Transcript PDAG - TherimuneX Pharmaceuticals, Inc

Characterization of a Unique, Naturally occurring Immunomodulatory
Lipopeptide: 1-peptidyl-2,3-diacylglyceride (PDAG)
Mitali Purohit*, Carol Artlett*, Sihem Sassi Gaha*, James D.
1*
Thacker ,
Richard F. Rest*
*Department of Microbiology and Immunology, Drexel University College of Medicine
1TherimuneX Pharmaceuticals, Inc., Doylestown, PA
Homology to TRPC-1
Abstract
conc. in pg
The PDAG peptide sequence is homologous with an extracellular loop of TRPC-1, a transmembrane Ca2+ channel protein.
Figure 1: Putative domain structure and topology of TRPC1. Percent
identity between TRPC1 and at least one other member of the TRP
family is indicated. The red circle indicates the PDAG peptide sequence.
Arrows indicate sites of known amino acid variance. Wes, et al. Proc.
Natl. Acad. Sci. USA Vol. 92, pp. 9652-9656
To test our hypothesis we sought to identify the effect of natural
PDAG on various cell types.
PDAG induces cytokine and
chemokine release from whole blood
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The evolution and rapid spread of resistant microorganisms are
significant problems in nosocomial infections and are of increasing
importance in community acquired infections. There is an urgent need
to look for alternatives to antimicrobials that can either have direct
microbicidal activity or that can exert their effects via up-regulation of
the innate immune system. One promising approach to provide
protection from infectious challenges is to modulate the innate
immune response. It is apparent that immunomodulatory peptides,
such as the defensins, stimulate a wide range of effects on innate
immune cells, including monocytes, macrophages, and neutrophils to
act as chemoattractants, promote histamine release from mast cells,
and enhance phagocytosis at the site of infection.
In our investigation of the low-molecular weight inflammatory
proteome in mammalian plasma and serum we identified a new
immunoactive lipopeptide with the general structure :
LPS Treatment 24 hrs
Untreated (13 hrs)
Untreated (24 hrs)
PDAG #5 (1:100) (13 hrs)
PDAG #5 (1:100) (24 hrs)
PDAG #5 (1:1000) (13 hrs)
PDAG #5 (1:1000) (24 hrs)
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600
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lps1ng
technical PDAG
1:100
Figure 4: 500,000 human monocyte derived macrophages (hMDMs)
[peripheral monocytes differentiated with M-CSF] were treated with
natural PDAG for 24 hours. IL-8 released in the supernatants was
measured by ELISA.
To determine if PDAG was a multi-functional modulator we treated
fibroblasts and keratinocytes with PDAG.
PDAG induces cytokine release
from human fibroblasts
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6
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mRNA
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(relative
level) 3
2
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0
Control
PDAG
MIP-1a
Conclusions
IL-1a
IL-1b
IL-8
IL-33
IL-18
700
0
IL-1β
IL-6
IL-8
TNF-α
MIP-1α
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1000
800
Untreated (24 hrs)
LPS Treatment 24 hrs
PDAG #5 (1:100) (24 hrs)
PDAG #5 (1:200) (24 hrs)
PDAG #5 (1:400) (24 hrs)
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400
•The chemokines/cytokines most substantially induced in
PDAG-treated blood were IL-6, IL-8, GM-CSF, G-CSF, MCP-1,
and MIP-1β.
•PDAG induced significant levels of IL-6, IL-8, and MIP-1b from
THP-1 macrophages.
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500
400
Control
•PDAG, but not the PDAG peptide (data not shown), activate an
innate immune response in human fibroblasts.
PDAG
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200
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0
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Figure 7: Swiss Webster mice were challenged with Salmonella
typhimurium (5 x 103 cfu/mouse) administered by intraperitoneal
injection. Mice were treated with natural PDAG in normal saline
(1.5 ug/mouse; n=15) or saline alone (n=15) by subcutaneous
injection), a day before challenging with the bacteria. Mortality
was monitored daily for ten days. By Day 10, all mice in the
control group were dead where as there was only 20% death in
the PDAG treated group. Administration of PDAG did not cause a
cytokine storm. Lymphopenia, neutrophilia, and monocytosis were
observed in the infected control mice, whereas circulating
neutrophils and monocytes in PDAG treated mice were at or near
normal levels. The bacterial load in the spleens of the mice was
50% less in PDAG treated mice on day 1 (data not shown).
•PDAG induces the expression by whole blood and
macrophages of a number of cytokines and chemokines, the
profile of which is clearly different from that induced by LPS.
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conc. (pg)
Introduction
conc. (pg)
1000
PDAG protects in a mouse
peritonitis model
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200
100
0
control
mRNA (relative level)
To limit disease caused by emerging antibiotic-resistant pathogens, or
genetically engineered agents of bioterrorism, there is a constant quest to
develop novel therapeutic agents and adjuncts to antimicrobials. A screen
of the low-molecular weight (< 10 kDa) inflammatory proteome in search
of immunoactive agents uncovered a new lipopeptide that was
characterized
as
1-peptidyl-2-arachidonoyl-3-stearoyl-sn-glyceride
(“PDAG”). We determined the unique amino acid sequence of the
peptidyl moiety – acetylALYDKGYTSKEQKDCVG
which is 100%
identical to a stretch of 17 amino acids of the third extracellular loop of
TRPC1, the
We have synthesized the complete lipopeptide and it
mimics the biological activity of natural PDAG, in vitro. Natural PDAG
stimulates the innate immune response resulting in activation of tissue
resident immune cells, recruitment of phagocytic cells, and clearance of
pathogens. Serum-derived, i.e., natural PDAG stimulates significant
release from whole human blood of proinflammatory cytokines IL-6, IL-8,
MCP-1, MIP-1α and MIP-1β. The cytokine profile induced by PDAG
differs from that induced by LPS, indicating a lack of endotoxin
contamination of the natural product, and a different mechanism of action.
Similarly, PDAG stimulates isolated human macrophages, neutrophils
and THP-1 macrophage-like cells to release IL-6 and IL-8 in a dose and
time dependent manner. Interestingly, PDAG also stimulates cultured
human fibroblasts and keratinocytes to secrete IL-6 and IL-8. We
determined whether PDAG activity was due to the entire molecule, the
PDAG peptide or DAG alone. Neither DAG nor synthetic PDAG peptide
alone appear to induce cytokine release from whole human blood or
isolated white cells. Thus, the whole molecule is required for activity. We
continue to study additional cell types responsive to PDAG and to
investigate the source(s) and mechanism of action of PDAG activity.
PDAG is an exciting new drug-like molecule that has many possible
clinical uses, including the modulation of inflammation or immediate
boosting of cellular and humoral antimicrobial responses. PDAG could
be used as an adjunct to antimicrobials, immunotherapeutic agents and
vaccines to help control infectious diseases, including those caused by
select agents. [This work was funded by Drexel University College of
Medicine]
PDAG induces IL-8 release
from hMDMs
MCP-1
NFkB
NALP-3
Caspase-1
IL-6
Future directions
0
PDAG isolation
PDAG induces IL-6 and IL-8
release from THP-1 macrophages
Amount of Cytokine (pg)
The lyophilized fraction extracted with methanol/chloroform, and the m/c
solution separated from un-dissolved solids by filtration. The m/c was
removed in vacuo by rotary evaporation.
MCP-3 MIP-1α MIP-1β
Figure 2: Fresh heparinized human blood was diluted five-fold with
RPMI 1640 medium and incubated with dilutions of purified natural
PDAG for 13 or 24 hrs. After incubation, samples were centrifuged and
plasma was assayed using the Excelarray Human Inflammation kit, or
Chemotaxis kit (Thermo Fisher Scientific). PDAG induced a dose and
time dependent release of cytokines.
Caprine serum dialyzed at 4° C
Dialysate reduced by lyophilization
MCP-1
The PDAG peptide was sequenced, and PDAG composition confirmed
by mass spectrometry.
PDAG induces cytokine release
from human keratinocytes
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Control
LPS 5ng/ml
PDAG (1:100)
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0
IL-6
Purified PDAG was prepared by preparative scale reversed phase
HPLC. The PDAG peak was collected and the solvent removed by
lyophilization. PDAG was >99% chromatographically pure.
Figure 5: Normal human fibroblasts were cultured in DMEM with
10% FBS at 37oC, with or without natural PDAG. RNA was purified
from treated and control fibroblasts, and assayed for the indicated
cytokines or signaling molecules by quantitative RT PCR. Values are
normalized to -actin expression.
IL-8
Cytokines
relative m-RNA level
wherein X1 is a linear peptide and X2 and X3 are arachidonoyl and
stearoyl respectively. The peptide is covalently linked to the
diacylglycerol by an ester bond at the C-terminus of the peptide. We
hypothesize that 1-peptidyl-2,3-diacylglyceride, “PDAG”, specifically
activates the innate immune cascade, activates tissue resident
immune cells at the site of injury, recruits professional phagocytic cells
to local tissue, clears pathogens, and abates disease progression.
IL-8
•Determine if PDAG induces an oxidative response
•Determine if synthetic PDAG or PDAG peptide engages
immune cells
•Determine if synthesis or release of PDAG is induced by
endotoxin, hyperthermia, or hypoxia
•Determine if either PDAG or PDAG peptide are microbicidal
•Identify the pathways via which PDAG signals.
Control
PDAG
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Figure 3: PMA-differentiated THP-1 cells (ATCC TIB-202) were
maintained in RPMI with heat-inactivated 10% FBS and 50 µM betamercaptoethanol at 37ºC in 5% CO2, and were treated with 1:100
natural PDAG for 24 hrs. Supernatants were assayed using the
Excelarray Human Inflammation kit. LPS is used as a positive control.
•Identify which blood cells respond to PDAG
IL-6
IL-8
Cytokines
Figure 6: PDAG treatment of keratinocytes induces increased
expression of IL-6 and IL-8 as determined by RT-PCR. Values are
normalized to β-actin.
FUNDING: Drexel University College of Medicine in a
cooperative agreement with TherimuneX
Pharmaceuticals, Inc.