An endogenous lipopeptide activates the inflammasome promoting the clearance of Methicillin-Resistant Staphylococcus aureus (MRSA) Skin and Soft Tissue Infections Carol M. Artlett 1, Sihem Sassi-Gaha 1, Mitali Purohit 1, Richard F. Rest 1, and James D. Thacker 1,2 1 Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA Therimunex Pharmaceuticals Inc., Doylestown, PA

MRSA, a Medical Burden Methicillin-resistant Staphylococcus aureus (MRSA) is classified due to its resistance to β-lactam antibiotics (methicillin, penicillin) More severe or life-threatening MRSA infections occur most frequently in the healthcare setting Patients with MRSA infections may be in the hospital 10 days longer than patients with Staph methicillin sensitive infections New Research Estimates MRSA Infections Cost U.S. Hospitals $3.2 Billion - $4.2 Billion Annually MRSA deaths are on the rise in the United States and around the world. A study published in mid-October in JAMA, the American Medical Association's journal, reported that there were an estimated 18,650 MRSA deaths in the U.S. in The MRSA death rate in the U.S. is now higher than the AIDS death rate

acALY18 Discovery We identified an immune activating lipopeptide (1-peptidyl-2- arachidonoyl-3-stearoyl glyceride); amino acid sequence acALYDKGYTSKEQKDCVGI “acALY18” (Thacker, et al., 2009) acALY18 alone is responsible for the biological effects and is uniquely encoded in Transient Receptor Potential Channel 1 protein We found it to be effective in treating parvovirus in dogs and reduced the bacterial burden in cows with mastitis acALY18 acts at cytokine-like molar concentrations (1.5 nM) to induce specific cytokine and chemokine production (IL-1β, IL-18, IL- 6, IL-8, MCP-1, MIP-1α (Thacker, et al., 2009) acALY18 response appears to be specific for fibroblasts and keratinocytes.

acALY18 Activates Caspase-1 Human fibroblasts were transfected with 3ng/ml acALY18, media collected after 72 h and assayed for secreted IL-1β, IL-18, and IL-33. Cell lysates were assayed for caspase-1 activity.

acALY18 Activity Requires the Inflammasome Mouse Fibroblasts were transfected with 3ng/ml acALY18, media collected after 72 h and assayed for IL-1β and IL-18 p= p=NS p=0.05 p=0.03

Gene Name Fold ChangeP Value CARD6Caspase recruitment domain family, member CARD18Caspase recruitment domain family, member NLRP12NLR family, pyrin domain containing MEFVMediterranean fever NAIPNLR family, apoptosis inhibitory protein PYCARDPYD and CARD domain containing NLRP4NLR family, pyrin domain containing NLRP3NLR family, pyrin domain containing NLRP5NLR family, pyrin domain containing AIM2Absent in melanoma NLRC5NLR family, CARD domain containing NOD2Nucleotide-binding oligomerization domain containing NLRP9NLR family, pyrin domain containing PYDC1PYD (pyrin domain) containing RIPK2Receptor-interacting serine-threonine kinase NLRP1NLR family, pyrin domain containing NLRC4NLR family, CARD domain containing XIAPX-linked inhibitor of apoptosis NLRX1NLR family member X acALY18 Alters Inflammasome Transcripts

acALY18 induces antigen specific IgM * * * * * * *p <0.01 Rabbits (n=9) were injected with incomplete adjuvant (IA) consisting of heat-killed and dried M. tuberculosis + acALY18 (100  g/Kg) or Freund’s Complete Adjuvant (FCA). Arterial test bleeds were taken on days 0, 3, 5, 7, 10, 12, 14, and 17. IgM specific for M. tuberculosis was measured by ELISA. FCA

acALY18 does not induce a cytokine storm A preliminary escalating intravenous-dose toxicity study was conducted in 6 female C57Bl/6 mice, 3 received 1 mg (0.1 mL) by a bolus intravenous injection, 3 female received 2 mg (0.1 mL). Control animals (n=3) received 0.1 mL of the vehicle. Body weights, and morbidity and mortality were recorded daily. Blood samples were taken from all animals prior to euthanasia for cytokine/chemokine analysis (IL1β, IL-2, IL-4, IL-5, IL-10, IL-12, TNF- α, IFN-  and CXCL1) and clinical hematology. All mice were euthanized 24 h post-dosing. Necropsy examinations were performed under procedures approved by a veterinary pathologist on all animals at the scheduled terminal necropsies groups. There was a possible test article-related effect of an increase in red cell mass, although the effects were seen only in one animal that received 1 mg of acALY18 and two animals that received 2 mg of acALY18

acALY18-Treated Fibroblasts Induce Monocyte Activation and Differentiation FL2-H % of Max 99.9 Unstained co-culture control 79.8 Unstained co-culture+acALY CD16 co-culture control 80.6 CD16 co-culture + acALY FL3-H % of Max 99.8 unstained co-culture control 79.8 unstained co-culture+acALY CD69 co-culture control 79.9 CD69 co-culture + acALY FL4-H % of Max 98.6 Unstained co-culture control79.8 unstained co-culture + acALY CD40 co-cult control 79.4 CD40 co-culture + acALY FL1-H % of Max 99.1 Unstained co-culture control 79.8 unstained co-culture + acALY CD14 co-culture control 79.8 CD14 co-culture + acALY1864.0

acALY18 Promotes Faster Clearance of MRSA Soft Tissue Infection Animals were treated 1 day prior to infection, 1 day post infection or with acALY18 or vehicle only. All animals were inoculated with 4 X 10 7 CFU/ml MRSA subcutaneous.

Bacterial Load is Reduced with acALY18

Summary 1-peptidyl-2-arachidonoyl-3-stearoyl glyceride is an endogenous lipopeptide uniquely from TRPC1 protein acALYDKGYTSKEQKDCVGI “acALY18” peptide moiety alone is responsible for the biological effects of parent lipopeptide acALY18 activates caspase-1 causing increased IL-1β, IL-18, and IL-33 secretion acALY18 requires a functional inflammasome and induces inflammasome gene transcripts acALY18 induces monocyte maturation and IgM specific antibodies acALY18 is not microbicidal and cleared MRSA infection in a mouse model of a skin and soft-tissue infection

Acknowledgements This work was funded by TherimuneX Pharmaceuticals, Inc., a grant from Drexel University College of Medicine, and the NIH/NIAD SBIR/AT Grant R43 AI award to TherimuneX Pharmaceuticals, Inc.