Gram-negative bacteria are shielded by an asymmetric outer membrane whose biogenesis depends on the lipopolysaccharide transport (Lpt) machinery. Therein, the essential membrane protein LptD mediates the final lipopolysaccharide insertion step, making it a prime target for antibiotics. Here, we determined five high-resolution cryo-electron microscopy structures of Escherichia coli and Pseudomonas aeruginosa LptDEM in complex with the peptide antibiotics murepavadin and thanatin, to resolve their modes of action. The structures reveal that both compounds target the distal β-jellyroll edge of LptD, blocking the essential interaction with the periplasmic bridge protein LptA. Despite converging on the same binding region, thanatin and murepavadin engage LptD via different interaction modes: thanatin binds via β-sheet augmentation, whereas murepavadin binds through side chain interactions. These alternative strategies explain the broad-spectrum activity of thanatin versus the species specificity of murepavadin and establish the distal β-jellyroll edge of LptD as a structural vulnerability in the Gram-negative envelope. Our findings thus provide a framework for rational design of antibiotics targeting this site.

The rise of antimicrobial resistance poses a substantial threat to our health system, and, hence, development of drugs against novel targets is urgently needed. The natural peptide thanatin kills Gram-negative bacteria by targeting proteins of the lipopolysaccharide transport (Lpt) machinery. Using the thanatin scaffold together with phenotypic medicinal chemistry, structural data, and a target-focused approach, we developed antimicrobial peptides with drug-like properties. They exhibit potent activity against Enterobacteriaceae both in vitro and in vivo while eliciting low frequencies of resistance. We show that the peptides bind LptA of both wild-type and thanatin-resistant Escherichia coli and Klebsiella pneumoniae strains with low-nanomolar affinities. Mode of action studies revealed that the antimicrobial activity involves the specific disruption of the Lpt periplasmic protein bridge.

Abstract The rapid rise of multi-resistant bacteria poses a significant threat to our health system. The development of drugs against novel targets is urgently needed to replenish the clinical arsenal with effective antibiotics. The naturally occurring peptide thanatin kills Gram-negative bacteria by targeting the periplasmic protein bridge, in particular the lipopolysaccharide transport protein A (LptA). Using the thanatin scaffold together with phenotypic medicinal chemistry and a target-focused approach, we developed potent antimicrobial macrocyclic peptides with drug-like properties that exhibited low frequencies for development of resistance both in vitro and in vivo. Binding affinities to Escherichia coli LptA of the thanatin analogs correlated well with minimal inhibitory concentrations for both parent and thanatin-resistant E. coli strains. Mode of action studies revealed that the antimicrobial activity of the antibiotics involves the specific disruption of the periplasmic Lpt protein bridge. Our studies validate the Lpt protein bridge as a novel target for macrocyclic peptide antibiotics that can avoid current mechanisms of drug resistance. The peptides warrant further preclinical and clinical studies addressing WHO priority 1 carbapenem-resistant Enterobacteriaceae, in particular for Klebsiella pneumoniae lung infections.

Polymyxins are last-resort antibiotics with potent activity against multi-drug resistant pathogens. They interact with lipopolysaccharide (LPS) in bacterial membranes, but mechanistic details at the molecular level remain unclear. Here, we characterize the interaction of polymyxins with native, LPS-containing outer membrane patches of Escherichia coli by high-resolution atomic force microscopy imaging, along with structural and biochemical assays. We find that polymyxins arrange LPS into hexagonal assemblies to form crystalline structures. Formation of the crystalline structures is correlated with the antibiotic activity, and absent in polymyxin-resistant strains. Crystal lattice parameters alter with variations of the LPS and polymyxin molecules. Quantitative measurements show that the crystalline structures decrease membrane thickness and increase membrane area as well as stiffness. Together, these findings suggest the formation of rigid LPS-polymyxin crystals and subsequent membrane disruption as the mechanism of polymyxin action and provide a benchmark for optimization and de novo design of LPS-targeting antimicrobials.

Background: Murepavadin, a novel peptidomimetic antibiotic, is being developed as an inhalation therapy for treatment of Pseudomonas aeruginosa respiratory infection in people with cystic fibrosis (CF). It blocks the activity of the LptD protein in P. aeruginosa causing outer membrane alterations. Objectives: To determine the in vitro activity of murepavadin against CF P. aeruginosa isolates and to investigate potential mechanisms of resistance. Methods: MIC values were determined by both broth microdilution and agar dilution and results compared. The effect of artificial sputum and Lung surfactant on in vitro activity was also measured. Spontaneous mutation frequency was estimated. Bactericidal activity was investigated using time-kill assays. Resistant mutants were studied by WGS. Results: The murepavadin MIC50 was 0.125 versus 4 mg/L and the MIC90 was 2 versus 32 mg/L by broth microdilution and agar dilution, respectively. Essential agreement was >90% when determining in vitro activity with artificial sputum or Lung surfactant. It was bactericidal at a concentration of 32 mg/L against 95.4% of the strains within 1-5 h. Murepavadin MICs were 2-9 two-fold dilutions higher for the mutant derivatives (0.5 to >16 mg/L) than for the parental strains. Second-step mutants were obtained for the PAO mutS reference strain with an 8xMIC increase. WGS showed mutations in genes involved in LPS biosynthesis (lpxL1, lpxL2, bamA2, lptD, lpxT and msbA). Conclusions: Murepavadin characteristics, such as its specific activity against P. aeruginosa, its unique mechanism of action and its strong antimicrobial activity, encourage the further clinical evaluation of this drug.

This review covers some of the recent progress in the field of peptide antibiotics with a focus on compounds with novel or established mode of action and with demonstrated efficacy in animal infection models. Novel drug discovery approaches, linear and macrocyclic peptide antibiotics, lipopeptides like the polymyxins as well as peptides addressing targets located in the plasma membrane or in the outer membrane of bacterial cells are discussed.

Abstract Despite availability of new therapies, outcomes for patients with relapsed metastatic breast cancer is poor with moderate median overall survival. High CXCR4 levels correlate with aggressive metastatic phenotypes and poor prognosis in breast cancer (BC). Anti-cancer mechanisms of CXCR4 antagonists include inhibition of angiogenesis and metastasis, activation of immunosurveillance and chemo sensitization. Balixafortide (POL6326) is a potent, selective inhibitor of the chemokine receptor CXCR4 in PhIII for metastatic HER2-negative breast cancer (BC) in combination with tubulin-binding eribulin (NCT03786094). Clinical proof-of-concept in combination with eribulin was demonstrated in a PhI single arm dose-escalation trial in patients with metastatic HER2-negative BC (NCT01837095). The objective response rate for a dose of 5.5mg/kg balixafortide and 1.4mg/m2 eribulin) was 38% (median duration 4.4 months), and the clinical benefit rate was 63% (median duration 8.1 months). Tolerability and safety of eribulin in combination with balixafortide was comparable to that of eribulin monotherapy. Balixafortide was combined with paclitaxel in a humanized BC patient-derived xenograft (PDX) model to explore the efficacy in combination with another tubulin-binding drugs. Human immunophenotyping confirmed successful human CD34+ cell engraftment and expansion of human immune cells prior implantation of an invasive ductal carcinoma (ypT1a ypN1a R0 G3 L1 V1), ER/PR/Her-2 negative tumor. 10mg/kg paclitaxel was given 2x weekly iv. Dosing of balixafortide was 20mg/kg bid sc for 5 days with 2 days break. Total treatment duration was 28 days. Partial remission was achieved in the combination arm balixafortide+paclitaxel (T/C 13%, 87% inhibition of tumor volume vs vehicle control) which was stat. significantly better than paclitaxel monotherapy (T/C 42%). There was no decrease in tolerability in balixafortide combination vs paclitaxel alone. This data suggests combination with balixafortide is more efficacious and equally tolerated to paclitaxel single agent treatment. Citation Format: Daniel Obrecht, Johann Zimmermann. Efficacy of Balixafortide (POL6326) and Paclitaxel alone and in combination in humanized breast cancer PDX [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 414.

OBJECTIVES:To determine the activity of murepavadin in comparison with tobramycin, colistin and aztreonam, against cystic fibrosis (CF) Pseudomonas aeruginosa isolates growing in biofilms. The biofilm-epidemiological cut-off (ECOFF) values that include intrinsic resistance mechanisms present in biofilms were estimated.METHODS:Fifty-three CF P. aeruginosa isolates from respiratory samples were tested using the Calgary (closed system) device, while 4 [2 clinical (one smooth, one mucoid) and 2 reference strains] were tested using the BioFlux, a microfluidic open model of biofilm testing. Biofilm was stained with SYTO9® and propidium iodide. The minimal biofilm inhibitory concentration (MBIC) and the minimal biofilm eradication concentration (MBEC) were determined. The MBIC-ECOFF and the MBEC-ECOFF were calculated.RESULTS:Colistin, tobramycin and murepavadin presented similar MBIC50/MBIC90 values (4/32, 8/64 and 2/32, respectively). Murepavadin exhibited the lowest MBEC90 (64 mg/L). Aztreonam MBIC and MBEC values were higher than those of the other antibiotics tested. Tobramycin and murepavadin had the lowest MBEC-ECOFF (64 and 128 mg/L, respectively), while those of aztreonam and colistin exceeded 512 mg/L. Using the BioFlux, for the PAO1, PAO mutS and the smooth clinical strain, a significant difference (P < 0.0125) was observed when comparing the fluorescence of treated and untreated biofilms. For the mucoid strain, only the biofilm treated with aztreonam (MBIC and MBEC) and tobramycin (MBEC) showed differences with respect to the untreated biofilm.CONCLUSIONS:Murepavadin demonstrated good activity against P. aeruginosa biofilms both in open and closed systems. The MBIC-ECOFF and the MBEC-ECOFF are proposed as new parameters to estimate the activity of antibiotics on biofilms.

Polyphor's macrocycle platform led to the discovery of novel antibiotics addressing specifically Gramnegative bacteria by targeting outer membrane proteins. Furthermore, POL6014, an inhibitor of neutrophile elastase and balixafortide, a CXCR4 inhibitor have been discovered and developed from the platform. Currently a combination of balixafortide and eribulin is in Phase III clinical trial for the treatment of patients with advanced metastatic HER2-negative breast cancer.

Pseudomonas aeruginosa is an opportunistic human pathogen and a leading cause of nosocomial infections. Due to its high intrinsic and adaptive resistance to antibiotics, infections caused by this organism are difficult to treat and new therapeutic options are urgently needed. Novel peptidomimetic antibiotics that target outer membrane (OM) proteins have shown great promise for the treatment of P. aeruginosa infections. Here, we have performed genome-wide mutant fitness profiling using transposon sequencing (Tn-Seq) to identify resistance determinants against the recently described peptidomimetics L27-11, compounds 3 and 4, as well as polymyxin B2 (PMB) and colistin (COL). We identified a set of 13 core genes that affected resistance to all tested antibiotics, many of which encode enzymes involved in the modification of the lipopolysaccharide (LPS) or control their expression. We also identified fitness determinants that are specific for antibiotics with similar structures that may indicate differences in their modes of action. These results provide new insights into resistance mechanisms against these peptide antibiotics, which will be important for future clinical development and efforts to further improve their potency.

Background: Balixafortide (B) is a highly selective antagonist of the chemokine receptor CXCR4. Clinical proof-of-concept in combination with eribulin (E) was achieved in a recent Phase 1 single arm dose-escalation trial in patients with metastatic HER2-negative breast cancer (NCT01837095). Safety and tolerability of B in combination with E was similar to that of B or E monotherapy. Anti-cancer effects of CXCR4 antagonists include sensitization of tumor cells to chemotherapy, suppression of metastatic spread, inhibition of angiogenesis, and activation of immune cells. Methods: Plasma samples from patients who received initial E monotherapy (1.4 mg/m2, run-in cycle) or combination of B and E (escalating doses of B intravenous up to 5.5 mg/kg plus E 1.4 mg/m2 in 21 days cycles) were immediately processed and stored at -80°C. Circulating levels of IFN-γ, an important marker of anti-tumor immune response, were determined by a high-sensitive S-Plex assay (Meso Scale Diagnostics). Absolute neutrophil counts (ANC) were measured by automated hematology instruments. Results: E monotherapy did not increase IFN-γ levels in the first 8 hours post dosing, but led to an increase after 24 hours. Combination with B modulated IFN-γ levels and led to a sustained ANC increase 5 days post dosing. The increase pre vs post treatment can be up to 100-fold and receded prior to the next treatment cycle. Conclusions: E treatment led to a strong, transient increase of plasma IFN-γ levels in breast cancer patients modulated by B. The ANC increase by B may balance possible neutropenic actions of E. Citation Format: Johann Zimmermann, Garry Douglas, Barbara Romagnoli, Debra Barker, Daniel Obrecht. Circulating levels of IFN gammaand neutrophil counts in breast cancer patients who received balixafortide and eribulin combination therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3965.

This review describes a selection of macrocyclic natural products and structurally modified analogs containing peptidic and non-peptidic elements as structural features that potentially modulate cellular permeability. Examples range from exclusively peptidic structures like cyclosporin A or phepropeptins to compounds with mostly non-peptidic character, such as telomestatin or largazole. Furthermore, semisynthetic approaches and synthesis platforms to generate general and focused libraries of compounds at the interface of cyclic peptides and non-peptidic macrocycles are discussed.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Murepavadin (POL7080) represents the first member of a novel class of outer membrane protein-targeting antibiotics. It specifically interacts with LptD and inhibits lipopolysaccharide (LPS) transport. Murepavadin is being developed for the treatment of serious infections by Pseudomonas aeruginosa. We determined the plasma protein binding and the pharmacokinetics of murepavadin in plasma and epithelial lining fluid (ELF; pulmonary) in infected animals, and we determined the exposure-response relationship. Treatment of CD-1 neutropenic mice was started 2 h after infection using murepavadin at different dosing frequencies for 24 h, and the number of CFU per lung was determined. The sigmoid maximum-effect model was used to fit the dose-response, and the pharmacodynamic index (PDI) response was used to determine the PDI values, resulting in a static effect and 1-log kill reduction. Using R-2 as an indicator of the best fit, the area under the concentration-time curve for the unbound fraction of the drug (fAUC)/MIC ratio correlated best with efficacy. The mean AUC required to provide a static effect was 36.83 mg h/liter (fAUC = 8.25 mg h/liter), and that to provide a 1-log reduction was 44.0 mg h/liter (fAUC = 9.86 mg h/liter). The mean static fAUC/MIC was determined to be 27.78, and that for a 1-log reduction was 39.85. These data may serve to determine doses in humans that are likely to be efficacious.

Abstract Background: Balixafortide is a potent and selective antagonist of the chemokine receptor CXCR4, and is in PhIII for metastatic HER2-negative breast cancer in combination with eribulin (NCT03786094). Clinical proof-of concept of the combination with comparable tolerability to that of eribulin monotherapy was demonstrated in a recent PhI single arm dose-escalation trial (NCT01837095). Balixafortide can inhibit tumor growth through several mechanisms including chemosensitization in combination with chemotherapy (e.g. eribulin), suppression of metastasis, and activation of immune cell response in the tumor microenvironment. In addition, data regarding the co-regulation of the angiogenic factor VEGF and CXCR4 in colorectal cancer (CRC) tissue suggest a possible anti-angiogenic activity of Balixafortide. Methods: VEGF alpha-induced migration of endothelial cells and vascular permeabilization were assessed in vitro. Changes in VEGF alpha levels were determined in CXF260 CRC PDX tumor samples by Nanostring and in CXF260 cells in vitro by qPCR. Results: Balixafortide potently inhibited VEGF alpha-induced migration and permeabilization of an endothelial monolayer in vitro and reduced VEGF alpha levels in both, cultured CXF260 tumor cells in vitro and CXF260 tumors in vivo. Conclusions: Balixafortide modulates angiogenic mechanisms in vitro and in vivo and suggests further investigation of balixafortide in anti-angiogenic therapies. Citation Format: Johann Zimmermann, Daniel Obrecht, Tobias Remus. Anti-angiogenic activity of the CXCR4 antagonist balixafortide [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A003. doi:10.1158/1535-7163.TARG-19-A003

Background: Balixafortide is a very potent, well tolerated and highly selective next generation CXCR4 antagonist derived over the past decade through multiple rounds of optimization starting from the natural product polyphemusin. Clinical proof-of-concept was achieved in a Ph 1/2 study in combination with Eribulin in metastatic HER2-neg breast cancer. The anti-cancer effects and pluripotent action of Balixafortide may include sensitization of tumor cells to chemotherapy, suppression of metastatic spread, inhibition of angiogenesis, and activation of the immune system. Methods: Balixafortide was tested in a HTRF-based CXCR4 ligand binding assay, in functional assays (Calcium flux and beta arrestin), and further profiled in a large panel of other receptors including CXCR7. Effects on tumor cell sensitization were followed with an intracellular pERK / pAKT signaling assay. Tumor cell migration was assessed by chemotaxis assays, and inhibition of angiogenesis was determined by HUVEC sprouting. Evidence for immune cell activation came from evaluation of corresponding marker such as interferon gamma. Balixafortide was in detail profiled in an extensive in vitro ADME panel. Results: Balixafortide binds CXCR4 with high affinity (IC50 < 10nM). It blocks beta arrestin recruitment and Calcium flux with IC50s < 10nM. A high 1000-fold selectivity window was demonstrated in a large panel of receptors including CXCR7. Balixafortide potently inhibits pERK / pAKT signaling in the lymphoma lines Namalwa (IC50 < 200 nM) and Jurkat (IC50 < 400 nM). Balixafortide efficiently blocks SDF-1 dependent chemotaxis of MDA MB 231 breast cancer cells (IC50 < 20 nM), Namalwa and Jurkat cells (IC50 < 10 nM). Receptor occupancy wash-out studies with competitive antibody 12G5 revealed prolonged binding of Balixafortide to CXCR4. In addition, Balixafortide was optimized for favorable mouse and human ADME properties with balanced plasma protein binding, greater plasma and microsomal stability. Conclusions: Balixafortide is a product of an extensive optimization process which started from polyphemusin and now represents a favorable balance between ADMET properties, potency and tolerability which allows high and frequent dosing of a CXCR4 antagonist in cancer patients. Legal entity responsible for the study: Polyphor Ltd. Funding: Has not received any funding. Disclosure: J. Zimmermann: Employee and shareholder: Polyphor Ltd. T. Remus, G. Lemercier, D. Barker, D. Obrecht, G. Douglas, G. Gambino: Employee: Polyphor Ltd.

Macrocyclic peptide-based natural products have provided powerful new antibiotic drugs, drug candidates, and scaffolds for medicinal chemists as a source of inspiration to design novel antibiotics. While most of those natural products are active mainly against Gram-positive pathogens, novel macrocyclic peptide-based compounds have recently been described, which exhibit potent and specific activity against some of the most problematic Gram-negative ESKAPE pathogens. This mini-review gives an up-date on recent developments.