An endogenous lipopeptide activates the inflammasome promoting the clearance of

Methicillin-Resistant Staphylococcus aureus (MRSA) Skin

and Soft Tissue Infections

Carol M. Artlett1, Sihem Sassi-Gaha1, Mitali Purohit1, Richard F. Rest1, and James D. Thacker1,2

1Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, PA 19129.

2Therimunex Pharmaceuticals Inc., Doylestown, PA 18902.

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 2005. 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=0.0006

p=NS

p=NSp=NSp=NSp=NS

p=NS

p=0.05 p=0.03

Gene NameFold

Change P Value

CARD6 Caspase recruitment domain family, member 6 1600.4365 0.052314

CARD18 Caspase recruitment domain family, member 18 213.5226 0.003097

NLRP12 NLR family, pyrin domain containing 12 164.9619 0.073774

MEFV Mediterranean fever 93.0715 0.047323

NAIP NLR family, apoptosis inhibitory protein 15.0489 0.00416

PYCARD PYD and CARD domain containing 12.2882 0.002462NLRP4 NLR family, pyrin domain containing 4 11.8586 0.008249NLRP3 NLR family, pyrin domain containing 3 10.3329 0.000419

NLRP5 NLR family, pyrin domain containing 5 8.5951 0.000711

AIM2 Absent in melanoma 2 7.7298 0.000146

NLRC5 NLR family, CARD domain containing 5 4.4139 0.000395

NOD2 Nucleotide-binding oligomerization domain containing 2 2.6371 0.001593

NLRP9 NLR family, pyrin domain containing 9 2.5632 0.00018

PYDC1 PYD (pyrin domain) containing 1 2.3065 0.121444

RIPK2 Receptor-interacting serine-threonine kinase 2 -2.3848 0.004472

NLRP1 NLR family, pyrin domain containing 1 -2.5978 0.00006

NLRC4 NLR family, CARD domain containing 4 -3.3116 0.001033

XIAP X-linked inhibitor of apoptosis -13.6292 0.000186NLRX1 NLR family member X1 -19.0134 0.000002

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

10 0 10 1 10 2 10 3 10 4FL2-H

0

20

40

60

80

100

% o

f Max

99.9

Unstained co-culture control 79.8Unstained co-culture+acALY18 64.4

CD16 co-culture control 80.6

CD16 co-culture + acALY18 63.7

100 101 102 103 104

FL3-H

0

20

40

60

80

100

% o

f Max

99.8

unstained co-culture control 79.8unstained co-culture+acALY18 64.4

CD69 co-culture control 79.9CD69 co-culture + acALY18 63.0

100 101 102 103 104FL4-H

0

20

40

60

80

100

% o

f Max

98.6

Unstained co-culture control 79.8unstained co-culture + acALY18 64.4CD40 co-cult control 79.4CD40 co-culture + acALY18 62.5

100 101 102 103 104FL1-H

0

20

40

60

80

100%

of M

ax

99.1

Unstained co-culture control 79.8unstained co-culture + acALY18 64.4

CD14 co-culture control 79.8CD14 co-culture + acALY18 64.0

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 107 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 AI077248-01 award to TherimuneX Pharmaceuticals, Inc.