Commercial-scale biotherapeutics manufacturing facility for plant-made pharmaceuticals.

Rapid, large-scale manufacture of medical countermeasures can be uniquely met by the plant-made-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plant-based manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever.

Determination of antiplasmodial activity and binding affinity of curcumin and demethoxycurcumin towards PfTrxR.

In our study, the inhibitory activity of curcuminoids towards Plasmodium falciparum thioredoxin reductase (PfTrxR) was determined using LC-MS-based functional assay and showed that only demethoxycurcumin (DMC) inhibited PfTrxR (IC50: 2 µM). In silico molecular modelling was used to ascertain and further confirm that the binding affinities of curcumin and DMC are towards the dimer interface of PfTrxR. The in vitro antiplasmodial activities of curcumin and DMC were evaluated and shown to be active against chloroquine (CQ)-sensitive (D6 clone) and moderately active against CQ-resistant (W2 clone) strains of Plasmodium falciparum while no cytotoxicity was observed against Vero cells.

Characterization of PfTrxR inhibitors using antimalarial assays and in silico techniques.

BACKGROUND: The compounds 1,4-napthoquinone (1,4-NQ), bis-(2,4-dinitrophenyl)sulfide (2,4-DNPS), 4-nitrobenzothiadiazole (4-NBT), 3-dimethylaminopropiophenone (3-DAP) and menadione (MD) were tested for antimalarial activity against both chloroquine (CQ)-sensitive (D6) and chloroquine (CQ)-resistant (W2) strains of Plasmodium falciparum through an in vitro assay and also for analysis of non-covalent interactions with P. falciparum thioredoxin reductase (PfTrxR) through in silico docking studies. RESULTS: The inhibitors of PfTrxR namely, 1,4-NQ, 4-NBT and MD displayed significant antimalarial activity with IC50 values of < 20 µM and toxicity against 3T3 cell line. 2,4-DNPS was only moderately active. In silico docking analysis of these compounds with PfTrxR revealed that 2,4-DNPS, 4-NBT and MD interact non-covalently with the intersubunit region of the enzyme. CONCLUSIONS: In this study, tools for the identification of PfTrxR inhibitors using phenotyphic screening and docking studies have been validated for their potential use for antimalarial drug discovery project.

Determination of antiplasmodial activity and binding affinity of selected natural products towards PfTrxR and PfGR.

In our study, the binding affinities of selected natural products towards PfTrxR, PfGR, human TrxR and human GR were determined using a mass spectrometry based ligand binding assay. The in vitro antimalarial activity and cytotoxicity of these ligands were also determined. Catharanthine, 11-(OH)-coronaridine, hernagine, vobasine and hispolone displayed antiplasmodial activity against PfK1 (IC50 = 0.996-3.63 microg/mL).

Development of liquid chromatography/mass spectrometry based screening assay for PfTrxR inhibitors using relative quantitation of intact thioredoxin.

RATIONALE: Plasmodium falciparum (Pf) thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin disulfide (Trx-S(2)) to thioredoxin dithiol (Trx-(SH)(2)) that is essential for antioxidant defense mechanism and DNA synthesis in the parasite and is a validated drug target for new antimalarial agents. METHODS: In this study, we have developed a liquid chromatography/mass spectrometry (LC/MS)-based functional assay to identify inhibitors of PfTrxR by quantifying the product formed (Trx-(SH)(2)) in the enzymatic reaction. Relative quantitation of the reaction product (intact Trx-(SH)(2)) was carried out using an Agilent 6520 QTOF mass spectrometer equipped with a positive mode electrospray ionization (ESI) source. RESULTS: The calibration curve prepared for Trx-(SH)(2) at concentrations ranging from 1.8 to 116.5 µg/mL was linear (R(2) >0.998). The limit of detection (LOD) and limit of quantification (LOQ) of Trx-(SH)(2) were at 0.45 and 1.8 µg/mL respectively. To validate the developed functional assay we have screened reference compounds 1, 2 and 3 for their PfTrxR inhibitory activity and ten natural compounds (at 10 µM) which were earlier identified as ligands of PfTrxR by a UF-LC/MS based binding assay. CONCLUSIONS: The developed LC/MS-based functional assay for identification of inhibitors of PfTrxR is a sensitive and reliable method that is also amendable for high-throughput format. This is the first representation of a relative quantitation of intact Trx-(SH)(2) using LC/MS.

Identification of oleamide in Guatteria recurvisepala by LC/MS-based Plasmodium falciparum thioredoxin reductase ligand binding method.

Our current research on applications of mass spectrometry to natural product drug discovery against malaria aims to screen plant extracts for new ligands to Plasmodium falciparum thioredoxin reductase (PfTrxR) followed by their identification and structure elucidation. PfTrxR is involved in the antioxidant defense and redox regulation of the parasite and is validated as a promising target for therapeutic intervention against malaria. In the present study, detannified methanol extracts from Guatteria recurvisepala, Licania kallunkiae, and Topobea watsonii were screened for ligands to PfTrxR using ultrafiltration and liquid chromatography/mass spectrometry-based binding experiments. The PfTrxR ligand identified in the extract of Guatteria recurvisepala displayed a relative binding affinity of 3.5-fold when incubated with 1 µM PfTrxR. The ligand corresponding to the protonated molecule m/z 282.2792 [M+ H]+ was eluted at a retention time of 17.95 min in a 20-min gradient of 95% B consisting of (A) 0.1%formic acid in 95% H2O-5% ACN, and (B) 0.1% formic acid in 95% ACN-5% H2O in an LC-QTOF-MS.Tandem MS of the protonated molecule m/z 282.2792 [M + H]+, C18H36NO (DBE: 2; error: 1.13 ppm) resulted in two daughter ions m/z 265.2516[M + H-NH3]+ (DBE: 3; error: 0.35 ppm) and m/z 247.2405 [M + H-NH3-H2O] +, (DBE: 4; error:2.26 ppm). The PfTrxR ligand was identified as oleamide and confirmed by comparison of the retention time, molecular formula, accurate mass,and double bond equivalence with the standard oleamide. This is the first report on the identification of oleamide as a PfTrxR ligand from Guatteria recurvisepala R. E. Fr. and the corresponding in vitro activity against P. falciparum strain K1 (IC50 4.29 µg/mL).

Screening of natural compounds for ligands to PfTrxR by ultrafiltration and LC-MS based binding assay.

In our study, we have screened 133 structurally diverse natural compounds from the MEGx® collection of AnalytiCon Discovery and three synthetic hispolone analogs for binding affinity to Plasmodium falciparum thioredoxin reductase (PfTrxR) using an ultrafiltration (UF) and liquid chromatography (LC/MS) based ligand-binding assay newly developed in our laboratory. PfTrxR catalyzes the reduction of thioredoxin (PfTrx) protein. In reduced form, PfTrx is essentially involved in the antioxidative defense and redox regulation of P. falciparum. Nine compounds (yohimbine (1), catharanthine (2), vobasine (3), gnetifolin E (4), quinidine N-oxide (5), 11-hydroxycoronaridine (6), hispolone (7), hispolone methyl ether (8), and hernagine (9)) displayed binding affinity for PfTrxR at 1µM. The ranking order of compound's binding affinities for PfTrxR is 7>6>2>4>5>8>1>9>3. On the other hand, compounds 6, 7, 2 and 8 demonstrated specific binding to the active site of PfTrxR, when ligands were tested in an equimolar mixture of 1 µM.