Mitali Purohit*, Carol Artlett*, Sihem Sassi Gaha*, James D. Thacker1*, Richard F. Rest* *Department of Microbiology and Immunology, Drexel University College of Medicine

1TherimuneX Pharmaceuticals, Inc., Doylestown, PA

Characterization of a Unique, Naturally occurring Immunomodulatory Lipopeptide: 1-peptidyl-2,3-diacylglyceride (PDAG)

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 :

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.

C H 2

H C

C H 2

O

O

O

X 1

X 2

X 3

The PDAG peptide was sequenced, and PDAG composition confirmed by mass spectrometry.

Caprine serum dialyzed at 4° C

Dialysate reduced by lyophilization

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.

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.

To test our hypothesis we sought to identify the effect of natural PDAG on various cell types.

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.

Figure 3: PMA-differentiated THP-1 cells (ATCC TIB-202) were maintained in RPMI with heat-inactivated 10% FBS and 50 µM beta-mercaptoethanol 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.

Abstract

Introduction

PDAG isolation

The PDAG peptide sequence is homologous with an extra-cellular 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

Homology to TRPC-1

PDAG induces cytokine and chemokine release from whole blood

Conclusions

Future directions

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]

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 protects in a mouse peritonitis model

•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.

•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.

•PDAG, but not the PDAG peptide (data not shown), activate an innate immune response in human fibroblasts.

•Identify which blood cells respond to PDAG

•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.

FUNDING: Drexel University College of Medicine in a cooperative agreement with TherimuneX Pharmaceuticals, Inc.

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.

PDAG induces IL-6 and IL-8 release from THP-1 macrophages

PDAG induces IL-8 release from hMDMs

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.

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.

To determine if PDAG was a multi-functional modulator we treated fibroblasts and keratinocytes with PDAG.

PDAG induces cytokine release from human fibroblasts

PDAG induces cytokine release from human keratinocytes