Semi-synthetic terpenoids with differential adjuvant properties as sustainable replacements for shark squalene in vaccine emulsions.

Synthetic biology has allowed for the industrial production of supply-limited sesquiterpenoids such as the antimalarial drug artemisinin and ß-farnesene. One of the only unmodified animal products used in medicine is squalene, a triterpenoid derived from shark liver oil, which when formulated into an emulsion is used as a vaccine adjuvant to enhance immune responses in licensed vaccines. However, overfishing is depleting deep-sea shark populations, leading to potential supply problems for squalene. We chemically generated over 20 squalene analogues from fermentation-derived ß-farnesene and evaluated adjuvant activity of the emulsified compounds compared to shark squalene emulsion. By employing a desirability function approach that incorporated multiple immune readouts, we identified analogues with enhanced, equivalent, or decreased adjuvant activity compared to shark squalene emulsion. Availability of a library of structurally related analogues allowed elucidation of structure-function relationships. Thus, combining industrial synthetic biology with chemistry and immunology enabled generation of sustainable terpenoid-based vaccine adjuvants comparable to current shark squalene-based adjuvants while illuminating structural properties important for adjuvant activity.

Kilo-Scale GMP Synthesis of Renewable Semisynthetic Vaccine-Grade Squalene.

Emulsions of the triterpene squalene ((6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, CAS 111-02-4) have been used as adjuvants in influenza vaccines since the 1990s. Traditionally sourced from shark liver oil, the overfishing of sharks and concomitant reduction in the oceanic shark population raises sustainability issues for vaccine adjuvant grade squalene. We report a semisynthetic route to squalene meeting current pharmacopeial specifications for use in vaccines that leverages the ready availability of trans-ß-farnesene ((6E)-7,11-dimethyl-3-methylene-1,6,10-dodecatriene, CAS 18794-84-8), manufactured from sustainable sugarcane via a yeast fermentation process. The scalability of the proposed route was verified by a kilo-scale GMP synthesis. We also report data demonstrating the synthesized semi-synthetic squalene's physical stability and biological activity when used in a vaccine adjuvant formulation.

Production of the antimalarial drug precursor artemisinic acid in engineered yeast.

Malaria is a global health problem that threatens 300-500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l(-1)) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

Monoacylglycerol lipase regulates 2-arachidonoylglycerol action and arachidonic acid levels.

The structure-activity relationships of organophosphorus (OP) and organosulfur compounds were examined in vitro and in vivo as inhibitors of mouse brain monoacylglycerol lipase (MAGL) hydrolysis of 2-arachidonoylglycerol (2-AG) and agonist binding at the CB1 receptor. Several compounds showed exceptional potency toward MAGL activity with IC(50) values of 0.1-10 nM in vitro and high inhibition at 10mg/kg intraperitoneally in mice. We find for the first time that MAGL activity is a major in vivo determinant of 2-AG and arachidonic acid levels not only in brain but also in spleen, lung, and liver. Apparent direct OP inhibition of CB1 agonist binding may be due instead to metabolic stabilization of 2-AG in brain membranes as the actual inhibitor.

Chemoproteomic Profiling of Acetanilide Herbicides Reveals Their Role in Inhibiting Fatty Acid Oxidation.

Acetanilide herbicides are among the most widely used pesticides in the United States, but their toxicological potential and mechanisms remain poorly understood. Here, we have used chemoproteomic platforms to map proteome-wide cysteine reactivity of acetochlor (AC), the most widely used acetanilide herbicide, in vivo in mice. We show that AC directly reacts with >20 protein targets in vivo in mouse liver, including the catalytic cysteines of several thiolase enzymes involved in mitochondrial and peroxisomal fatty acid oxidation. We show that the fatty acids that are not oxidized, due to impaired fatty acid oxidation, are instead diverted into other lipid pathways, resulting in heightened free fatty acids, triglycerides, cholesteryl esters, and other lipid species in the liver. Our findings show the utility of chemoproteomic approaches for identifying novel mechanisms of toxicity associated with environmental chemicals like acetanilide herbicides.

Each lipase has a unique sensitivity profile for organophosphorus inhibitors.

Lipases sensitive to organophosphorus (OP) inhibitors play critical roles in cell regulation, nutrition, and disease, but little is known on the toxicological aspects in mammals. To help fill this gap, six lipases or lipase-like proteins are assayed for OP sensitivity in vitro under standard conditions (25 degrees C, 15 min incubation). Postheparin serum lipase, lipoprotein lipase (LPL) (two sources), pancreatic lipase, monoacylglycerol (MAG) lipase, cholesterol esterase, and KIAA1363 are considered with 32 OP pesticides and related compounds. Postheparin lipolytic activity in rat serum is inhibited by 14 OPs, including chlorpyrifos oxon (IC50 50-97 nM). LPL (bovine milk and Pseudomonas) generally is less inhibited by the insecticides or activated oxons, but the milk enzyme is very sensitive to six fluorophosphonates and benzodioxaphosphorin oxides (IC50 7-20 nM). Porcine pancreatic lipase is very sensitive to dioctyl 4-nitrophenyl phosphate (IC50 8 nM), MAG lipase of mouse brain to O-4-nitrophenyl methyldodecylphosphinate (IC50 0.6 nM), and cholesterol esterase (bovine pancreas) to all of the classes of OPs tested (IC50 < 10 nM for 17 compounds). KIAA1363 is sensitive to numerous OPs, including two O-4-nitrophenyl compounds (IC50 3-4 nM). In an overview, inhibition of 28 serine hydrolases (including lipases) by eight OPs (chlorpyrifos oxon, diazoxon, paraoxon, dichlorvos, and four nonpesticides) showed that brain acetylcholinesterase is usually less sensitive than butyrylcholinesterase, liver esterase, cholesterol esterase, and KIAA1363. In general, each lipase (like each serine hydrolase) has a different spectrum of OP sensitivity, and individual OPs have unique ranking of potency for inhibition of serine hydrolases.

Mapping Proteome-Wide Targets of Environmental Chemicals Using Reactivity-Based Chemoproteomic Platforms.

We are exposed to a growing number of chemicals in our environment, most of which have not been characterized in terms of their toxicological potential or mechanisms. Here, we employ a chemoproteomic platform to map the cysteine reactivity of environmental chemicals using reactivity-based probes to mine for hyper-reactive hotspots across the proteome. We show that environmental contaminants such as monomethylarsonous acid and widely used pesticides such as chlorothalonil and chloropicrin possess common reactivity with a distinct set of proteins. Many of these proteins are involved in key metabolic processes, suggesting that these targets may be particularly sensitive to environmental electrophiles. We show that the widely used fungicide chlorothalonil specifically inhibits several metabolic enzymes involved in fatty acid metabolism and energetics, leading to dysregulated lipid metabolism in mice. Our results underscore the utility of using reactivity-based chemoproteomic platforms to uncover novel mechanistic insights into the toxicity of environmental chemicals.

Chemical genetics screening reveals KIAA1363 as a cytokine-lowering target.

Inflammation is a hallmark of many human diseases, including pain, arthritis, atherosclerosis, obesity and diabetes, cancer, and neurodegenerative diseases. Although there are several successfully marketed small molecules anti-inflammatory drugs such as cyclooxygenase inhibitors and glucocorticoids, many of these compounds are also associated with various adverse cardiovascular or immunosuppressive side effects. Thus, identifying novel anti-inflammatory small molecules and their targets is critical for developing safer and more effective next-generation treatment strategies for inflammatory diseases. Here, we have conducted a chemical genetics screen to identify small molecules that suppress the release of the inflammatory cytokine TNFα from stimulated macrophages. We have used an enzyme class-directed chemical library for our screening efforts to facilitate subsequent target identification using activity-based protein profiling (ABPP). Using this strategy, we have found that KIAA1363 is a novel target for lowering key pro-inflammatory cytokines through affecting key ether lipid metabolism pathways. Our study highlights the application of combining chemical genetics with chemoproteomic and metabolomic approaches toward identifying and characterizing anti-inflammatory smal molecules and their targets.

A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3).

Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450(BM3) from Bacillus megaterium. Using a computer model, we illustrate how key P450(BM3) active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450(BM3) enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L(-1) in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.

Platelet-activating factor acetylhydrolase: selective inhibition by potent n-alkyl methylphosphonofluoridates.

Platelet-activating factor (PAF) is a potent endogenous phospholipid modulator of diverse biological activities, including inflammation and shock. PAF levels are primarily regulated by PAF acetylhydrolases (PAF-AHs). These enzymes are candidate secondary targets of organophosphorus (OP) pesticides and related toxicants. Previously known OP inhibitors of other serine hydrolases were tested with PAF-AH from mouse brain and testes of established functional importance compared with the structurally different human plasma enzyme. Several key OP pesticides and their oxon metabolites were very poor inhibitors of mouse brain and human plasma PAF-AH in vitro but moderately active for mouse brain and blood PAF-AH in vivo (e.g., tribufos defoliant and profenofos insecticide, presumably following oxidative bioactivation). OP compounds were then designed for maximum in vitro potency and selectivity for mouse brain PAF-AH vs. acetylcholinesterase (AChE). Lead compounds were found in a series of benzodioxaphosphorin 2-oxides. Ultrahigh potency and selectivity were achieved with n-alkyl methylphosphonofluoridates (long-chain sarin analogs): mouse brain and testes IC50 < or = 5 nM for C(8)-C(18) analogs and 0.1-0.6 nM for C(13) and C(14) compounds; human plasma IC50 < or = 2 nM for C(13)-C(18) analogs. AChE inhibitory potency decreased as chain length increased with maximum brain PAF-AH/AChE selectivity (>3000-fold) for C(13)-C(18) compounds. The toxicity of i.p.-administered PAF (LD50 ca. 0.5 mg/kg) was increased less than 2-fold by pretreatment with tribufos or the C(13)n-alkyl methylphosphonofluoridate. These studies with a mouse model indicate that PAF-AH is not a major secondary target of OP pesticide poisoning. The optimized PAF-AH inhibitors may facilitate investigations on other aspects of PAF metabolism and action.