WO2024123712A1 - Analyse de compositions cytotoxiques candidates - Google Patents

Analyse de compositions cytotoxiques candidates Download PDF

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Publication number
WO2024123712A1
WO2024123712A1 PCT/US2023/082388 US2023082388W WO2024123712A1 WO 2024123712 A1 WO2024123712 A1 WO 2024123712A1 US 2023082388 W US2023082388 W US 2023082388W WO 2024123712 A1 WO2024123712 A1 WO 2024123712A1
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Prior art keywords
polymyxin
outer membrane
binding
omvs
lps
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PCT/US2023/082388
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English (en)
Inventor
John Gerard Quinn
Steven Thomas RUTHERFORD
Kerry Renee BUCHHOLZ
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Genentech, Inc.
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Publication of WO2024123712A1 publication Critical patent/WO2024123712A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

Definitions

  • the principal lipid component of the outer leaflet of the outer membrane is lipopolysaccharide (LPS), a complex glycolipid composed of a conserved lipid A anchor, a solvent-facing core oligosaccharide, and a variable, extracellular O-antigen.
  • LPS lipopolysaccharide
  • the polymyxin class of antibiotics which include polymyxin B and colistin, are last-resort antibiotics for the treatment of multi-drug resistant Gram-negative bacterial infections and target lipid A.
  • These electropositive amphiphilic antibiotics can establish multiple electrostatic interactions with phosphate groups of lipid A in addition to hydrogen bonds and lipophilic interactions with the hydrophobic anchor and the associated cooperativity leads to enhanced binding avidity.
  • the present disclosure is directed to methods of identifying a composition capable of inducing the death of Gram-negative bacterial cells, comprising: contacting the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom, with the composition; determining that the composition complexes with a lipopolysaccharide integral with the outer membrane; contacting the complex with a wash; determining that the composition is retained in the outer membrane after contact with the wash; repeating steps a-d; and determining that the composition accumulates in the outer membrane and is retained in the outer membrane, e.g., for at least about 10 minutes; wherein said accumulation of the composition in the outer membrane and its retention in the outer membrane, e.g., for at least 10 minutes, identifies the composition as capable of inducing the death of Gram-negative bacterial cells.
  • the outer membrane is an outer membrane of an outer membrane vesicle derived from a Gram-negative bacterial cell.
  • the composition accumulates in the outer membrane, e.g., to a mass fraction greater than 10% (weight of accumulated composition / weight of outer membrane).
  • the determination that the composition complexes with a lipopolysaccharide integral with the outer membrane is performed by a SPR analysis.
  • the determining that the composition is retained in the outer membrane after contact with the wash is performed by SPR analysis.
  • the determining that that the composition accumulates in the outer membrane and is retained in the outer membrane, e.g., for at least about 10 minutes, is performed by SPR analysis.
  • the Gram-negative bacterial cell or outer membrane vesicle derived therefrom is immobilized on an SPR sensor substrate.
  • the Gram-negative bacterial cell or outer membrane vesicle derived therefrom is immobilized on an SPR sensor substrate via an affinity coupling.
  • the affinity coupling comprises amine-coupled polymyxin B immobilized on the SPR sensor substrate.
  • the Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom has lipopolysaccharide with variable length sugars.
  • the Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom is polymyxin B resistant.
  • the composition is a candidate antibacterial.
  • the candidate antibacterial is a polypeptide or polypeptide analog.
  • the candidate antibacterial is a polymyxin.
  • FIG. 1A Cartoon of SPR chip surface with bound whole cells or OMVs, which present the outer membrane in a near-native state.
  • Figure 1B Transmission electron micrograph of wild-type OMVs (wt-OMVs) exposed to polymyxin B (right) or buffer (left). Representative images shown. Scale bar 100 nm.
  • Figure 1C Scanning electron micrograph of wt-OMVs affinity captured by polymyxin B pre-coupled to the planar surface. Representative images shown. Scale bar 300 nm.
  • Figure 1D SPR binding curves showing a stepwise increase in binding response upon exposure of wt-OMVs to eight serial-doubling concentrations of polymyxin B from 39 nM to 5 ⁇ M.
  • Figure 1E Same plot shown in ( Figure 1C) but illustrating the approximately 1:2 fraction of weakly bound polymyxin B to tightly bound polymyxin B.
  • Figures 2A-2I provide SPR kinetic traces for binding of antibiotics to whole bacterial cells or OMVs. Each serial injection profile was recorded as outlined in Figures 1A-1E with a maximum concentration of 625 nM.
  • Figure 3A-3B illustrates two versions of the three-state model for LPS-targeting antibiotics.
  • FIG.3A The first more approximate version is shown in FIG.3A, Initial State: Affinity network consisting of tightly packed LPS (black circles, L) stabilized by divalent cations (red dots) is shown.
  • State 1 Polymyxin B (PMB, blue) binds to L transiently to form PMB- L.
  • State 2 A fraction of PMB-L slowly form lipophilic interactions with the membrane Active 108465066.1.
  • DOCX 3 00B206.1385 producing a more stable complex, nPMB-L.
  • State 3 LPS phase transition is triggered forming hexagonal clusters when sufficient PMB-L are bound.
  • the model describes LPS-catalyzed accumulation of polymyxin B clusters, cPMB through phase separation mediated by transition state intermediates.
  • PMBi is injected polymyxin B; PMB is polymyxin B located at the LPS coated surface; L is LPS; PMBL is a transient polymyxin B-LPS complex; n indicates available membrane insertion sites (open circles); nPMBL is the membrane-inserted polymyxin B-LPS species; tPMB is a transient nPMBL dimer in complex with LL which is a transient LPS dimer; and cPMB is a phase- separated monomer of polymyxin B that exists in clusters containing multiples of this monomer.
  • k t is a mass transport rate that supplies PMBi to the LPS-coated surface annotated as PMB where the concentration of PBM is less than the concentration of PMBi due to depletion. Depletion results from the surface reactions initiated by binding to L; KD1 is an effective affinity constant for binding of PMB to L.
  • Figures 4A-4C illustrate the three-state model from Fig.3A fitted to experimental SPR binding curves (Fig. 4A- Fig. 4B) and associated simulations (Fig. 4C). Model fit to binding curves for a maximum polymyxin B concentration of 0.625 ⁇ M with ( Figure 4A) wt-OMVs and ( Figure 4B) over resistant-OMVs.
  • FIG.4A bottom panel and Fig.4B bottom panel A species component analysis of the eight-step binding curves (Fig.4A bottom panel and Fig.4B bottom panel) associated with fitting the three-state model fit of Fig.3A reveals the fraction of each species present over time for polymyxin B.
  • Figure 4A upper panel and polymyxin B nonapeptide Fig.4B upper panel
  • Fig.4C right panel: Expected long-term time-dependency of binding. Simulations were performed based on the estimated rate constants in Table 2 assuming k6 3.25 x 10-5 M-1s-1 (Table 4).
  • FIG. 5A Representative TEM images of wt-OMVs exposed to buffer (left), PMBN (middle), or polymyxin (right) for 40 minutes. Scale bars 500 nm.
  • Figure 5B Uncropped TEM images from Figure 1B of wt-OMVs exposed to buffer (left) or polymyxin B (right). Scale bars 200 nm
  • Figure 5C Additional representative images of wt-OMVs exposed for polymyxin B for 40 minutes. Scale bars 200 nm. White arrows point to sites of micro- vesiculation or tubules.
  • Figures 6A-6G illustrate SPR analysis of mammalian vesicles and bacterial OMVs using lipophilic LP chips and via amine-coupled polymyxin B to the surface of a C1 chips.
  • Figure 6A Representative SPR sensorgram of mammalian extracellular vesicles loading onto LP chips and regeneration.
  • Figure 6B Representative SPR sensorgram of wt-OMVs loading onto LP chips and regeneration.
  • Figure 6C Representative SPR sensorgram of polymyxin (39 nM to 5 ⁇ M) over mammalian vesicles on LP chip. “Referenced” indicates subtraction of binding on blank chip surface from binding to EV-loaded surface.
  • FIG. 6D Representative SPR sensorgram of wt-OMVs loading onto C1 chip via amine-coupled polymyxin B followed by regeneration.
  • Figure 6E SPR binding curves showing a stepwise increase in binding response upon exposure of affinity-captured wt-OMVs to eight serial- doubling concentrations of colistin from 39 nM - 5 ⁇ M.
  • Figure 6F Representative SPR sensorgram of polymyxin B (39 nM to 5 ⁇ M) over wt-OMVs on LP chip.
  • Figure 6G Representative SPR sensorgram of resistant-OMVs loading onto amine-coupled polymyxin B-C1 chip and regeneration.
  • FIGS 7A-7G provide Lipid A R1 MS- extracted ion chromatograms (EIC) of lipid A and modifications thereof as annotated (top).
  • EIC mass ranges, peak intensity, and identification are as follows: ( Figure 7A) EIC m/z 884.07 – 884.10 EIC, 3.16E4, unmodified lipid A; ( Figure 7B) EIC m/z 898.08 – 898.12, 1.40E5, unmodified lipid A + C2H4; (Figure 7C) EIC m/z 919.60 – 919.64, 2.73E5, unmodified lipid A + C5H10; ( Figure 7D) EIC m/z Active 108465066.1.DOCX 5 00B206.1385 945.07 – 949.11, 1.14E5, singly modified lipid A; ( Figure 7E) EIC m/z 959.58 – 959.62, 8.40E5, singly modified lipid A + C2H4
  • Lipid A R1 MS- mass spectrum exhibiting relative intensities of doubly charged lipid A and modified ions (bottom). Relative ratios of modified versus unmodified lipid A were compared by extracted ion chromatography peak area.
  • Figure 8 provide chemical structures of polymyxin B and a tail-less polymyxin B derivative, polymyxin B nonapeptide (PMBN) that lacks antibacterial activity.
  • Figure 10 provides SPR kinetic traces for binding of polymyxin B to wt-OMVs in the presence of the metal chelator EDTA. Each serial injection profile was recorded as outlined in Figure 1 with a maximum concentration of 625 nM. Representative SPR sensorgram of polymyxin B binding to wt-OMVs in the presence of 4 mM of EDTA. Figures 11A-11B depicts measuring apparent kinetic constants for polymyxin B. ( Figure 11A) Chaser analysis of the stable, long-lived interaction between polymyxin B and wt-OMVs. Representative sensorgram of wt-OMV capture, polymyxin B saturation, incubation, and re-saturation with polymyxin B to visualize decreased occupancy.
  • FIG. 11B Apparent KD of the reversible, saw-tooth binding interaction of polymyxin B by saturating the stable binding component.
  • Figure 12 depicts measuring apparent kinetic constants for Brevicidine and Ogiopeptin.
  • Figure 13A-13H depicts binding of polymyxin B and PMBN to E. coli cells, OMVs, and LPS and fitting to approximate kinetic models.
  • 13A and 13B Affinity analysis of PMBN binding to (13A) E. coli cells and (13B) OMVs using a 1:1 kinetic boundary model fit (Eqn (S3)) to estimate affinity (13C).
  • the fitted SPR curves are shown in the upper panels with SPR data (black) and model fit (red) together with decomposition of one of these binding curves into component species (lower panels) with PMBL (pink), nPMBL (turquoise), tPMB (dark purple), cPMB (light purple), composite (black).
  • the fitted model is near superimposable upon the experimental SPR binding curves (14A and 14B, upper panels).
  • 14C and 14D) 2D fitspace analysis associated with each fitted data set are shown (right panels). Binding constants were constrained to global values per curve set and the resulting parameter values, standard error associated with the fit, confidence intervals, and ⁇ 2 values are summarized in Table 2.
  • the presently disclosed subject matter relates to SPR methodologies to record kinetic binding data associated with the interaction of compositions and Gram-negative bacterial cell outer membranes and/or OMVs.
  • This approach can be used to study any composition for its ability to bind to or alter the outer membrane barrier of Gram-negative bacteria and the kinetic analysis outlined herein can provide insight into the mechanisms of action and resistance to such compositions.
  • the detailed description is divided into the following subsections: 1. Definitions 2. Outer Cell Membranes & Outer Membrane Vesicles 3. SPR to Monitor the Binding of Compositions to Bacterial Cells and/or OMVs 4. Examples 1.
  • the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art.
  • “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • pathogenic Gram-negative bacteria that find use in connection with the methods of the present disclosure include, but are not limited to: Escherichia coli, Klebsiella pneumoniae, Active 108465066.1.DOCX 8 00B206.1385 Pseudomonas aeruginosa, Enterobacter cloacae, Enterobacter aerogenes, Burkholderia cepecia, Proteus mirabilis, Salmonella, Vibrio cholerae, and Acinetobacter baumannii.
  • the Gram-negative bacteria employed in the context of the methods described herein are wild type (wt) Gram-negative bacteria.
  • such wt Gram-negative bacteria will exhibit one or more wt phenotype.
  • exemplary wt phenotypes include, but are not limited to, wt antibiotic sensitivities.
  • the wt Gram-negative bacteria can exhibit polymyxin sensitivity.
  • the wt-Gram-negative bacteria is sensitive to one or more polymyxin, e.g., polymyxin B.
  • the Gram-negative bacteria employed in the context of the methods described herein are Gram-negative bacteria that exhibit one or more mutant, i.e., non-wt, phenotype.
  • such mutation may impact a biosynthetic enzyme, e.g., ADP-l-glycero-d-manno-heptose-6-epimerase (waaD).
  • waaD ADP-l-glycero-d-manno-heptose-6-epimerase
  • such Gram-negative bacteria will exhibit resistance to one or more antibiotic.
  • such resistant Gram-negative bacteria can exhibit resistance to a polymyxin.
  • the resistant-Gram-negative bacteria is resistant to polymyxin B.
  • the presently disclosed subject matter relates to SPR methodologies to record kinetic binding data associated with the interaction of compositions and outer membrane vesicles (OMVs).
  • OMVs outer membrane vesicles
  • the OMVs of the instant disclosure are membrane spheres with diameters of about 20 nm to about 250 nm. While the exact composition of OMVs can vary, e.g., based on species and/or culture conditions, they generally capture both the protein and lipid constituents of the outer membrane from which they are derived.
  • the OMVs are prepared from Gram-negative bacteria.
  • the methods of the present disclosure are applicable to the use of OMVs derived from Gram-negative bacteria generally, although the methods find particular use in connection with OMVs derived from pathogenic Gram-negative bacteria.
  • the OMVs that find use in connection with the methods of the present disclosure include, but are not limited to, those derived from: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Enterobacter aerogenes, Burkholderia cepecia, Proteus mirabilis, Salmonella, Vibrio cholerae, and Acinetobacter baumannii. Active 108465066.1.DOCX 9 00B206.1385
  • the OMVs employed in the context of the methods described herein are derived from wild type (wt) Gram-negative bacteria.
  • such OMVs will exhibit one or more wt Gram-negative bacteria phenotype.
  • Exemplary wt Gram-negative bacteria phenotypes include, but are not limited to, antibiotic sensitivities.
  • the wt-OMV can exhibit polymyxin sensitivity.
  • the wt-Gram-negative bacteria is sensitive to one or more polymyxin, e.g., polymyxin B.
  • the OMVs employed in the context of the methods described herein are derived from Gram-negative bacteria that exhibit one or more non-wt phenotype.
  • such OMVs will exhibit resistance to one or more antibiotic.
  • resistant-OMVs can exhibit resistance to a polymyxin.
  • the resistant-OMV is resistant to polymyxin B. 3. SPR to Monitor the Binding of Compositions to Bacterial Cells and/or OMVs
  • the present disclosure is directed to methods of identifying a composition capable of binding to a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom.
  • the methods described herein comprise contacting the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom, with a candidate composition and determining whether the composition complexes with a lipopolysaccharide or other component integral with the outer membrane.
  • the determination of whether the composition complexes with a lipopolysaccharide or other component integral with the outer membrane is made using SPR.
  • the present disclosure is directed to methods to monitor the binding of a candidate composition to the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom, over time.
  • the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom can be contacted with a candidate compound and two or more determinations of whether the composition complexes with a lipopolysaccharide or other component integral with the outer membrane can be made separated in time.
  • the determinations of whether the composition complexes with a lipopolysaccharide or other component integral with the outer membrane are made using SPR.
  • the present disclosure is directed to methods to monitor the binding of a candidate composition to the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom, over time and when the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom, and the candidate compound are exposed to physical manipulation, e.g., a wash, between the first time point and a subsequent time point.
  • physical manipulation e.g., a wash
  • the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom can be contacted with a candidate compound and two or more determinations of whether the composition complexes with a lipopolysaccharide or other component integral with the outer membrane can be made separated in time, where the physical manipulation, e.g., a wash, occurs between the time of the first determination and the time of the second determination.
  • the determinations of whether the composition complexes with a lipopolysaccharide or other component integral with the outer membrane are made using SPR.
  • the present disclosure is directed to methods to monitor the binding of a candidate composition to the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom, over time, where the outer membrane of a Gram-negative bacterial cell, or an outer membrane vesicle derived therefrom, and the candidate compound are exposed to physical manipulation, e.g., a wash, between the first time point and a subsequent time point.
  • the methods disclosed herein encompass determining whether the candidate compound and is retained in the outer membrane over time.
  • the methods of the present disclosure encompass determining whether the candidate compound is retained for up to about 1 to about 120 minutes, up to about 1 to about 90 minutes, up to about 1 to about 60 minutes, or up to about 1 to about 30 minutes.
  • the methods of the present disclosure encompass determining whether the candidate compound is retained up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 minutes, about 61, about 62, about 63, about 64, about 65, about 66,
  • the methods described herein encompass determining whether the candidate compound is retained for at least about 1 to about 120 minutes, about 1 to about 90 minutes, about 1 to about 60 minutes, or about 1 to about 30 minutes. In certain embodiments, the methods of the present disclosure encompass determining whether the candidate compound is retained at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 minutes, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about
  • the binding of the candidate composition induces the death of the Gram-negative bacterial cell.
  • the retention of the candidate composition in the outer membrane identifies the composition as capable of inducing the death of Gram-negative bacterial cells.
  • such cytotoxic retention is up to about 1 to about 120 minutes, up to about 1 to about 90 minutes, up to about 1 to about 60 minutes, or about 1 to about 30 minutes, e.g., up to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about Active 108465066.1.DOCX 12 00B206.1385 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52
  • such cytotoxic retention is at least about 1 to about 120 minutes, at least about 1 to about 90 minutes, at least about 1 to about 60 minutes, or at least about 1 to about 30 minutes, e.g., at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 minutes, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about
  • the methods disclosed herein encompass determining whether the candidate compound and accumulates in the outer membrane over time.
  • the methods of the present disclosure encompass determining whether the candidate compound accumulates in the outer membrane to a mass fraction of between about 1% to about 30% (weight of accumulated composition / weight of outer membrane).
  • the methods of the present disclosure encompass determining whether the candidate compound accumulates in the outer membrane to a mass fraction of up to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, Active 108465066.1.DOCX 13 00B206.1385 about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.
  • the methods described herein encompass determining whether the candidate compound accumulates in the outer membrane to a mass fraction of at least about 1% to about 30%, e.g., at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.
  • the accumulation of the candidate composition in the outer membrane identifies the composition as capable of inducing the death of Gram-negative bacterial cells.
  • such cytotoxic accumulation is up to about 1% to about 30%, e.g., up to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.
  • such cytotoxic accumulation is at least about 1% to about 30%, e.g., at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.
  • the methods disclosed herein encompass determining whether the candidate compound and accumulates in the outer membrane over time.
  • the methods of the present disclosure encompass determining whether the candidate compound accumulates in the outer membrane to a mass fraction of between about 1% to about 30% (weight of accumulated composition / weight of outer membrane). In certain embodiments, the methods of the present disclosure encompass determining whether the candidate compound accumulates in the outer membrane to a mass fraction of up to about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%.
  • the methods described herein encompass determining whether the candidate compound is retained for at least about 1 to about 120 minutes, about 1 to about 90 minutes, about 1 to about 60 minutes, or about 1 to about 30 minutes.
  • the methods of the present disclosure encompass determining whether the candidate compound is retained at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, or about 60 minutes, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81,
  • the Gram-negative bacterial cell, or OMV derived therefrom is immobilized on an SPR sensor substrate via an affinity coupling.
  • the affinity coupling comprises amine-coupled polymyxin B immobilized on the SPR sensor substrate.
  • polymyxin B can be covalently attached to a sensor substrate, e.g., a C1 chip (Series S Sensor Chip C1, Cytiva), using an amine coupling kit (Cytiva).
  • reagents N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride can be mixed and added to the gold chip surface, e.g., for about 2 min, washed, e.g., with distilled deionized water, and dried.
  • Sufficient polymyxin B e.g., 1 mM in 1 M HEPES buffer, pH 8
  • 1 M ethanolamine hydrochloride-NaOH, pH 8.5 for about 1 min.
  • Chips e.g., C1 chips, can be Active 108465066.1.DOCX 15 00B206.1385 used for multiple runs and discarded upon removal from the SPR device, e.g., a Biacore S200.
  • the SPR experiments e.g., experiments using a Biacore S200 SPR device and C1-chips
  • running buffer e.g., Dulbecco's phosphate-buffered salt solution 1x without calcium or magnesium (Fisher Scientific), pH 7.4, with 0.0005% tween-80 (Sigma) passed through a 0.2 ⁇ m filter.
  • MgCl2 can be added as indicated throughout the figures and examples.
  • inclusion of tween-80 can prevent loss of candidate compositions, e.g., polymyxins, to the plastics.
  • the candidate composition can be suspended in DMSO, and DMSO, e.g., 0.125% DMSO, can be included in running buffer.
  • DMSO e.g. 0.125% DMSO
  • the analysis and compartment temperature of the SPR device can be set to 25°C or 37°C.
  • OMVs can be diluted from frozen stocks to approximately 20-30 ⁇ g/ml protein in OMV buffer. Capture of OMVs can be performed at low flow rate, e.g., 5 ⁇ l/ml for 300 sec over the test channel(s), followed by a stabilization period, e.g., about 300 sec stabilization period.
  • All subsequent steps can be performed at a higher flow rate, e.g., at a flow rate of 40 ⁇ l/ml.
  • two-fold dilutions can be injected over the channel(s) loaded with OMVs and a reference channel without OMVs, e.g., for about 30 sec contact and about 480 sec dissociation.
  • detergent e.g. 0.5% SDS (desorb 1, Cytiva)
  • SPR on whole bacterial cells can be performed and analyzed as described for OMVs on C1 chip, but using a lipophilic chip (e.g., LP (Xantec), or L1 (Cytiva)), and with an additional carry-over wash prior to the capture.
  • Regeneration of the chip after whole cell binding can also include additional steps (e.g., 40 ⁇ l/ml flow rate): 1) PBS supplemented with 32 mM MgCl2 for 120 sec, 2) 2.5 M NaCl for 30 sec, 3) 0.5% SDS (Desorb 1, Cytiva) for 60 s with carry over controls between.
  • the apparent KD of the reversible binding event occurring between the candidate composition and the Gram-negative bacteria or OMV derived therefrom, single-cycle kinetics were performed.
  • an appropriate amount of candidate composition e.g., 5 ⁇ M polymyxin B
  • the base-to-peak value of each trace can then be determined Active 108465066.1.DOCX 16 00B206.1385 from double-referenced traces (reference 1: - OMVs/ + compound channel; reference 2: + OMVs/ - compound) exported from the SPR software, e.g., Biacore S200 evaluation software into PRISM 9 software.
  • the change in RU with each pulse can be plotted over the concentration and the KD determined by fitting a non-linear regression, one-site total function with background parameter set to 0 (GraphPad Prism version 9.3.1 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com).
  • determination of the apparent KD can occur without any pre-saturation step prior to kinetic pulses.
  • the determination of the residence time of a candidate compound with OMVs can be performed using the ‘chaser method’ to account for drift that can occur in the system over long incubation times.
  • OMVs can be loaded onto the chip as described above then the candidate composition, e.g., 5 ⁇ M polymyxin B, can be injected using the ‘low-sample consumption’ setting, e.g., for 300 sec (5 ⁇ l/ml flowrate), followed by a 240 sec dissociation time.
  • a second dose of the candidate composition e.g., 5 ⁇ M polymyxin B
  • the RUs of associated the candidate composition can be determined after the start of the dissociation, e.g., after 120 seconds.
  • the candidate composition is a candidate cytotoxic composition.
  • the candidate cytotoxic composition is a candidate antibacterial.
  • the candidate antibacterial is a polypeptide antibacterial or polypeptide analog antibacterial.
  • the candidate antibacterial is a polymyxin.
  • the candidate composition is natural product antibiotic.
  • the natural product antibiotic is brevicidine, ogipeptin, or pedopeptin.
  • SPR is used to kinetically interrogate the interactions between polymyxins and LPS in bacterial cells as well as OMVs and pure LPS films, which are effective surrogates that capture the natural complexity of the outer membrane and afford more experimental robustness.
  • the information-rich SPR data presented herein enables application of a mechanistic model showing that LPS catalyzes super-stoichiometric accumulation of polymyxins via three-states: (1) transient binding to the lipid A anchor of LPS facilitates (2) membrane insertion of polymyxins and promotes (3) phase-separation of long-lived polymyxin B clusters that reach a high density.
  • a proposed microscopic mechanism is consistent with the kinetic measurements as well as previous observations and is generalizable to other lipid A-targeting antibacterial molecules, including brevicidine.
  • Bacterial killing is not observed when the outer membrane fails to accumulate polymyxin clusters as observed for polymyxin B nonapeptide, which lacks an acyl tail, and when lipid A is modified, leading to polymyxin resistance.
  • Transient binding to LPS is sufficient for outer membrane permeabilization, whereas cluster formation upon lipid A binding is critical for cell killing.
  • OMV-based SPR is a useful platform for dissecting interactions with the outer membrane able to yield mechanistic insight that enables efforts to discover LPS- targeting antibiotics among other cytotoxic compositions.
  • Polymyxin B targets the conserved lipid A anchor of LPS and exhibits potent and selective Gram-negative antibacterial activity, which was confirmed by determining the minimal inhibitory concentration (MIC), or the concentration of compound required to completely inhibit bacterial growth, for polymyxin B.
  • MIC minimal inhibitory concentration
  • An MIC of 0.08 nM for polymyxin B was measured against wild-type E. coli, a model Gram-negative species, whereas this antibiotic had no detectable activity at the highest tested concentration against a Gram- Active 108465066.1.
  • MICs Minimum Inhibitory Concentrations (MICs) of polymyxin B and rifampicin potentiation against wild-type and polymyxin-resistant strains.
  • MIC ( ⁇ M) 1 species background Polymyxin B PMBN 2 Brevicidine
  • MICs Minimal Inhibitory Concentrations: lowest concentration of antibiotic that completely inhibits bacterial growth 2 PMBN, polymyxin B nonapeptide 3 Strain carrying a plasmid encoding the mcr-1 gene 4 Rifampicin present at 1.56 ⁇ M, which has no effect on growth of E. coli or polymyxin-resistant E. coli strains tested
  • SPR was employed to monitor binding to either whole E. coli cells or OMVs that were linked to a planar surface (Fig.1A).
  • OMVs are useful surrogates for the Gram-negative outer membrane, and when treated with polymyxin B they exhibited vesiculation and tubules (Fig.1B and Figs.5A-5C) similar to those perturbations observed on polymyxin-treated bacterial cells. While the sensitivity of SPR decays exponentially with distance from the planar surface and is reduced to about 30% at 270 nm, it is sufficient to detect binding to both whole E. coli cells (diameter of approximately 0.5 ⁇ m to about 1 ⁇ m along their shorter axis) and OMVs (diameters of about 20 nm to about 250 nm) range, which fit entirely within the sensitivity range of the sensor (Fig.1A).
  • SPR optically probes the volume within this sensitivity depth to produce an averaged refractive index change, represented as response units (RUs), that is proportional to a change in average concentration.
  • the response is also weakly sensitive to mass redistribution within this volume because detection sensitivity decays exponentially from the surface.
  • Membrane vesicles from Expi293 cells and Gram-negative bacterial OMVs were initially immobilized non-specifically on the surface of a lipophilic chip, however this approach was not compatible with detergents needed to reduce non-specific interactions, exhibited poor Active 108465066.1.DOCX 19 00B206.1385 regeneration, and was complicated by interactions between polymyxin B and the chip surface (Figs.6A-6C).
  • OMVs were immobilized via amine- coupled polymyxin B to the surface of a C1 chip. OMVs were stably bound to this chip surface in the presence of 0.0005% tween-80 and OMVs bound on the surface could be regenerated by standard methods, leaving the amine-coupled polymyxin B intact for subsequent OMV capture (Fig. 6D). Electron microscopy showed discretely bound wild- type OMVs (wt-OMVs) that retained their spherical shape when attached to the planar surface (Fig.1C). To measure the interactions of polymyxin B with immobilized vesicles and cells, single-cycle kinetic (SCK) injection and multicycle injection formats were employed.
  • SCK single-cycle kinetic
  • SCK finds particular use in cases where there is an accumulation of long-lived bound species as it is possible to obtain a full dose-response range in a single binding curve.
  • Adapting the SPR format, contact time, and dosing regimen allowed the complex kinetic processes of polymyxin B binding to the outer membrane to be resolved (Fig.1D and 1E).
  • Binding response curves for serial injection of increasing concentrations of polymyxin B (Fig. 1D and 1E) or colistin (Fig. 6E) over captured wt-OMVs revealed a binding profile dominated by the accumulation of tightly bound polymyxin B and a superimposed saw-tooth profile associated with transient binding.
  • polymyxin B still potentiated the activity of rifampicin, an antibiotic normally excluded by the outer membrane, consistent with outer membrane disruption (Table 1).
  • OMVs isolated from polymyxin-resistant bacteria were composed of modified lipid A (Table 3 and Fig.7A-7G) but could still be immobilized via amine-coupled polymyxin B to the surface of a C1 chip (Fig.6G).
  • Fig.2A serial injection of increasing concentrations of polymyxin B over captured resistant-OMVs exhibited only weak, saw-tooth binding (Fig.2B).
  • brevicidine exhibits antibacterial activity against strains with polymyxin- resistant lipid A modifications (Table 1). Strikingly, and consistent with its antibacterial activity, brevicidine binding to polymyxin-resistant-OMVs was indistinguishable from its binding to wt-OMVs (Fig.2I). e. Polymyxins Bind to OMVs with a Long Half-Life Binding of polymyxin B to OMVs was too complex for kinetic constants to be extracted using standard modeling.
  • the interaction models introduced here are annotated in terms of binding of polymyxins (polymyxin B [PMB]) to LPS but also apply to any other affinity binding pair.
  • R G.MW.[PMB]
  • MW the molecular weight of PMB
  • G mass to response conversion factor
  • a simple 1:1 model for binding of PM to LPS to form an affinity complex PML is given by the simple 1:1 pseudo-first-order model defined by Eqn (S1).
  • DI/dt (kon.[PMB]i.(Rmax-R(t)) -koff.R(t))
  • Eqn (S1) Where R is the SPR response for accumulation of affinity complex PMBL.
  • [PMB]i is the injected concentration of PM and Rmax is the saturation response assuming full target occupancy.
  • This model assumes that mass transport of PMB within the flow cell, which governs the rate of exchange of PMB between the bulk liquid and the sensing surface, is non-limiting.
  • multicycle kinetics i.e., one concentration per sensorgram
  • any compound i.e., one concentration per sensorgram
  • the multicycle SPR curves of PMBN binding to whole cells and OMVs show kinetic curvature that resembles binding kinetics but is instead caused by the development, and decay, of a mass transport-limited boundary, reporting the binding reaction at quasi- steady-state (Fig. 13A and 13B).
  • the high-quality fit to the boundary layer model (Eqn (S3)) confirms that under these conditions, PMBN (and polymyxin B as described below) binds at an extremely high kinetic rate despite the presence of divalent cations.
  • a finite element based-numerical model shows that such mass transport dominance also applies to single cells, which is relevant to in vivo milieus.
  • Active 108465066.1.DOCX 26 00B206.1385 The binding mechanism that produces both reversible states and more stable polymyxin B bound states by comparing differences in affinity, binding capacity, and stoichiometry after full saturation with polymyxin B was next investigated.
  • the affinity constant and binding capacity for PMBN binding were estimated from the corresponding fitting equation and indicated a two-fold drop in both affinity and binding capacity when LPS was pre-saturated with polymyxin B. This implies that only a fraction of the polymyxin B binding events can transition to the more stable bound state(s) (t1/2 of >6 hours).
  • Polymyxin B binding to LPS exhibited exponential binding kinetics towards a defined saturation limit (Fig.13D) that is consistent with high occupancy of available LPS.
  • Direct binding of polymyxin B to E. coli cells, OMVs, and LPS (Fig. 13E-13G) showed tight polymyxin B binding for all three targets.
  • the apparent association rate constant which is driven by transport kinetics and binding affinity, when averaged for cells, OMVs, and LPS was in good agreement (ka 2.1 ( ⁇ 0.78) x 105 M-1s-1).
  • the dissociation is dominated by a moderate rate that was relatively consistent ( ⁇ 3-fold variation) when averaged over all three targets.
  • PMBL interacts lipophilically with n to produce a membrane-inserted species nPMBL which is the second bound state.
  • the model assumes that all polymyxin B-bound states are described on a monomer basis other than the transition state intermediates that require a dimeric state to trigger phase separation to the third state, cPMB.
  • the transition state begins with self- association of nPMBL complexes through interactions between each respective polymyxin B contained in the dimeric nPMBL. These interactions displace pre-existing interactions between each PMB and its paired L thereby forming transient membrane-inserted polymyxin B dimers (tPMB) and LPS dimers (LL).
  • the transition state intermediate tPMB is fundamentally a form of nucleate and, therefore, it may be expected to share the same dissociation constant (k 4 ). This was the case when fitting the model as dissociation of the LL intermediate state gated release of cPMB and matched the dissociation of nPMB from the acyl LPS matrix to form PMBL.
  • the rate constant (k8) for dissociation of cPMB from tPMB had no effect on the data and was therefore non-limiting and held constant at an arbitrary high non-limiting value (>1).
  • tPMB does not accumulate significantly because it dissociates irreversibly into phase-separated cPMB.
  • [PMBi](t) is the injected concentration profile and [PMB] t is the concentration profile at the sensing surface.
  • [PMBi] (t) follows a serial-doubling concentration of injected analyte, with a concentration profile defined by a serial step function, where each injection step represents a discrete concentration followed by a dissociation step without injected analyte and this is repeated for each concentration in the SCK dosing series.
  • Kintek Explorer V9.5 was used to build and fit the three-state model. This program employs its own numerical integrator, which reports the change in concentration of each species over time.
  • the SPR responses for concentrations of each accumulating PMB-species estimated from Eqn (8 - 15) are summed over time in Eqn (1), repeated here.
  • MW is the molecular weight of the injected analyte, in this case PMB (1203.48 Da).
  • the constant, G is a unit conversion factor that converts protein concentration (g/L) to response (RU) and is typically 100 (units, RU.L/g).
  • Biacore SPR systems are calibrated such that 1 RU is equivalent to a change of 1x10 -6 refractive index units (RIU), which is equivalent to a 2D concentration of 1 pg/mm 2 protein when the mass is distributed uniformly within a 100 nM hydrogel.
  • ROU refractive index units
  • the average height for OMVs bound to the surface is assumed to follow solution phase size measurements of approximately 100 nm and therefore G is assumed to apply as a reasonable approximation.
  • Eqn (S3) the rate equation for the diffusion boundary model (Eqn (S10) was substituted into Eqn. (S10), effectively with KD as shown in Eqn (S16) below.
  • polymyxin B binds to LPS, approximated as a simple 1:1 complex PMB-L, with a relatively weak apparent KD of approximately 1.3 ⁇ M. Formation of PMB-L leads to outer membrane barrier disruption through displacement of divalent metal ions.
  • polymyxin liberated through dissociation of PMB-L either exits from the outer membrane or undergoes lipid A-mediated membrane insertion to form nucleates, nPMB-L.
  • the fitted model shows that nucleates remain stoichiometrically associated with LPS such that LPS can co-exist in three populations of various fractions: free L, PMB-L, and nPMB- L.
  • nPMB-L coalesce into long-lived, lipid A-free polymyxin B clusters, cPMB.
  • the binding constants for these steps were well-resolved because of the wide variation in residence time between the three-bound states, which represent transiently, moderately, and highly stable species.
  • the model is consistent with “self-promoted uptake” wherein polymyxin B induces the lipid A layer to act as a catalyst that promotes accumulation of super-stoichiometry concentrations of polymyxin B into clusters.
  • weak polymyxin B binding to Active 108465066.1.DOCX 33 00B206.1385 lipid A in the first step is necessary to allow subsequent phase separation as tight binding in this step would trap polymyxin B into a 1:1 stoichiometric complex that would be rendered incapable of forming clusters.
  • This three-state model quantitatively defines the changes in mass of each species in real-time and reveals the total mass of polymyxin bound.
  • weak polymyxin B binding to LPS in the first step is necessary to allow the subsequent phase separation as tight binding would trap polymyxin B into a 1:1 stoichiometric complex that would be rendered incapable of forming cPMB clusters.
  • polymyxin B outnumbered LPS by an approximately 2:1 molar ratio (Fig. 1E, where the reversible fraction represents the total LPS), suggesting that outer membrane stretching might be expected.
  • the excess of cPMB clusters likely cause the vesiculation and tubules observed on cells and OMVs upon polymyxin exposure (Fig. 2B).
  • the three-state model suggests that polymyxin B clustering is catalyzed from transient interactions between polymyxin B and LPS and formation of clusters correlates with bactericidal activity was observed.
  • the instant model also provides insight into the differences in binding that occur when polymyxin B or lipid A are modified.
  • PMBN lacks a hydrophobic tail and does not kill bacterial cells.
  • This polymyxin B variant exclusively produces rapidly reversible binding. Strikingly, PMBN binding to OMVs is identical to polymyxin B binding when polymyxin B clusters are pre-saturated and this recapitulated with pure LPS, indicating that while PMBN can form PMBL, the lack of an acyl tail prevents further transitions.
  • nPMBL small nucleates
  • nPMBL small nucleates
  • cluster formation can promote cell killing by enabling a transmembrane flux of polymyxin B at the stretched phase boundary of cluster sites.
  • Active 108465066.1.DOCX 34 00B206.1385 The generalizability of the instant model was explored by monitoring binding of brevicidine, a distinct lipid A-binding antibacterial natural product.
  • Brevicidine exhibits a binding pattern similar to that observed for polymyxin B with SPR traces showing accumulation of a tightly bound species and a superimposed saw-tooth profile associated with transient binding, suggesting polymyxin B and brevicidine might utilize a common approach for interacting with lipid A in the outer membrane.
  • the complex formed by brevicidine (t 1/2 >1 hour) was less stable than those formed by polymyxin B (t 1/2 >6 hours) and this could account for the less potent antibacterial activity of brevicidine (Table 1).
  • brevicidine displayed an identical binding pattern with wt- OMVs and resistant-OMVs, consistent with the antibacterial activity of brevicidine against polymyxin-resistant mutants (Table 1).
  • soluble LPS is highly polymorphic and the oligosaccharide chain length has a significant impact on the packing density of LPS.
  • the area per LPS molecule can range from 8-16 nm depending on applied pressure.
  • LPS has been Active 108465066.1.DOCX 35 00B206.1385 observed in nanocrystalline formations by EM.
  • tubules and vesiculations have been observed in the absence of Mg2+ while hexagonal LPS clusters were seen in the presence of Mg2+.
  • Each hexagonal cluster in the lattice is composed of an LPS molecule on each side with an LPS-free center.
  • the lattice constant of 14 nm matches that of the hexagonal LPS lattice in the presence of polymyxin recently observed by AFM.
  • the LPS network becomes destabilized due to polymyxin displacement of divalent cations, while a Mg2+-dependent LPS transition to a crystalline lattice has now been shown to be highly destabilizing in vitro. Taking these into account, it is proposed that at the onset of exposure, polymyxin B will first displace metal ions bound to the core saccharide while leaving lipid A-bound Mg2+.
  • Polymyxin B interactions with the core saccharide should thus lower charge repulsion, increase the excluded volume, and thereby lower solvation, which destabilizes the LPS barrier function to increase permeability.
  • Surface energy minimization triggers a rapid phase change that rearranges LPS from a linearly interlinked network into clusters that pack densely into a hexagonal lattice. Once formed, the lipid A region of the LPS crystal lattice is less permeable to polymyxin B, preventing dissolution of the lattice through competition with the remaining Mg2+.
  • the LPS phase transition causes the outer membrane to expand laterally and thin, facilitating the accumulation of polymyxin B monomers unbinding from LPS.
  • polymyxin B is capable of interacting with other available sites, such as phosphorylated sugars in the core oligosaccharide, and it is these interactions that are sufficient to induce outer membrane permeability. This also indicates that divalent cation stabilization is not fully inhibited by polymyxin B.
  • polymyxin induced outer membrane permeability through core oligosaccharide binding explains why the reversible binding events for polymyxin B and PMBN with wt-OMVs and resistant OMVs are indistinguishable (Fig.
  • Polymyxin B and PMBN bind to the phosphate groups of the lipid A, however, conditions within a native LPS film favor a heterogeneous ensemble of bound states that changes over time, involving phosphates in the core saccharide. Polymyxin B induced Mg2+ displacement from lipid A can be hindered by increased avidity for Mg2+ ions due to lipid A bridging and diminished access due to tight packing around the lipid A region. f.
  • polymyxin B and colistin are both polymyxins, thus it was not unexpected that they would utilize a common mechanism (Fig.2A and 6E)
  • brevicidine a distinct LPS- binding antibacterial natural product, also exhibits a similar binding pattern. Accumulation of a tightly bound species and a superimposed saw-tooth profile associated with transient binding were observed (Fig.2I), suggesting polymyxin B and brevicidine share a common mechanism of action and indicate the generalizability of this three-state model.
  • the model supports “self-promoted uptake” wherein polymyxin B induces the LPS layer to act as a catalyst to promote accumulation of super-stoichiometric concentrations of polymyxin B.
  • transient binding of polymyxin B to LPS is a necessary first step that enables subsequent phase separation as tight binding would saturate available LPS and prevent catalytic accumulation.
  • how polymyxin B interacts with LPS in the context of the phospholipid inner membrane a step proposed to be necessary for cell killing, remains to be determined.
  • cluster-driven antibacterial activity is supported by these findings, the possibility that LPS-targeting antibiotics that do not require clustering can exist or can be developed cannot be excluded.
  • LB growth media was prepared according to manufacturer’s instructions and supplemented with 0.2% arabinose and carbenicillin (50 ⁇ g/mL) for the strains containing pBAD24-mcr1 plasmid. Cultures were started by inoculating 3 ml LB with 1-2 colonies from fresh overnight plates and grown at 37°C until in log phase. Modified minimal inhibitory concentration (MIC) assays were Active 108465066.1.DOCX 39 00B206.1385 performed in 96-well round bottom polystyrene plates (Corning) with a final volume of 100 ⁇ l in LB supplemented with tween-80 at 0.0005%.
  • MIC minimal inhibitory concentration
  • OMVs were isolated as follows.1 L cultures of bacteria were grown in LB overnight (approximately 16 h) at 37°C with aeration. Cells were pelleted by centrifugation at 15,000 rpm for 30 min at 4°C. Supernatants were filtered through a 0.45 ⁇ M PVDF filter (VWR) and concentrated via tangential flow filtration to a volume of approximately 50 mL. OMVs were pelleted by ultracentrifugation at 40,000 rpm for 2 h at 4°C.
  • VWR 0.45 ⁇ M PVDF filter
  • OMV pellets were washed in 50 ml of OMV buffer (phosphate buffered saline [PBS, Fisher Scientific] plus 200 mM NaCl, 1 mM CaCl2, and 0.5 mM MgCl2) by ultracentrifugation as described above.
  • the washed OMV pellet was resuspended in 1.5 ml of OMV buffer and passed through a 0.45 ⁇ M PVDF syringe filter.
  • OMV preparations were quantified using a standard Bradford protein assay. Aliquots were stored at 4°C or at -80°C To compare the composition of the OMVs to outer membranes, E. coli ⁇ tolQ, E. coli ⁇ tolQ pmrA G53E , and E.
  • coli ⁇ tolQ with pBAD24-mcr1 were grown as described for OMV isolation and cell pellets frozen at -20.
  • the cell pellet was brought up in ice-cold Active 108465066.1.
  • pellets were suspended in 25 mM HEPES buffer, pH 7.4, with 2% sodium lauroyl-sarcosinate (Sigma), incubated with rotation at room temperature for 30 min, and centrifuged as before.
  • the outer membrane protein containing pellet was suspended in OMV buffer and quantified using Bradford protein assay.
  • 0.5 ⁇ g of each sample prepared with BOLT LDS sample buffer and reducing agent (Invitrogen) was separated on a 4-12% NuPAGE gel in 1x MOPS buffer (Invitrogen) and stained for 1h with InstantBlue Protein Stain (Novus Biologicals).
  • C1 Chip Preparation and OMV Capture Polymyxin B was covalently attached to a C1 chip (Series S Sensor Chip C1, Cytiva) using an amine coupling kit (Cytiva). Briefly, equal amounts of reagents N- hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride were mixed as per manufacturer’s instructions and immediately added to the gold chip surface for 2 min, washed with distilled deionized water, and dried.
  • Capture of OMVs was performed at low flow rate (5 ⁇ l/ml) for 300 sec over the test channel(s) followed by a 300 sec stabilization period. All subsequent steps were at a flow rate of 40 ⁇ l/ml.
  • Active 108465066.1.DOCX 41 00B206.1385 kinetics two-fold dilutions were injected over the channel(s) loaded with OMVs and a reference channel without OMVs for 30 sec contact and 480 sec dissociation.
  • 0.5% SDS desorb 1, Cytiva
  • SPR on whole bacterial cells was performed and analyzed as described for OMVs on C1 chip, but with an additional carry-over wash prior to the capture. Regeneration of the chip after whole cell binding also required additional steps (40 ⁇ l/ml flow rate): 1) PBS supplemented with 32 mM MgCl2 for 120 sec, 2) 2.5 M NaCl for 30 sec, 3) 0.5% SDS (Desorb 1, Cytiva) for 60 s with carry over controls between. Capturing sufficient RUs of whole bacterial cells required additional injection optimization and exhibited high experiment-to-experiment variability. e.
  • LP Chip LP chips (2D carboxymethyldextran surface, partially alkyl derivatized, Xantex Bioanalytics) were cleaned with two-20 sec pulses of 40 mM CHAPS (3-((3- cholamidopropyl) dimethylammonio)-1-propanesulfonate) with 10 sec dissociation at 30 ⁇ l/ml flowrate as recommended by the manufacturer. All LP-chip experiments were performed in 0.2 ⁇ m filtered 1x Dulbecco's PBS without CaCl2 or MgCl2 (Fisher Scientific), pH 7.4. Tween-80 was not compatible with this system, increasing the loss of polymyxin B due to non-specific binding. Analysis and compartment temperature were set to 25°C.
  • Mammalian vesicles from Expi293 cells were isolated and diluted to 0.1 mM in PBS. OMVs diluted in OMV buffer (described above) or mammalian vesicles were captured onto the chip for 60 sec (5 ⁇ l/ml flow rate) followed by a 300 sec stabilization period. Single cycle kinetics were performed as described above for the C1 chip. To regenerate the chip, 40 mM CHAPS was injected to all channels for 180 sec (40 ⁇ l/ml), washed, 50 mM NaOH for 60 sec (40 ⁇ l/ml), buffer washed again, and finally four carry-over control steps.
  • the base-to-peak value of each trace was determined from double-referenced traces (reference 1: - OMVs/ + compound channel; reference 2: + OMVs/ - compound) exported from the Biacore S200 evaluation software into PRISM 9 software.
  • the change in RU with each pulse was plotted over the concentration and the KD determined by fitting a non-linear regression, one-site total function with background parameter set to 0 (GraphPad Prism version 9.3.1 for Mac, GraphPad Software, San Diego, California USA, www.graphpad.com).
  • To determine the apparent KD of nonapeptide and polymyxin B on resistant-OMVs the same approach was taken as described above but without any pre-saturation step prior to kinetic pulses.
  • the ‘chaser method’ was used because it accounts for drift that can occur in the system over long incubation times.
  • OMVs were loaded onto the chip as described above then 5 ⁇ M polymyxin B was injected using the ‘low-sample consumption’ setting for 300 sec (5 ⁇ l/ml flowrate), followed by a 240 sec dissociation time.
  • a second dose of 5 ⁇ M polymyxin B was then injected for 60 sec (30 ⁇ l/ml) to assure saturation followed by a 2 h dissociation time prior to the ‘chaser’, a 60 sec pulse of 5 ⁇ M polymyxin B.
  • the RUs of associated polymyxin B were determined 120 sec after the start of the dissociation.
  • cloudy colloid suspension (above its CMC, ⁇ 1 mg/ml) contains LPS micelles that were injected and captured.
  • sample buffer and running buffer were composed of Dulbecco's phosphate-buffered salt solution containing 0.0005% tween-80 (Sigma), 1 mM calcium and 0.5 mM magnesium (Fisher Scientific), pH 7.4. Analysis and compartment temperatures were both set to 37°C.
  • Freshly prepared OMV-coated or whole cell-coated sensing surfaces were employed for each injection of polymyxin B while LPS-coated sensing surfaces could be fully regenerated by injecting 50 mM CHAPS.
  • Negative Staining and TEM Imaging OMVs were incubated in OMV buffer (as above) plus 0.0005% tween-80 and then polymyxin B, PMBN, or an equal volume of OMV buffer, added for a final ratio of 0.5:1 polymyxin B (or PMBN) to LPS for either 1 min or 40 min.
  • the amount of LPS in the OMV preparations was determined using fluorescently labeled LPS.
  • the suspensions were then adsorbed to the surface of formvar and carbon coated TEM grids (100 Mesh) for 60 sec, quickly rinsed in ultrapure water and stained twice for 60 sec with 2% aqueous uranyl acetate. Excess staining solution was blotted off and grids were air dried.
  • Lipid A was extracted from OMVs by using the following method. To begin, 50 ⁇ l ( ⁇ 50-75 ⁇ g) of the OMV preparation was suspended in 5 ml PBS and Lipid A extracted. Dried samples were brought up in 1 ml of 0.25% n-Dodecyl-B-D-Maltoside (DDM) detergent in water by heating to 42°C and bath-sonicating.
  • DDM n-Dodecyl-B-D-Maltoside
  • Lipid A analysis was performed on an LC-ESI-MS/MS instrument with a Thermo qExactive orbitrap mass spectrometer with Active 108465066.1.DOCX 45 00B206.1385 an electrospray ionization source in negative mode.
  • the LC was a Thermo Ultimate 3000 and LC eluent was split between the qExactive MS and a Thermo charged aerosol detector (CAD).
  • the LC separation was performed at 40 °C on a Phenomenex Luna 5 ⁇ m C8100 ⁇ , 50 x 2 mm column.
  • a 15 minute gradient utilized solvent A: 10 mM Ammonium Acetate in H2O and solvent B: isopropyl alcohol:acetone:ethanol 2:1:1.

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Abstract

La présente invention concerne des méthodologies de résonance plasmonique de surface pour enregistrer des données de liaison cinétique associées à l'interaction de compositions et de membranes externes de cellule bactérienne Gram-négatif et/ou de vésicules de membrane externe. Cette approche peut être utilisée pour étudier la capacité de n'importe quelle composition à se lier à la barrière de la membrane externe des bactéries Gram-négatives ou à l'altérer, et l'analyse cinétique présentée dans la présente invention peut donner un aperçu des mécanismes d'action et de résistance à de telles compositions.
PCT/US2023/082388 2022-12-04 2023-12-04 Analyse de compositions cytotoxiques candidates WO2024123712A1 (fr)

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