receptor ligands
Weterings, J.J.
Citation
Weterings, J. J. (2008, November 27). Synthesis and evaluation of peptide and nucleic acid based Toll-like receptor ligands. Bio-organic Synthesis, Leiden Institute of Chemistry, Faculty of Science, Leiden University. Retrieved from https://hdl.handle.net/1887/13284
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Chapter 1
General Introduction
Toll like receptors1 (TLRs) are type I transmembrane receptors that continuously scour their direct surroundings for pathogen associated molecular patterns (PAMPs) of bacterial, viral or fungal origin. A total of 13 TLRs are currently known in mammals of which 11 can be found in humans. Their specific PAMPs are directly related to the location where the receptor resides. TLRs 1, 2, 4, 5, 6 and 11 are situated on the outside of the cell membrane where they come into direct contact with membrane components of pathogens. TLRs 3, 7, 8 and 9 can be found intracellularly and their ligands mainly consist of viral components released after cellular uptake and degradation of the invader. A link between TLRs and the innate immune system was envisaged after a search towards receptors orthologous to Drosophila Melanogaster (fruit fly) Toll led to the discovery of Toll-like receptors in humans. The Toll receptor in fruit flies was originally found to influence the dorsal-ventral patterning development of fly embryo’s2,3 and was later linked to the immune system of Drosophila. A mutation in the gene responsible for Toll resulted in underdevelopment of the ventral portion of the fly larva and was commented on by Christiane Nüsslein-Volhard: “Das war ja toll”. A decade later a second function of Toll in Drosophila was found. Flies lacking the Toll receptor were susceptible to fungal infections. It turned out that the antifungal peptide, spatzle, was no longer generated by the immune system of Drosophila genetically modified to lack the Toll gene and the connection between pathogen detection and the Toll receptor was made. The discovery of highly homologous Toll genes in humans led to the discovery of the TLRs and their involvement in human immunity.4 The innate immune system5 which is fixed in the genome was selected over evolutionary time and triggering of it leads to immediate activation of effectors and release of co-stimulatory molecules such as cytokines (IL-1β, IL-6) and chemokines (IL-8). Pathogen-recognition receptors (PRRs) of the innate immune system of which the TLRs are part recognize conserved molecular patterns such as
lipopolysaccharide (LPS), lipoteichoic acid (LTA) and glycans. Three different kinds of PRRs participate in the innate immunity: soluble extracellular receptors such as complements, membrane associated receptors like Toll-like receptors, and nucleotide binding oligomerisation domain proteins (NODs).6 The Toll-like receptors are the most important PRRs in the human innate immune system and are expressed on various immune and non immune cell types, including monocytes, macrophages, dendritic cells, neutrophils, epithelial cells and keratinocytes. Binding of the TLR to their corresponding ligand leads to a signaling cascade involving a number of proteins, such as MyD88 and IRAK.7 This signal transduction leads to activation of the transcription factor NF-κB after which secretion of pro- inflammatory cytokines and effector cytokines leads to the induction of the adaptive immunesystem. TLR may exist either as homodimers (TLR3) or heterodimers (TLR1/2), and may alsoform complexes with other factors (TLR4/MD2).8 Binding occurs at the leucine rich repeats (LRRs) which together with cysteine rich regions make up the large and divergent ligand-binding ectodomain of the TLR. Furthermore the structure consists of a transmembrane region and a conserved cytoplasmic domain that is highly homologous among the TLRs. Induction of TLRs results in the formation of ‘m’ shaped TLR dimers in which the C-termini of the extracellular domains converge in the middle.9 To date several crystal structures of TLRs and their respective ligand have been published.10 This chapter describes the score of natural and synthetic TLR ligands available to date with a focus on well-defined synthetic compounds. As described earlier triggering of the TLR signaling cascade is effected by binding of the TLR to its specific ligand. The resulting immune response can be manipulated beneficially by the use of synthetic TLR ligands. When used as well-defined adjuvants, their combination with antigen may increase the immunogenicity of the antigen itself. This so-called vaccine principle may lead to a desired long-lasting adaptive immune response or humoral memory. If applied to human beings, the prerequisites for an adjuvant would be well-defined in structure, free of contaminants and non-toxic. Synthetic ligands should fit this description. Furthermore the development and use of synthetic TLR ligands can help to achieve a better understanding of the immune system. Synthetic TLR ligands11 can be combined with an antigen to perform vaccine studies, used in binding studies, ligated to fluorescent labels to follow trafficking, combined with other TLR ligands to investigate synergism and further developed as leads in medicinal chemistry.
TLR1
Toll-like receptor 1 is located on the outside of the cell membrane and forms heterodimers with TLR2 and TLR6. Most diseases involving TLR1/TLR2 are related to bacterial and fungal infections. Natural ligands are triacyllipopeptides derived from bacteria and mycobacteria such as Borrelia burgdorferi and neisseria and mycobacteria derived lipoproteins. A selection of synthetic TLR1/TLR2 and TLR1/TLR6 ligands is shown in Figure 1.1.
NH
Se r-Lys-L ys-Lys- Lys
O S 1 4
1 4
P am3CysS K4, 1 O
O O
O O
1 4
NH
(Val-Pr o-Gly-Val-G ly)4Va l-Pro-G ly-L ys-Gly
O S 6
6
P amO ct2Cys(VPGV G)4VPGK G, 5 O
O O
O O
1 4 NH
Ser-L ys-Lys- Lys-Lys
O S 10
10
N-Pam-S-La u2CysSK4, 2 O
O O
O O
14
NH
Se r-Lys-L ys-Lys- Lys
O S 1 2
1 2
Myr3CysSK4, 3 O
O O
O O
1 2
NH
Se r-Lys-L ys-Lys- Lys
O S 1 0
1 0
L au3CysSK4, 4 O
O O
O O
1 0
NH S O O
O C11H23
C11H23
O O
C15H31
O H N
NH O
S O
O ONa
O N
H S
O
O ONa
JBT3002 , 7 NH
Se r-OH
O S O O
O O
O O
O
O Cl
Cl Cl
Cl Cl Cl
Cl Cl
Cl
Troc3CysSer , 6
Figure 1.1: Overview of TLR1 ligands
The extensively studied TLR1/TLR2 ligand Pam3CysSK4 1 is the most used synthetic ligand to activate its designated TLR heterodimer.12 It consists of the oligopeptide sequence Cys-Ser- Lys-Lys-Lys-Lys, with L-cysteine containing one N-terminal palmitoyl group, and features a dipalmitoyl moiety coupled to the thiol function via the glycerol moiety. Potency can be gained by extension of the peptide portion (5).13 Furthermore the palmitoyl tails can be replaced by for instance lauryl14, 15 or myristyl15 as shown in 2, 3 and 4 or replaced by groups not resembling a fatty acid in any way 6.16 The overall conclusion for this type of ligands is that, having S chirality in either cysteine and/or the glycerol moiety17 removal of the thiol in cysteine18 or reducing the fatty acids length below 8 atoms all result in loss of activity towards
TLR1. Immunomodulating lipopeptide JBT300215, 19, 20 (7), a drug used for anticancer therapy, has also shown to be a TLR1/TLR2 dependent immune system activating ligand.
TLR2
Toll-like receptor 2, which is located on the cell membrane, recognizes the largest diversity of ligands of all TLRs. It forms heterodimers with TLR1 and TLR6 and the possible existence of a TLR2/TLR10 dimer is under investigation.21 Recent studies suggest that the function of the different TLR2 dimers is to enlarge the amount of ligands to which it responds, rather than to yield more differentiated outcomes of the immune response after activation. Natural ligands include diacylated and triacylated proteins, lipoproteins, peptidoglycan (PGN), glycolipids, GPI anchors, outer membrane protein A, porins, heat shock proteins, lipoteichoic acids, lipoarabinomannans22, lipomannans23, alginates and zymosan.24 TLR2 plays a role in mycobacterial and fungal infections. The last few years show a tremendous amount of research towards synthetic ligands of which a selection is displayed in Figures 1.2-1.4.
NH2
Gly-Asp-Pro -Lys-His-Pro-L ys-Ser -Phe
O S 1 4
1 4
FSL- 1 O O
O
O
NH2
G ly-A sn-A sn-A sp -Glu-Ser -Asn-Ile -Ser-Ph e-Lys-G lu-Lys
O S 14
14
MALP-2 O
O O
O
NH2
Thr -Gly-Ile- Gln-Ala-A sp- Leu-Arg -Asn-Leu-Ile-Lys
O S 1 4
1 4
MPP L-1 O
O O
O NH2
Ser- Lys-Lys-Lys-Lys
O S 14
14
Pam2CysSK4 O
O O
O
8 9
10
11
Figure 1.2: a selection of TLR2 ligands
Having the N-terminal palmitoyl group removed Pam2CysSK4 813 still closely resembles Pam3CysSK4 1 in structure but binds preferentially to TLR2/TLR6 rather than TLR1/TLR2.
In the light of recent discoveries this makes sense. In binding9 of Pam3CysSK4 to TLR1/TLR2, the dipalmitoyl moiety inserts itself into a pocket located in TLR2, while the third palmitoyl fits into a hydrophobic channel in TLR1. The TLR1/TLR2 heterodimer is then further stabilized by hydrogen-bonding to generate an activation signal. Pam2CysSK4 lacking
the third fatty acid can no longer facilitate the TLR1/TLR2 interaction and signaling occurs from the TLR2/TLR6 heterodimer instead. This structural difference also plays an important role in the initial recognition of microbial lipoproteins. FSL-1 913 and MALP-21025 contain a di-palmitoylated cysteine residue whereas bacterial lipoproteins contain a tri-acylated one.
Therefore FSL-1 and MALP-2 are recognized by TLR2/TLR6 whereas bacterial lipoproteins are recognized by TLR1/TLR2. As can clearly be seen all synthetic ligands portrayed in Figure 1.2 lack the third fatty acid. Main difference between the ligands shown (Pam2CysSK4
8, FSL-1 9, macrophage activating lipopeptide-2; MALP-2 10 and MPPL-1 11) is the variation and length of the C-terminal peptide sequence. The mean congener is that the described dipalmitoyl constructs all resemble N-terminal parts of lipoproteins. FSL-1 9, Lipoprotein 44 of Mycoplasma salivarium which stimulates a MyD88 dependent signaling cascade, MALP-2 10 Mycoplasma fermentans and MPPL-1 Mycoplasma pneumoniae26 are all examples of N-terminal parts of lipoproteins. FSL-1 is a stronger stimulatory agent than both MALP-2 and MPPL-1 regarding levels of IL-6 and IL-8. MALP-2 having the R chirality in the glycerol moiety is a stronger activator than S-MALP-2.27
Peptidoglycan, lipoteichoic acid and alginate fragments that are part of pathogen membranes have been shown to facilitate an activation signal via the TLR2/TLR6 pathway.
O O O
NHA c HO
HO O
HO
NHA c O O
NH
O O
HN O
OH
O n
NH O
NH
R1
NHR2 O
HN O H2N
2
3
R1= H (Lys) Gr am pos. PGN R1= CO OH (Dap) Gram neg. PG N R2= H or Cross-l inkin g
HO O
HO
NHA c OH O
R O
13a. R =L-Ala- D-isoGlu 13b. R =L -Ala-D-isoGlu-L-L ys 13c. R =L-Ala- D-isoGlu -L-Lys-D-Ala 13d. R =L -Ala-D-isoGlu-L-L ys-D-Ala-D-Ala 13e. R =L-Ala- D-isoGlu -Da p
13f. R =L-Ala-D-isoGlu-Dap-D-Al a 13g. R =L -Ala-D-isoGlu-Dap-D-Ala- D-A la
12
13
Figure 1.3: Overview of TLR2 ligands continued, 12 general PGN structure, 13 PGN derivatives
Peptidoglycan (PGN) 12 of which the general structure is shown in Figure 1.328 was first described as a PAMP in 1999. Peptidoglycan can be found as a layer in bacterial cell walls of Gram-positive and Gram-negative bacteria as well as in mycobacteria. It is composed of a linear polysaccharide of alternating N-acetyl glucosamine and N-acetyl muramic acid residues
connected via peptide bridges to form macromolecular structures. The mechanism of signaling via TLR2 remains unclear28. Even though TLR2 knockout mice show abolished immune responses, signaling remains intact in TLR1 and TLR6 deficient mice. This could indicate that PGN is recognized by TLR2 alone or by co-receptors besides TLR1 or TLR6.
TLR10 is suggested as a candidate21. Recently peptidoglycan was questioned29 as a TLR2 ligand, but this observation remains arguable30. Several synthetic derivatives of PGN 13 have been prepared (Figure 1.3)28 but their immunostimulatory properties have not yet been determined.
Lipoteichoic acids (LTA) are found in most Gram-positive bacteria and may activate the immune system via TLR2. Its general structure consists of an amphiphilic, negatively charged glycolipid31 14as shown in Figure 1.4. The first fully active synthetic LTA 15 was prepared by Schmidt et al., in 2003.32, 33 After Schmidt’s publication several other studies towards synthetic lipoteichoic acid have been undertaken and the biological evaluation of these reveals that the minimal LTA structure needed for TLR2 induced cytokine induction is an anchor with two fatty acids and a short backbone of three glycerolphosphate subunits.34, 35, 32 This minimal structure is shown in Figure 1.4 (14, R1= R2 =H, n = 3).
O
O O
O
O O OH HO
O HO O O
HO HO
2´
P O- O O O
O OH HO
HO
AcHN
O P O
O O
O O NH3+
O- P
O O
O- OH HO
4
O
O R1 O R1
O
O O OH HO
O HO O O
HO HO
2 ´ P
O- O O O
OH OH
O R2
H R1= alkyl and/or branched alkyl chain
R2=
NH3+ Me
(D-Ala, 70% )
R2= H, 15 %
R2=
O
AcNH OH
HO HO
(alpha -D-G lcNAc, 15% ) G eneral LTA structu re 1 4
First synthetic LTA 15 n
O
n = 40-5 0
Figure 1.4: Overview of TLR2 ligands continued
Alginates consist of linear polysaccharide chains of 1,4-linked β-D-mannuronic acid (M) and its C5-epimer α-L-guluronic acid (G)36. The general structure of alginates is shown in Figure 1.5. Alginates are produced by bacteria from the Azobacter and Pseudomonas class and by marine algae36. The composition of alginates varies highly depending on the species they originate from. Alginate polymers and alginate oligosaccharides analogs have been shown to be immune stimulators via the TLR2/TLR4 pathway.36 This observation led to the believe that short oligosaccharides from alginates could also serve as a TLR2/TLR4 agonist. The small number of synthetic alginates available to date is due to the fact that the synthesis of the1,2- cis-glycosidic linkage of D-mannuronic acid and L-guluronic acid is challenging. In 2006 van den Bos et al.37 were the first to present a synthetic alginate trisaccharide 17. Brenk et al.36 added a lipidated version in 2007 after which the same group published another analogue having a longer saccharide 19 stretch in 2008.38 The immuno-stimulatory properties of the synthetic alginates have not been assessed to date.
O HOOC H O
RO OR
O O
HO OC
RO OR
O O
m HOOC
RO OR
O O
HO OC OH
O OH
n
P oly-G M P oly-M
O HOOC
OH
O H
O O
OH
O H
HOOC O H
k
R = H, Ac
Poly-G
O O
OH O
HO O HO O
HO
HO HO
OH O
OH
O O
O O
OH O
HO O HO O
O
HO HO
OH O
OH
O O O
OH HO O
HO HO
Br ent et al. 2 007
Br ent et al. 2 008 18
19
Ge neral Algina te structure 16
17
van den Bos et al . 2006
O O
OH O
HO O HO O
O
HO HO
OH O O
OH HO O
HO HO
NH3+Cl-
Figure 1.5: Overview of TLR2 ligands continued
TLR3
TLR 3 is an intracellular homodimeric receptor located on endosomal membranes and double stranded RNA (dsRNA) of viral origin is its natural ligand.39, 40 One of the three different synthetic ligands for TLR 3 is Poly I:C12U (Ampligen ®) and has potent antiviral and immunomodulatory properties.41 The structure consists of polyriboinosine (poly I) hybridized to a complementary polyribocytosine strand containing a uridine residue statistically at every 13th position. Polyinosine-polycytidylic acid (poly (I:C)) is the second available synthetic TLR 3 ligand.42-46 Also Polyinosine (poly I) has been reported as TLR3 ligand.47
NH
N N N
O
O
OH O H O
N NH2
O N O
OH OH O NH
O
O N O
OH OH O
20 21 22
P HO
O
OH P
HO O
O H P
HO O
OH
Figure 1.6: 5’-uridylic acid 20, 5’-inosinic acid 21, 5’-cytidylic acid 22
Poly (I:C), originally identified as an IFN inducer, was selected out of a series of natural and synthetic dsRNAs for being the most potent amongst the set48. A dsRNA with a minimum molecular weight of 2.7x105 Da, that is stable at physiological temperatures is the required element for IFN production. Alexopoulou et al40 were the first to link synthetic (poly(I:C)) dsRNA to TLR3. Poly(I:C12U), a non-toxic derivative of poly(I:C), showed similar IFN induction as its parent compound and is nowadays known as Ampligen ® produced by Hemispherx. Another Hemispherx developed drug namely Polyadenure® (a Poly (A:U)) has been reported as TLR3 agonist and is used as treatment for hepatitis B or C and cancer.49
TLR4
TLR4 is the first mammalian TLR characterized and its discovery led to the identification of the other TLRs.50 Activation via TLR4 requires the recruitment of the MD-2 protein complex.51 In addition it is the only TLR that can activate two distinct signaling pathways namely the TIRAP-MyD88 pathway, and the TRAM-TRIF pathway.30, 52 Even the possibility of recruiting a third protein exists, resulting in a TLR4/MD2/CD14 cluster.51 TLR4 is localized at plasma membranes and endosomal vesicles.52a, 52b The most well-known TLR4 ligand is lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria which can activate the immune system at very low concentrations.51 Exposure to LPS can lead
to septic shock and it is important to develop TLR4 antagonists that can prevent septic shock from happening.53 Besides LPS (also known as endotoxin), TLR4 is triggered by several other natural ligands including mannan30, glucuronoxylmannan30, glycoinositolphospholipids30, envelope proteins30, heat shock proteins30 (hsp60 and hsp70), hyaluronic acid54, heparin sulfate55, β-defensin 255 and fibrinogen.56 Almost all available synthetic TLR4 ligands can be traced back to LPS of which lipid A (Figure 1.7) is the active constituent. Lipid A has very good immunostimulatory properties but its high toxicity prevents it from being used in the clinic. Some of the toxicity of lipid A can already be subdued by removal of the anomeric phosphate group resulting in monophosphoryl lipid A (MPL®), which is momentarily being tested as prophylactic and therapeutic vaccine. Synthetic ligands57 based on lipid A consist of lipid A mimics from several bacterial sources. Other derivatives of lipid A are analogues in which the sugar backbone is gradually removed finally ending up at hexa-acylated acyclic structures. Recently a synthetic peptide has been found to act as a TLR4 ligand. TLR4 is involved in bacterial infections, chronic inflammation, autoimmune disorders, atherosclerosis and cancers.53, 58
O
O O HO
HN O
OH O
O
OH NH
O
O O
OPO3H2
H2O3PO O O
O O O
O O HO
HN O
OH O
O
OH NH
O
O O
OPO3H2
H2O3PO O O
O O
K DO -KDO Hep Hep G lu-Gal G al G lu-GluNac G al Rha Man-Ab e
n
O -anti gen
Cor e
L ipid A
O
O O HO
HN
E Co li Lip id A
O
OH O
O
OH NH
O
O O
OR HO
H2O3PO O O
O O L ipopolysacchar ide structur e 23
Re-Type L PS E Coli Lipid A
24
2 5 R = H Mo nophosphor yl lipid A (MP L) 2 6 R =P(O)(O H)2Lipid A
O
O COOH OH OH OH O
O COO H OH OH
OH
HO K DO dimer
Figure 1.7: Lipopolysaccharide (LPS) 23, Re-Type LPS 24, MPL 25, Lipid A 26
Lipopolysaccharide is located on the cell membrane of gram-negative bacteria and consists of three parts as can be seen in 23, Figure 1.7. These are: 1) polysaccharide side chain (O- antigen), 2) a core saccharide part containing unusual sugars (e.g. 3-deoxy-D-manno-2- octurosonic acid and heptose and 3) lipid A. The lipid A part is responsible for most of the activity of LPS. By synthesising several constituents of LPS the minimal requirements for
TLR4 stimulating properties have been determined.59a, 59b As mentioned before, lipid A (26) which was first synthesised in 1987 by Shiba et al60 still has toxic properties. However hydrolysis of the anomeric phosphate yields monophosphoryl lipid A 25 (MPL) as a non toxic analogue. Several studies61 have been performed to elucidate the biological and physicochemical role of the sugar moieties linked to lipid A62; for example by the synthesis of Re-LPS 24.63
O
O O HO
NH
S . Min neso ta Lipid A NH
O HO
H2O3PO O
P O
OH O OH
O O O
HO O O
O O O
O O
O NH HO
H2O3PO O
O
O O O
O O
RC5 29, 28
CRX-527 GLA- 60
O
NH
O
O O
O NH HO
H2O3PO O
O
O O O
O O
O
NH
O
O O
OH O
O NH HO
H2O3PO O
O
HO O
O O
OH 2 7
2 9 3 0
Figure 1.8: Lipid A S. Minnesota, RC529, CRX-527 and GLA60
Furthermore lipid A structures from several bacterial sources have been determined, synthesised and their biological activity assessed. Variation in the structure of synthetic lipid A analogs comprises the number of acyl groups, the substitution pattern in the acyl groups and the length of the alkyl chain. Figure 1.8 shows lipid A as present in S. Minnesota from which the synthetic TLR4 ligands CRX64 and RC were derived.65, 66 These so called AGPs (aminoalkyl glucosaminide 4-phosphates)67 were identified as a new class of monosaccharide lipid A derivatives and were shown to have immunostimulatory properties.68a GLA-60 is a LPS antagonist with TLR4 agonistic activity.68b CRX-527 29 developed by Corixa is in the preclinical stage and is tested as a drug to prevent infection.69
NH NH
O
O O
O
HO
OM-174 O
O O HO
HO
OP(O)(O H)2
(HO )2P(O)O
HO HO
P O
N
H O NH2
O
NH O
NH O
OH
O
O O
O
HO
OM-197 -MP -AC
HO P O
N
H O
NH O
NH O
OH
O
O O
O
HO
OM-294-DP OM-294 -MP
P OH O
OH HO
P O
N
H OH
NH O
NH O
OH
O
O O
O
HO
3 1 3 2
3 3 3 4
Figure 1.9: OM-174, OM-197-MP-AC, OM-294-DP, OM-294-MP
Figure 1.9 shows OM-174 3170 a synthetic TLR4 ligand derived from E. Coli, with only 3 acyl spacers instead of the usual six. OM-174 induces IL-12 and TNFα production in human DCs and results in a Th1 response when added to naïve CD4+ T cells. OM-197-MP-AC71-73 (32), has the same configuration as OM-174 and mimics the lipid A structure by replacement of the disaccharide backbone by a pseudo-peptide backbone. OM-197-MP-AC 32 can induce maturation of human DCs, promotes a primary T cell response and activates antigen presenting cells via TLR4, suggesting that it can be used as adjuvant in vaccines. OM-294-DP 33 and OM-294-MP75 34 also derived from OM-174 have either immunostimulating or modulating activities.
O
HN NH
O
P
HO O
O
HN O
O O
O O
P O H O
O
NH O
O O
O O
ER-112022
HN NH
P
HO O
O
HN O
O O
O O
P O H O
O
NH O
O O
O O
ER-804057 O
R R
R R
R R
RS RS
35 36
Figure 1.10: ER112022, ER804057
ER11202275 35and ER80405776, 77 36 are a new class of TLR4 ligands that show agonistic properties (Figure 1.10). Compared to the previous described TLR4 ligands ER112022 does not have a carbohydrate or pseudo-peptide backbone. It is made up out of two phospholipid components connected via an acyclic linkage. Furthermore the phosphate groups are located inside the structure instead of on one or two terminal positions as in for example 33. Also the symmetry of the compounds 35 and 36 is a key feature. Structurally related ER804057 has a small difference with 35 namely the backbone length has been decreased to just a carbonyl.
Compound ER804057 36 has the R configuration in all 4 chiral centers which makes it more potent than related compounds having any other combination of R or S stereochemistry.78 Several other lipid A mimics provided with TLR4 agonistic activities have been synthesized.
Relevant examples are DT-546179, C50680 and ONO-4007.81
Besides the use of TLR4 agonists it is interesting to note that in the light of the LPS-septic shock relationship several synthetic TLR4 antagonists are available such as E5531 37 and E556482 38(Figure 1.11).
O
O O HO
HN
E556 4
O
O NH O
O OPO3H2
H3CO H2O3PO
O
H3CO O
O O HO
HN O
O NH O
O OPO3H2
H3CO H2O3PO
O
O
E553 1 O OH O
37
38
Figure 1.11: E5531 and E5564
Also ligands (not even) remotely related to lipid A exist for TLR4. One of these ligands the α- galactosylceramide analog CCL-3483 (40) Figure 1.12. α-Galactosylceramide 39 is a marine sponge Agelas mauritanius derived glycolipid which does not act on TLR4 itself but studies towards synthetic derivatives have revealed TLR4 agonistic activities amongst its analogs.
CCL-34 (40), is able to activate NF-κB in a TLR4 dependent fashion.
O O H OH
HO O HO
HN
O HN O
CCL-34 40 O
O H OH
HO O HO
HN
OH O
OH
alpha-Ga lCer 39
Figure 1.12: alpha-GalCer 39 and CCL-34 40
Taxol84, 85 also known as Paclitaxel 42, has been reported as a TLR4 ligand and its synthesis has been described. Also flavolipin84 41, a di-acylated dipeptide, has been shown to have TLR4 agonistic properties.
O H
O H O N H O H N O O O
11
11
AcO
O OA c O OH
OH O O
O
OH NH O
41 42 O
Figure 1.13: Flavolipin, Paclitaxel
Besides the synthetic TLR4 ligands given in Figure 1.7 to 1.13 several approaches have been developed to the synthesis of key components of natural TLR4 ligands, such as hyaluronic acid54,lipomannan86 and lipoarabinomannan.87
TLR5
Toll like receptor 5 is a homodimeric receptor that can be found on the cell surface. Its natural ligand is flagellin88, the major component of the bacterial flagellar filament of Gram-positive and Gram-negative bacteria. Recently a tridecameric peptide peptide has been described to be the first known synthetic ligand for TLR5.89 Bacterial infections are linked to TLR5 stimulation. VaxIne® developed by VaxInnate is a flu related fusion protein.11 This TLR5 binder is in the preclinical stage.
TLR6
TLR6 is a cell surface receptor which forms heterodimers with TLR2.13 Natural ligands include diacylated lipoproteins related to mycoplasm bacteria. Synthetic ligands include Pam2CysSK4, FSL-1, MALP-2 and MPPL-1 which are shown in Figure 1.2 and described in the TLR2 section.
TLR7
TLR7 is an intracellularly located homodimeric receptor which can recognize GU rich short single stranded RNA (ssRNA).90-92 Synthetic ligands are mainly based on the family of imidazoquinolines.93, 94 Imiquimod (43, S26308, R837)95-97, resiquimod (44, S28463, R848)98-
101, S-27609102 (45), 3M-012103 (46),gardiquimod (47), sotirimod (48, R-850), CL097 (49), S- 28826 (50), 852A, (51, 3M-001)104-106,3M-011107 (52), 3M-003 (53) and loxoribine108, 109 (54) (Figure 1.16) have been reported to activate the immune system via TLR7. Also ANA245 (55, isatoribine), ANA975110 (56), Bropirimine111 (57),7-Deaza-dG (58), 7-Deaza-G (59), 7- thia-8-oxo-G (60, TOG) 112, 113 are known TLR7 ligands (Figure 1.14). Recently the focus on small synthetic molecules for TLR7 has shifted from imidazoquinolines to 7-hydro-8-oxo- adenines114 such as UC-1V150115 (61)and SM3603211 (62) (Figure 1.14). Imiquimod 43 is the only imidazoquinoline to date that has been FDA approved and is the active component of Aldara, an ointment used to treat superficial basal carcinomas. Oral administering imiquimod resulted in severe side effects and therefore Hirota et al., screened a library of purines and pyrimidines for alternatives.114 From this library the class of 8-hydroxyadenines was shown to function as TLR7 ligand and through lead optimalisation several 8-hydroxyadenines were found with good TLR7 stimulating properties. Besides small molecules also several synthetic single strand RNAs have been reported as TLR7 ligand for example R1354.101 It is an AU rich oligonucleotide with the following sequence: 5’-UUAUUAUUAUUAUUAUUAUU-3’.
N N N NH2
N N N NH2
OH
O N
N N NH2
OH HN
HN
N N
N O
O H2N
O OH
OH
OH O
Imiquimod, S26 308, R837 re siqu imod , S28463 , R84 8 Gard iquimod 4 7
Loxor ibine 54
HN
N O
B r
H2N
B ropir imin e 57 N
N N NH2
HN S O O
852A 51
N N N NH2
OH O
3M-003
N
N H N NH2
O
CL097 N
N N NH2
Sotirimod, R-850
N
N N
S
H2N
O
O OAc
OAc HO
A NA 975 56
N
N H N NH2
O O
O
SM360 32 N
N N NH2
S-28 828 50 O
HN
N N
S
H2N
O
O OH
OH HO
ANA245, Isator ibine O
HN
N N
O
H2N
O OH
OH 7-Deaza-dG
HN
N N
O
H2N
O OH
OH
OH 7-DeazaG
HN
N N
S O
O H2N
O OH
OH
OH
7-Thial-8-oxo-G (TOG)
N
N N
H N NH2
O O
H
O UC-1V15 0
O N
N N NH2
3M-012 46 O
NH2 N
N N NH2
OH
S-27 609 45
N N N NH2
3M-011 52 O
NH S
O O
43 44
48
49
53
55
58 59
60
61
62
Figure 1.14: TLR7 overview
TLR8
Homodimeric receptor TLR8 is located intracellularly on endosomal membranes, uses ssRNA as natural ligand and is involved in viral infections.116 Several RNA fragments and small molecules have been reported for TLR8.116 GU rich short stranded ssRNA, PolyU, but also small synthetic molecules such as imidazoquinolines can be used to activate the immune system via TLR8. Resiquimod98-101, S27609102, 3M-003, 3M011107, 3M-012103, loxoribine108,
109 and bropirimine111 (Figure 1.14) are besides TLR7 activating agents also reported to be TLR8 ligands. CL075 63 also known as 3M-002117 is the only known imidazoquinoline to act solely on TLR8 (Figure 1.15).
N S N NH2
3M-002 CL0 75 6 3
Figure 1.15: 3M-002
TLR9
TLR9 is an intracellular homodimeric receptor located on endosomal membranes118. It can distinguish mammalian DNA from bacterial or viral DNA by recognition of specific unmethylated CpG-oligodeoxynucleotides. TLR9 is involved in bacterial and viral infections, atopic disorders, autoimmune diseases and cancer. Several synthetic versions of CpG-DNA have been prepared and are currently being tested in different stages of clinical trials. The backbone of the synthetic DNA is phosphorothioate protected to improve the biological stability. Recently it has been shown that the phosphorothioate functions are required for the immunostimulatory properties of CpG-DNA itself.119
N NH2
O N O
O P O
S- O
NH N N
O
NH2
N O
O P O
O O
S-
P hosphorothioate CpG 64
N N NH2
O NH
N N O
NH2
HO
7 -deaza-dG 65
5 -hyd roxy-d C 66
Figure 1.16: CpG fragment of phosphorothioate DNA
Three main classes can be discerned for CpG DNA namely class A/D, class B/K and class C.118 The different classes and their primary structures are shown in Figure 1.17.
5'-G G G G N N N N N N C G N N N N N N G G G G-3' phosphoro thioa te phosphor oth ioate
phosphod iester
Class A/D
5'-N N C G N N N N C G N N N N C G N N N N-3' phosphor oth ioate
Class B/K
5'-T C G N N A A C G T T N N C G N N N N -3' phosphor oth ioate
Class C
N= Nucleobase
Figure 1.17: CpG DNA classes
Palindromic sequences including CpG dinucleotides having a phosphodiester backbone and flanking phosphorothioate dG stretches are known as class A/D. Immune responses triggered by class A/D are linked to its ability to form secondary structures. Class B/K comprises of CpG dinucleotides having a phosphorothioate backbone within a specific sequence context. It can not form secondary structures. Class C, the latest addition, mimics the activities of class A/D and B/K, has a phosphorothioate backbone, contains palindromic and non-palindromic DNA sequences and forms secondary structures. Structure-activity studies on TLR9 activation have revealed the requirement that any CpG DNA ligand must have a free 5’end.118 Oligomers linked via their 3’-terminus and having two free 5’-ends show enhanced immuno- stimulating properties compared to oligonucleotides having only one free 5’end.118 Furthermore the location of the CpG in the sequence and the nucleotides flanking the CpG motif are of importance. In the CpG motif, the 2-keto, 3-imino, 4-amino groups of cytosine, and the 1-imino, 2-amino and 6-keto group of guanine, are key features responsible for the TLR9 activity.118 Several alternative nucleobases have been reported to replace either C (e.g.
5-hydroxycytidine120 66or G (e.g. 7-deazaguanine 65120 (Figure 1.16) in CpG after which the CpX or YpG motif still retains immunostimulatory activity. Several drugs based on CpG- DNA are currently in development. Promune (Coley/Pfizer) has completed the phase II clinical trial in treatment of non-small cell lung cancer. Actilon (Coley/Pfizer) is a synthetic C-class CpG oligonucleotide in phase Ib trials against HCV. Also AVE7279, AVE0675 (Sanofi-aventis), IMOxine, HBY2093 (Novartis) and dSLIM (Mologen) are TLR9 drugs currently being tested in different stages of clinical trial121.
TLR10
Since Toll like receptor 10 is highly homologous to both TLR1 and TLR6, it is a candidate to form a pair with TLR2 to recognize peptidoglycan (PGN)21. TLR10 is expressed in lung and in B-lymphocytes and is an asthma candidate gene122. To this date no specific natural or synthetic ligand has been assigned to TLR10122.
Contents of this thesis
The aim of the studies described in this thesis is the preparation of well defined TLR2, TLR7 and TLR9 ligands either alone or linked to antigenic peptide. The synthetic constructs have been assessed on their TLR activating properties and their possible use as a starting point for vaccine development has been evaluated. In Chapter 2 the design and synthesis of 2- azidoalkoxy substituted 7-hydro-8-oxo-adenines is described, along with their IL-12 stimulating properties. One of these compounds forms the basis of the studies presented in Chapter 3, in which the synthesis and biological evaluation of TLR7-L-peptide epitope conjugates is discussed. Chapter 4 describes the preparation of (Pam3CysSK4) TLR2-L- peptide epitope and (CpG-DNA) TLR9-L-peptide epitope constructs as well as their fluorescent equivalents. Antigen presentation and IL-12 induction of all synthesized conjugates were determined. Furthermore the trafficking of the fluorescent conjugates was evaluated. The glycerol moiety in Pam3Cys which was used in the prepared TLR2-L-peptide conjugates was studied in Chapter 5. The two enantiomers of the glycerol (R and S) were built into Pam3Cys which was subsequently coupled to a peptide. IL-12 production, antigen presentation and specific T cell induction were determined. Chapter 6 summarizes this thesis and describes unfinished research as well as future prospects.
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