Recent Advances and Perspectives in Small-molecule TLR Ligands

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Recent Advances and Perspectives in Small-molecule TLR Ligands and Their Modulators Nikunj M. Shukla, Michael Chan, Tomoko Hayashi, Dennis A. Carson, and Howard B. Cottam* Moores Cancer Center, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0695, United States

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ABSTRACT: Activation of Toll-like receptors (TLRs) located on immune cells leads to induction of immune responses that can be useful in vaccines for infectious diseases, cancer immunotherapy, and autoimmune diseases. Novel TLR signaling pathway modulators can further enhance the efficacy of TLR ligands.

KEYWORDS: Toll-like receptors, TLR, vaccine, infectious disease, cancer, autoimmune, modulator

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nnate immune receptors such as Toll-like receptors (TLRs) belong to a class of pattern recognition receptors (PRR) that recognize patterns present in molecules that are broadly shared by pathogens. These receptors function to detect microbial and viral components and thereby direct the innate and adaptive immune response toward not only fighting the infection but also generating immunological memory. There are more than 10 functional TLRs known in the human genome and the natural ligands for these TLRs include lipotechoic acid (LTA) from Gram-positive bacteria or MALP2 originally isolated from Mycoplasma fermentans for TLR1, 2, and 6; lipopolysaccharide (LPS) from Gram-negative bacteria for TLR4 and flagellin for TLR5. However, the natural ligand for TLR3 is double-stranded RNA, while TLR7 and 8 recognize single stranded RNA, and TLR9 recognizes unmethylated CpG motifs of bacterial DNA. These are membrane-spanning receptors that can be broadly classified based on their localization in cells. TLR1, 2, 4, 5, and 6 are located primarily on the plasma membrane, while TLR3, 7, 8, and 9 are found in the endosomal compartment.1 These receptors are highly expressed on immune cells; however, their presence and distribution vary by immune cell type. Some TLRs are also expressed in certain nonimmune cells. TLR Agonists. Over the past two decades since the discovery of TLRs, several agonists of these receptors have been reported. While some of the TLRs are activated by only macromolecules such as TLR3, 5, and 9, synthetic smallmolecule agonists have been identified for TLR2, 4, 7, and 8. The structures of several of these agonists are shown in Figure 1. The most studied TLR2 agonists include palmitoylated peptides based on the structure of MALP-2 bearing a S-[2,3bis(palmityloxy)-(2R)-propyl-cysteinyl group or its additional palmitoyl derivative, which is amide linked to a pentapeptide (Ser-Lys-Lys-Lys-Lys) to obtain Pam2CSK4 and Pam3CSK4, respectively (Figure 1). The structure−activity relationship © XXXX American Chemical Society

Figure 1. Structures of selected small-molecule TLR agonists. TLR2 agonists shown in red, TLR4 in blue, and TLR7/8 in green.

(SAR) studies on these series of compounds show that the bis palmitoylation as in Pam2CSK4 leads to heterodimerization of TLR2 and TLR6 necessary to activate TLR2, while the additional palmitoylation of the cysteine amine as in Pam3CSK4 leads to heterodimerization of TLR2 with TLR1, whereas TLR6 cannot accommodate the extra lipid chain that fits very well into the pocket of TLR1. The cysteine part of the molecule is a necessary part of the pharmacophore, while the

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DOI: 10.1021/acsmedchemlett.8b00566 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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serine can be modified to accommodate other small amino acids such as glycine. The rest of the lysine tetrapeptide does not seem to play any role in binding to TLR2, but enhances the solubility of these highly lipophilic compounds.2 Next, TLR4 has been identified to be the target receptor for several endogenous and exogenous nonspecific ligands; however, we will focus on the specific ligands derived from the structure of LPS and some novel nonlipid-like small-molecule agonists. Monophosphoryl Lipid A (MPLA), which is now FDA approved, has structural remnants of LPS with elimination of the O-antigen and one phosphate group. Thus, MPLA is the lipid A portion of the LPS consisting of keto-deoxyoctulosonate (KDO) disaccharide, one phosphate group, and six acyl chains (Figure 1). The MPLA is either extracted from LPS obtained from Salmonella Minnesota R595 strain or chemically synthesized. The synthesized MPLA is highly pure and activates only TLR4 receptor. A small-molecule TLR4 specific agonist that belongs to the class of pyrimidoindoles (1Z105) was identified using high-throughput screening for smallmolecules that activate the NF-κB pathway in THP-1 cells. In this series N5-methylated compound 1Z105 was identified as a nontoxic TLR4 agonist, and the second-generation SAR showed that modification at position C8 to include C8phenyl significantly enhanced the TLR4 agonistic activity in both murine and human cells.3 In another screening of peptidomimetic compounds for activation of primary mouse peritoneal macrophage cultures, Neoseptin-3 was identified as a murine-specific TLR4 agonist.4 Neoseptin-3 exhibited strict SAR in which addition of an O-alkyl group at the ortho position on the aniline ring to obtain ether-linked substitution retained activity as in Neoseptin-4. Small-molecule TLR7 and 8 agonists share the structural motif consisting of heteroaromatic amidine functionality that binds to 2 or 3 different aspartate residues on the receptors as shown by cocrystallization studies. The TLR7 versus TLR8 agonistic profiles for these small-molecule ligands are difficult to separate, and many of the compounds activate both TLR7 and 8. Figure 1 shows structures of a few molecules of the vast compendium of different chemotypes that activate these receptors including quinoline derivatives such as thiazoloquinoline CL075 and imidazoquinoline IMDQ. A ring-expanded quinoline as in VTX-2337 displayed stronger TLR8 agonistic activity relative to TLR7 activity. Early work involving purine derivatives as type I interferon (IFN) inducers, particularly certain guanine nucleosides, were eventually identified as agonists of TLR7.5 Moreover, certain adenine derivatives were shown to be potent antiviral agents by virtue of their type I IFN inducing activity mediated by TLR7 agonism and modulated by IMPDH inhibition.6 A related adenine derivative 1V270 that also possesses TLR7 specific agonistic activity was designed with two lipophilic oleoyl groups aimed at providing a depot effect. Applications of TLR Agonists. Activation of TLRs leads to induction of cytokines such as IL-12, IL-10, IL-6, TNF-α, and types I and II IFN. In addition, it also enhances the activation of CD4+ helper T cells and CD8+ cytotoxic T cells (CTL), which leads to induction of Th1 and Th2 responses. Upon stimulation with TLR agonists, induction of an inflammatory response then activates feedback pathways resulting in downregulation of inflammatory responses represented by the gray line curve in Figure 2. This immune activation phenomenon is widely utilized for obtaining vaccine adjuvant effects. Vaccination using TLR agonists combined with appropriate antigen(s) helps generate Th1, Th2, and

Figure 2. Theoretical kinetic profiles for immune stimulation.

Th17 driven antibody and cell-mediated responses. The selection of the appropriate TLR agonist to provide a Th1, Th2, or balanced Th1/Th2 response depends on the pathogen targeted by the vaccine. Generally, for extracellular organisms, humoral responses (antibody) provide the most important adaptive mechanism of host defense, and therefore, a strong Th2 response is desired. For intracellular organisms such as mycobacteria, a cellular mechanism of defense is desired, and therefore, a Th1 (and/or Th17) response is more important. TLR2 agonists have been shown to produce Th2-biased responses while TLR7 and 8 agonists induce more Th1-biased responses. MPLA in combination with alum or saponin QS-21 has been approved for human use in human papillomavirus (HPV) and varicella zoster virus (VZV) vaccines, respectively. For many infectious agents, a combined adjuvant comprising TLR4 and TLR7 agonists, for example, provides a much more effective vaccine than either agonist alone.7 This was demonstrated in influenza virus vaccination studies in mice where a strong Th1 response appeared to be important to provide cross protection against infection by heterologous strains of virus, and therefore, enhancement of both Th1 and Th2 responses was preferred.8 In conditions such as cancer, there are a number of mechanisms by which tumors avoid immune recognition or evade an immune response once it is generated leading to immune escape and malignant cell proliferation. Examples are low immunogenicity, tumor-induced immune suppression, loss of surface tumor antigens, etc. However, a robust inflammatory response that includes dendritic cell maturation and antigen cross-presentation, together with secretion of inflammatory cytokines, could efficiently prime cytotoxic T cells, resulting in effective antitumor immune responses. Theoretical kinetics of inflammatory response in the tumor microenvironment can be represented by the blue line curve in Figure 2. Thus, boosting the immune response using TLR agonists could help immune cells to overcome tumor-induced suppression and recognize malignant tumor antigens, leading to direct lysis of tumor cells as well as inhibition of metastatic tumor growth.9 This premise has been explored to identify TLR agonists that can be used for cancer immunotherapy. While several small-molecule TLR agonists are being explored for cancer immunotherapy, MPLA has been approved for prophylaxis of HPV-associated cervical cancer and is in clinical trials for colorectal cancer. Also, the imidazoquinoline derivative TLR7 agonist imiquimod has been approved for therapy of basal cell carcinoma, and other analogs are being tested in clinical trials for melanoma. B

DOI: 10.1021/acsmedchemlett.8b00566 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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Another promising application for these TLR agonists may be in the treatment of chronic inflammatory and autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, atherosclerosis, chronic hepatitis, and diabetes, among many others. The theoretical inflammatory profile in Figure 2 (in red) shows the relatively enhanced inflammatory response particularly directed against self-antigens. A strategy to downregulate the response to a more normal level can be achieved by treatment with TLR agonists. Recent studies indicate that repeated administration of a potent TLR7 agonist in the oxoadenine class attenuated the inflammatory response and was neuroprotective in in vivo experimental models of type 1 diabetes and multiple sclerosis in mice.10 The mechanism was proposed to be the induction of tolerogenic dendritic cells that can produce negative immune regulatory factors and favor the production of T regulatory cells. Other studies targeting TLR4 have shown that repeated administration of TLR4 agonists was effective in reducing target organ damage in two different mouse models of sterile inflammation.11 This treatment strategy might be a new modality to treat chronic inflammation and T cell-mediated autoimmune diseases. TLR Pathway Modulators. TLRs, upon activation by their ligands, signal through a variety of receptor complexes leading to activation of transcription factors and cytokine production. A signaling pathway modulator that does not directly interact with the TLRs but modulates the downstream signaling pathway could potentially enhance the effects of these TLR agonists. One such application of this modulation is the potentiation of antiviral activity provided by TLR ligands that induce interferon production. As mentioned above, the use of IMPDH inhibitors with a TLR7 agonist was shown to potentiate the anti-hepatitis C virus activity of the TLR7 agonist.6 Another application involves the area of vaccine adjuvant development where a small-molecule could serve as a coadjuvant to prolong the stimulus from a TLR ligand, possibly by inhibiting negative regulatory factors that are normally induced after an immune stimulus as part of the resolution process. To identify such small-molecule modulators, a high throughput screen of a compound library was designed to identify compounds that sustained an initial TLR4 stimulus by modulating negative feedback signaling. These studies led to the identification of four structural classes of compounds that did not directly interact with TLR4 but sustained the activation over a prolonged time.12 One of the chemotypes identified was the pyrimidoindoles, as in 1Z105, but with N3-alkyl substitution. Subsequent SAR studies identified potent compounds in this class, which enhanced the adjuvant activity of MPLA in vaccine studies in mice. In conclusion, this field has led to the identification of several potentially distinct single-component small molecule TLR agonists, and recent studies point in the direction of using combinations of two or more components for achieving more efficient therapeutics than can be obtained with a single agent. These combinations can be mixtures of multiple TLR agonists or TLR agonists with appropriate pathway modulators.

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Howard B. Cottam: 0000-0001-7382-0585 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

Views expressed in this editorial are those of the authors and not necessarily the views of the ACS. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by contracts from the National Institute of Allergy and Infectious Diseases (HHSN272200900034C and HHSN272201400051C) of the National Institutes of Health to D.A.C.

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ABBREVIATIONS CTL, cytotoxic T-lymphocyte; HPV, human papillomavirus; IFN, interferon; IL, interleukin; IMPDH, inosine monophosphate dehydrogenase; KDO, keto-deoxyoctulosonate; LPS, lipopolysaccharide; LTA, lipotechoic acid; MALP-2, macrophage-activating lipopeptide-2; MPLA, monophosphoryl lipid A; PRR, pattern recognition receptor; SAR, structure− activity relationship; TLR, Toll-like receptor

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (858) 534 5424. Fax: (858) 246-1586. ORCID

Nikunj M. Shukla: 0000-0002-5150-7827 C

DOI: 10.1021/acsmedchemlett.8b00566 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

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(9) Shi, M.; Chen, X.; Ye, K.; Yao, Y.; Li, Y. Application potential of toll-like receptors in cancer immunotherapy: Systematic review. Medicine 2016, 95 (25), e3951. (10) Hayashi, T.; Yao, S.; Crain, B.; Chan, M.; Tawatao, R. I.; Gray, C.; Vuong, L.; Lao, F.; Cottam, H. B.; Carson, D. A.; Corr, M. Treatment of autoimmune inflammation by a TLR7 ligand regulating the innate immune system. PLoS One 2012, 7 (9), e45860. (11) Hayashi, T.; Crain, B.; Yao, S.; Caneda, C. D.; Cottam, H. B.; Chan, M.; Corr, M.; Carson, D. A. Novel synthetic toll-like receptor 4/MD2 ligands attenuate sterile inflammation. J. Pharmacol. Exp. Ther. 2014, 350 (2), 330−340. (12) Chan, M.; Ahmadi, A.; Yao, S.; Sato-Kaneko, F.; Messer, K.; Pu, M.; Nguyen, B.; Hayashi, T.; Corr, M.; Carson, D. A.; Cottam, H. B.; Shukla, N. M. Identification of Biologically Active Pyrimido[5,4b]indoles That Prolong NF-kappaB Activation without Intrinsic Activity. ACS Comb. Sci. 2017, 19 (8), 533−543.

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DOI: 10.1021/acsmedchemlett.8b00566 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX