Structure-Activity Relationship of Synthetic Toll-like Receptor 4 Agonists (2004)

Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators. Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators. The ability of an organism to protect itself from microbial challenge requires the rapid recognition of “pathogen-associated molecular patterns,” conserved structural motifs found throughout the microbial world. Toll-like receptors are a family of structurally related surface receptors that play an important role in transducing an inflammatory signal in response to pathogen-associated molecular pattern recognition. Toll was originally identified as a Drosophila gene essential for embryonic development and antifungal immunity (1Lemaitre B. Nicolas E. Michaut L. Reichhart J.M. Hoffmann J.A. Cell. 1996; 86: 973-983Abstract Full Text Full Text PDF PubMed Scopus (2998) Google Scholar). Ten mammalian orthologues of Toll-like receptors (TLRs) 1The abbreviations used are: TLRToll-like receptorILinterleukinSACsecondary acyl chainAGPaminoalkyl glucosaminide phosphateLPSlipopolysaccharideELISAenzyme-linked immunosorbent assayTNFtumor-necrosis factorRANTESregulated on activation normal T cell expressed and secretedMCP1macrophage chemoattractant protein 1MIPmonocyte inhibitory protein. have been identified (2Rock F.L. Hardiman G. Timans J.C. Kastelein R.A. Bazan J.F. Proc. Natl. Acad. Sc. i U. S. A. 1998; 95: 588-593Crossref PubMed Scopus (1451) Google Scholar, 3Takeuchi O. Kawai T. Sanjo H. Copeland N.G. Gilbert D.J. Jenkins N.A. Takeda K. Akira S. Gene (Amst.). 1999; 231: 59-65Crossref PubMed Scopus (353) Google Scholar, 4Chuang T. Ulevitch R.J. Biochim. Biophys. Acta. 2001; 1518: 157-161Crossref PubMed Scopus (323) Google Scholar). While all TLRs have similar structural features (e.g. extracellular leucine-rich repeats and cytoplasmic domains with homology to the interleukin-1 (IL-1) receptor), each TLR alone as a homodimer (e.g. TLR4, TLR5, TLR7, and TLR9) or in combination with another TLR as a heterodimer (e.g. TLR1, TLR2, and TLR6) is capable of recognizing distinct pathogen-associated molecular patterns due to unique structural motifs (5Takeda K. Kaisho T. Akira S. Annu. Rev. Immunol. 2003; 21: 335-376Crossref PubMed Scopus (4751) Google Scholar). Interestingly several natural and synthetic TLR ligands have been demonstrated to have significant clinical utility both as vaccine adjuvants and as stand-alone immunomodulatory agents (6Persing D.H. Coler R.N. Lacy M.J. Johnson D.A. Baldridge J.R. Hershberg R.M. Reed S.G. Trends Immunol. 2002; 10: S32-S37Scopus (222) Google Scholar, 7Martin M. Michalek S.M. Katz J. Infect. Immun. 2003; 71: 2498-2507Crossref PubMed Scopus (158) Google Scholar, 8Krieg A.M. Vaccine. 2000; 19: 618-622Crossref PubMed Scopus (142) Google Scholar). Toll-like receptor interleukin secondary acyl chain aminoalkyl glucosaminide phosphate lipopolysaccharide enzyme-linked immunosorbent assay tumor-necrosis factor regulated on activation normal T cell expressed and secreted macrophage chemoattractant protein 1 monocyte inhibitory protein. One of the best studied interactions between a microbial pathogen-associated molecular pattern and a TLR is the recognition of lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, by TLR4 (for a review, see Ref. 9Kimbrell D.A. Beutler B. Nat. Rev. Genet. 2001; 2: 256-267Crossref PubMed Scopus (495) Google Scholar). Beutler and colleagues (10Poltorak A. He X. Smirnova I. Liu M.Y. Van Huffel C. Du X. Birdwell D. Alejos E. Silva M. Galanos C. Freudenberg M. Ricciardi-Castagnoli P. Layton B. Beutler B. Science. 1998; 282: 2085-2088Crossref PubMed Scopus (6451) Google Scholar) first identified TLR4 as the “LPS receptor” by positional cloning studies in the LPS hyporesponder mouse strains C3H/HeJ and C57BL/10ScCr. The critical role of TLR4 in the transduction of an inflammatory signal by LPS has been confirmed in studies using mice with a deletion of TLR4. In addition, two additional proteins have been identified that interact with TLR4 and are implicated in LPS recognition, MD-2 and CD14. MD-2 is a secreted molecule that physically interacts with TLR4 (11da Silva Correia J. Soldau K. Christen U. Tobias P.S. Ulevitch R.J. J. Biol. Chem. 2001; 276: 21129-21135Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar), and genetic data (12Poltorak A. Ricciardi-Castagnoli P. Citterio S. Beutler B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2163-2167Crossref PubMed Scopus (396) Google Scholar) suggest that this molecule is required for LPS signaling via TLR4. CD14 is a glycosylphosphatidylinositol-linked membrane protein (devoid of a cytoplasmic signaling domain) with LPS binding activity. Collectively TLR4, MD-2, and CD14 form a trimolecular LPS receptor complex. The delineation of the precise molecular events associated with LPS binding and initiation of the signaling cascades via TLR4 remains an area of intense investigation. Lipid A is the active component of the LPS molecule that stimulates the TLR4 receptor complex (13Baker P.J. Hraba T. Taylor C.E. Myers K.R. Takayama K. Qureshi N. Stuetz P. Kusumoto S. Hasegawa A. Infect. Immun. 1992; 60: 2694-2701Crossref PubMed Google Scholar). Lipid A structures show considerable variability between bacterial species, and individual Gram-negative species can modulate the structure of the lipid A on their surface in the context of infection (14Bishop R.E. Gibbons H.S. Guina T. Trent M.S. Miller S.I. Raetz C.R. EMBO J. 2000; 19: 5071-5080Crossref PubMed Scopus (280) Google Scholar, 15Guo L. Lim K.B. Poduje C.M. Daniel M. Gunn J.S. Hackett M. Miller S.I. Cell. 1998; 95: 189-198Abstract Full Text Full Text PDF PubMed Scopus (522) Google Scholar). These structural differences, in particular the degree of acylation, can dramatically modify their proinflammatory capacity. In general, highly acylated (e.g. hexa-acylated lipid A from Escherichia coli or Salmonella species) tend to be potent inflammatory stimulators, while less acylated lipid A structures (e.g. lipidIVA and lipid A from certain periodontal bacteria such as Porphyromonas gingivalis) tend to be less inflammatory or even display LPS antagonist activity (16Mullarkey M. Rose J.R. Bristol J. Kawata T. Kimura A. Kobayashi S. Przetak M. Chow J. Gusovsky F. Christ W.J. Rossignol D.P. J. Pharmacol. Exp. Ther. 2003; 304: 1093-1102Crossref PubMed Scopus (263) Google Scholar). Because these studies have generally used natural biological preparations of LPS, detailed structure-function studies of the interaction between lipid A and the TLR4 complex have not been possible. Important questions remain regarding the precise region of the lipid A molecule that is required to stimulate the TLR4 complex and the effect of specific changes in the lipid A molecule on CD14 and/or MD-2 dependence of TLR4 stimulation and the transcriptional responses that ensue. To address these questions, we have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates (AGPs) (17Johnson D.A. Sowell C.G. Johnson C.L. Livesay M.T. Keegan D.S. Rhodes M.J. Ulrich J.T. Ward J.R. Cantrell J.L. Brookshire V.G. Bioorg. Chem. 1999; PubMed Scopus Google Scholar). The of are lipid A mimetics of a active hexa-acylated component in the used lipid A Because are by we have been to a of closely related with in the acyl chain Using transcriptional profiling of human responses of TLR4 receptor complex cell transfectants, and in vivo and we to a detailed structure-function of these hexa-acylated lipid A we demonstrate a clear dependence of TLR4 agonist activity on normal chain length with changes in of the secondary acyl chains a effect on of the than changes in the secondary acyl we the utility of certain of this novel of compounds as stand-alone on their TLR4 agonist activity. of compounds used in this are in 1 and as (17Johnson D.A. Sowell C.G. Johnson C.L. Livesay M.T. Keegan D.S. Rhodes M.J. Ulrich J.T. Ward J.R. Cantrell J.L. Brookshire V.G. Bioorg. 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PubMed Scopus Google Scholar) from to of these compounds and in the by the for as the response from in the of in response to the as a of the secondary acyl chain of cells with carbons or in length and in the of In the compounds with less than carbons and not the of by the cells alone or with of the cells with not of to the cells in to the the of not the between acyl chain length and activity not We found that the with the was the most potent active a of less than or to 1 Compounds with of and less potent than and a clear in the to The was seen these compounds for the of using the and cell not These data a clear dependence on length with to the of synthetic lipid A the that activity in these and was active less than or to 1 we compounds in which acyl chains each in These in to the structural features of the lipid A molecule required for macrophage in of or two chains dramatically reduced the activity of the molecule with to by human of of by data demonstrate the ability of to in human we in the associated with the complex transcriptional response following stimulation with To this we a inflammatory with each in to and on each from human with individual for was used as a to or used for to the In the was to in the best signal to of the of not cells with LPS from E. coli as a in LPS the of than for each with the of gene seen in the The of the macrophage gene on this confirmed the dependence on length seen with the induction of by these not on the While not in the by we the induction of eight on the following stimulation with this in the A in the transcriptional activation was seen with by and and by and We the transcriptional responses by the compounds using the in the of two chains with chains dramatically in a of activity with the of the with the several of the compounds with substitutions of a the transcriptional responses this response was seen with and compounds in which the left-sided was In was the of seen with the compounds or The of by is in I. Because stimulation with in the most significant transcriptional response of all of the we on the that or by with a of less than or to of the data by D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar) a gene was and is in The of gene induction by LPS and are similar with LPS a to this are the and the by the of these are than as by with LPS and While may not be in the due to of the data set using a of in general, we not to by of the alone and that not by The transcriptional of and are highly and with LPS, that these two and LPS the of the and between the of these two the and with the of Using the TLR4 is expressed on the surface as of a trimolecular complex with both MD-2 and CD14 Silva Correia J. Ulevitch R.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). MD-2 has been to physically with TLR4 in the is secreted and interacts with the extracellular of TLR4 expressed on the cell surface A. A. J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar). on and genetic MD-2 is required for stimulation via TLR4. CD14 is a glycosylphosphatidylinositol-linked protein to which LPS has been to be via protein in the P.S. Ulevitch R.J. PubMed Scopus Google Scholar). We have demonstrated that cells to TLR4, MD-2, and CD14 following to LPS Silva Correia J. Ulevitch R.J. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). We have used a a portion of the known to be by LPS via the activation of as in this cells with TLR4 or in combination with MD-2 and/or and with of in cells with TLR4 and MD-2 to LPS and of CD14 the response and of cells for the of by and a similar pattern of response was not In addition, cells with Drosophila Toll or or with alone not by LPS or the not The ability of the to stimulate the was on the length of the with closely those seen with the human of CD14 in the was not required for stimulation responses to the and with the compounds with (e.g. and to the human macrophage and we not see activation by which in all of the secondary These data demonstrate the TLR4 dependence and of stimulation with the We a set of compounds in this TLR4 seen in the of human monocytes we found that compounds with a acyl chain in the or and in the context of the structure had significant activity in the The with a of a acyl the left-sided which activation via was to stimulate the Interestingly of CD14 with TLR4 and MD-2 to and the responses to and with the data from the the compounds in which two for had to in the These data the of the left-sided acyl chain length in lipid A structures in stimulation via the TLR4 complex. To TLR1, TLR2, or to the with cells in the or of and not cells TLR2, TLR1, or alone or in combination In these S. a potent of TLR2, was used as In addition, cells with Drosophila Toll or not by LPS or the not of following in of the response of human and TLR4 complex to the compounds has demonstrated the potent activity of these compounds and a dependence on the activity with To these to an in vivo we the effect of administration of these compounds on and in with of and was for of mouse and of and following administration are in and of the patterns with all of the cytokines and chemokines In all was a dependence on length that closely mirrored that seen in human and the TLR4 or induction was with and activity was seen with The between and in vivo induction the of In this we studied a of synthetic lipid A mimetics to the critical between the acyl chain length in the lipid A structure and activation via TLR4. Using both human and a TLR4 we demonstrate that a family of hexa-acylated a secondary acyl chain length of eight is for TLR4 and a length of is of this acyl chain length was critical for the secondary on the of the lipid A The responses of these compounds in using cell-based and TLR4 with their in vivo activity. The development of the family of lipid A mimetics was on with lipid A a of LPS from Salmonella used as an in human (6Persing D.H. Coler R.N. Lacy M.J. Johnson D.A. Baldridge J.R. Hershberg R.M. Reed S.G. Trends Immunol. 2002; 10: S32-S37Scopus (222) Google Scholar). a from and of S. LPS, is a of and The most active was to be a hexa-acylated species in both human and studies. These data are in with the from A.M. Miller S.I. Nat. Immunol. 2002; PubMed Scopus Google Scholar) in which natural hexa-acylated species of lipid A the inflammatory response from human cells with less acylated lipid A Interestingly that identified a region specific to human TLR4 itself that the ability to between and hexa-acylated lipid A with these the synthetic hexa-acylated species used in studies potent than the and a of synthetic compounds not M. K. Kusumoto S. H. M. T. 2003; PubMed Scopus Google Scholar) antagonist activity in using a synthetic hexa-acylated molecule on the structure of E. coli lipid A not the reduced that the induction of several inflammatory cytokines in human cells by lipid is to that the structure of these compounds in the hexa-acylated structure to be associated with TLR4 agonist activity K. M. H. M. M. H. U. Biophys. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). a family of synthetic hexa-acylated compounds with an we to a dependence on secondary acyl chain length and TLR4 agonist activity. a secondary acyl chain length of eight carbons or was to inflammatory and a length of carbons was to acyl chains of or the substitutions of acyl chains the or secondary acyl chain in TLR4 agonist activity, substitutions or changes in the left-sided acyl chain be that the response to even the most potent was on TLR4 and MD-2 not CD14 in the cell While we the that CD14 was in the and to the responses seen in the cell transfectants, was clear that CD14 was capable of the response to the compounds with acyl chains (e.g. and These data suggest that CD14 may to lipid A that are not by the the of that can an inflammatory response from a cell TLR4 and We that the of the from the monocytes most closely mirrored the data from the both to or agonist activity (e.g. with that was not as was from The from the data the that the synthetic TLR4 a transcriptional response to that of the transcriptional responses the family of compounds that the a similar response to the or LPS, reduced and/or a of While the data a highly of using in which than by LPS as have the G. M. E. D. H. and M. Interestingly the from the in with the biological of the in vivo in the context of The for is as an vaccine which both and T cell responses to has been to than in the context of and vaccine the (6Persing D.H. Coler R.N. Lacy M.J. Johnson D.A. Baldridge J.R. Hershberg R.M. Reed S.G. Trends Immunol. 2002; 10: S32-S37Scopus (222) Google Scholar), has been used in a clinical with a we have been to demonstrate the ability of of the family to or challenge in the of of the itself or microbial J. A. G. D. H. and M. in the potent induction and ability to challenge in the of the that TLR4 may be used as stand-alone agents in and/or The of synthetic H. Kaisho T. O. S. Sanjo H. K. T. H. Takeda K. Akira S. Nat. Immunol. 2002; PubMed Scopus Google Scholar) and S. H. S. Akira S. H. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google Scholar) as stand-alone in clinical development a clear for this and the for immunomodulatory these synthetic compounds have in structure-function relationships to the interaction between lipid A and TLR4 and will to be useful compounds in a of cell-based and studies TLR4.