Macaque antibodies targeting Marburg virus glycoprotein induced by multivalent immunization.

Marburg virus infection in humans is associated with case fatality rates that can reach up to 90%, but to date, there are no approved vaccines or monoclonal antibody (mAb) countermeasures. Here, we immunized Rhesus macaques with multivalent combinations of filovirus glycoprotein (GP) antigens belonging to Marburg, Sudan, and Ebola viruses to generate monospecific and cross-reactive antibody responses against them. From the animal that developed the highest titers of Marburg virus GP-specific neutralizing antibodies, we sorted single memory B cells using a heterologous Ravn virus GP probe and cloned and characterized a panel of 34 mAbs belonging to 28 unique lineages. Antibody specificities were assessed by overlapping pepscan and binding competition analyses, revealing that roughly a third of the lineages mapped to the conserved receptor binding region, including potent neutralizing lineages that were confirmed by negative stain electron microscopy to target this region. Additional lineages targeted a protective region on GP2, while others were found to possess cross-filovirus reactivity. Our study advances the understanding of orthomarburgvirus glycoprotein antigenicity and furthers efforts to develop candidate antibody countermeasures against these lethal viruses. IMPORTANCE: Marburg viruses were the first filoviruses characterized to emerge in humans in 1967 and cause severe hemorrhagic fever with average case fatality rates of ~50%. Although mAb countermeasures have been approved for clinical use against the related Ebola viruses, there are currently no approved countermeasures against Marburg viruses. We successfully isolated a panel of orthomarburgvirus GP-specific mAbs from a macaque immunized with a multivalent combination of filovirus antigens. Our analyses revealed that roughly half of the antibodies in the panel mapped to regions on the glycoprotein shown to protect from infection, including the host cell receptor binding domain and a protective region on the membrane-anchoring subunit. Other antibodies in the panel exhibited broad filovirus GP recognition. Our study describes the discovery of a diverse panel of cross-reactive macaque antibodies targeting orthomarburgvirus and other filovirus GPs and provides candidate immunotherapeutics for further study and development.

Glycoengineered recombinant alpha1-antitrypsin results in comparable in vitro and in vivo activities to human plasma-derived protein.

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

Particle Metrology Approach to Understanding How Storage Conditions Affect Long-Term Liposome Stability.

Lipid nanoparticles are a generic type of nanomaterial with broad applicability in medicine as drug delivery vehicles. Liposomes are a subtype of lipid nanoparticles and, as a therapeutic platform, can be loaded with a genetic material or pharmaceutical agents for use as drug treatments. An open question for these types of lipid nanoparticles is what factor(s) affect the long-term stability of the particles. The stability of the particle is of great interest to understand and predict the effective shelf-life and storage requirements. In this report, we detail a one-year study of liposome stability as a function of lipid composition, buffer composition/pH, and storage temperature. This was done in aqueous solution without freezing. The effect of lipid composition is shown to be a critical factor when evaluating stability of the measured particle size and number concentration. Other factors (i.e., storage temperature and buffer pH/composition) were shown to be less critical but still have some effect. The stability of these particles informs formulation and optimal storage requirements and assists with future developmental planning of a NIST liposome-based reference material. This work also highlights the complex nature of long-term soft particle storage in biopharmaceutical applications.

A round-robin approach provides a detailed assessment of biomolecular small-angle scattering data reproducibility and yields consensus curves for benchmarking.

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å-1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å-1, but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H2O and in D2O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å-1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set.

Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration.

Lipid Nanoparticles (LNPs) are used to deliver siRNA and COVID-19 mRNA vaccines. The main factor known to determine their delivery efficiency is the pKa of the LNP containing an ionizable lipid. Herein, we report a method that can predict the LNP pKa from the structure of the ionizable lipid. We used theoretical, NMR, fluorescent-dye binding, and electrophoretic mobility methods to comprehensively measure protonation of both the ionizable lipid and the formulated LNP. The pKa of the ionizable lipid was 2-3 units higher than the pKa of the LNP primarily due to proton solvation energy differences between the LNP and aqueous medium. We exploited these results to explain a wide range of delivery efficiencies in vitro and in vivo for intramuscular (IM) and intravascular (IV) administration of different ionizable lipids at escalating ionizable lipid-to-mRNA ratios in the LNP. In addition, we determined that more negatively charged LNPs exhibit higher off-target systemic expression of mRNA in the liver following IM administration. This undesirable systemic off-target expression of mRNA-LNP vaccines could be minimized through appropriate design of the ionizable lipid and LNP.

Design of a native-like secreted form of the hepatitis C virus E1E2 heterodimer.

Hepatitis C virus (HCV) is a major worldwide health burden, and a preventive vaccine is needed for global control or eradication of this virus. A substantial hurdle to an effective HCV vaccine is the high variability of the virus, leading to immune escape. The E1E2 glycoprotein complex contains conserved epitopes and elicits neutralizing antibody responses, making it a primary target for HCV vaccine development. However, the E1E2 transmembrane domains that are critical for native assembly make it challenging to produce this complex in a homogenous soluble form that is reflective of its state on the viral envelope. To enable rational design of an E1E2 vaccine, as well as structural characterization efforts, we have designed a soluble, secreted form of E1E2 (sE1E2). As with soluble glycoprotein designs for other viruses, it incorporates a scaffold to enforce assembly in the absence of the transmembrane domains, along with a furin cleavage site to permit native-like heterodimerization. This sE1E2 was found to assemble into a form closer to its expected size than full-length E1E2. Preservation of native structural elements was confirmed by high-affinity binding to a panel of conformationally specific monoclonal antibodies, including two neutralizing antibodies specific to native E1E2 and to its primary receptor, CD81. Finally, sE1E2 was found to elicit robust neutralizing antibodies in vivo. This designed sE1E2 can both provide insights into the determinants of native E1E2 assembly and serve as a platform for production of E1E2 for future structural and vaccine studies, enabling rational optimization of an E1E2-based antigen.

Single-Molecule 3D Images of "Hole-Hole" IgG1 Homodimers by Individual-Particle Electron Tomography.

The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more than one antigen. Optimization of engineered IgG requires knowledge of three-dimensional (3D) structure of synthetic IgG. However, due to flexible nature of the molecules, their structural characterization is challenging. Here, we use our reported individual-particle electron tomography (IPET) method with optimized negative-staining (OpNS) for direct 3D reconstruction of individual IgG hole-hole homodimer molecules. The hole-hole homodimer is an undesired variant generated during the production of a bispecific antibody using the knob-into-hole heterodimer technology. A total of 64 IPET 3D density maps at ~15 Å resolutions were reconstructed from 64 individual molecules, revealing 64 unique conformations. In addition to the known Y-shaped conformation, we also observed an unusual X-shaped conformation. The 3D structure of the X-shaped conformation contributes to our understanding of the structural details of the interaction between two heavy chains in the Fc domain. The IPET approach, as an orthogonal technique to characterize the 3D structure of therapeutic antibodies, provides insight into the 3D structural variety and dynamics of heterogeneous IgG molecules.

Spectral-Based Screening Approach Evaluating Two Specific Maize Lines With Divergent Resistance to Invasion by Aflatoxigenic Fungi.

In an effort to control aflatoxin contamination in food and/or feed grains, a segment of research has focused on host resistance to eliminate aflatoxin from susceptible crops, including maize. To this end, screening tools are key to identifying resistant maize genotypes. The traditional field screening techniques, the kernel screening laboratory assay (KSA), and analytical methods (e.g., ELISA) used for evaluating corn lines for resistance to fungal invasion, all ultimately require sample destruction. A technological advancement on the basic BGYF presumptive screening test, fluorescence hyperspectral imaging offers an option for non-destructive and rapid image-based screening. The present study aimed to differentiate fluorescence spectral signatures of representative resistant and susceptible corn hybrids infected by a toxigenic (SRRC-AF13) and an atoxigenic (SRRC-AF36) strain of Aspergillus flavus, at several time points (5, 7, 10, and 14 days), in order to evaluate fluorescence hyperspectral imaging as a viable technique for early, non-invasive aflatoxin screening in resistant and susceptible corn lines. The study utilized the KSA to promote fungal growth and aflatoxin production in corn kernels inoculated under laboratory conditions and to provide actual aflatoxin values to relate with the imaging data. Each time point consisted of 78 kernels divided into four groups (30-susceptible, 30-resistant, 9-susceptible control, and 9-resistant control), per inoculum. On specified days, kernels were removed from the incubator and dried at 60°C to terminate fungal growth. Dry kernels were imaged with a VNIR hyperspectral sensor (image spectral range of 400-1000 nm), under UV excitation centered at 365 nm. Following imaging, kernels were submitted for the chemical AflaTest assay (VICAM). Fluorescence emissions were compared for all samples over 14 days. Analysis of strain differences separating the fluorescence emission peaks of resistant from the susceptible strain indicated that the emission peaks of the resistant strain and the susceptible strains differed significantly (p < 0.01) from each other, and there was a significant difference in fluorescence intensity between the treated and control kernels of both strains. These results indicate a viable role of fluorescence hyperspectral imaging for non-invasive screening of maize lines with divergent resistance to invasion by aflatoxigenic fungi.

Small-angle X-ray and neutron scattering demonstrates that cell-free expression produces properly formed disc-shaped nanolipoprotein particles.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.

Temporal Effects on Internal Fluorescence Emissions Associated with Aflatoxin Contamination from Corn Kernel Cross-Sections Inoculated with Toxigenic and Atoxigenic Aspergillus flavus.

Non-invasive, easy to use and cost-effective technology offers a valuable alternative for rapid detection of carcinogenic fungal metabolites, namely aflatoxins, in commodities. One relatively recent development in this area is the use of spectral technology. Fluorescence hyperspectral imaging, in particular, offers a potential rapid and non-invasive method for detecting the presence of aflatoxins in maize infected with the toxigenic fungus Aspergillus flavus. Earlier studies have shown that whole maize kernels contaminated with aflatoxins exhibit different spectral signatures from uncontaminated kernels based on the external fluorescence emission of the whole kernels. Here, the effect of time on the internal fluorescence spectral emissions from cross-sections of kernels infected with toxigenic and atoxigenic A. flavus, were examined in order to elucidate the interaction between the fluorescence signals emitted by some aflatoxin contaminated maize kernels and the fungal invasion resulting in the production of aflatoxins. First, the difference in internal fluorescence emissions between cross-sections of kernels incubated in toxigenic and atoxigenic inoculum was assessed. Kernels were inoculated with each strain for 5, 7, and 9 days before cross-sectioning and imaging. There were 270 kernels (540 halves) imaged, including controls. Second, in a different set of kernels (15 kernels/group; 135 total), the germ of each kernel was separated from the endosperm to determine the major areas of aflatoxin accumulation and progression over nine growth days. Kernels were inoculated with toxigenic and atoxigenic fungal strains for 5, 7, and 9 days before the endosperm and germ were separated, followed by fluorescence hyperspectral imaging and chemical aflatoxin determination. A marked difference in fluorescence intensity was shown between the toxigenic and atoxigenic strains on day nine post-inoculation, which may be a useful indicator of the location of aflatoxin contamination. This finding suggests that both, the fluorescence peak shift and intensity as well as timing, may be essential in distinguishing toxigenic and atoxigenic fungi based on spectral features. Results also reveal a possible preferential difference in the internal colonization of maize kernels between the toxigenic and atoxigenic strains of A. flavus suggesting a potential window for differentiating the strains based on fluorescence spectra at specific time points.

Isotopic labeling of proteins in Halobacterium salinarum.

It is often necessary to obtain isotopically labeled proteins containing (15)N, (13)C, or (2)H for nuclear magnetic resonance; and (2)H for small-angle neutron scattering or neutron diffraction studies. To achieve uniform isotopic labeling, protein expression is most commonly performed in Escherichia coli or yeast using labeled media. However, proteins from extreme halophiles sometimes require a cellular environment with high ionic strength and cannot be heterologously expressed in E. coli or yeast in functional form. We present here methods for the cultivation of Halobacterium salinarum in isotopically labeled rich media, using commercially available isotopically labeled hydrolysates. The methods described here are both technically simple and relatively inexpensive.

Genome Sequence of Aspergillus flavus NRRL 3357, a Strain That Causes Aflatoxin Contamination of Food and Feed.

Aflatoxin contamination of food and livestock feed results in significant annual crop losses internationally. Aspergillus flavus is the major fungus responsible for this loss. Additionally, A. flavus is the second leading cause of aspergillosis in immunocompromised human patients. Here, we report the genome sequence of strain NRRL 3357.

ZmGns, a maize class I ß-1,3-glucanase, is induced by biotic stresses and possesses strong antimicrobial activity.

Plant ß-1,3-glucanases are members of the pathogenesis-related protein 2 (PR-2) family, which is one of the 17 PR protein families and plays important roles in biotic and abiotic stress responses. One of the differentially expressed proteins (spot 842) identified in a recent proteomic comparison between five pairs of closely related maize (Zea mays L.) lines differing in aflatoxin resistance was further investigated in the present study. Here, the corresponding cDNA was cloned from maize and designated as ZmGns. ZmGns encodes a protein of 338 amino acids containing a potential signal peptide. The expression of ZmGns was detectible in all tissues studied with the highest level in silks. ZmGns was significantly induced by biotic stresses including three bacteria and the fungus Aspergillus flavus. ZmGns was also induced by most abiotic stresses tested and growth hormones including salicylic acid. In vivo, ZmGns showed a significant inhibitory activity against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 and fungal pathogen Botrytis cinerea when it overexpressed in Arabidopsis. Its high level of expression in the silk tissue and its induced expression by phytohormone treatment, as well as by bacterial and fungal infections, suggest it plays a complex role in maize growth, development, and defense.

Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs.

Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.

Co-inoculation of aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus to study fungal invasion, colonization, and competition in maize kernels.

A currently utilized pre-harvest biocontrol method involves field inoculations with non-aflatoxigenic Aspergillus flavus strains, a tactic shown to strategically suppress native aflatoxin-producing strains and effectively decrease aflatoxin contamination in corn. The present in situ study focuses on tracking the invasion and colonization of an aflatoxigenic A. flavus strain (AF70), labeled with green fluorescent protein (GFP), in the presence of a non-aflatoxigenic A. flavus biocontrol strain (AF36), to better understand the competitive interaction between these two strains in seed tissue of corn (Zea mays). Corn kernels that had been co-inoculated with GFP-labeled AF70 and wild-type AF36 were cross-sectioned and observed under UV and blue light to determine the outcome of competition between these strains. After imaging, all kernels were analyzed for aflatoxin levels. There appeared to be a population difference between the co-inoculated AF70-GFP+AF36 and the individual AF70-GFP tests, both visually and with pixel count analysis. The GFP allowed us to observe that AF70-GFP inside the kernels was suppressed up to 82% when co-inoculated with AF36 indicating that AF36 inhibited progression of AF70-GFP. This was in agreement with images taken of whole kernels where AF36 exhibited a more robust external growth compared to AF70-GFP. The suppressed growth of AF70-GFP was reflected in a corresponding (upto 73%) suppression in aflatoxin levels. Our results indicate that the decrease in aflatoxin production correlated with population depression of the aflatoxigenic fungus by the biocontrol strain supporting the theory of competitive exclusion through robust propagation and fast colonization by the non-aflatoxigenic fungus.

Fluorescence imaging spectroscopy (FIS) for comparing spectra from corn ears naturally and artificially infected with aflatoxin producing fungus.

In an effort to address the problem of rapid detection of aflatoxin in grain, particularly oilseeds, the current study assessed the spectral differences of aflatoxin production in kernels from a cornfield inoculated with spores from 2 different strains of toxigenic Aspergillus flavus. Aflatoxin production in corn from the same field due to natural infestation was also assessed. A small corn plot in Baton Rouge, La., U.S.A., was used during the 2008-growing season. Two groups of 400 plants were inoculated with 2 different inocula and 1 group of 400 plants was designated as controls. Any contamination detected in the controls was attributed to natural infestation. A subset of each group was imaged with a visible near infra red (VNIR) hyperspectral system under ultra violet (UV) excitation and subsequently analyzed for aflatoxin using affinity column fluorometry. Group differences were statistically analyzed. Results indicate that when all the spectral data across all groups were averaged, any potential differences between groups (treated and untreated) were obscured. However, spectral analysis based on contaminated "hot" pixel classification showed a distinct spectral shift/separation between contaminated and clean ears with fluorescence peaks at 501 and 478 nm, respectively. All inoculated and naturally infected control ears had fluorescence peaks at 501 nm that differed from uninfected corn ears. Results from this study may be useful in evaluating rapid, noninvasive instrumentation and/or methodology for aflatoxin detection in grain.

Breeding aflatoxin-resistant maize lines using recent advances in technologies - a review.

Aflatoxin contamination caused by Aspergillus flavus infection of corn is a significant and chronic threat to corn being used as food or feed. Contamination of crops at levels of 20 ng g(-1) or higher (as regulated by the USFDA) by this toxin and potent carcinogen makes the crop unsalable, resulting in a significant economic burden on the producer. This review focuses on elimination of this contamination in corn which is a major US crop and the basis of many products. Corn is also "nature's example" of a crop containing heritable resistance to aflatoxin contamination, thereby serving as a model for achieving resistance to aflatoxin contamination in other crops as well. This crop is the largest production grain crop worldwide, providing food for billions of people and livestock and critical feedstock for production of biofuels. In 2011, the economic value of the US corn crop was US$76 billion, with US growers producing an estimated 12 billion bushels, more than one-third of the world's supply. Thus, the economics and significance of corn as a food crop and the threat to food safety due to aflatoxin contamination of this major food crop have prompted the many research efforts in many parts of the world to identify resistance in corn to aflatoxin contamination. Plant breeding and varietal selection has been used as a tool to develop varieties resistance to disease. This methodology has been employed in defining a few corn lines that show resistance to A. flavus invasion; however, no commercial lines have been marketed. With the new tools of proteomics and genomics, identification of resistance mechanisms, and rapid resistance marker selection methodologies, there is an increasing possibility of finding significant resistance in corn, and in understanding the mechanism of this resistance.

A single ligand is sufficient to activate EGFR dimers.

Crystal structures of human epidermal growth factor receptor (EGFR) with bound ligand revealed symmetric, doubly ligated receptor dimers thought to represent physiologically active states. Such complexes fail to rationalize negative cooperativity of epidermal growth factor (EGF) binding to EGFR and the behavior of the ligandless EGFR homolog ErbB2/HER2, however. We report cell-based assays that provide evidence for active, singly ligated dimers of human EGFR and its homolog, ErbB4/HER4. We also report crystal structures of the ErbB4/HER4 extracellular region complexed with its ligand Neuregulin-1ß that resolve two types of ErbB dimer when compared to EGFR:Ligand complexes. One type resembles the recently reported asymmetric dimer of Drosophila EGFR with a single high-affinity ligand bound and provides a model for singly ligated human ErbB dimers. These results unify models of vertebrate and invertebrate EGFR/ErbB signaling, imply that the tethered conformation of unliganded ErbBs evolved to prevent crosstalk among ErbBs, and establish a molecular basis for both negative cooperativity of ligand binding to vertebrate ErbBs and the absence of active ErbB2/HER2 homodimers in normal conditions.

Tight control of mycotoxin biosynthesis gene expression in Aspergillus flavus by temperature as revealed by RNA-Seq.

To better understand the effect of temperature on mycotoxin biosynthesis, RNA-Seq technology was used to profile the Aspergillus flavus transcriptome under different temperature conditions. This approach allowed us to quantify transcript abundance for over 80% of fungal genes including 1153 genes that were differentially expressed at 30 and 37 °C. Eleven of the 55 secondary metabolite clusters were upregulated at the lower temperature, including aflatoxin biosynthesis genes, which were among the most highly upexpressed genes. On average, transcript abundance for the 30 aflatoxin biosynthesis genes was 3300 times greater at 30 °C as compared with 37 °C. The results are consistent with the view that high temperature negatively affects aflatoxin production by turning down transcription of the two key transcriptional regulators, aflR and aflS. Subtle changes in the expression levels of aflS to aflR appear to control transcription activation of the aflatoxin cluster.

Biomimetic syntheses and antiproliferative activities of racemic, natural (-), and unnnatural (+) glyceollin I.

A 14-step biomimetic synthetic route to glyceollin I (1.5% overall yield) was developed and deployed to produce the natural enantiomeric form in soy, its unnatural stereoisomer, and a racemic mixture. Enantiomeric excess was assessed by asymmetric NMR shift reagents and chiral HPLC. Antiproliferative effects were measured in human breast, ovarian, and prostate cancer cell lines, with all three chiral forms exhibiting growth inhibition (GI) in the low to mid µM range for all cells. The natural enantiomer, and in some cases the racemate, gave significantly greater GI than the unnatural stereoisomer for estrogen receptor positive (ER(+)) versus ER(-) breast/ovarian cell lines as well as for androgen receptor positive (AR(+)) versus AR(-) prostate cancer cells. Surprisingly, differences between ER(+) and ER(-) cell lines were not altered by media estrogen conditions. These results suggest the antiproliferative mechanism of glyceollin I stereoisomers may be more complicated than strictly ER interactions.