Herman F.
Staats
*abc,
Shaun M.
Kirwan
a,
Hae Woong
Choi
a,
Christopher P.
Shelburne
a,
Soman N.
Abraham
abde,
Gulice Y. C.
Leung
f and
David Y.-K.
Chen
*g
aDepartment of Pathology, Duke University Medical Center, Box 3712, Durham, NC 27710, USA. E-mail: herman.staats@duke.edu
bDepartment of Immunology, Duke University Medical Center, Durham, NC 27710, USA
cDepartment of Medicine, Duke University Medical Center, Durham, NC 27710, USA
dDepartment of Molecular Genetics & Microbiology, Duke University Medical Center, Durham, NC 27710, USA
eProgram in Emerging Infectious Diseases, Duke-National University of Singapore Graduate Medical School, Singapore 169547
fOrganic Chemistry @ Helios, Institute of Chemical and Engineering Sciences (ICES), Agency for Science, Technology and Research (A*STAR), 11 Biopolis Way, The Helios Block, #03-08, Singapore 138667
gDepartment of Chemistry, Seoul National University, Gwanak-1 Gwanak-ro, Gwanak-gu, Seoul 151-742, South Korea. E-mail: davidchen@snu.ac.kr
First published on 21st June 2012
The development and use of vaccines and their ability to prevent infection/disease is a shining example of the benefit of biomedical research. Modern vaccines often utilize subunit immunogens that exhibit minimal immunogenicity and require the use of adjuvants to maximize the induction of protective immune responses. We recently described a novel class of vaccine adjuvants, mast cell (MC) activators, that exhibit safe and effective vaccine adjuvant activity when administered by intranasal or intradermal routes. A compound library containing 580 functionalized benzopyrans, a structural motif found in a diverse array of natural and designed bioactive compounds, was screened using a MC degranulation assay to identify novel MC activating compounds for future evaluation as novel vaccine adjuvants. This approach identified 12 novel MC degranulating compounds. Therefore, MC degranulation can be used to reliably detect novel compounds for evaluation as adjuvants for use in mucosal vaccine strategies.
Recently, several studies have elucidated a role for mast cells (MCs), which are granulated tissue resident leukocytes in the development of adaptive immunity. MC release of pro-inflammatory mediators such as TNF or IL-1 was found to affect the development of both humoral and cellular components of immunity in mice after challenge with diverse stimuli including bacteria, the TLR7 ligand COMPOUND LINKS
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Download mol file of compoundimiquimod or contact sensitizers such as FITC.8–10 Moreover, targeted activation of MCs with the small molecule MC activator compound 48/80 (c48/80) was found to induce safe and robust systemic and mucosal immune responses against various protein antigens when co-administered with vaccine antigens at mucosal surfaces.11,12 In all cases, MCs were suggested to affect adaptive immunity by enhancing the recruitment of dendritic cells (DCs) and lymphocytes into local lymphoid tissues, a prerequisite for optimal immune responses. These data suggest that MCs may be a common denominator in the modulation of immunity to many diverse agents and molecules capable of activating MCs are potentially highly effective adjuvants. Therefore, MCs might be used as sensors to screen chemical libraries to detect novel compounds with mucosal adjuvant activity.
In this study, we describe the development of a novel screen using the MC degranulation response as a sensitive monitor to detect potential mucosal adjuvant compounds. We report that this methodology successfully detected 12 novel MC degranulating compounds from a library containing 580 functionalized benzopyrans, a privileged structural motif found in a diverse array of natural and designed bioactive compounds.13–18 Therefore, MC degranulation can be used to reliably detect novel compounds which may greatly expand our current lexicon of potential adjuvant compounds for use in mucosal vaccine strategies.
Fig. 1 MC screening assay plate layout. Internal plate controls will include MC/9 cells treated with Tyrode's buffer alone (background), a maximum value control treated with the detergent Triton-X 100 (Triton-X 100), and MC/9 cells treated with the known MC activator c48/80. Each compound was evaluated in duplicate. Percent (%) degranulation will be calculated using the following equation: percent (%) degranulation = (experimental β-hex release − vehicle control β-hex release)/(Triton-X-100 β-hex release − vehicle control β-hex release) × 100. Compounds that elicit 5% degranulation of MC/9 cells will be selected for further evaluation. |
In this scheme, chemical compounds are mixed with the murine MC/9 MC line,19–24 and evaluated for their ability to induce the release of MC granule contents, a process called degranulation. Degranulation, a reliable marker of MC activation, is assayed by examining cell culture supernatants for the presence of β-hexosaminidase (β-hex), an enzyme located in MC granules. Release of this enzyme implies that MCs have released their granule components, whereas its absence suggests that the chemical compounds do not have the ability to activate MCs. To perform this assay, we modified a procedure described by others.26,28 MC/9 cells were aliquoted in sterile Tyrode's buffer and added to each well of a sterile V-bottom plate at 5 × 105 cells per well in a 100 μl volume. For each 96-well plate, internal plate controls included MCs treated with the positive control degranulating agent compound 48/80 (c48/80; doses of 49.6, 198.3 and 793.5 μM corresponding to 31.25, 125 and 500 μg ml−1). C48/80 is a cationic mast cell stimulating chemical compound first reported for its ability to induce histamine secretion.29 Compound 48/80 appears to activate mast cells by a receptor-independent mechanism that activates G-proteins required for mast cell exocytosis.30–35 Other studies, however, suggest that Mas-related gene (Mrg) receptors on mast cells are involved in the mast cell activating activity of compound 48/80.36 Additional assay controls included a vehicle control (COMPOUND LINKS
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Download mol file of compoundDMSO) to evaluate background β-hex release and a maximum β-hex release control, which is cells treated with the detergent Triton X-100. Potential adjuvant compounds are added to each well in 100 μl volume at a final concentration of 100 μM.
Fig. 2 General synthetic scheme for the construction of the benzopyran library. |
The compounds were examined for their ability to induce β-hex release from MC/9 cells in a total of 17 plates. As shown in Fig. 3, our quality control results from these plates generated consistent minimal background β-hex release (average mean = 0.096 ± 0.02) (Fig. 3A) and maximal β-hex release measurements (average mean = 2.26 ± 0.50) (Fig. 3A) which are well within the quality control limits. Moreover, we found that treatment of MC/9 cells with the positive control mast cell degranulator c48/80 generated consistent results within three standard deviations from the mean of all assays (Fig. 3B). Collectively, these data demonstrate the reproducibility of the assay and that a result from any given plate should be comparable with results on a separate plate.
Fig. 3 The MC activation screen for novel adjuvants is a consistent high throughput assay. (A) Background β-hex release values for all 17 plates evaluated in this assay. Note that all values fall below the quality control criteria of 0.2. Maximal β-hex release values from MC/9 cells treated with the detergent Triton-X 100 from all 17 plates evaluated in the assay. Note that all values are greater than the quality control criteria of 1.0. (B) Average percent β-hex release from MC/9 cells treated with the MC activator c48/80 (doses of 49.6, 198.3 and 793.5 μM corresponding to 31.25, 125 and 500 μg ml−1) from all 17 plates evaluated in the assay. The individual values represent the calculated % degranulation from each assay plate and demonstrate that the assay performed reproducibly since the calculated % degranulation for each plate was within 3 standard deviations of the mean for each concentration of c48/80 tested. |
Rank | Compound number | % Degranulation | Rank | Compound number | % Degranulation |
---|---|---|---|---|---|
1 | 10 | 10.1 | 26 | 117 | 3.7 |
2 | 293 | 8.4 | 27 | 471 | 3.6 |
3 | 360 | 7.8 | 28 | 428 | 3.3 |
4 | 272 | 7.4 | 29 | 327 | 3.2 |
5 | 43 | 7.4 | 30 | 511 | 3.2 |
6 | 90 | 7.3 | 31 | 260 | 3.1 |
7 | 254 | 6.7 | 32 | 177 | 3.1 |
8 | 309 | 6.5 | 33 | 280 | 3.1 |
9 | 479 | 6.4 | 34 | 348 | 3.0 |
10 | 290 | 5.7 | 35 | 289 | 3.0 |
11 | 379 | 5.6 | 36 | 16 | 3.0 |
12 | 37 | 5.5 | 37 | 302 | 2.9 |
13 | 133 | 4.9 | 38 | 242 | 2.9 |
14 | 433 | 4.9 | 39 | 459 | 2.9 |
15 | 278 | 4.7 | 40 | 319 | 2.8 |
16 | 376 | 4.6 | 41 | 358 | 2.8 |
17 | 328 | 4.3 | 42 | 369 | 2.8 |
18 | 340 | 4.2 | 43 | 95 | 2.7 |
19 | 38 | 4.2 | 44 | 324 | 2.7 |
20 | 427 | 4.0 | 45 | 337 | 2.7 |
21 | 113 | 3.9 | 46 | 291 | 2.7 |
22 | 380 | 3.8 | 47 | 274 | 2.6 |
23 | 356 | 3.8 | 48 | 89 | 2.6 |
24 | 11 | 3.8 | 49 | 152 | 2.6 |
25 | 326 | 3.7 | 50 | 245 | 2.6 |
Fig. 4 Notable structural classes of the benzopyran library found to elicit degranulation of MC/9 cells. |
Fig. 5 Structure–activity relationship of the benzylamine subset of the benzopyran library (compound identification number over % MC/9 degranulation). |
(a) The benzylamines represent the major sub-group within the top 50 MC activating compounds (34%, 17 compounds) with compound 10 and compound 360 the most active members of this family. Further structural analysis of compound 10 suggested the necessity of its COMPOUND LINKS
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Download mol file of compoundbenzyl ether and secondary COMPOUND LINKS
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Download mol file of compoundbenzylamine bearing an unsubstituted methylene linker, since compounds 287, 147, and 16, 17, 578 and 580 all showed significant loss of MC activation capability. For lead compound 360, both the para-methoxy benzamide (cf. compounds 358 and 359) and the indanyl substitutions (cf. compounds 181 and 227) appear to be critical, where the carefully balanced steric environment is likely to be responsible for the observed MC activation capability (Fig. 5).
(b) The cyanostilbenes represent 14% (7 compounds) of the top 50 MC activating compounds with compound 272 being the most active member of this family (Fig. 6). Structure–activity analysis indicated that the regiochemistry of the cyanostilbene substitution was crucial for the observed activity (cf. compound 349), together with marginal tolerability in the substitution of the two aromatic systems (cf. compounds 7, 32, 36 and 37).
Fig. 6 Structure–activity relationship of the cyanostilbene subset of the benzopyran library (compound identification number over % MC/9 degranulation). |
(c) Lastly, the chalcone family of compounds within the benzopyran library also displayed a MC activation property that warrants additional attention (Fig. 7). This class appeared to demonstrate a wider structure–activity window, in which compounds 293, 309, 479 and 290 all exhibited comparable potencies. Substitution at the non-benzopyran bearing aromatic ring appears to be an essential element for the observed activity (cf. compound 2), and a variety of substituents including but not limited to halogen, COMPOUND LINKS
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Download mol file of compoundhydroxyl, methoxy and methylmercaptan groups (cf. compounds 293, 309, 290 and 289) were well tolerated. Interestingly, the chalcone olefin could be replaced with an analogous conformation restricting pyrazole functionality with good retention of MC activation capability (cf. compound 479), suggesting that suitable functional handles at this location could be used to further improve the potency and pharmacological properties of this family of compounds.
Fig. 7 Structure–activity relationship of the chalcone subset of the benzopyran library (compound identification number over % MC/9 degranulation). |
Finally, the major MC activating compounds shortlisted and discussed herein showed little cross-structural resemblance. Although we have demonstrated the utility of the benzopyran library in the bioassay-guided identification of bioactives, it was not possible to formulate a unified structure–activity relationship proposal. Nevertheless, the structural diversity exhibited by COMPOUND LINKS
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Download mol file of compoundbenzylamine, cyanostilbene and COMPOUND LINKS
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Download mol file of compoundchalcone presents new avenues for further structural modifications and biological investigations.
In this study we sought to develop a novel strategy to facilitate the detection of potential mucosal adjuvant compounds from a privileged-structure inspired chemical library based on their ability to activate MCs. MCs have recently been described to play a role in the development of adaptive immunity in several pro-inflammatory scenarios. Importantly, targeted activation of MCs using the small molecule MC activator c48/80 was found to induce robust systemic and mucosal immune responses in mice without any harmful side effects. Therefore, we reasoned that MCs might be used to identify novel agents that might be effective adjuvant compounds in vivo. The MC based screen is a high throughput strategy that has the capacity to screen thousands of chemical compounds from virtually any source. The high reproducibility of the assay based on internal plate controls that evaluate total β-hex, baseline β-hex release and a positive control using the MC activator compound 48/80 ensures that results from plate to plate can be compared and ranked based on MC degranulating activity as we described herein.
Others have used a semi-high-throughput screening assay37 to identify possible adjuvant compounds based on induction of interleukin 8 (IL-8) by bovine epithelial cells. Screening a natural compound library containing 280 compounds revealed 3 compounds that consistently induced IL-8 production by bovine intestinal epithelial cells. Our mast cell degranulation screening assay also utilizes live cells but has an important difference when compared to the epithelial cell screening assay used by others.37 The epithelial screening assay depends upon the incubation of the cells with the test compound for 24 hours followed by testing of the culture supernatant for IL-8 produced in response to the test compound. Our mast cell degranulation is a rapid assay that tests the ability of the test compound to induce mast cell degranulation and release of granule contents into the tissue culture after a short incubation time of 30 minutes.
Evaluating novel compounds for cytotoxicity in cell-based screens is a common step in screening assays. However, we intentionally did not monitor cell viability due to the recent observations that COMPOUND LINKS
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Download mol file of compoundalum, a US FDA approved vaccine adjuvant, involves cell death in its mechanism of adjuvant activity.38 COMPOUND LINKS
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Download mol file of compoundAlum has been suggested to be an inflammasome activator. The inflammasome is an intracellular multiprotein complex that is activated by a variety of microbial pathogens or COMPOUND LINKS
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Download mol file of compoundalum that may result in “pyroptotic cell death”.39 Cell death via the pyroptotic pathway results in the release of the cytosolic contents as well as interleukin 1β (IL-1β) secretion and both events are expected to be inflammatory.39 While cytotoxicity is typically viewed as an adverse event and an outcome that should be avoided, the recent observation that COMPOUND LINKS
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Download mol file of compoundalum provides effective and safe vaccine adjuvant activity via a mechanism that requires cell death38 suggests that controlled cell death may be an effective strategy to mimic infection that ultimately provides safe and effective adjuvant activity. For this reason, cell viability was not measured since cell death may be an indicator of inflammasome activation and also an indicator of effective adjuvant activity. Mast cells have recently been reported to produce IL-1β via an inflammasome-dependent mechanism.40 Therefore, additional studies beyond the scope of this screening assay will be required to determine if the mast cell activating compounds identified in this screen: (1) provide effective adjuvant activity in vivo and (2) activate the inflammasome to provide IL-1β production and/or pyroptotic cell death.
Cumulatively, in this screen, we evaluated 580 compounds from a benzopyran-based chemical library and successfully identified 12 compounds that could induce at least 5% MC/9 degranulation. Based on their structures and functionalities, these compounds fall into three distinct categories namely, the benzylamine derivatives, the cyanostilbenes and the chalcones. Although comparative analysis mindful of c48/80 (Fig. 8) revealed several essential structural features within each of the categories, a definitive structure–activity relationship conclusion cannot be reached at this juncture. This finding may suggest that these compounds mediate MC activation by engaging different receptors along the relevant biochemical pathway, and the structural diversity presented by the three classes of compounds opens up new avenues for further molecular interrogations in the search for small molecule(s) with mucosal adjuvant activity.
Fig. 8 Chemical structure of compound 48/80. |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2md20073b |
This journal is © The Royal Society of Chemistry 2013 |