Combining Multidrug-Resistant Bacteria Using Chemokine-Derived Antimicrobial Peptides. Inventors: Molly Hughes, Borna Mehrad

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1 Combining Multidrug-Resistant Bacteria Using Chemokine-Derived Antimicrobial Peptides Inventors: Molly Hughes, Borna Mehrad

2 Clinical Value: Antibiotic Resistance is an Urgent Global Health Concern and New Antibiotic Strategies are Critically Needed Clinical importance and market value: -In U.S., >2 million infections due to MDR organisms with ~23,000 deaths per year. -Worldwide, >700,000 deaths/year due to MDR bacterial infections. If left unchecked, it is estimated that by 2050, ~10 million deaths/year with reduction in world s GDP by trillions of dollars annually due to MDR infections Urgent campaigns Problematic bacteria for drug resistance identified as ESKAPE pathogens: Lack of new antibiotics in the drug discovery pipeline Enterococcus faecium Staphylococcus aureus Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa 2 Enterobacter spp. CXCL10-derived peptides kill ESKAPE pathogens and MDR Superbugs

3 Project Background Information: CXC Chemokine CXCL10 as Novel Antimicrobial Agent Very potent at recruiting immune cells to sites of inflammation via interaction with its human receptor CXCR3 Direct antimicrobial activity against variety of bacteria (Yang et al. and other groups) CXCR3 receptor interaction responsible for chemotaxis Resemblance to antimicrobial peptides (cationic, amphipathic helix) Adapted from: Frederick, Expert Rev. Mol. Med., 3(19):1-18; 2001 Team members: Dr. Hughes and Dr. Mehrad = collaborators for 12 years on CXC chemokine antimicrobial activity (Hughes) and host immune response / in vivo models of infection (Mehrad) 3

4 CXCL10 kills MDR Gram-negative clinical isolates MDR Acinetobacter baumannii clinical isolate Untreated control CXCL10 treatment Survival of MDR bacteria following exposure to recombinant CXCL10 or buffer alone. Viability was measured by CFU determination, as demonstrated. 4

5 CXCL10 peptide library screen: N- and C-terminal peptides have antibacterial activity against multidrug resistant (MDR) bacteria VPLSRTVRCT CISISNQPVN PRSLEKLEII PASQFCPRVE IIATMKKKGE KRCLNPESKA IKNLLKAVSK ERSKRSP Peptide 1 and its variants = antibacterial activity +/- chemotactic (immunostimulatory) Peptide 9 and its variants = antibacterial activity with no chemotactic activity Goal is to optimize these immunomodulatory antimicrobial peptides for customized applications Killer experiment = Test efficacy of optimized leading candidate peptides in an in vivo infection model (murine wound infection model or murine pneumonia model) 5

6 Peptide antimicrobial activity against multidrug-resistant bacteria % survival (log 10 ) E. faecium ** S. aureus K. pneumoniae nd A. baumannii P. aeruginosa E. cloacae N. gonorrhoeae S.Typhi S. flexneri peptide: P1 P5 P9 6 Main take-home points: Peptide 1: antimicrobial activity against Gram-positive and Gram-negative organisms Peptide 5: negative control (no antimicrobial activity) Peptide 9: antimicrobial activity against many Gram-negative organisms, no Gram-positive activity

7 Antimicrobial activity of CXCL10-derived peptides CXCL10-derived peptides were tested for bactericidal activity against Gram-negative and Gram-positive organisms (Y=antimicrobial activity (85-95% killing), N=no antimicrobial activity). Peptide 1 exerted broad-spectrum bactericidal activity against all organisms and Peptide 9 was effective at killing Gram-negative bacteria. 57

8 Alanine Scan of Peptide 1 Reveals Key Amino Acids in Sequence: CRE K. pneumoniae data P1: VPLSRTVRCTCISI APLSRTVRCTCISI VALSRTVRCTCISI VPASRTVRCTCISI VPLARTVRCTCISI VPLSRAVRCTCISI VPLSRTARCTCISI VPLSRTVACTCISI VPLSRTVRATCISI VPLSRTVRCACISI VPLSRTVRCTAISI VPLSRTVRCTCASI VPLSRTVRCTCIAI VPLSRTVRCTCISA P5: EIITMKKKGEKRCL <---VPLSATVRCTCISI not yet synthesized K. pneumoniae AKU % BL13802 Control, fluorescence (NDM1 CRE) Survival as % of Control 100

9 Minimal length of Peptide 1 derivative determined to be 8 amino acids CRE Klebsiella pneumoniae P5: EIIATMKKKGEKRCL P1: VPLSRTVRCTCISI P60: RTVRCTCI Peptide concentration [5.6 µm] n = 2 experiments P65: RTVRCTC P66: RTVCRTCI (SCRAMBLED) P67: RVCTICRT (SCRAMBLED) Survival as % Control, fluorescence 160 9

10 % Control, Fluorescence K. pneumoniae AKU BL13802 (NDM1 CRE) Survival, % Control Chimeric Peptide P1-GGG-P9 Kills Klebsiella pneumoniae CRE Isolate P55 (P1-GGG-P9): VPLSRTVRCTCISIGGGPESKAIKNLLKAVSKERSKRSP P1 P55 P5 mm 10

11 Antimicrobial activity in traditional low salt hypotonic buffer versus RPMI tissue culture medium 10mM KPB/1%TSB hypotonic buffer RPMI tissue culture medium, buffered with HEPES Survival as % Control P1 P9 P1 + P9 P1-GGG-P9 P9-GGG-P1 Peptide 9 loses activity in RPMI (it only has activity in hypotonic buffer) Peptide 1 retains activity in RPMI but has solubility issues at higher concentrations Chimeric peptides retain activity in RPMI; no solubility issues noted so far Chimeric peptides are more effective than just Peptide 1 and Peptide 9 added to same well P5 Peptide concentration [mm] 11

12 Initial toxicity studies: Human red blood cell (RBC) hemolysis assay Melittin (bee venom) and Triton X-100 (detergent): positive controls for RBC hemolysis the negative controls 12

13 Summary of peptide antibacterial activity data P1 exhibits bacterial killing activity in RPMI tissue culture medium, but its use at higher concentrations is limited by solubility issues P9 exhibits bacterial killing only in hypotonic, low salt buffer; activity abolished in medium with physiological salt concentrations P1 and P9, when added separately into the same wells, do not exhibit the same potency as the conjugate peptides Conjugate peptides: P1-GGG-P9 and P9-GGG-P1 each exhibit antimicrobial activity in RPMI tissue culture medium with physiological salt concentrations; no solubility issues so far Shortest P1-derived peptide that retains activity is 8 amino acids: RTVRCTCI (includes core amino acids determined by the alanine scan) 13 Initial toxicity testing promising: <4% hemolysis of human red blood cells

14 Relevant Publications PNAS Oct 11;108(41) Hughes MA, et. al. PLoS Pathogens Nov;6(11)e Hughes MA, et. al. Infect Immun Apr;77(4) Hughes MA, et. al. Relevant IP U.S. Provisional Application 62/702,557 filed 7/24/18 U.S. Provisional Application 62/743,662 filed 10/10/18 614