Technologies

Drug Discovery : Drug production & design

Technologies

Enhanced Endotoxin Detection: New Advantages in Liquid Crystal Assays for Gram-Negative Pathogens

UW–Madison researchers have now demonstrated enhanced endotoxin detection in the presence of masking agents in their previous liquid crystal system.

Unlike the LAL assay, the LC-based method does not suffer from LER or any loss of sensitivity due to the presence of cations (e.g., Ca2+ or Mg2+), buffers (e.g., citrate), surfactants (e.g., SDS), chelating agents (e.g., EDTA), proteins or nucleic acids (e.g., DNA or RNA). Thus, the LC-based method provides faster and cheaper detection of endotoxin when compared to existing methods, such as the LAL assay.
P160072WO01

New System for Producing Fungal Secondary Metabolites

UW–Madison researchers have developed a new system for producing fungal secondary metabolites using test plasmids and a genetically modified strain of Aspergillus nidulans (TPMW2.3). The strain begins producing secondary metabolites when a gene promoter in the plasmid is triggered by culture conditions. This allows researchers to induce or repress production.
P150029US02

Yeast-Based Intein Platform for Drug Production

UW–Madison researchers have engineered non-self-cleaving Mxe GyrA inteins shown to significantly improve the production of fusion proteins from Saccharomyces cerevisiae. The novel inteins were developed through directed evolution, and they enhance fusion protein display (up to 3x) and secretion levels (up to 30x) compared to the wild type intein. The new yeast-based platform provides a robust alternative to bacterial intein expression systems.
P150056US02

Small Molecule Catalysts of Oxidative Protein Folding

UW–Madison researchers have synthesized novel catalysts for use in the production of high value recombinant proteins including antibodies and other biologics. The small molecules contain binding features that mimic PDI and are nearly optimal for catalyzing disulfide bond formation. Namely, pendant “R” groups provide affinity for the hydrophobic substrate (unfolded protein) but not the product (folded protein), enabling the catalysts to operate like a true enzyme.

The novel catalysts and method can be used in vivo or in vitro.
P150010US02

Peptide Mimics Last Longer, Target Protein-Protein Interactions

UW–Madison researchers have developed modified Z-domain peptides that last longer in vivo while retaining strong binding properties. The researchers removed one of the helices and stabilized the remaining two with a disulfide bond. They substituted some residues with alpha and beta amino acid residues; the latter helps resist degradation by proteolytic enzymes.

The α/β-peptide mimics (or foldamers) can be tailored to target a variety of different proteins and protein-protein interactions. Given their small size (39 amino acids) relative to full-length Z-domains (59 amino acids), the new peptide mimics are easier to synthesize and modify.
P140148US02

Easier, More Flexible Synthesis of Therapeutic and Promising Compounds

UW–Madison researchers have developed efficient processes to prepare asymmetric, heteroatom-bearing stereotriads and tetrads via allene oxidation. The number and type of heteroatoms stereoselectively introduced into the hydrocarbon chain or ring is flexible, and the methods allow for the transfer of chirality to three new carbon-heteroatom bonds. The new triads and tetrads may be incorporated into biologically active molecules, including modified aminoglycoside and neuraminidase inhibitors.

Any reaction products can be further oxidized, reduced or hydrolyzed to form other compounds and intermediates, notably synthetic motifs containing three contiguous carbon-heteroatom bonds. Bicyclic methylene aziridines can be formed and altered to provide therapeutically-promising N,N-aminals in a one pot reaction.
P110299US02

Safer Influenza Vaccine from Replication-Knock Out Virus

UW–Madison researchers have developed methods for generating novel influenza vaccines that can elicit robust immune response without the risk of symptoms or genetic reversion.

The recombinant influenza A virus is made to lack the genetic sequence necessary for replication in normal host cells. Specifically, the coding region of PB2 viral RNA can be deleted, disrupted or replaced with a harmless reporter gene useful for tracing during cell culture preparation. With the mutant gene segment, the virus is ‘biologically contained’—capable of replicating only in specially developed PB2-expressing cells.
P110003US02

VeA, a Global Regulator of Secondary Metabolism, Can Increase Production of Secondary Metabolites

UW-Madison researchers now have identified another global regulator of secondary metabolism, called VeA.  VeA is a conserved protein that interacts with LaeA in an as yet unknown mechanism.  Overexpression of veA upregulates secondary metabolism in A. flavus to a greater degree than overexpression of laeA.  This gene could be used to increase the production of important natural products, including novel products with medicinal value.
P09056US02

Better Living Through Peptides; Improved Approach to HIV Therapy

UW–Madison researchers have developed a new method to fabricate combination alpha and beta peptides for the treatment of HIV and other disorders.

Because beta amino acids are non-natural, they are resistant to proteolysis. Substituting beta amino acids for some of the alpha amino acids in fusion inhibitors increases resistance to proteolysis with little effect on efficacy. The resulting α/β-peptide combination lasts longer and is less likely to cause drug resistance, leading to improved outcomes for HIV patients. This technique is also applicable to other peptide-based therapeutics.
P08414US03

Biosynthetic Gene Cluster That Produces the Natural Antibiotics Platensimycin and Platencin

UW-Madison researchers have isolated and sequenced the gene clusters encoding the biosynthetic machineries for platensimycin and platencin production. The cloned pathways provide a means of synthesizing a library of novel platensimycin and platencin analogs. They also provide a means of producing large amounts of platensimycin and platencin by microbial fermentation.
P07515US

Efficient Generation of Influenza Virus with Adenoviral Vectors

UW-Madison researchers now have modified the reverse genetics approach to make it possible to introduce viral genetic information into more cell types. Instead of multiple plasmids, adenoviral vectors are used to introduce the viral genes and other sequences needed for replication and transcription into the cells. Because these vectors are highly efficient at transferring genes, they allow vaccine viruses to be generated in a greater variety of cell lines than the plasmids used in the original system.
P06444US

New Capreomycin Derivatives for the Treatment of Multidrug-Resistant Tuberculosis

UW-Madison researchers have isolated and sequenced the capreomycin gene cluster from the bacteria S. mutabillis subspecies capreolus. They transformed the genetically tractable bacteria Streptomyces lividans, which do not naturally produce capreomycin, by using a vector containing this gene cluster. The transformed bacteria were then able to produce capreomycin, making metabolic engineering of capreomycin and capreomycin derivatives possible for the first time. 
 
P07320US

Universal Nucleotidyltransferases Expand Sugar Substrate Families Available for Glycorandomization

The UW-Madison researcher has discovered that the nucleotidyltransferase protein RmlA is capable of utilizing all naturally occurring NTPs.  He used X-ray crystallographic protein structures to design variations of RmlA with mutations at a specific amino acid in the active site of the enzyme.  These mutants have an increased purine/pyrimidine bias in NTP substrate specificity.  They can be used in the production of diverse purine-based sugar nucleotide libraries.
P07375US

Type 1 Polyketide Synthase Extender Units

UW-Madison researchers have discovered two novel extender units that can be used in the creation of new polyketide derivatives. This invention also includes the enzymes needed to form the extender units and the PKSs needed to incorporate the units into the polyketide backbone.

One or both of the extender units can be introduced into a desired polyketide to create new structural derivatives. For example, the PKSs involved in the production of polyketides such as erythromycin, rifamycin, rapamycin, FK520 or zwittermicin can be engineered to incorporate these units. The resulting polyketide derivatives can then be used as bioactive molecules or as lead compounds for further modification.
P06464US

Method for Assessing 3-D Crystal Structures from in Situ Digital Images

UW–Madison researchers now have developed a 3-D image analysis method to automatically extract information from developing crystal populations. The method is based on object recognition and can extract crystal size and shape distributions from low-quality in situ images in which the particles are overlapping, out of focus, randomly oriented or poorly illuminated. 

The method segments, or separates, objects from the background portion of the image. It uses a 3-D wireframe model to more accurately segment each particle.  Once the objects are removed from the image, shape, size, orientation and other relevant information can be determined for individual particles.  This information then can be used to control processes associated with the crystals, maximizing manufacturing efficiency.
P06449US

Improved Asymmetric Hydroformylation of Therapeutics Using Novel Bisphosphines

UW–Madison researchers have developed a method for using a new class of bisphosphines as ligands in catalytic transformations of alkenes. The method involves subjecting an alkene to an asymmetric reaction such as hydroformylation, which is carried out in the presence of a bisphosphine ligand catalyst. The bisphosphine ligand is synthesized according to the formula depicted in the figure, or as the opposite enantiomer of the formula. Using this process, asymmetric hydroformylation reactions resulting in 90 percent or greater of the desired enantiomer are possible. The ligand structure has a modular design, which can be varied systematically to obtain the best results for any given alkene substrate, and can be synthesized efficiently from readily available raw materials.
P04447US

Neoglycorandomization and Digitoxin Analogs

UW-Madison researchers have now developed a different approach to glycosylating natural compounds to create a large library of diverse compounds for high throughput screening. This approach, neoglycorandomization, uses a universal chemical glycosylation method that employs reducing sugars and requires no protection or activation. It is based on the chemoselective formation of glycosidic bonds between the reducing sugars and an aglycon with a secondary alkoxyamine.

To form a neoglycoside, the alkoxyamine-containing aglycon is reacted with at least one reducing sugar selected from the group consisting of L-sugars, D-sugars, amino-sugars, deoxy-sugars, dideoxy-sugars, glucose epimers, substituted sugars, uronic acids, and oligosaccharides.
P04455US

Microfluidics Platform and Method That Mimic the Cellular Environment

UW-Madison researchers have developed a microfluidics-based platform for mimicking the environment within a cell. This model environment is simpler than a cell, yet captures the basic characteristics of the cellular nano-environment, including charge, crowding, water content and structure. It has many advantages over current systems, including the fact it uses much less protein, can detect weaker interactions and requires less time for experiments.

The platform includes a microfluidic device that contains a chamber. At least one hydrogel post is positioned within the chamber. Each post may contain a different density of polymers or a different cross-linker to simulate various crowding or caging effects. A solution containing proteins of interest is introduced into the chamber and the proteins diffuse into the hydrogel posts. The interactions between the proteins are then observed inside the posts.
P04153US

High Titer Recombinant Influenza Viruses for Vaccines and Gene Therapy

A UW-Madison researcher has developed an efficient technique and system for producing high titer influenza A virus in vertebrate cells in the absence of helper virus. The technology takes advantage of a reverse genetics system created by Dr. Kawaoka that allows efficient production of influenza virus for vaccines and gene therapy applications (see WARF reference number P03252US). In the technology described here, the inventor developed a new set of plasmids for use with the reverse genetics system. The plasmids contain cDNAs from a high titer influenza virus isolate; the promoter for RNA polymerase I or RNA polymerase II; and the terminator sequence for RNA polymerase I. When these constructs are transfected into host cells, the cells consistently generate high yields of infectious influenza particles.
P03322US

Recombinant Influenza Vectors with a Pol II Promoter and Ribozymes for Vaccines and Gene Therapy

UW-Madison resarchers have developed an improved reverse genetics system for producing influenza virus in vertebrate cells in the absence of helper virus. The system starts with a set of plasmids containing viral genome cDNAs flanked by ribozymes. Each plasmid carries a cDNA for one of the eight influenza A viral RNA segments. On each plasmid, the cDNA sits between a polymerase II promoter and a poly-A addition signal at the 3-prime end. When the plasmids are transfected into a vertebrate cell, the host cell’s RNA polymerase II transcribes each construct into a capped viral RNA with a proper poly-A tail. The flanking ribozyme RNAs then undergo site-specific, self-catalyzed cleavage to precisely trim each end of the viral RNA. Next, viral polymerase, which is provided by a protein expression plasmid, acts upon the viral RNAs, resulting in replication and mRNA synthesis. This system does not require a helper virus and allows the creation of transfectants with mutations in any gene segment.
P03252US

Methods for Engineering Influenza Viruses to Carry Defined Mutations

A UW-Madison reseasrcher has developed a method of preparing viruses with defined mutations. The method uses a reverse genetics system created by Dr. Kawaoka (see WARF reference number P03252US), which consists of plasmids containing the promoter for RNA polymerase I or RNA polymerase II; a cDNA for each of the influenza virus RNA segments; and the terminator for RNA polymerase I. These plasmids are transfected into cells along with protein expression plasmids to generate live virus.

The technology featured here allows mutations to be introduced into any of the cDNAs to generate viruses with defined mutations. For example, viruses lacking the NB protein -- an integral membrane glycoprotein that promotes efficient replication in vivo -- were created with this method. The NB knockout viruses replicated as efficiently as wild type virus in cell culture, but were attenuated in mice.
P03251US

A Global Regulator of Secondary Metabolite Biosynthesis in Fungi

UW-Madison researchers have developed a global regulator of secondary metabolism, called LaeA, in fungi. LaeA was identified in the fungus Aspergillus nidulans and subsequently in A. fumigatus. LaeA exerts global control over gene clusters for secondary metabolite biosynthesis, including the penicillin and lovastatin clusters.

The researchers developed methods for increasing or decreasing the production of secondary metabolites in transformed organisms by modulating the expression of laeA in these organisms. For example, overexpression of the gene laeA greatly increases penicillin production in A. nidulans and lovastatin production in A. terreus. Deletion of laeA in A. fumigatus eliminates the production of gliotoxin and other secondary metabolites, and decreases the virulence of this human pathogen.

LaeA could also provide a novel tool for identifying new secondary metabolites. For example, microarray analysis of deletion and overexpression in laeA mutants has identified several secondary metabolite gene clusters in A. nidulans.
P02379US

Use of Glycorandomization to Produce Novel Glycosylated Products for Drug Discovery

A UW-Madison researcher has developed combinatorial methods for rapidly generating a diverse library of novel glycosylated compounds for use in drug discovery. Glycorandomization is a chemoenzymatic strategy that overcomes the limitations in natural product derivatization associated with both chemistry-based approaches and in vivo engineering.

In the methods, at least one aglycon and a pool of nucleoside diphosphate (NDP)-sugars are incubated with a glycosyltransferase enzyme to produce glycorandomized structures. The pool of NDP-sugars may include difficult-to-produce or never-before-produced sugars, which can now be made easily and efficiently through a one-step nucleotidyltransferase-catalyzed conversion developed by the inventors. The glycorandomized structures may be incubated with the sugars and glycosyltransferase multiple times to generate additional compounds. The glycorandomized structures may also be incubated with at least one chemoselectively ligatable moiety to generate chemoselectively-ligated compounds.
P04020US

Replicating Influenza Mutants with Reduced Sialidase Activity

A UW-Madison researcher has developed mutant cells useful for propagating influenza A virus mutants with reduced sialidase activity. The mutant cells have decreased levels of sialic acid and sialic acid-containing cell receptors as compared to wild type cells.

To generate the mutant cells, the researcher started with cells that supported the growth of influenza virus. These cells were incubated with a growth-inhibiting agent. Surviving cell colonies were cloned and infected with influenza virus variants with known sialic acid receptor-linkage specificity. A small percentage of cells, designated as MaKS cells, continued to grow without any evidence of virus production, and showed lowered levels of sialic acid-containing receptors. Two influenza virus variants that were able to grow well in these cells after several rounds of selection showed large deletions in the gene for NA as compared to the parent viruses.
P01273US

Methods for Synthesizing Natural and “Unnatural” UDP- and TDP-Nucleotides

UW-Madison researchers have developed methods of modifying nucleotidylyltransferases to vary their substrate specificity in a directed manner. The researchers discovered a nucleotidylyltransferase from Salmonella enterica, called LT2 alpha-D-glucopyranosyl phosphate thymidylyltransferase (Ep), that displays unexpected promiscuity toward both its nucleotide triphosphate (NTP) and sugar phosphate substrates. Ep can convert a wide variety of phosphates to their corresponding dTDP- and UDP-nucleotide sugars.

The researchers used the 3-D structure and other molecular details of Ep substrate recognition to design nucleotidylyltransferase mutants with varying substrate specificity. The nucleotidylyltransferases are preferably mutated at one or more amino acids in their active sites, divalent cation binding sites and/or auxiliary sites.
P04021US

Recombinant Influenza Viruses for Vaccines and Gene Therapy

UW-Madison researchers have now developed a method to efficiently generate fully constructed, artificial influenza virus. The method involves transforming a host cell with 10 DNA plasmids, each containing a DNA copy of a segment from the influenza viral genome. Nine of the plasmids express proteins needed for viral replication and assembly, while the tenth contains a transgene inserted between an RNA pol I promoter and a terminator, for gene expression.
P99264US

Microfabricated Microbial Growth Assay Method and Apparatus

UW-Madison researchers have developed a microbial growth assay that can be used to rapidly screen substances for their effect on cell growth. The invention includes microbial growth assay wells containing 30 microliters or less of liquid. Two electrodes are placed in each well to measure the capacitance across the liquid. Capacitance or resistance in each well is used to determine the extent of bacterial growth.

This technology represents a significant improvement over commercially available growth assays, most of which rely on flow-through chambers, large sample sizes and long incubation periods. It should provide an economical way to screen potential antibiotics on a massive scale.
P00224US

Increasing Secondary Metabolite Production in Fungus for Drug Development

The researchers now have developed a set of genetically modified Aspergillus nidulans strains with increased secondary metabolite production. The strains overexpress one or both of the global regulators previously implicated in secondary metabolite production. Moreover, naturally occurring gene clusters in the strains are deleted to reduce competition for the desired genes.
P130203US01