Synthetic TLR4-active Glycolipids as Vaccine ... - Ingenta Connect

Current Topics in Medicinal Chemistry, 2008, 8, 64-79

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Synthetic TLR4-active Glycolipids as Vaccine Adjuvants and Stand-alone Immunotherapeutics David A. Johnson* GlaxoSmithKline Biologicals, 553 Old Corvallis Road, Hamilton, Montana 59840, USA Abstract: The design of vaccine adjuvants and stand-alone immunotherapeutics has historically been a mix of alchemy and accident partly because of the complex nature of the molecular mechanisms involved in immune system function. The recent discovery of pattern recognition receptors and toll-like receptors (TLRs) in particular on cells of the immune system has shown the important role that stimulation of these cell receptors by microbial products plays in both innate and adaptive immune responses. Considerable effort has been directed at developing pharmaceutically acceptable mimetics of many TLR-active natural products, including the main cell-surface component of Gram-negative bacteria: lipopolysaccharide (LPS). LPS and its active principle, lipid A, are potent stimulators of host defense systems via their interaction with TLR4. However, the profound pyrogenicity and lethal toxicity of LPS and lipid A have precluded their medicinal use. Structure/activity investigations on natural S. minnesota R595 lipid A and its derivatives have led to the development of a novel class of synthetic lipid A mimetics known as aminoalkyl glucosaminide phosphates (AGPs). This review discusses the evolution of the AGPs and related TLR4-active glycolipids with emphasis on structure/activity relationships in the AGP series and pre-clinical/clinical development of selected AGPs, including the potent vaccine adjuvant RC-529.

Keywords: TLR4 agonist, aminoalkyl glucosaminide phosphate, AGP, RC-529, CRX-527, innate immunity, adaptive immunity, lipid A mimetic. I. INTRODUCTION The recent discovery of toll-like receptors (TLRs) on cells of the immune system has led to a great deal of interest in developing TLR agonists and antagonists to manipulate innate and adaptive immune responses [1-3]. Targeting TLR receptors and cognate intracellular pathways could potentially lead to more effective vaccines and novel therapeutic approaches to treat immune and inflammatory diseases. Although the addition of microbial components to vaccines has long been known to enhance adaptive immune responses, the molecular mechanisms involved have not been well understood. Only recently were TLRs on cells involved in immune surveillance, such as epithelial and dendritic cells (DCs), shown to engage many of these microbial products via so-called “pathogen-associated molecular patterns” (PAMPs). There is increasing evidence that most vaccine adjuvants and stand-alone immunomodulators interact with members of the toll-like receptor family. The TLR family of pattern recognition receptors is now known to consist of at least 13 distinct receptors that recognize conserved structural components on most if not all pathogens. Of the 10 known TLRs that have been identified in humans, five are associated with the recognition of bacterial components: TLR4 and TLR5 recognize lipopolysaccharide (LPS, endotoxin) and flagellin, respectively, and TLRs 1, 2 and 6 cooperate in the recognition of lipopeptides/proteins and glycolipids from Gram-positive bacteria [4]. The TLRs involved in the recognition of bacterial ligands are type-1 transmembrane proteins which contain leucine-rich extracellular domains that likely play a major role in PAMP recognition. In contrast, TLRs 3, 7, 8 and 9 appear to be *Address correspondence to this author at GlaxoSmithKline Biologicals, 553 Old Corvallis Road, Hamilton, Montana 59840, USA; Tel: (406) 3752134; Fax: (406) 363-6129; E-mail: [email protected]

1568-0266/08 $55.00+.00

restricted to cytoplasmic compartments and are involved in the detection of viral RNA (TLR3, 7, 8) and unmethylated DNA (TLR9) common to both bacteria and viruses [4]. All members of the TLR family of pattern recognition receptors possess conserved cytoplasmic domains which share amino acid homology with the IL-1 receptor (IL-1R). The Toll/IL1R (TIR) domain regulates intracellular signaling pathways and leads to gene expression via interaction with intracellular adapter molecules such as MyD88, TRIF, TIRAP, and TRAM [5,6]. These adapter molecules can differentially regulate the expression of inflammatory cytokines/chemokines and type I interferons (IFN/), which can lead to the preferential enhancement of non-specific resistance (NSR) against bacterial and viral infections or antigen-specific humoral and cell-mediated immune responses [7]. The recognition of microbial ligands by TLR receptors triggers a “danger signal” that alerts antigen presenting cells (APCs) to the presence of a foreign antigen or invading pathogen. This initial alert results in the release of antimicrobial defensins and reactive oxygen intermediates, as well as the secretion of inflammatory cytokines. The release of pro-inflammatory cytokines and chemokines, such as IL-1, IL-12, TNF-, and IFN-, by activated immune cells leads to the recruitment and activation of APCs—principally macrophages and dendritic cells—as well as the activation of effector B and T lymphocytes involved in antigen-specific immunity. While the initial innate immune response serves to reduce pathogen levels immediately following exposure, the antigen-specific acquired immune response is needed to eliminate all traces of the infection and provide immunological memory and long-term immunity. This adaptive immune response initially involves presentation of exogenous antigens to T-cell receptors on naive T-helper (Th) cells in the context of class II major histo-compatibility complex (MHC) proteins. It has been suggested the efficiency of this © 2008 Bentham Science Publishers Ltd.

Synthetic TLR4-active Glycolipids as Vaccine Adjuvants

process is enhanced by the up-regulation of classical costimulatory molecules such as CD80 (B7-1) and CD86 (B72) on the surface of APCs leading to preferential development of Th1 and Th2 cells, respectively, via stimulation of CD28 on T cells in lymphoid tissue [8]. However, recent evidence suggests that other B7 members play a role in regulating effector T-cell functions at peripheral sites of infection via binding to CD28 homologs on activated T cells [9]. And, while Th1 cells have historically been associated with cell-mediated immunity via the production of proinflammatory cytokines and cytotoxic T-lymphocytes (CTLs), and Th2 cells with the production of IL-4 and IL-10 and antibody (humoral) responses, certain of these novel B7 co-stimulatory pathways are important for T-cell/B-cell collaboration in generating both humoral and CTL responses. In addition, the so-called follicular helper T cells (Tfh) present in lymph nodes and lacking distinct Th1 or Th2 cytokine profiles appear to be critical to antibody responses via B7 ligation as well as expression of CXCR5, a chemokine receptor that allows Tfh cells to enter B-cell areas in lymph nodes and enhance antibody production [10]. Further, in the case of CD28 ligands B7-1 and B7-2, the picture is complicated by the fact that Th1/Th2 polarization depends on the dose of the antigen [11]. While the interrelationship and function of different T cell populations remains complex, it is clear that TLR-mediated stimulation of APCs and up-regulation of B7 co-stimulatory molecules on T cells plays an integral role in triggering adaptive immune responses to infectious agents as well as determining the nature of the response to heterologous vaccine antigens. While most conventional vaccines employ killed/attenuated microbes or microbial fragments as antigens to stimulate a protective immune response, problems associated with vaccine manufacture and the presence of non-essential components have resulted in considerable effort to refine vaccines as well as to develop well-defined synthetic antigens using chemical and recombinant techniques [12,13]. However, the refinement and simplification of microbial vaccines and the use of synthetic antigens have resulted in a concomitant loss in vaccine potency. These observations have led to investigations on co-administering adjuvants with vaccine antigens to potentiate the activity of vaccines and the weak immunogenicity of synthetic epitopes. Although initially coined in the 1920’s to describe an additive that enhances an antibody response to a vaccine antigen, the term “adjuvant” is now used to describe compounds that induce Th1 (cell-mediated) and/or Th2 (antibody) immune responses. More recent findings have also implicated the T-regulatory phenotype, which involves enhanced IL-10 production and the suppression of T-cell proliferation to parasitic antigens, as a possible adjuvant strategy in the case of allergy vaccines [14,15]. Currently, the only adjuvant licensed for human use in the United States is a group of aluminum salts known as alum; but alum is not without side-effects and generally enhances humoral (Th2) immunity only [12,16]. The recognition that cell-mediated immune responses—particularly the induction of CTLs, which typically requires presentation of endogenous antigens via class I MHC complexes—are crucial for generating protective immunity in the case of many intracellular pathogens and cancers has prompted

Current Topics in Medicinal Chemistry, 2008, Vol. 8, No. 2

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efforts to develop new vaccine adjuvants that enhance both antibody and T-cell responses. Thus, new adjuvants are needed to help control the magnitude, direction, and duration of the immune response against antigens. The discovery of mammalian Toll-like receptors on innate immune cells, and DCs in particular, has led to the identification of several TLR-active natural product leads potentially useful in the design of new adjuvants. For example, the main cell-surface component of Gram-negative bacteria, lipopolysaccharide, and its active principle, lipid A, are ligands for TLR4 and accessory molecules such as MD-2 on dendritic and other immune cells [17,18]. As a result, many lipid A molecules are potent stimulators of host defense systems, both as adjuvants for vaccine antigens [13] and as inducers of non-specific resistance to infection in animal models [19]. However, the profound pyrogenicity and lethal toxicity of LPS and lipid A have precluded their use as vaccine adjuvants and monotherapeutic agents. Thus, considerable effort has been directed towards the development of semi-synthetic and synthetic lipid A mimetics with simplified structures and improved toxicity/activity profiles for use as vaccine adjuvants and stand-alone therapeutics [20-22]. Due to the pathophysiology of LPS—which includes septic shock, organ failure, and death—the TLR4 receptor has also been an attractive target for the development of LPS antagonists for the treatment of endotoxinrelated diseases [1]. Inhibiting the toxic effects of LPS by blocking its cell-surface receptor with synthetic antagonists has led to the clinical development of disaccharide antagonists based on the structure of R. sphaeroides lipid A. This and other approaches for alleviating endotoxin-mediated disease were reviewed recently [1]. The scope of this review will be limited primarily to synthetic glycolipid-based TLR4 agonists as vaccine adjuvants and stand-alone immunomodulators, with particular emphasis on the synthesis and biological activity of synthetic mimetics of S. minnesota R595 lipid A II. DISACCHARIDE MINNESOTA LIPID A

DERIVATIVES

OF

S

Several years ago, it was shown that the toxic effects of Salmonella minnesota R595 lipid A 1 (Fig. 1) could be ameliorated by selective hydrolysis of the 1-O-phosphono and (R)-3-hydroxytetradecanoyl groups [23]. The resulting chemically modified natural lipid A product, monophosphoryl lipid A (MPL) adjuvant, is an effective adjuvant in prophylactic and therapeutic vaccines and shows an excellent safety profile in humans [24]. MPL adjuvant is the first TLR4 agonist to be approved for use in a human vaccine (FENDrix® vaccine for hepatitis B in Europe). Structural analysis of the 3-O-desacyl monophosphoryl lipid A derived from Re mutants of S. minnesota R595 indicated that, in addition to the major hexaacyl component 2, MPL comprised several less highly acylated congeners, including pentaacyl component 3 (Fig. 1) [24]. The structural heterogeneity in MPL is primarily due to partial acyl substitutions in the cognate LPS as well as intrinsic microheterogeneity in fatty acid chain length. To confirm the chemical structure of putative components 2 and 3 and the postulate that the biological activity of MPL is due to these materials and not to the

66 Current Topics in Medicinal Chemistry, 2008, Vol. 8, No. 2

P

HO

O

OH

O HO

David A. Johnson

O

O O

O

HO

O HO O

NH

NH

O

O

O

O

HO

O

O

P

O

O O

O

O

O

O

P

HO

O

OH

NH

OH

O

NH

O O

OH

O

HO HO

O

O

O O

O

OH

RO

O

2 R = hexadecanoyl 3 R=H

1 S. minnesota lipid A

Fig. (1) S. minnesota R595 lipid A (1) and two of the major components present in MPL

presence of other bioactive substances, we prepared compounds 2 and 3 by independent chemical synthesis (Scheme 1) [25]. We also anticipated that evaluating the structureactivity relationships (SAR) of discrete components present in MPL could potentially lead to the design of pharmaceutically acceptable mimetics with improved activity/ toxicity profiles and other beneficial attributes. Our highly convergent synthesis of compounds 2 and 3, which was patterned after Shiba’s synthesis of E. coli monophosphoryl lipid A [26], exploited the N-2,2,2-trichloroethoxycarbonyl (Troc) method and silver ion catalysis to construct the -(16)disaccharide linkage from glycosyl chloride donor 5 and glycosyl acceptors 7 [25]. The 2-(trimethylsilyl)ethyl (TMSEt) group was chosen for anomeric protection of the glycosyl donor progenitor because of the facility with which the TMSEt group could be converted directly into 1-chloro derivatives as well as the chemical stability of carbamate-protected glycosaminyl chlorides and their efficiency in silver ion-mediated glycosylations. Koenigs-Knorr coupling of glycosyl donor/acceptor pairs 5/7, readily obtained from the known amino alcohols 4 and 6 and the appropriate (R)-3-hydroxy- and/or (R)-3-alkanoyloxytetradecanoic acids, followed by further elaboration/ deprotection afforded the desired MPL components 2 and 3, which were isolated and characterized as their triethylammonium salts. The synthesis of the requisite (R)-3-hydroxy- and alkanoyloxytetradecanoic acid starting materials 8 and 9 was initially achieved by Ru(II)-Binap-catalyzed hydrogenation of keto ester 10 in the key step and subsequent enantiomeric enhancement by crystallization of the dicyclohexylam-

monium (DCHA) salt of 11 (Scheme 2) [27]. Reproducibility problems with commercial Ru(II) catalysts as well as difficulties in removing the DCHA-HBr by-product in the preparation of p-bromophenacyl (PAc) ester 12 were overcome by using Genet’s in-situ-Ru(II) asymmetric hydrogenation method [28] and e.e. enhancement (to >99.8%) by crystallization of 12 [29]. Other methods for the preparation of 11 were also examined, including copper-catalyzed Grignard addition to epoxybutyrate 13 (99.9% e.e., prepared in three steps from maltose) [30], but generally found to be less suitable, particularly for larger scale preparations. However, this latter method proved useful for the preparation of unsaturated derivatives of 11, which are inaccessible via catalytic hydrogenation [31]. Reverse-phase HPLC comparison of the synthetic and natural materials using spiking experiments showed a direct correlation of synthetic 2 and 3 with the two MPL components whose structures had been established previously by FAB-MS [32]. Comparison of the biological activities of synthetic 2 and 3 revealed that the hexaacyl derivative 2 was equivalent to 3 with respect to adjuvant activity for a peptide-tetanus toxoid conjugate, but up to 20-times more active than 3 in terms of its ability to induce nitric oxide synthase in murine macrophages [24]. Consistent with this observation, underacylated MPL constituents isolated by fractionation of MPL also exhibited low iNOS activity as well as low cytokine activity (production of TNF- and IL1 in human peripheral monocytes) relative to compound 2 [24]. These observations corroborate other lipid A studies showing that hexaacylation is prerequisite to the full expression of certain endotoxic activities, and that underacylated lipid A components tend to be much less


PhO

O

O

O HO

O

OTCBOC

P

O

PhO

OTMSEt


O O

O

OH O

O O

O

NH Cl Troc

NH

OH

NH

O O

n-C13H27

O

HO HO O

O

n-C11H23

O

O

O

O 4

P

HO

O

NH2

O HO

67

RO

5

OH O

O

HO TrocO

O

O HO

OBn

OBn

NH2

NH O

6 RO n-C11H23 TCBOC = CO2C(Me)2CCl3 Troc = CO2CH2CCl3 TMSEt = CH2CH2SiMe3

2 R = n-C15H31CO 3 R=H

7 R = Troc or n-C15H31CO

Scheme (1). Synthesis of two major components present in S. minnestota-derived monophosphoryl lipid A. Br OH

O CO2Me

n-C11H23

OH CO2H

n-C11H23

n-C11H23

O

R O O

11

10

n-C11H23

O CO2H

12

95-99% e.e.

>99.8% ee

8 R=H 9 R = n-alkanoyl

O maltose

CO2Et 13 99.9% ee

Scheme (2). Synthesis of (R)-3-hydroxy- and alkanoyloxytetradecanoic acids

active [33,34] and may even play a role in immune evasion and bacterial virulence [35]. While removing the (R)-3-hydroxytetradecanoyl group from the 3-position of heptaacyl S. minnesota monophosphoryl lipid A (MLA) 14 to form 2 reduces pyrogenicity [23], a comparison of the pyrogenic activity of S. minnesota MLA derivative 14 (LA-16-PH) with that of E. coli MLA 15 (LA-15-PH) shows that eliminating the -fatty acyl residue from the adjacent position has just the opposite effect: the fever response is enhanced for the hexaacyl species 15 (Table 1).* Thus, biological comparison of the highly immunostimulatory hexaacyl MLA derivatives 2 and 15 demonstrates that certain undesirable properties (pyrogenicity) can be separated from beneficial immune-stimulating effects (iNOS, cytokine induction) on the basis of fatty acid distribution. *

Myers, K.R.; Johnson, D.A.; Ulrich, J.T.; Gustafson, G.L. Monophosphoryl Lipid A Species with Variable Endotoxicity Profiles: The Relative Importance of Fatty Acid Content Versus Location. 2nd Biennial Conference of the International Endotoxin Society, Vienna, Austria, 1992

Since minor variations in acyl chain length that occur naturally in lipid A do not appear essential to biological activity [37], we also evaluated the activity of the synthetically simpler MLA derivative 16, in which the normal or secondary fatty acids are identical but contain the same absolute number of carbon atoms as in MPL component 2. Compound 16, which was prepared via a modification of the method in Scheme (1) involving simultaneous introduction of the two N-linked acyl groups, was very similar in activity to MPL component 2 with respect to febrile response in rabbits (Table 1) as well as other models of toxicity (lethal toxicity in mice) and efficacy (iNOS, cytokine induction, adjuvan-ticity) examined [36]. These results are in agreement with other studies suggesting that the hydrophobe-hydrophile balance in lipid A analogues may play a role in determining certain biological activities [38]. The effect of fatty acid structure on endotoxicity in the MLA series was further evaluated with a series of chain length homologs (4 to 12 secondary acyl carbon atoms) of MLA derivative 16 [36]. Secondary fatty acid chain length


David A. Johnson

Table 1. O

O HO

P

HO

OH O

O O

O

NH O

O

HO O

HO

O

HO O

NH

R1

O

O O

HO

O

NH

O

O

HO HO

O

NH

O

O

OH

O

O

O O

O

NH

OH

O

O

O

OH O O

O NH

O

P

HO

O

HO HO

O

OH

O O

O

OH

O

O

O

P

O O

O

O

O

R2

17 16

iNOS

Fever Responsea, ºC Compound

Fatty acyl groups

R1

inductionb

R2 2.5 g/kg

10 g/kg

ED50 (ng/mL)

14 (LA-16-PH)

7

C14-OH

C16

0.2

1.7

28

15 (LA-15-PH)

6

C14-OH

H

3.4

4.1

12

c

68 (10)d

2

6

H

C16

0.2

0.6

16

6

-

-

0.3e

0.6e

13 (5)d

17

6

-

-

4.7

4.1

(0.05)d

C14-OH = (R)-3-hydroxytetradecanoyl; C16 = hexadecanoyl Relationship between structure and pyrogenic and iNOS activity of 4’-monophosphoryl lipid A compounds. Assay details are described in reference [36]. LA-16-PH and LA-15-PH were obtained from ICN Biomedicals and Osaka University, respectively. aTotal temperature rise for three rabbits at dose shown. bED50 values represent the concentration of the test article required to produce a half-maximal response. cAverage of two tests. dResults from separate experiment.[36] eAverage of four tests with two separate synthetic preparations.

was found to have a profound bimodal effect on the expression of various endotoxic activities, including TLR4mediated cytokine induction in peripheral blood mononuclear cells (PBMCs). Short chain derivatives with 4 or 6carbon acyl chains were inactive, whereas the 10-carbon homolog 17 exhibited the highest levels of iNOS and cytokine induction and greatest pyrogenicity; the 14-carbon derivative 16 was intermediate in activity and toxicity. A threshold chain length was observed for activity in both the murine iNOS (8 carbons) and human monocyte (10 carbons) models, indicative of strict but slightly different structural and/or conformational requirements for the two bioactivities and likely attributable to the known species differences between the structure of the glycoprotein MD-2, a TLR4 accessory molecule required for LPS responsiveness, in mice and humans [39,40]. MD-2 is thought to possess a hydrophobic binding pocket which is sensitive to both the degree of acylation and acyl chain length, as well as contain conserved amino acid sequences critical for the association with the ectodomain of TLR4 [41]. Despite the high endotoxicity of 17 in these models, the longer chain derivatives 2 and 16 both induced significantly higher tetanus toxoid-specific antibodies of all classes of immunoglobulins tested including complement fixing IgG2a

and IgG2b isotypes than 17 in murine vaccine models [36]. These data suggest that fatty acid chain length, which is known to influence molecular conformation as well as solution aggregate structure, is more important than the presence or absence of the 1-phosphate and 3-hydroxytetradecanoyl groups in determining the biological activity in the MLA series. Clearly, the reduced toxicity of MPL component 2 relative to that of parent lipid A 1 cannot be reconciled with the high pyrogenicity and lethal toxicity of compound 17 on the basis of deacylation/ dephosphorylation alone. Further, rhodobacter lipid A which possesses both an anomeric phosphate and -hydroxyfatty acyl group on the 3position as well as unique acyl groups, is not only low in toxicity but also an effective antagonist of enterobacterial lipid A [42]. Biophysical characterization of synthetic lipid A molecules has revealed that the molecular shape and supramolecular structures of TLR4 agonists and antagonists are very different, with compounds adopting a conical confirmation and non-lamellar aggregates being more active than compounds that adopt a cylindrical shape and lamellar aggregates [43]. Nevertheless, it has also been suggested that solution aggregation properties are not important determinants of TLR4 agonist activity per se and that the monomeric LPS:MD-2 complexes formed from underacylated variants



such as rhodobacter lipid A are just less adept at activating TLR4 and triggering oligomerization [44]. Oligomerization or aggregation of TLR4 molecules into lipid rafts is thought to be prerequisite to signal transduction and the elicitation of cellular activities associated with the innate immune response. Another detoxified lipid A product closely related to MPL adjuvant and containing heptaacyl derivative 18 (Fig. 2) as a major component is being employed as an adjuvant in therapeutic cancer vaccines against non-small-cell lung (NSCL) and prostate cancer [45]. Biomira, the developer of this and other therapeutic cancer vaccines, recently reported that the synthetic hexa- and hepta-fatty alkyl/acyl MLA derivatives 19 and 20, which possess hydrolytically more stable alkoxy substituted acyl residues and a unique tri-lipid moiety, induce T-cell proliferation and levels of IFN- comparable to those induced by the detoxified lipid A 18 (as congeneric mixture) in experimental MUC-1 vaccines in murine models [46]. MPL adjuvant has also been shown to possess activity similar to that of ether lipids 19 and 20 and the detoxified MLA preparation containing 18 in experimental models for NSCL and prostate cancer [45,46]. Compounds 19 and 20 were constructed via a highly convergent strategy similar to that outlined in Scheme 1 utilizing the benzyl group for global protection and Schmidt glycosylation to couple the glycosyl donor/acceptor pair 21/22, followed by subsequent elaboration/deprotection (Scheme 3). The unique ether acid 23 was prepared via Oalkylation of the PAc ester 24, followed by deprotection and esterification of the resulting acid 25 with the homoallylic alcohol 26 and oxidation of the terminal olefin.

O HO

P

HO

O

O O

O

NH O

The pre-clinical and clinical results with these monophosphoryl lipid A derivatives suggest that the structural requirements for adjuvanticity are different, and perhaps less stringent, than for other endotoxicities and that fatty acid chain length is a critical determinant of TLR4 agonist activity. But factors besides TLR4 agonism such as aqueous solubility and stability, solution aggregation, and other physicochemical properties may also be important to adjuvanticity in the MLA series. III. MONOSACCHARIDE MINNESOTA LIPID A

OH O

O O

O

O

NH

O

P

HO

O

HO O

OH

O

NH

O

O

HO HO

NH

O O

O

O

O

RO

HO

O

S

OF

Synthetic analogs of either the reducing or non-reducing glucosamine moieties of lipid A containing up to five fatty acids and lacking an aglycon unit typically exhibit low biological activity. In fact, monosaccharide derivatives of the

HO O

MIMETICS

Numerous subunit derivatives of bacterial lipid A have also been prepared with the aim of separating toxic properties from beneficial immunostimulatory effects. These molecules typically have been synthetic analogs of either the reducing or non-reducing glucosamine moieties of lipid A [47] or analogs in which one of the saccharide units or the entire disaccharide backbone has been replaced with an acyclic scaffold [20,21,48]. In addition, subunit analogs have been prepared in which the labile anomeric phosphate has been replaced with a stable “bioisosteric” acidic group [49,50] and/or the ester-branched acyl groups replaced with ether- or alkyl-branched lipid groups [21,51,52]. Such subunit analogs also provide a structural motif more amenable to systematic SAR investigations than more complex disaccharide derivatives.

O

OH

69

O

O

O

O

O

18

Fig. (2). Heptaacyl S. minnesota monophosphoryl lipid A (18) and ether lipid analogs

19 R = H 20 R = n-C12H25

OH


HO

BnO

P

O

O HO

HO

O

O

O

O O

HO HO

NH

NH

O

OH

O RO

n-C11H23

O HO

O

O O

O

O n-C11H23

OH

O

NH OC(NH)CCl3 Troc

OAll

23

P

O

O O

BnO NH2

O

OBn

O 25

O

Ph

David A. Johnson

O

21

O

OH

O O

HO BnO

NH OBn NH2

O

OBn

O O

n-C11H23 19 R = H 20 R = n-C12H25

O n-C11H23 n-C11H23 22 OH

OH n-C11H23

O

R O

n-C11H23

O

n-C11H23

CO2H

O 24

26 R

O

O O

n-C11H23 n-C11H23

25 R = n-C11H23

CO2H

23

Scheme (3). Synthesis of monophosphoryl lipid A ether lipid derivatives.

reducing glucosamine often exhibit TLR4 antagonist properties. However, certain monosaccharides particularly nonreducing subunit analogs possessing three acyl residues possess significant endotoxic activity yet low pyrogenicity and lethal toxicity in comparison to lipid A. For example, GLA-60 (27, Fig. 3) showed the strongest B-cell activation and adjuvant activities among various non-reducing subunit analogs that have been examined [47] and has been shown to non-specifically protect mice against cytomegalovirus virus [53]. SAR investigations showed the importance of normal fatty acid chain length, stereochemistry, and the number, type and relative position of the fatty acyl moieties in this series. However, despite its low pyrogenicity, GLA-60 was two orders of magnitude less active than E. coli lipid A (28) with respect to its ability to induce TNF- production in human mononuclear cells. Biophysical characterization of 27 showed that it adopts a slightly conical molecular conformation and mainly unilamellar aggregate structures, which is consistent with its reduced bioactivity [54]. Adding a fatty acid onto the side-chain hydroxyl group of 27 to form tetraacyl derivative 29 (GLA-47), which corresponds to the lefthand side of E. coli (28) and S. minnesota lipid A (1), abolishes TNF- and IL-6 induction in human U937 cells and PBMCs [47]. Thus, increasing or decreasing the number of acyl residues from the optimum of six in disaccharides and three in monosaccharides reduces endotoxic activity [37,38].

Since the spacial arrangement, chain length, and number of fatty acyl residues in lipid A and other bacterial amphiphiles appeared important for immunostimulant activity [55,56], we designed a novel class of lipid A mimetics known as aminoalkyl glucosaminide 4-phosphates (AGPs) and investigated structure-activity relationships within this series [57]. The AGPs, which possess general structure 30 (Fig. 3), are synthetic mimetics of MPL component 2 in which the reducing sugar of 2 has been replaced with a conformationally flexible N-acyl aglycon unit. We speculated that coupling a flexible N-acylated aglycon to the structurally more conserved non-reducing sugar of MPL component 2 would permit energetically favored close packing of the six fatty acyl chains present in compound 2 as well as facilitate the intercalation of the acyl residues into the hydrophobic pocket of the TLR4 accessory molecule MD-2. The known immunostimulating ability of naturally occurring N-(3-acyloxylacyl)amino acids such as flavolipin [58,59], which was recently shown to require MD-2 and TLR4 [60], made the evaluation of seryl -O-glycosides of particular interest. It was also anticipated that the seryl carboxyl group could serve as a stable bioisostere of the labile anomeric phosphate of lipid A. The ionic interaction between the two phosphate groups of lipid A and the lysine residues along the edge of the hydrophobic pocket of MD-2 appear to be important for the formation of monomeric endotoxin–MD-2 complexes capable of binding TLR4 [61].

Synthetic TLR4-active Glycolipids as Vaccine Adjuvants O HO

P

HO

O

OH HO

O

O O

OH

P

NH

O

OH

O

O

HO O

HO O

NH

NH

O

O

O

O

27 R = H (GLA-60) 29 R = n-C13H27CO (GLA-47)

71

R4

O

O O

O n O

NH

NH

O

P OH

O

R

O

OH

O

OH

HO

O

P

HO O

O

O

O O

O

O

O O

HO

O


RO

O

O

R1

O

R3

R2

30

28 R = H (E. coli lipid A) 1 R = n-C15H31CO (S. minnesota lipid A)

AGP generic structure

Fig. (3). Non-reducing end subunit analogs of lipid E. coli and S. minnesota lipid A.

Two general methods have been used to prepare compounds in the AGP series (Schemes 4 and 5). Our initial synthesis was similar to what was used to prepare the MLA disaccharide derivatives and convergent with respect to the left and right halves of the molecule [57]. Compounds were constructed from glycosyl chlorides 31 and glycosyl acceptors 32 using the N-Troc method for -glycosylation in the key step (Scheme 4), which permitted the incorporation of the same or different fatty acids. A variation of this synthesis was also developed in which the glycosyl chlorides were obtained from more readily accessible 1-O-tbutyldiphenysilyl (TBDPS) glycoside starting materials using a modification of Magnusson’s method for direct conversion of functionalized 1-O-silyl glycoside precursors to the chlorides 31 [62]. However, the general approach outlined in Scheme 4, while allowing ready access to a variety of compounds possessing different aglycon and fatty acyl units, suffers from a number of drawbacks. Most notably, incorporating two of the three chiral acyl residues onto the AGP scaffold early in synthesis leads to multiple synthetic and chromatographic steps involving sensitive glycolipid intermediates. This method also precludes the use of a late-stage

intermediate for the synthesis of AGPs comprising the same sugar and aglycon units but different fatty acid substituents. Accordingly, an alternate synthesis was developed convergent with respect to the AGP backbone and the fatty acid substituents (Scheme 5) [29]. The choice of N-protecting groups depends on the nature of functionality in the aglycon -substituent R4 as well as the need to chemically differentiate the two amino groups. For many AGPs containing three identical fatty acyl residues such as ethanolamine-based RC529 (35) and CRX-524 (36), the common advanced intermediate 34 (R4=H) is best prepared via hydrogenolysis of the di-Cbz derivative 33 (PG1=PG2 =Cbz, R4=H; path A, Scheme 5). Selective triacylation of 34 followed by phosphorylation/deprotection provides the target molecules. However, for certain other derivatives such as seryl-based CRX-527 (38) the fatty acids are more expediently introduced in two separate acylation steps beginning with the selective 3-O-acylation of intermediate 33 (PG1= PG2=Troc, R4=CO2Bn; path B, Scheme 5); this is partly due to the formation of complexed zinc during Troc deprotection, which is difficult to remove by aqueous work-up, leading to emulsions and low recoveries of diamino diol 34 (R4= CO2Bn). O

R4 OTCBOC

O O O HO

O OPG NH Troc

PhO P PhO

n O

O O

OR3

HO N H

n-C11H23 32

P

HO

OH

O

NH Cl Troc n-C11H23

R 1O

R4

O

O O

O

NH

O

R1O PG=TMSEt or TBDPS

O HO

O

n-C11H23 n-C11H23

31 30

Scheme (4). “Left-right” convergent synthesis of aminoalkyl glucosaminide phosphates (AGPs)

NH

R3O

R2O n-C11H23

n O


David A. Johnson

R4 OAc AcO AcO

HO

O OAc NHPG1

A NHPG2

OTBDMS

OTBDMS HO HO

PG1=PG2

R4

O O

NHPG2

NHPG1

R4

O

HO HO

O

NH2

NH2

PG1 = Troc, Aoc, Cbz, etc 34

33 B

OTBDMS HO

R4

O O

O

NHPG1

O

O NHPG2

HO

P

HO

OH O

O O

NH NH

O

R1O

R4 O O

O R1O

n-C11H23

R3O

R2O n-C11H23

n-C11H23 n-C11H23

37 35 R1=R2=R3= n-C13H27CO, R4 = H (RC-529) 36 R1=R2=R3= n-C9H19CO, R4 = H (CRX-524) 38 R1=R2=R3= n-C9H19CO, R4 = CO2H (S) (CRX-527)

Scheme (5). “Top-bottom” convergent synthesis of aminoalkyl glucosaminide phosphates (AGPs)

Several members of this class of lipid A mimetics, including prototypical AGPs RC-529 (35), CRX-524 (36), and CRX-527 (38), have been shown to improve humoral and cell-mediated immune responses to a variety of different antigens in mice [57,63-65] as well as enhance non-specific resistance in mice to viral and bacterial infections [62,66]. Protection against influenza virus or Listeria monocytogenes challenge showed a profound dependence on both fatty acid and aglycon chain length as well as the nature of the substituent in the aglycon. The highest levels of protection are observed when the secondary fatty acids (R1, R2, R3) are 10 to 12 carbons in length, the aglycon is 2-carbons long (n=1), and the aglycon -substituent is a carboxyl group (e.g., CRX-527 (38)). The finding that certain AGPs protect against an intranasal challenge of influenza suggests that the accessibility of TLR4 and MD-2 on mucosal surfaces may provide a unique way of providing short-term resistance to infectious disease. To further demonstrate the prophylactic anti-infective effects with an AGP, CRX-527 was evaluated in a preclinical model for respiratory syncytial virus (RSV) [63]. RSV is a significant pathogen in humans which causes severe pathology in the lungs of infants, immunocompromised patients, and the elderly, and has been implicated in acute exacerbations of chronic-obstructive pulmonary disease (COPD). CRX-527 was found to effectively inhibit RSV replication in BALB/c mice when intranasally administered 24 hours prior to infection via the same route. The preclinical results with CRX-527—particularly when considered alongside the progress made with therapeutics such as resiquimod (TLR7/8) and CpG-ODNs (TLR9) that target other TLR receptors [67]—support the advancement

of CRX-527 or a related AGP to human clinical trials for infectious diseases. The induction of pro-inflammatory cytokines, chemokines, and iNOS by the AGPs generally paralleled the protective effects in the murine models, showing both a striking chain length (normal fatty acid, aglycon) and stereochemical dependence. For example, cytokine induction and protective responses are abolished when the secondary acyl group is 6-carbons in length (CRX-526 (39, Fig. 4)) or shorter, and when the aglycon contains 6 carbon atoms (n=5) or more (cf. compound 40, Fig. 4). The decreased activity with longer aglycon units can likely be ascribed to a change in spacial orientation of the three acyl groups, making close packing of the lipids difficult, as well as to steric hindrance of the aglycon chain to an effective drug-receptor (AGP– MD-2) interaction. Stimulation of the innate immune response by AGPs appears to involve activation of TLR4 because the AGPs failed to elicit protective effects in C3H/HeJ mice having a defect in TLR4 signaling or induce significant cytokine levels in C3H/HeJ spenocytes. A comparison of the ability of representative stimulatory (CRX527, RC-529) and non-stimulatory (CRX-526) AGPs to induce iNOS in murine macrophages and protect against Listeria challenge in mice is given in Figure (4). Transcriptional profiling of human monocytes and responses to TLR4 cell transfectants also showed a clear dependence of secondary acyl chain length on TLR4 activation in the seryl series [68]. Seryl AGPs having secondary acyl chains less than 8 carbons in length exhibited dramatically reduced activity. The same potency relationship


Current Topics in Medicinal Chemistry, 2008, Vol. 8, No. 2 O

O HO

P

HO

O

OH CO2H O

O O

HO

O

HO

NH

O

CO2H O

O O

O O

O

O

O O

O n

O

NH

NH

O

O O

O

O

OH

O

NH

O

O O

P

HO

O

O

O O

HO

NH

O

O

NH

P

OH

73

O

O

O

O O

O

O

O

38 (CRX-527) iNOS ED50 0.06 ng/mL % survival 100%

39 (CRX-526) iNOS ED50 >10,000 ng/mL % survival 0%

35 n=1 (RC-529) iNOS ED50 100 ng/mL % survival 75%

40 n=7 iNOS ED50 >10,000 ng/mL % survival 0%

Fig. (4). Comparison of iNOS induction in peritoneal macrophages and protection against Listeria challenge in mice for selected AGPs.

was observed when these compounds were tested for the production of TNF- using U937 and THP1 monocyte/ macrophage cell lines. An evaluation of a “chemical factorial” of fatty acyl hybrid molecules in the seryl series containing all possible combinations of 6- and 10-carbon secondary acyl chains revealed that the left-hand acyl residue has the greatest effect on activity. For example, the seryl compound having a single hexanoyl in this position (R1 = hexanoyl, compound 30, Fig. 3) was essentially nonstimulatory, and similar in non-responsiveness to the seryl compound containing three 6-carbon secondary residues (CRX-526). Conversely, the seryl compound having a hexanoyl on the far right (R3 = hexanoyl, compound 30, Fig. 3) stimulated IL-8 production in TLR4/MD-2 HeLa cell transfectants similar to that of CRX-527. Interestingly, coexpression of CD14, a protein involved in shuttling LPS to MD-2 and required for MyD88-independent signaling [69], in the transfectants was not required for activity but did enhance responses, particularly for low stimulatory AGPs. The response to the most potent compound (CRX-527) was strictly dependent on TLR4 and MD-2 but not CD14, suggesting that CRX-527 may be able engage both MyD88 and MyD88-independent (TRIF) pathways in the absence of CD14 [68]. Similar results were obtained in murine infectious disease models with these hybrid molecules [66]. These observations are consistent with the conserved nature of the non-reducing sugar among natural lipid A variants, as well as with the postulate that CD14 increases responsiveness to low endotoxicity lipid A molecules, thus potentially expanding the range of molecules recognized by the TLR4– MD-2 receptor complex. The greater importance of the lefthand fatty acid on endotoxicity in the AGP series may also be related to the ability of human acyloxyacyl hydrolase to detoxify lipid A by cleaving certain secondary fatty acids at faster rates [70].

The potent immunostimulary activity of CRX-527 in both murine and human model systems suggests that the seryl carboxyl group is an effective bioisostere for the anomeric phosphate of lipid A. This observation is consistent with the postulate that the two negatively charged groups of lipid A-like molecules are needed to interact with the positively charged lysine groups in the MD-2 binding site for maximal activity [66]. This is further supported by the observation that increasing the distance between the anionic groups (e.g. compound 30, R4 = CH2 CH2CO2H, CH2OPO3H) diminishes activity, suggesting that the distance between ionizable groups is a critical determinant for binding to MD2 [66]. Biophysical characterization of MLA mimetic CRX527 shows that the individual molecules adopt a conical shape and non-lamellar, inverted hexagonal HII aggregate structure in solution, which is consistent with its high agonist activity and in accord with data on other TLR4 agonists.† In comparison, physicochemical analysis of CRX-526, a 6carbon secondary acyl homolog of CRX-527, revealed that it forms lamellar aggregates in solution, which is characteristic of inactive or antagonistic molecules. In marked contrast to CRX-527, CRX-526 (39) has potent antagonist activity and can block the induction of proinflammatory cytokines induced by LPS both in vivo and in vitro [71]. Since CRX-526 has LPS-antagonist activity similar to the disaccharide E5564 (Eritoran), a TLR4 antagonist related to R. sphaeroides lipid A currently in Phase III clinical trials, and related molecules [1]. it is likely that CRX-526 binds to the TLR4–MD-2 complex in a similar manner and may have utility in immune disorders where blocking TLR4 signaling would be beneficial. †

Myers, K.R.; Johnson, D.A.; Andrä, J.; Howe, J.; Müller, M.; Garidel, P.; Seydel, U.; Brandenburg, K. Physicochemical Characterization and Endotoxic Activity of Synthetic Monophosphoryl Analogues of Lipid A, 8th Biennial Conference of the International Endotoxin Society, Kyoto, Japan, 2004


David A. Johnson

Recently, CRX-526 was shown to inhibit the development of moderate-to-severe disease in two murine models of inflammatory bowel disease (IBD) [71]. While the exact role for TLR4 in IBD remains controversial, human patients with IBD have increased TLR4 expression on intestinal epithelium, suggesting that TLR4 may offer a new therapeutic target for this disease.

cytokine- and NSR-inducing ability of RC-529 in comparison to CRX-527, these two AGPs exhibited equivalent IgG2a/IgG2b inducing ability in a tetanus toxoid vaccine model and induced comparable CTL activity against EG.7ova target cells (an ovalbumin gene-transfected cell line which expresses the ovalbumin CTL epitope) [57]. In addition, when formulated with hepatitis B surface antigen (HBsAg), RC-529 was comparable to MPL adjuvant with respect to antibody titers and T-cell mediated immune responses [74]. The reduced pyrogenicity of RC-529 in comparison to other AGPs and MPL adjuvant suggests that RC-529 may be a useful adjuvant for pediatric vaccines, where adjuvant pyrogenicity must be minimized. The potent adjuvant activity of RC-529 in preclinical models together with its low pyrogenicity in rabbits make RC-529 an attractive candidate for clinical development. In fact, RC-529 was recently shown to be a safe and effective adjuvant in a pivotal phase III vaccine trial with a recombinant hepatitis B antigen.§ The HBsAg vaccine formulated with RC-529 mediated four-fold higher geometric mean antibody titers and increased levels of seroprotection compared to the HBsAg absorbed to alum alone. This clinical evidence indicates that RC-529 can induce protective responses faster and with fewer doses than conventional vaccines and thus should provide substantial cost savings as well as increased compliance, particularly in developing countries where relative vaccine costs are high and compliance is low.

As mentioned above, lipid A molecules are presumably metabolized in part by the action of acyloxyacyl hydrolase enzyme, which selectively cleaves the secondary fatty acyl residues in vivo, as well as by non-specific esterases. To overcome the chemical and metabolic instability of the esterlinked secondary fatty acids present in CRX-527 and CRX526, and further evaluate structural modifications in the AGP series, we prepared the corresponding secondary ether lipid analogs 41 and 42 following methodology similar to that used for the synthesis of CRX-527 and alkoxy acid 25 (Scheme 6).‡ Selective carbodiimide-mediated 3-O-acylation of advanced intermediated 43 with alkoxy acids 44, prepared in 2 steps from p-bromoPAc ester 12, followed by further elaboration/deprotection gave the desired ether lipid derivatives 41 and 42. Despite literature reports showing that the introduction of ether linkages can eliminate endotoxic activities in both mono- and disaccharide derivatives [72,73], CRX-527 and its ether lipid 41 were virtually indistinguishable with respect to their ability to induce cytokine production in human cells and non-specific resistance to Listera and influenza challenges in mice. Glycolipid 42, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models-and in fact was weakly agonistic. This species specific agonist/antagonist activity of 42 is reminiscent of what has been reported for lipid IVa, a biosynthetic precursor of lipid A, and is likely attributable to species differences in the structure of MD-2 [39,40].

The expression of TLR4 on mucosal surfaces [75] together with the anti-infective effects of the AGPs upon intranasal administration, suggested that certain AGPs may also be active when administered in a vaccine delivered via intranasal and oral routes. Intranasal administration of influenza vaccines adjuvanted with serinol-based AGP RC544 (46), whose synthesis is shown in Scheme 7 and a variation of the method in Scheme 5 (path B), resulted in the development of both systemic (IgG) and mucosal (IgA) immune responses to influenza hemagglutinin [76]. In addition, cell-mediated responses-including cytotoxic Tlymphocytes-were induced following mucosal administration of other model antigens adjuvanted with RC-544 or other AGPs. One AGP in particular, RC-529 (35), has been

As observed for disaccharide derivatives of MPL major component 2, the structural requirements for adjuvanticity among the AGPs appear to be much less stringent than those for other endotoxicities. For example, despite the moderate 44 R = n-C9H19 or n-C5H11 R n-C11H23

OTBDMS HO HO

O

O HO

CO2H

OTBDMS

O

NHTroc

CO2Bn

O

HO

CO2Bn

O

P

O

O

NHTroc

HO

OH O

O O

NH NH

O

O

O

NHTroc

R O

R

43 R

O

O

NHTroc

O

CO2H O

O

n-C11H23

n-C11H23

R

n-C11H23

n-C11H23

45 R = n-C9H19 or n-C5H11

41 R = n-C9H19 42 R = n-C5H11

Scheme (6). Synthesis of ether lipid derivatives of CRX-527 and CRX-526.

‡

Bazin, H.G.; Bess, L.A.; Baldridge, J.R.; Evans, J.T.; Cluff, C.W.; Probst, P.; McGowan, P.; Fling, S.P.; Johnson, D.A. Synthesis of the Secondary Ether Analogs of Glycolipids CRX-526 and CRX-527, and Evaluation of their TLR4 Antagonist and Agonist Activity, 23rd International Carbohydrate Symposium, Whistler, BC, 2006

§

Dupont, J.-C.; Altclas, J.; Sigelchifer, M.; Von Eschen, K.; Timmermans, I.; Wagener, A. Efficacy and safety of AgB/RC529: a novel two-dose adjuvant vaccine against hepatitis B, 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA, USA, 2002



75

OBn OAc AcO AcO

O OAc NHAoc

HO

O

NHAoc O O HO

OBn O O NHAoc

HO

P

HO NHAoc

O

O O

O NH

NH

O RO

OH

OH

O

O RO

RO n-C11H23

n-C11H23 n-C11H23

46 R = n-C11H23CO (CRX-544)

Scheme (7). Synthesis of serinol-based AGP CRX-544.

extensively evaluated for its potential as a mucosal vaccine adjuvant. For example, RC-529 was shown to induce antigen-specific serum and mucosal IgG and IgA antibodies and reduce nasal colonization in mouse intranasal challenge models for both Neisseria meningitidis and non-typeable Haemophilis influenzae (NTHi) [64,65]. In each case, the serum IgG isotypes were predominantly IgG2a and IgG2b, indicative of bactericidal activity and a Th1 response. NTHi and N. meningitidis are the major causes of bacterial otitis media and bacterial meningitidis, respectively, particularly among young children. RC-529 has also been shown to elicit functional antibodies in nasopharyngeal challenge models for Streptococcus pneumoniae, another leading cause of otitis media and meningitis as well as other diseases [77]. In view of the exceptional safety profile of RC-529 in animal models and humans, these results suggest that intranasal immunization with recombinant proteins combined with RC529 may offer effective pediatric vaccine strategies against bacterial meningitidis and otitis media, which are two significant health problems worldwide. While the precise mechanism by which less inflammatory/toxic TLR4 agonists like RC-529 and MPL adjuvant exert their profound adjuvant activity in comparison to highly endotoxic molecules like CRX-527 and LPS is not known, recent evidence suggests that MPL adjuvant and RC529 are just as effective in promoting CD4+ T-cell activation and initial clonal expansion of CD4+ T cells as S. minnesota R595 LPS [78]. The increase in CD4+ T-cell clonal expansion was correlated with the expression of two chemokines—IP-10 and MCP-1—which have been implicated in the MyD88-independent pathway involving the TRIF adapter molecule [7]. RC-529, which up-regulated IP-10 and MCP-1 to the same extent as LPS in this study, is known to induce far lower levels of inflammatory cytokines than LPS or CRX-527. Thus, TRIF-selectivity coupled with threshold levels of MyD88-dependent cytokines may preferentially enhance adaptive immunity and the memory response, whereas strong MyD88-dependent inflammatory signaling may preferentially lead to increased NSR and certain toxic effects. Additional evidence for the uncoupling of the beneficial adjuvant effects of RC-529 from toxic properties when administered in a vaccine setting is provided by the observation that RC-529 and MPL adjuvant lead to greatly reduced levels of the acute-phase protein SAA in serum in comparison to LPS. SAA is the major acute phase protein in mice (and analogous to the acute-phase protein C-reactive protein in humans) that is produced in the liver in response

to inflammatory cytokines and used as a measure of acute toxicity. The ability of low-endotoxicity AGPs like RC-529 to enhance adaptive immunity preferentially over the innate immune response may relate, in part, to the efficiency of the interaction between RC-529 and the TLR4 accessory molecule CD14, which is required for MyD88-independently signaling. By increasing the immune system’s responsiveness to low-endotoxicity lipid A derivatives, CD14 effectively expands the range of molecules recognized by the TLR4–MD-2 receptor complex and thus may play an important role in the adaptive immune response against certain pathogens. It could be that CD14 has evolved in mammalian hosts as a counter measure to attempts by pathogens to evade the immune system through the expression of less-endotoxic PAMPs. The structure of CD14, which was determined recently by X-ray diffraction [79], contains a strikingly large hydrophobic pocket at the Nterminus with the apparent capacity to accommodate glycolipids with long-chain fatty acids such as those present in RC-529 and other lipid A derivatives exhibiting low endotoxicity. The large pocket and nearby grooves are thought to play an important role in ligand binding and subsequent TLR4/MD-2-mediated signaling, and thus are likely responsible for the broad ligand specificity of CD14. As further testimony to the viability of this approach to adjuvant design using synthetic subunit derivatives of MLA, the lipid A mimetics 47 and 48 (Scheme 8) possessing an Nacylated pentaerythritol aglycon unit and the same acyl residues as RC-529 (35) were found to induce cytokines in human adherent APCs and antigen-specific T-cell proliferation in an experimental MUC-1 liposomal vaccine [22]. Interestingly, the monophosphate 47 induced higher levels of TNF-, IL-6, and IL-8 in human APCs than the corresponding diphosphate 48. This observation corroborates other studies showing that the distance between anionic groups and/or spacial relationship of the aglycon phosphate/ phosphate bioisostere to the aglycon lipid moiety are important to TLR4 agonist activity [80]. These compounds were constructed via a strategy similar to that used in the preparation of disaccharides 19 and 20, except that the two amide-linked acyl groups were introduced simultaneously in the penultimate step. Due to the prochiral nature of the glycosyl acceptor 49, the diphosphate 48 was isolated and tested as a 1:1 mixture of diastereomers. However, it is important to note that chirality in the aglycon or glucosamine


David A. Johnson

HO O O HO

BnO

O

P

BnO NH2

O

HO

OBn

HO

NHTroc

O

O

OH O

O

O

O O

OR

NH NH

O

O

O

O O

NH OC(NH)CCl3 Troc

OAll

P

49

OBn

O Ph

OH

O

OH

O

O

O O

O O

n-C11H23 n-C13H27

47 R = H 48 R = PO3H2

Scheme (8). Synthesis of pentaerythritol-based lipid A mimetics.

mimetic can have a profound effect on bioactivity in monosaccharide and acyclic lipid A derivatives [21,57]. IV. FUTURE PERSPECTIVES FOR SYNTHETIC TLR4 AGONISTS Structure-activity investigations within the AGP class of lipid A mimetics have demonstrated that many of these TLR4-active glycolipids are not only potent inducers of nonspecific resistance against bacterial and viral infection but also capable of inducing strong humoral and cell-mediated immune responses to a variety of different vaccine antigens. These studies, which originated with natural product investigations on the complex MLA mixture derived from S. minnesota R595 lipid A, have led to the clinical development and commercialization of RC-529 (35) as a safe and effecttive adjuvant in a hepatitis B vaccine. Other low toxicity AGPs such as CRX-544 (46) act as mucosal adjuvants which mediate strong adaptive immune responses to co-administered vaccine antigens including influenza. Still other AGPs such as CRX-524 (36) and CRX-527 (38) show promise as stand-alone therapeutics for providing short-term nonspecific resistance against bacterial and viral infections within a few hours of administration. Exploitation of both these TLR4-agonist properties short-term innate immunity and mucosal adaptive immunity may yield vaccines that overcome the one major drawback to conventional vaccines: the time needed to build up antibodies to a specific threat. Thus, novel vaccine strategies can be envisioned that provide short-term, non-specific protection against pathogens as well as durable, antigen-specific acquired immunity. A “rapidacting vaccine” approach such as this may provide protection in critical times of exposure such as during a pandemic influenza outbreak or acts of bioterrorism. Another promising approach toward optimizing immune responses, particularly as our understanding of the immuno-

biology of TLR4 and other TLR receptors expands, is the combination of TLR4 agonists with other TLR agonists or molecular adjuvants to simultaneously stimulate multiple TLRs and other immune cell receptors in a synergistic manner [81,82]. For disease indications like malaria, HIV, and cancer that have proven resistant to traditional vaccine strategies, the combination of TLR4 agonists like the AGPs with other immune response modifiers could yield new therapeutic and prophylactic vaccines for a variety of human diseases. The full potential of lipid A mimetics as adjuvants and stand-alone immunotherapeutics is only now being realized. The manipulation of the toll-like receptors on cells of the immune system will likely have far reaching consequences on human health and how we live out our lives. To paraphrase John Donne: ‘For whom the immune cell tolls; it tolls for thee’. ACKNOWLEDGMENTS The author would like to thank Drs. Marcelle P. Van Mechelen and Jory R. Baldridge for their critical reading of this manuscript and helpful discussions. This work was supported in part by the National Institute of Allergy and Infectious Diseases (NIAID) under contract HHSN 266200400008C/N01-AI-40008. Any opinions, findings and conclusions or recommendations expressed in this article are those of the author and do not necessarily reflect the views of the NIAID. REFERENCES [1]

[2]

Hawkins, L.D.; Christ, W.J.; Rossignol, D.P. Inhibition of Endotoxin Response by Synthetic TLR4 Antagonists. Curr. Top. Med. Chem. 2004, 4, 1147-1171. Jiang, Z-H.; Koganty, R.R. Synthetic Vaccines: The Role of Adjuvants in Immune Targeting. Curr. Med. Chem. 2003, 10, 1423-1439.

Synthetic TLR4-active Glycolipids as Vaccine Adjuvants [3] [4] [5] [6] [7]

[8] [9]

[10]

[11]

[12]

[13] [14]

[15] [16] [17]

[18]

[19] [20]

[21]

[22]

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Received: December 18, 2006

Accepted: May 1, 2007

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