic monomers and two initiators: providing two different hydrocarbon chain tail lengths (C2 and C12). The polymers exhibited structure-dependent antibacterial activity when tested against a selection of bacteria, viz, Staphylococcus aureus ATCC 29213, Klebsiella pneumoniae ATCC 13883, Acinetobacter baumannii ATCC 19606, and Pseudomonas aeruginosa ATCC 27853 as a representative palette of Gram-positive and Gram-negative ESKAPE pathogens. The five best-performing polymers were identified for additional testing against the polymyxin-resistant A. baumannii ATCC 19606R strain. Polymers having the lowest DP and a C12 hydrophobic tail were shown to provide the broadest antimicrobial activity against the bacteria panel studied as evidenced by lower minimum inhibitory concentrations (MICs). An optimal polymer composition was identified, and its mechanism of action investigated via membrane permeability testing against Escherichia coli. Membrane disruption was identified as the most probable mechanism for bacteria cell killing.

Antibacterial and antifungal screening of natural products sourced from Australian fungi and characterisation of pestalactams D-F.

Eighteen natural products sourced from Australian micro- or macro-fungi were screened for antibacterial and antifungal activity. This focused library was comprised of caprolactams, polyamines, quinones, and polyketides, with additional large-scale isolation studies undertaken in order to resupply previously identified compounds. Chemical investigations of the re-fermented culture from the endophytic fungus Pestalotiopsis sp. yielded three caprolactam analogues, pestalactams D-F, along with larger quantities of the known metabolite pestalactam A, which was methylated using diazomethane to yield 4-O-methylpestalactam A. The chemical structures of the previously undescribed fungal metabolites were determined by analysis of 1D/2D NMR and MS data. The structure of 4-O-methylpestalactam A was confirmed following single crystal X-ray diffraction analysis. The antibacterial and antifungal activity of all compounds was assessed, which identified three compounds, (1S,3R)-austrocortirubin, (1S,3S)-austrocortirubin, and 1-deoxyaustrocortirubin with mild activity (100 µM) against Gram-positive isolates and one compound, 2-hydroxy-6-methyl-8-methoxy-9-oxo-9H-xanthene-1-carboxylic acid, with activity against Cryptococcus neoformans and Cryptococcus gattii at 50 µM.

Activity and Predicted Nephrotoxicity of Synthetic Antibiotics Based on Polymyxin B.

The polymyxin lipodecapeptides colistin and polymyxin B have become last resort therapies for infections caused by highly drug-resistant Gram-negative bacteria. Unfortunately, their utility is compromised by significant nephrotoxicity and polymyxin-resistant bacterial strains. We have conducted a systematic activity-toxicity investigation by varying eight of the nine polymyxin amino acid free side chains, preparing over 30 analogues using a novel solid-phase synthetic route. Compounds were tested against a panel of Gram-negative bacteria and counter-screened for in vitro cell toxicity. Promising compounds underwent additional testing against primary kidney cells isolated from human kidneys to better predict their nephrotoxic potential. Many of the new compounds possessed equal or better antimicrobial potency compared to polymyxin B, and some were less toxic than polymyxin B and colistin against mammalian HepG2 cells and human primary kidney cells. These initial structure-activity and structure-toxicity studies set the stage for further improvements to the polymyxin class of antibiotics.

Synthesis of Norbornane Bisether Antibiotics via Silver-mediated Alkylation.

A small series of norbornane bisether diguanidines have been synthesized and evaluated as antibacterial agents. The key transformation-bisalkylation of norbornane diol 6-was not successful using Williamson methodology but has been accomplished using Ag2O mediated alkylation. Further functionalization to incorporate two guanidinium groups gave rise to a series of structurally rigid cationic amphiphiles; several of which (16d, 16g and 16h) exhibited antibiotic activity. For example, compound 16d was active against a broad range of bacteria including Pseudomonas aeruginosa (MIC = 8 µg/mL), Escherichia coli (MIC = 8 µg/mL) and methicillin-resistant Staphylococcus aureus (MIC = 8 µg/mL).

Flemingin-Type Prenylated Chalcones from the Sarawak Rainforest Plant Desmodium congestum.

In an ongoing program to identify new anti-infective leads, an extract derived from whole plant material of Desmodium congestum collected in the Sarawak rainforest was found to have anti-MRSA activity. Bioassay-guided isolation led to the isolation of two new prenylated chalcones, 5'-O-methyl-3-hydroxyflemingin A (1) and 5'-O-methylflemingin C (2), which were closely related to the flemingins previously isolated from various Flemingia species. Chalcones 1 and 2, which were determined to be 4:6 enantiomeric mixtures by chiral HPLC, exhibited moderate activity against a panel of Gram-positive bacteria and were also cytotoxic to the HEK293 human embryonic kidney cell line.