28th Annual CBB Conference
"Expanding the Frontiers in Biocatalytic Science"
October 22, 2019
The University of Iowa
Iowa Memorial Union, Iowa City, IA
James A. Ankrum, Ph.D., Assistant Professor, Department of Biomedical Engineering, College of Engineering, The University of Iowa, Iowa City, IA
“The role of biomanufacturing decisions on the therapeutic function of Mesenchymal Stromal Cells (MSC)”
Mesenchymal stromal cells (MSCs) are being explored as a cell based therapy for the treatment of devastating immune conditions that are refractory to standard of care anti-inflammatory therapies. With both academic health centers and commercial entities launching clinical studies of the utility of MSCs to treat graft versus host disease, Crohn’s disease, and other chronic inflammatory conditions, the diversity of MSC products has grown substantially. However, while MSCs share common base characteristics, as a living therapeutic, they are responsive to stresses and cues within their local environment. Thus, while MSCs from different companies or labs may at first look similar, at the functional level, their phenotypes could be highly divergent. Decisions in the biomanufacturing process from selection of donors, outgrowth strategy, and cryopreservation technique all contribute the ultimate therapeutic utility of MSC therapies and need to be actively chosen to tailor the product to the clinical indication. In this talk we will examine the influence of individual bioprocessing decisions have on the immunomodulatory potential and resiliency of the resultant MSC product.
Laura Shawver, Ph.D., President and CEO, Synthorx, Inc., La Jolla, CA
“THOR-707 – An Engineered “Not Alpha” Interleukin 2 using an Expanded Genetic Alphabet”
Aldesleukin is a recombinant form of IL-2 approved for metastatic melanoma and renal cell carcinoma that induced complete, durable remissions in certain patients. Yet, its use is infrequent because of vascular leak syndrome, a severe dose-limiting adverse event stemming from the engagement of the high affinity IL-2 receptor (IL-2R) alpha chain in type 2 innate lymphoid cells, eosinophils and vascular endothelial cells. THOR-707 is a site-directed, singly pegylated form of IL-2 completely lacking IL-2R alpha chain engagement yet retaining normal binding to the intermediate affinity IL-2R beta-gamma signaling complex expressed by natural killer (NK) and CD8+ T tumor-killing cells. Site-directed pegylation is enabled by utilizing the Synthorx Expanded Genetic Alphabet. A new DNA base pair (X-Y) creates new distinct codons that code for novel amino acids. The X-Y pair is fully functional for replication, transcription and translation in E. coli. The new amino acid placed in the THOR-707 polypeptide chain allows for covalent attachment of a PEG via click chemistry. THOR-707 is now in a Phase 1/2 dose escalation and expansion clinical trial.
Jennifer Fiegel, Ph.D., Associate Professor, Department of Chemical and Biochemical Engineering, College of Engineering, The University of Iowa, Iowa City, IA
“Protein Coronas Formed in BALF and Serum Differentially Impact Nanoparticle Stability and Cell Uptake”
Foreign material entering the human body first interacts with the bodily fluids, where proteins and other biomolecules can readily adsorb to the materials’ surface. This thin biomolecular shell, often called the protein corona, directly interacts with cells and tissues in the body and helps determine the materials’ fate. Nonspecific protein adsorption to particles can cause particle aggregation, impair the ability of particles to cross biological barriers, and induce clearance as part of the foreign body response. The majority of protein corona studies have focused on interactions with blood proteins, which have good clinical relevance for injected therapeutics, but is not compositionally representative of all bodily fluids. Therefore, we have compared the impact of protein coronas formed in human serum and concentrated bronchoalveolar lavage fluid (BALF) on particle stability and cellular responses. Overall, studies conducted in serum are not strong predictors of particle interactions in BALF, as the responses in each fluid are distinct. We have further been developing zwitterionic polymer coatings as nonfouling coatings to inhibit protein adsorption. We have observed that polymer coatings help maintain nanoparticle stability in BALF or serum, likely due to strong hydration of the polymers and reduced protein adsorption. However, while pMPC-coated particles exposed to serum experienced 3-to-10 fold increase in particle uptake compared to bare particles, no differences were observed with particles exposed to BALF. This suggests that differences in protein composition or protein abundance between the two fluids results in a distinct protein corona that influences cell interactions.
Kevin D. Walker, Ph.D., Associate Professor, Department of Chemistry and Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI
“Biocatalysis of Paclitaxel Analogs and Hydroxy Amino Acids”
This presentation will summarize the biocatalysis of paclitaxel analogs and the repurposed application of an aminomutase to make hydroxy amino acids from epoxides. Paclitaxel (Taxol®) is a widely used chemotherapeutic drug with additional medical applications in drug-eluting stents as an anti-restenosis treatment. Paclitaxel is a structurally complex natural product with an excellent scaffold for designing analogs with improved pharmacological properties. Plant cell fermentation methods produce paclitaxel and larger quantities of its precursors 10-deacetylbaccatin III (10-DAB) and baccatin III (Bac). The complexity of the purported ~19-step paclitaxel biosynthetic pathway limits bioengineering the complete pathway in a chassis organism. However, the abundance of 10-DAB and Bacc and access to enzymes working on the latter parts of the pathway make a semi-biocatalytic approach to paclitaxel analogs possible. We have designed a short biocatalytic cascade capable that highlight its potential broader application toward making paclitaxel or value-added analogs of pharmacological interest. The success of this biocatalytic cascade centers on various enzymes co-opted from Taxus cell cultures, a baccatin III 3-amino-13-O-phenylpropanoyl CoA transferase (BAPT) and N-debenzoyltaxol-N-benzoyltransferase (NDTNBT); and CoA ligases from Bacillus, a (2R,3S)-phenylisoserinyl CoA ligase (PheAT) and from Rhodopseudomonas, a benzoate CoA ligase (BadA) to produce paclitaxel analogs. Here we show by proof-of-principle the biocatalysis of a paclitaxel analog, N-2-furanyl-N-debenzoylpaclitaxel, via a shortened assembly pathway.
Over several decades, many accounts on methylidene imidazolone (MIO)-dependent biocatalysts described how these enzymes add ammonia (at high concentrations from ammonium salts) across arylacrylate double bonds to make α- and, more recently, β-arylalanines. β-Amino acids are excellent building blocks of valuable bioactive compounds. These biocatalysis efforts have even drawn contributions from Nobel Laureate (Ben Feringa) to help dissect the dynamics of the catalysis among this family of enzymes. We diverge from this fixed program of aminating cinnamic acids and describe a first account of employing an MIO-aminomutase (TcPAM) from Taxus plants to convert previously untested epoxy acid substrates to bifunctional hydroxy amino acids. Arylserines were made more abundantly over the arylisoserines when styrylalanine was used instead of ammonium salts, to selectively transaminate the MIO group of TcPAM. This mild amine group delivery to the oxirane avoided nonenzymatic ring-opening of the epoxides.
Brent H. Shanks, Ph.D., Mike and Jean Steffenson Chair, Anson Marston Distinguished Professor in Engineering, Department of Chemical and Biological Engineering, Iowa State University, Ames, IA
“Developing Novel Chemicals through Bioprivileged Molecules”
Much of the effort in converting biomass to biobased chemicals has been driven by the opportunistic synthesis/isolation of a specific molecule or the retrosynthesis of target molecules. While these are reasonable approaches for a technology area in its infancy, the realization of viable biobased chemical development will require more systematic strategies that are robust in the face of realistic constraints. Importantly, these postulated strategies will dictate the research questions that will need to be addressed. Presented will be a new strategy of synthesizing “bioprivileged molecules,” which are biology-derived chemical species that can be readily converted to a diversity of chemical products including drop-in replacements and novel species while be discussed. The dual potential for bioprivileged molecule can help create value from biomass since innovative bioproducts represents a powerful driver for the development of biobased chemicals beyond just replacing fossil carbon with renewable carbon. Ongoing efforts involving combining synthetic routes with a computational framework will be discussed and several examples presented.
Pratik Rajesh Chheda, Ph.D. candidate, Department of Pharmaceutical Sciences and Experimental Therapeutics, Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA
"Allosteric inhibitors of apicoplast DNA polymerase: New antimalarials that bind a novel allosteric pocket"
Plasmodium spp. are the causative agents of malaria, killing nearly 600,000 people each year. Resistance of Plasmodium to current therapies accentuates the need for new drugs that target novel aspects of the parasite’s biology. Parasites in the phylum Apicocomplexa have an unusual organelle; apicoplast, which participates in the biosynthesis of fatty acids, heme, iron-sulfur clusters, and isoprenoids. Any defect in apicoplast metabolism or its failure to replicate leads to the death of the parasite. Additionally, lack of a human counterpart makes apicoplast a promising drug target. The apicoplast genome is replicated by select DNA replication enzymes, of which apicoplast DNA polymerase (apPOL) is unique to the parasite. The apPOLs from P. falciparum and P. vivax have 84% homology, while the most similar human DNA polymerases are the lesion bypass polymerases theta and nu (23 and 22% identity, respectively). Towards identifying inhibitors of apPOL, a high throughput screen of 400 compounds from the Open Malaria Box provided by (Medicines for Malaria Venture (MMV) identified a sub-micromolar inhibitor of apPOL. Our studies indicate that MMV666123 is specific for apPOL, with no inhibition of human DNA Pol or E. coli DNA Pol I. Additionally, being a malaria-box compound substantiates the anti-malarial activity of MMV666123. Presented here are our current design, synthesis, crystallographic, and in vitro evaluation efforts toward understanding the structural requirements of MMV666123 for inhibition of apPOL, identifying the binding site and designing more potent and drug-like apPOL inhibitor derivatives.
Danielle T. Webb, Ph.D. candidate, Department of Civil and Environmental Engineering, Environmental Engineering and Sciences, College of Engineering, University of Iowa, Iowa City, IA
“Sorption of Neonicotinoid Insecticides and their Metabolites to Granular Activated Carbon during Drinking Water Treatment: Implications for Human Exposure, Treatment, and Biofilm Transformations”
Neonicotinoid pesticides are the most widely used insecticides in the world. The widespread application of neonicotinoids has led to their proliferation in waters across the U.S., including those used as drinking water sources. Although neonicotinoids exhibit relatively low toxicity towards mammals, formation of toxic metabolites is an exposure concern. Our prior studies identified granular activated carbon (GAC) as a method for neonicotinoid removal from drinking water. The objective of the present study is to determine if imidacloprid and its mammalian toxic metabolite, desnitro-imidacloprid, adsorbed to black carbon can be mineralized into less toxic products by coupling the sorptive and redox capabilities of black carbon with biofilm metabolism. Desorption tests conducted using five-year-old used GAC from the Iowa City DWTP to determine the likelihood of residual neonicotinoid desorption and provide further evidence that neonicotioids are removed from drinking water by the GAC filter. Imidacloprid adsorbs to GAC and desorbs in solvents of intermediate polarity (despite high polarity / solubility), but desnitro imidacloprid was not desorbed in any condition, indicating that this metabolite is not adsorbed to GAC or may be degraded following initial sorption. To probe possible imidacloprid and desnitro imidacloprid degradation on black carbon associated with biofilms, we are conducting experiments with biofilm coated corn biochar. Soil microorganisms were extracted grown on corn biochar until a biofilm was formed. Batch experiments show that imidacloprid is rapidly adsorbed to the spent IC DWTP GAC. Microorganisms desorbed from the spent GAC surface can degrade imidacloprid, forming the mammalian toxic desnitro imidacloprid metabolite, indicating the adsorbed neonicotinoid may be degraded on the black carbon surface. Similarly, imidacloprid is removed from solution in the presence of the bioaugmented corn biochar, though to a lesser extent than the GAC. Nevertheless, the bioaugmented biochar showed more rapid imidacloprid degradation and toxic metabolite formation than the GAC system. These results show that environmentally relevant microorganisms associated with black carbon are capable of degrading neonicotinoids into mammalian toxic metabolites. Sacrificial batch experiments are underway to determine the rates and possible mechanisms behind these biotransformation reactions. Further experiments will also elucidate whether the mammalian toxic metabolite is a dead-end product or if the metabolite can be further transformed by the bioaugmented black carbons. If these insect and mammalian toxic compounds can be further degraded, bioaugmented black carbon may be a promising method to remove water in natural and engineered systems.
Nathan Delvaux, Ph.D. candidate, Department of Pharmaceutical Sciences and Experimental Therapeutics, Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City, IA
“Overcoming the Cell Membrane Barrier in Non-Viral Gene Delivery with Modular Pore-Forming Peptide Conjugates”
Although significant advancements in the field of non-viral gene therapy have been made towards the design of polymers that can effectively stabilize DNA in biological systems, bypassing the cell membrane remains a challenge for many vectors. Active targeting of DNA nanoparticles with ligands directed towards specific endocytic cell surface receptors can be an effective approach to this obstacle; however, receptor-mediated uptake faces its own set of problems. Receptor endocytosis is saturable, limiting the maximal rate of migration across the cell membrane, as well as generally dependent on nanoparticle size and surface charge, potentially requiring optimization of the nanoparticle for each receptor. As non-specific cell entry alternatives, cell-penetrating peptides have found use in non-viral gene delivery. Melittin is a peptide derived from bee venom that forms pores in lipid bilayers. Several melittin analogues have been designed with increased pore-forming potency and decreased overall charge, facilitating control over nanoparticle surface charge. To capitalize on the pore-forming characteristics of these analogues, we conjugated them to a polyacridine DNA binding peptide with a variety of bioconjugate linkers to form DNA nanoparticles with inherent cell-penetrating properties. These conjugates were tested in vitro for the ability to transfect HepG2 and primary mouse hepatocytes using luciferase expression as a readout. Direct conjugation of the melittin analogues to the polyacridine peptide resulted in luciferase expression almost equivalent to polyethylenimine, the gold standard for in vitro gene transfection. Thus, these pore-forming peptides can be used to facilitate cell entry and transfection, and further work is being conducted on translating the systems to in vivo models.