From the Bench to the Pharmacy: Protecting Innovation During Vaccine Development and Commercialization.

Patentable inventions may be made during the development of a vaccine, and patents on such inventions can help to protect the vaccine from competition. This chapter introduces several patent law concepts, including patent eligible subject matter, written description, enablement, novelty, and nonobviousness, by following a hypothetical vaccine development timeline that begins with the discovery of a previously unknown virus and ends with the commercial launch of a vaccine against the virus. Regulatory exclusivity, freedom to operate, and lifecycle management considerations are also discussed.

Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins.

Elucidating the structures of membrane proteins is essential to our understanding of disease states and a critical component in the rational design of drugs. Structural characterization of a membrane protein begins with its detergent solubilization from the lipid bilayer and its purification within a functionally stable protein-detergent complex (PDC). Crystallization of the PDC typically occurs by changing the solution environment to decrease solubility and promote interactions between exposed hydrophilic surface residues. As membrane proteins have been observed to form crystals close to the phase separation boundaries of the detergent used to form the PDC, knowledge of these boundaries under different chemical conditions provides a foundation to rationally design crystallization screens. We have carried out dye-based detergent phase partitioning studies using different combinations of 10 polyethylene glycols (PEG), 11 salts, and 11 detergents to generate a significant amount of chemically diverse phase boundary data. The resulting curves were used to guide the formulation of a 1536-cocktail crystallization screen for membrane proteins. We are making both the experimentally derived phase boundary data and the 1536 membrane screen available through the high-throughput crystallization facility located at the Hauptman-Woodward Institute. The phase boundary data have been packaged into an interactive Excel spreadsheet that allows investigators to formulate grid screens near a given phase boundary for a particular detergent. The 1536 membrane screen has been applied to 12 membrane proteins of unknown structures supplied by the structural genomics and structural biology communities, with crystallization leads for 10/12 samples and verification of one crystal using X-ray diffraction.

His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.

Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.

Purification, crystallization and preliminary X-ray diffraction analysis of pathogen-inducible oxygenase (PIOX) from Oryza sativa.

Pathogen-inducible oxygenase (PIOX) is a heme-containing membrane-associated protein found in monocotyledon and dicotyledon plants that utilizes molecular oxygen to convert polyunsaturated fatty acids into their corresponding 2R-hydroperoxides. PIOX is a member of a larger family of fatty-acid alpha-dioxygenases that includes the mammalian cyclooxygenase enzymes cyclooxygenase 1 and 2 (COX-1 and COX-2). Single crystals of PIOX from rice (Oryza sativa) have been grown from MPD using recombinant protein expressed in Escherichia coli and subsequently extracted utilizing decyl maltoside as the solubilizing detergent. Crystals diffract to 3.0 angstroms resolution using a rotating-anode generator and R-AXIS IV detector, and belong to space group P1. Based on the Matthews coefficient and self-rotation function analyses, there are presumed to be four molecules in the asymmetric unit related by noncrystallographic 222 symmetry.