Letter Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Cell-Based Kinetic Target-Guided Synthesis of an Enzyme Inhibitor Henrik Antti and Magnus Sellstedt* Department of Chemistry, Umeå University, 901 87 Umeå, Sweden S Supporting Information *
ABSTRACT: Finding a new drug candidate for a selected target is an expensive and time-consuming process. Target guided-synthesis, or in situ click chemistry, is a concept where the drug target is used to template the formation of its own inhibitors from reactive building blocks. This could simplify the identification of drug candidates. However, with the exception of one example of an RNA-target, target-guided synthesis has always employed purified targets. This limits the number of targets that can be screened by the method. By applying methods from the field of metabolomics, we demonstrate that target-guided synthesis with protein targets also can be performed directly in cell-based systems. These methods offer new possibilities to conduct screening for drug candidates of difficult protein targets in cellular environments. KEYWORDS: Target-guided synthesis, in situ click chemistry, enzyme catalysis, drug discovery
K
The most common reaction employed for kinetic targetguided synthesis is the dipolar cycloaddition of alkynes and azides, which is often referred to as in situ click chemistry.12 Azides and alkynes are relatively stable in cells and their reaction is bio-orthogonal.13 The compatibility of this reaction with cellular environments makes it suitable for proving the potential of KTGS in cell-based systems. However, performing KTGS in cellular environments poses several challenges. First, reported KTGS reactions have used target concentrations orders of magnitude higher than what is typically found in cells. Second, in cells the building blocks will compete with natural ligands and can thus interact with unintended targets as well, meaning that higher building block concentrations might be required compared to cell-free KTGS. These combined effects could result in higher levels of background reactions resulting in smaller differences in amounts of product assembled by the target-catalyzed and by the uncatalyzed reaction. In addition, the produced amount of product from KTGS is usually small, and if high concentrations of cell-derived material are simultaneously eluting during mass analysis, the detector might be oversaturated, resulting in worse sensitivity for the KTGS product. To overcome these effects, we have employed techniques from the field of metabolomics.14,15 Multireaction monitoring (MRM)16 mass spectroscopy was used to detect the product. MRM is a highly sensitive MS/MS method that filters the data on both product and fragment mass, giving low background levels. This is ideal for detecting products in complex mixtures of compounds present in biological samples. MRM have previously been used with KTGS but was then not combined with chromatography.17,18 To avoid oversaturation of the detector, it was necessary to remove a high proportion of the cell-material through a chloroform-aqueous phase extrac-
inetic target-guided synthesis (KTGS) is a method in drug discovery that employs the intended biological target to catalyze the formation of its own inhibitor from two sets of building blocks with complementary reactivity. This method has the potential to expedite the identification of drug candidates with less synthetic efforts since only the building blocks and not all combinations thereof have to be made (Figure 1). Since an early example in 1991,1 this strategy has
Figure 1. Kinetic target-guided synthesis. An active target selects building blocks with high affinity for the target, and the formation of product from these building blocks is amplified compared to the uncatalyzed background reaction.
proven successful with numerous protein targets,2−8 but also DNA-fragments9 and even bacterial ribosomes.10 In 2014 Disney and co-workers showed that special RNA-repeats can catalyze the formation of their own inhibitor in cells.11 Performing KTGS in cells offer significant advantages over in vitro models with purified targets since sensitive targets that require a cellular environment to maintain their active structure also can be screened. Here we show that KTGS also can be performed with an enzyme target in a cell-based system, enabling screening for protein inhibitors using KTGS in a more natural context. © XXXX American Chemical Society
Received: December 22, 2017 Accepted: March 4, 2018
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DOI: 10.1021/acsmedchemlett.7b00535 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
ACS Medicinal Chemistry Letters
Letter
runs in the presence and absence of the carbonic anhydrase inhibitor (Table 1, entry 7). This was also subsequently confirmed in an independent run (ratio 1.26, p-value 0.029). Performing the reaction with unlysed RBCs also gave similar results. However, lysis occurred under these reaction conditions and at the end of the run complete lysis was observed. PBS and blood serum did, as expected, not catalyze the reaction between 1 and 2 (Table 1, entries 6 and 10). This data shows that kinetic target-guided synthesis of the carbonic anhydrase inhibitor 3 can be performed in a cell-based system. An increase in concentration of azide 1 compared to published cell-free conditions was needed to reach significant results. This could be due to partial unspecific binding of 1 to various cell components and suggests that KTGS-screening in cell-based systems could benefit from using slightly higher compound concentrations than in cell-free systems. Figure 2 shows an example of KTGS with lysed RBCs. Along with the formation of the anti-trizaole 3, the syn-isomer was also
tion step. This also required the use of a deuterium-labeled analog of the KTGS-product as internal standard to minimize variation due to the workup procedure. Bovine carbonic anhydrase II (bCAII) catalyzes the formation of triazole 3 from azide 1 and alkyne 2 (Scheme 1).17 Since bCAII is an abundant enzyme in red blood cells Scheme 1. Previously Described KTGS Reaction That Employs bCAII
(RBCs), we decided to use this as a model system to demonstrate that KTGS of protein inhibitors can be performed in cell-based environments. As previously described for cell-free KTGS employing bCAII,17,19,20 we used the carbonic anhydrase inhibitor ethoxzolamide to outcompete building block binding to the target. In the presence of ethoxzolamide, a significantly lower amount of 3 should be detected as compared to DMSO controls; otherwise, the detected amount of 3 is mainly from the background reaction between 1 and 2. Initial attempts using bovine blood diluted with phosphate buffered saline (PBS) and concentrations of building blocks previously used for bCAII-mediated KTGS17,19 failed to show significant differences between runs with and without the carbonic anhydrase inhibitor ethoxzolamide after 2 days of incubation at 37 °C (Table 1, entries 1 and 2).21 Neither was
Figure 2. Example of chromatograms of KTGS with lysed RBCs, 800 μM 1, and 40 μM 2.
observed. In the absence of the inhibitor ethoxzolamide, the formation of the anti-isomer increased, showing that bCAII favored the formation of this isomer. The uncatalyzed background reaction, however, appears to give a mixture of both isomers. The amount of the syn-isomer was decreased in the absence of ethoxzolamide. This is likely because in the absence of a competing inhibitor, a large fraction of the alkyne 2 is bound to bCAII, giving a lower unbound concentration of 2 available for the uncatalyzed reaction. This does to some extent obscure the catalytic effect of the enzyme since the contribution to the formation of 3 from the uncatalyzed reaction is smaller in the absence of ethoxzolamide. Although the concentration of carbonic anhydrases in red blood cells is high (probably some ten micromolar), this particular KTGS reaction is inefficient, both with cells and in the presence of purified enzyme. The produced concentration of product 3 is estimated to approximately 50 nM, only a fraction of the enzyme concentration. Many other reported KTGS reactions give product concentrations in similar levels as the target concentration. This suggests that the methods described here could be employed for targets of much lower abundance than carbonic anhydrase as well. In summary, cell-based KTGS with a protein target have been demonstrated by using sensitive LC−MS/MS techniques and a deuterium-labeled internal standard of the analyte, combined with liquid−liquid extraction of the samples to remove much of the bulk cell-matrix prior to analysis. This method opens up screening for inhibitors of specific proteins using KTGS in cells, e.g., by comparing genetically modified cells that overexpress/do not express the intended target. This could allow KTGS identification of inhibitors of “difficult”
Table 1. Results from Cell-Based KTGS Performed in Triplicates with and without 250 μM of the Carbonic Anhydrase Inhibitor Ethoxzolamide entry
conc. 1 (μM)
conc. 2 (μM)
1
300
300
2
400
60
3 4 5
400 400 400
60 60 60
6 7 8 9 10
400 800 800 800 800
60 40 40 40 40
conditions blood/PBS 1:2 blood/PBS 3:4 blooda lysed RBCs PBS + 17 μM bCAII PBSa lysed RBCs lysed RBCsb RBCs Blood serum
ratio of 3 without/ with inhibitor
p-value
0.97
0.85
1.05
0.25
0.93 1.17 1.19
0.41 0.13 0.070
1.01 1.25 1.32 1.26 0.90
0.90 0.026 0.056 0.045 0.17
a
Duplicates. bAlternative workup procedure, see Supporting Information.
the use of undiluted blood successful. However, by using freeze lysed packed red blood cells, thereby further concentrating the target protein in the samples, the data started to trend toward a difference, giving results almost comparable to samples with purified carbonic anhydrase II (Table 1, entries 4 and 5). Further optimization of the conditions by changing the building block concentrations finally gave a clear difference between B
DOI: 10.1021/acsmedchemlett.7b00535 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
ACS Medicinal Chemistry Letters
Letter
(8) Jaegle, M.; Steinmetzer, T.; Rademann, J. Protein-templated formation of an inhibitor of the blood coagulation factor Xa through a background-free amidation reaction. Angew. Chem., Int. Ed. 2017, 56, 3718−3722. (9) Panda, D.; Saha, P.; Das, T.; Dash, J. Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor. Nat. Commun. 2017, 8, 16103. (10) Glassford, I.; Teijaro, C. N.; Daher, S. S.; Weil, A.; Small, M. C.; Redhu, S. K.; Colussi, D. J.; Jacobson, M. A.; Childers, W. E.; Buttaro, B.; Nicholson, A. W.; MacKerell, A. D.; Cooperman, B. S.; Andrade, R. B. Ribosome-templated azide-alkyne cycloadditions: synthesis of potent macrolide antibiotics by in situ click chemistry. J. Am. Chem. Soc. 2016, 138, 3136−3144. (11) Rzuczek, S. G.; Park, H.; Disney, M. D. A toxic RNA catalyzes the in cellulo synthesis of its own inhibitor. Angew. Chem., Int. Ed. 2014, 53, 10956−10959. (12) Lewis, W. G.; Green, L. G.; Grynszpan, F.; Radic, Z.; Carlier, P. R.; Taylor, P.; Finn, M. G.; Sharpless, K. B. Click chemistry in situ: acetylcholinesterase as a reaction vessel for the selective assembly of a femtomolar inhibitor from an array of building blocks. Angew. Chem., Int. Ed. 2002, 41, 1053−1056. (13) Prescher, J. A.; Bertozzi, C. R. Chemistry in living systems. Nat. Chem. Biol. 2005, 1, 13−21. (14) Dettmer, K.; Aronov, P. A.; Hammock, B. D. Mass spectrometry-based metabolomics. Mass Spectrom. Rev. 2007, 26, 51−78. (15) Fiehn, O.; Kopka, J.; Dormann, P.; Altmann, T.; Trethewey, R. N.; Willmitzer, L. Metabolite profiling for plant functional genomics. Nat. Biotechnol. 2000, 18, 1157−1161. (16) Lu, W.; Bennett, B. D.; Rabinowitz, J. D. Analytical strategies for LC-MS-based targeted metabolomics. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2008, 871, 236−242. (17) Wang, Y. J.; Lin, W. Y.; Liu, K.; Lin, R. J.; Selke, M.; Kolb, H. C.; Zhang, N. G.; Zhao, X. Z.; Phelps, M. E.; Shen, C. K. F.; Faull, K. F.; Tseng, H. R. An integrated microfluidic device for large-scale in situ click chemistry screening. Lab Chip 2009, 9, 2281−2285. (18) Shelke, S. V.; Cutting, B.; Jiang, X.; Koliwer-Brandl, H.; Strasser, D. S.; Schwardt, O.; Kelm, S.; Ernst, B. A fragment-based in situ combinatorial approach to identify high-affinity ligands for unknown binding sites. Angew. Chem., Int. Ed. 2010, 49, 5721−5725. (19) Mocharla, V. P.; Colasson, B.; Lee, L. V.; Roper, S.; Sharpless, K. B.; Wong, C. H.; Kolb, H. C. In situ click chemistry: enzyme-generated inhibitors of carbonic anhydrase II. Angew. Chem., Int. Ed. 2005, 44, 116−120. (20) Mocharla, V. P.; Walsh, J. C.; Padgett, H. C.; Su, H.; Fueger, B.; Weber, W. A.; Czernin, J.; Kolb, H. C. From in situ to in vivo: an in situ click-chemistry-derived carbonic anhydrase II imaging agent for positron emission tomography. ChemMedChem 2013, 8, 43−48. (21) The integrated peak data was normalized against a deuterated analog of 3 used as internal standard, and p-values were determined using two-sided unpaired Student t tests assuming equal variances.
targets that require a cellular environment to maintain their function.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.7b00535. Experimental data and procedures; copies of spectral and chromatographic data (PDF) Additional data (XLSX)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Henrik Antti: 0000-0003-1423-9517 Magnus Sellstedt: 0000-0001-8839-286X Author Contributions
The manuscript was written through contributions of all authors. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the foundation Stiftelsen Olle Engkvist Byggmästare (SOEB) (M.S.), the Swedish Research Council (grant number 2014-04495) (H.A.), and the Swedish Cancer Society (grant number CAN 2016/741) (H.A.) for financial support. The Swedish Metabolomics Centre’s Open Access Lab is acknowledged for access to and assistance with mass spectroscopy instruments.
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ABBREVIATIONS bCAII, bovine carbonic anhydrase 2; KTGS, kinetic targetguided synthesis; MRM, multi reaction monitoring; PBS, phosphate buffered saline; RBC, red blood cell
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REFERENCES
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DOI: 10.1021/acsmedchemlett.7b00535 ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
