Table 2.

Structure-activity relationships of Lipid A analogs binding to TLR4.

2 a) TLR4 activity as a function of number and distribution of acyl chains
Description		Lipid A [Benchmark] Compound 2	Penta-acylated Lipid A	Penta-acylated Lipid A	Tetra-acylated Lipid A	Lipid IVA Compound 1
NF-κB	Activation	+++++	ND	+++++	++++	ND
MyD88	TNF-α	+++++	ND	++	++	N/A
	IL-1β	+++++	+	+++	++	N/A
	IL-8	+++++	++	+++++	+++++	N/A
TRIF	G-SCF	+++++	ND	++	+	N/A
	RANTES	+++++	+	++	++	N/A
	MCP-1	+++++	+	+++	+++	N/A
Conditions	Lipid A analog cone.	100ng/mL	100ng/mL	100ng/mL	100ng/mL	100ng/mL
	Cell type	THP-1	THP-1	THP-1	THP-1	THP-1
	Reference	[9]	[9]	[9]	[9]	[29]

2 b) TLR4 activity as a function of number and distribution of phosphate substituents
Description		Bis-phosphorylated Lipid A [Benchmark]	4′- phosphorylated Lipid A	1- phosphorylated Lipid A
NF-κB	Activation	++	++	+++
Conditions	Lipid A analog cone.	100ng/mL	100ng/mL	100ng/mL
	Cell type	HEK293 hTLR4/hMD-2	HEK293 hTLR4/hMD-2	HEK293 hTLR4/hMD-2
	Reference	[32]	[32]	[32]

2 c) TLR4 activity as a function of secondary acyl chain length
Description	Secondary acyl chain length	4′- phosphorylated Lipid A [Benchmark]	14-14-14	12-12-12	10-10-10	8-8-8
MyD88	TNF-α	+	+	+++	++	ND
	IL-1β	+	++	++	+++++	ND
Conditions	Lipid A analog conc.	10μg/mL	10μg/mL	10μg/mL	10μg/mL	10μg/mL
	Cell type	Human whole blood	Human whole blood	Human whole blood	Human whole blood	Human whole blood
	Reference	[30]	[30]	[30]	[30]	[30]

2 d) TLR4 activity as a function of combined structural changes
Description		Lipid A [Benchmark] Compound 2	Hexa-acylated, monophosphorylated Lipid A	Tetra-acylated, monophosphorylated Lipid A	Hexa-acylated, monophosphorylated Lipid A	Penta-acylated Lipid A without phosphates
Modification enzymes involved		Benchmark	PagP, PagL, LpxE	LpxE, PagL, LpxR	LpxM, LpxE, PagP	LpxM, LpxE, LpxF
VF-κB	Activation	+++++	+++++	+++++	++++	++
MyD88	TNF-α	+++++	++	+	+	ND
	IL-1β	+++++	+++	+	+	ND
	IL-9	+++++	+++++	++++	+++++	+++
TRIF	G-SCF	+++++	++	+	ND	ND
	RANtes	+++++	++	+	+	ND
	MCP-1	+++++	++	+	++	+
Conditions	Lipid A analog conc,	100ng/ml	l00ng/mL	100ng/mL	100ng/mL	100ng/mL
	Cell type	THP-1	THP-1	THP-1	THP-1	THP-1
	Reference	[9]	[9]	[9]	[9]	[9]

^a‘+’ symbols correspond to the signaling output intensity. All activity intensities are normalized with respect to the benchmark (Column 1, Tables (2a, 2b, 2c, 2d)). The greater the number of ‘+’ symbols, the greater the signaling intensity.

^bSignaling intensity of ‘ND’ corresponds to baseline/ negligible activity output

^cN/A: not available