i. In Vitro Pathway Engineering
The enormous pool of chemical diversity found in nature serves as an excellent inventory for accessing biologically active compounds. This chemical inventory, primarily found in microorganisms and plants, is generated by a broad range of enzymatic pathways under precise genetic and protein-level control. In vitro pathway reconstruction can be used to characterize individual pathway enzymes, identify pathway intermediates, and gain an increased understanding of how pathways can be manipulated to generate natural product analogs. Moreover, through in vitro approaches, it is possible to achieve a diversification that is not restricted by toxicity, limited availability of intracellular precursors, or preconceived (by nature) regulatory controls. Additionally, combinatorial biosynthesis and high-throughput techniques can be used to generate both known natural products and analogs that would not likely be generated naturally.
Advances in DNA sequencing technology, genome mining, and microbial metagenomic research provide a powerful toolbox to identify natural product biosynthetic pathways and open up a vast portion of the unexploited natural product space. In vitro reconstruction of natural product biosynthetic pathways (Fig. 1) can be used to: firstly, generate highly pure biosynthetic intermediates and final products; secondly, limit competing side reactions and cell metabolites (including some that are potentially toxic) that often occur in vivo, thereby complicating pathway understanding; thirdly, overcome unknown or poorly controlled regulatory constraints, including posttranslational limitations such as protein–protein interactions; and finally, enable unnatural synthetic building blocks to be used.
The tremendous breadth of biosynthetic enzymes, natural and unnatural pathway precursors, and chemical diversity that can be achieved using the aforementioned biosynthetic pathways is conceptually similar to combinatorial chemistry. This begs the question of how one can access the potential breadth of natural product biosynthesis in a manner consistent with the identification of highly bioactive agents. A major advantage of biocatalysis is that enzymes typically function under rather similar conditions. This has facilitated the design of in vitro biosynthesis coupled with high-throughput technology, and has led to a potentially new platform for the synthesis and identification of novel biologically active compounds and their analogs.
Figure 1. Schematic of in vitro reconstitution and biosynthetic pathway engineering for generating (unnatural) terpenoids, polyketides and nonribosomally generated peptides. Enzyme notation: terpenoid synthase (TS); halogenase (Hal); ligase (L); thioesterase (TE); aromatase (ARO); cyclase (CYC); methyltransferase (MT); oxygenase/oxidase (Ox); reductase (Re); glycosyltransferase (GT); epimerase (E); peroxidase (PO); prenyltransferase (PT) and carbamoyltransferase (CT).
This microarray-based, in vitro biosynthetic approach allows the in situ generation of natural product libraries by printing different enzymes and substrates (Fig. 2). The scaffold-generating PKS and TS enzymes, plus the large number of tailoring enzymes, are ideally suited for a combinatorial, high-throughput biosynthesis coupled with chemical or cellular screening for ‘hits’. The number of screenable compounds is immense when one considers that multiplicative combinatorial variables include precursor substrates (acyl-CoA starter and some flexibility in extender substrates of PKS and NRPS, and chain elongation and cyclization manipulation via engineered FPPase and GGPPase, etc., coupled with engineered cyclases in TSs), and the vast range of tailoring enzymes capable of functionalizing natural product core structures. Beyond natural tailoring enzymes, numerous biotransformations are possible, including C-, O-, and N-glycosylations, prenylation, acyltransfers, hydroxylations, epoxidations, halogenation, transamination, nitrile formation and desaturation. Hence, a new and potentially powerful platform technology can be used to exploit a combinatorial route to construct artificial biosynthetic pathways1.
Figure 2. Microarray-based construction of in vitro biosynthetic pathway for generating natural product libraries, which are amenable to HTS.
ii. Polyketide Synthesis on a Chip Leading to Highly Potent Anticancer Candidates
The generation of biological diversity by engineering the biosynthetic gene assembly of metabolic pathway enzymes has led to a wide range of “unnatural” variants of natural products. However, current biosynthetic techniques do not allow the rapid manipulation of pathway components and are often fundamentally limited by the compatibility of new pathways, their gene expression, and the resulting biosynthetic products and pathway intermediates with cell growth and function. To overcome these limitations, we have developed an entirely in vitro approach to synthesize analogs of natural products in high throughput6. Using several type III polyketide synthases (PKS) together with oxidative post-PKS tailoring enzymes, we performed 192 individual and multienzymatic reactions on a single glass microarray. Subsequent array-based screening with a human tyrosine kinase led to the identification of three compounds that acted as modest inhibitors in the low micromolar range. This approach, therefore, enables the rapid construction of analogs of natural products as potential pharmaceutical lead compounds.
Figure 3 highlights the central location of the type III PKS in aromatic metabolism, and in particular the connections among polyphenols, lignin biosynthesis, and oligophenol biosynthesis.
Figure 3. Central metabolic pathways involving phenolic compounds.
Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poor clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets (Figure 4). We have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogues with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme A (CoA) and methylmalonyl-CoA as extender substrates7. The resulting polyketide analogues possessed a similar structural polyketide backbone (aromatic-2-pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT-474 cells resulted in identification of several trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of nontransformed MCF-10A breast cells.
Figure 4. Type III PKS generation of highly potent anticancer compounds. Upper left is a cartoon depicting the potential interaction of a trifluormethylcinnamoyl-based pyrone against the HER receptor tyrosine kinase isoforms. Right panels depict very low IC50 values (~30 nM) against BT-474 (HER-overexpressing cell line) vs. non-HER overexpressing cell line (MCF-7) and a non-transformed cell line (MCF-10A).
iii. Tools for High Throughput Screening
We have begun to develop new tools to interrogate the effects of small molecules on biological function (Figure 1). These include cell- and protein-based high-throughput screening5,6 on chip immunoassays7,8 and high-throughput biocatalysis on a microarray platform9. With respect to the cell-based screening platform, we have developed (in collaboration with Dough Clark’s group at UC Berkeley, and scientists at Solidus Biosciences, Inc.) a miniaturized three-dimensional (3D) cellular array chip (the DataChip, Figure 2) that has been used for in vitro toxicological assessment of drug candidates and other chemicals in high-throughput. Although recent emphasis has been placed on understanding cellular metabolism in 3D, relatively little effort has been spent using 3D cultures to study cytotoxicity, particularly at very small volumes consistent with a high-throughput screening technique.
Figure 1. New high-throughput tools being developed. Upper left is the DataChip (Data Analysis Toxicology Assay Chip) for high throughput 3D cell culture screens. Upper right is the MetaChip (Metabolizing Enzyme Assay Chip for high-throughput interrogation of enzyme function. In this case, CYP450 inhibition assays. Lower left is a modification of the DataChip to enable on-chip, in-cell immunofluorescence assays and quantitative analysis of protein expression levels within cells. Lower right is a schematic of an Artificial Golgi, which includes an image of a digital microfluidic device.
Figure 2. The DataChip. Upper left represents the hemispherical spots generated by microarray spotting of cells in alginate solution, which upon contact with poly-L-lysine and BaCl2 results in nearly immediate gelation. The cells grow well up to 5 days in culture (upper right). Lower left shows an actual image of a stained DataChip (live-dead assay) and representative dose response curves for drug candidates and chemicals in the presence or absence of metabolizing enzymes contained within a MetaChip and stamped on top of the DataChip. Lower right is the output of the on-chip, in-cell immunofluorescence assay. The dose response to an activator of Hif-1a in a pancreatic tumor cell line (triangles represent standard Western blotting analysis on mL scale and open squares represent the on-chip immunofluorescence assay in volumes as low as 30-nL)
iv. Transfected Enzyme and Metabolism Chip (TeamChip)
Differential expression of various drug-metabolizing enzymes (DMEs) in the human liver may cause deviations of pharmacokinetic profiles, resulting in interindividual variability of drug toxicity and/or efficacy. We developed the ‘Transfected Enzyme and Metabolism Chip’ (TeamChip), which predicts potential metabolism-induced drug or drug-candidate toxicity10. The TeamChip is prepared by delivering genes into miniaturized three-dimensional cellular microarrays on a micropillar chip using recombinant adenoviruses in a complementary microwell chip (Figure 1). The device enables users to manipulate the expression of individual and multiple human metabolizing-enzyme genes (such as CYP3A4, CYP2D6, CYP2C9, CP1A2, CYP2E1, and UGT1A4) in THLE-2 cell microarrays. To identify specific enzymes involved in drug detoxification, we created 84 combinations of metabolic-gene expressions in a combinatorial fashion on a single microarray (Figure 2). Thus, the TeamChip platform can provide critical information necessary for evaluating metabolism-induced toxicity in a high-throughput manner.
Figure 1. TeamChip schematics and photographs. (a) Micropillar/microwell chip components in relation to a standard glass microscope slide. (b) The micropillar chip containing THLE-2 cells encapsulated in matrigel droplets. (c) The microwell chip containing recombinant adenoviruses carrying genes for drug-metabolizing enzymes (the red colour indicates the no-virus control and the two colours represent different viruses). (d) Stamping of the micropillar/microwell chips for drug-metabolizing gene expression. (e) Experimental procedure for use of the TeamChip.
Figure 2. Effects of combinatorial expression of human drug-metabolizing enzymes on the toxicity of tamoxifen. (a) Layout of the microwell chip containing 84 combinations of multiple recombinant adenoviruses (three sets of recombinant adenoviruses dispensed sequentially) to prepare the TeamChip for high-throughput gene transduction, and an additional microwell chip containing 200 mM tamoxifen for metabolism-induced toxicity screening. (b) Scanned image of THLE-2 cells expressing 84 combinations of multiple drug-metabolizing enzymes on the chip exposed to 200 mM tamoxifen for 48 h (top) and normalized THLE-2 cell viability at different drug-metabolizing enzyme expression levels (bottom). The viability of multiple drug-metabolizing enzyme-expressing THLE-2 cells exposed to tamoxifen was normalized by the fluorescent intensity of THLE-2 cells incubated in the absence of compound. The least toxic region in the scanned image is highlighted in a yellow box, and the red circle in the graph designates normalized THLE-2 cell viability calculated from the least toxic region.
- S.-J. Kwon, M. Mora-Pale, M.-Y. Lee, and J.S. Dordick (2012), "Expanding Nature's Small Molecule Diversity via in Vitro Biosynthetic Pathway Engineering", Curr. Opin. Chem. Biol. 16, 186-195.
- S.J. Kwon, M.Y. Lee, B. Ku, D.H. Sherman, and J.S. Dordick (2007), “High-Throughput, Microarray-Based Synthesis of Natural Product Analogues via in Vitro Metabolic Pathway Construction”, ACS Chem. Biol. 2, 419-425.
- M.-I. Kim, S.-J. Kwon, and J.S. Dordick (2009), "In Vitro Precursor-Directed Synthesis of Polyketide Analogues with Coenzyme A Regeneration for the Development of Antiangiogenic Agents", Org. Lett. 11, 3806-3809.
- S.J. Kwon, M.-I. Kim, B. Ku, L. Coulombel, J.-H. Kim, J.H. Shawky, R.J. Linhardt, and J.S. Dordick (2009), “Unnatural Polyketide Analogs Selectively Target the HER Signaling Pathway in Human Breast Cancer Cells.” ChemBioChem 11, 573-580.
- M.-Y. Lee, R.A. Kumar, S.M. Sukumaran, M.G. Hogg, D.S. Clark, and J.S. Dordick (2008), “Three-Dimensional Cellular Microarray for High-Throughput Toxicology Assays”, Proc. Natl. Acad. Sci. USA 105, 59-63
- M.-Y. Lee, C.-B. Park, J.S. Dordick, and D.S. Clark (2005), “Metabolizing Enzyme Toxicology Assay Chip (MetaChip) for High-Throughput Microscale Toxicity Analyses”, Proc. Natl. Acad. Sci. USA 102, 983-987. Cover Article.
- T.G. Fernandes, S.-J. Kwon, M.-Y. Lee, M.M. Diogo, D.S. Clark, J.M.S. Cabral, and J.S. Dordick (2010), “Three-Dimensional Cell Culture Microarrays for High-Throughput Studies of Stem Cell Fate”, Biotechnol. Bioeng. 106, 106-118.
- T.G. Fernandes, S.-J. Kwon, M.-Y. Lee, D.S. Clark, J.M.S. Cabral, and J.S. Dordick (2008), “An On-Chip, Cell-Based Microarray Immunofluorescence Assay for High-Throughput Analysis of Target Proteins”, Anal. Chem. 80, 6633-6639.
- S.M. Sukumaran, B. Potsaid, M.-Y. Lee, D.S. Clark, and J.S. Dordick (2009), “Development of a Fluorescence-Based, Ultra High-Throughput Screening Platform for Nanoliter-Scale Cytochrome P450 Microarrays”, J. Biomol. Screen. 14, 668-678.
- S.J. Kwon, D.W. Lee, D.A. Shah, B. Ku, S.Y. Joon, K. Solanki, J.D. Ryan, D.S. Clark, J.S. Dordick, and M.Y. Lee (2014), "High-Throughput and Combinatorial Gene Expression on a Chip for Metabolism-Induced Toxicology Screening", Nat. Commun. 5, 3739.