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Assay for Integration Inhibitors Using Pre-Integration Complexes (S97022B.pdf)

Inventors
Frederic Bushman, Tal Kafri, Mark Hansen

Applications
Infection, HIV, Drug Discovery
A screen for viral integrase inhibitors

The invention describes an assay for viral integration inhibitors using pre-integration complexes (PICs). A cell line expressing high titers of HIV-based vectors has been developed. The vector particles produced have been used to generate PICs containing HIV integrase and other HIV proteins. This technique provides a source of PICs that are much safer to handle than live virus derived from Molt IIIB. Integrase is one of three enzymes encoded by HIV. Inhibitors of reverse transcriptase and protease have been shown to be effective in treatment of HIV infection. Similarly integrase is a promising, non-toxic target for drug therapy since there are no similar proteins known to be important for normal cell function. Despite extensive efforts, no clinically useful integrase inhibitors have been developed. In this invention, vector-based PICs were used to devise a microtiter plate-based assay for integration directed by PICs. The response to inhibitors by PICs is more authentic than purified recombinant integrase making them attractive for use in screens for inhibitors. Purified integrase protein can be inhibited by diverse compounds, complicating the identification of promising leads. However, assays with PICs are more resistant to inhibition and display a response that more closely matches the response of virus in vivo. Microtiter assay results can be obtained by PCR and gel analysis or use of the TaqMan gene quantification method..

References
Nat Biotechnol 17(6):578-82 (June 1999)

Patent Status:

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Method of Rapidly Identifying Inhibitors of Topoisomerase DNA Religation (S00014.pdf)

Inventors
Frederic Bushman and Young Hwang

Applications
Infection, Oncology, Antibacterials, Antifungal, Antiviral, Drug Discovery and Development
High throughput screen for compounds that modulate topoisomerase activity

Topoisomerases play a central role in nucleic acid metabolism and are important in a variety of biological processes related to cell division, DNA replication, chromosome structure and gene expression. Compounds that act as effective cellular inhibitors of topoisomerases are expected to act as cytotoxic agents through the disruption of the normal cell division process. Such compounds can be effective and selective antibacterial, antifungal and antiviral agents. And because cell division is an important characteristic of cancers and other proliferative diseases, agents that inhibit topoisomerases are also useful as antineoplastic agents. The invention provides methods for identifying topoisomerase activity modulators in both solid and liquid phase formats. High throughput screening methods, compositions, kits and integrated systems for performing the assays are provided. The invention represents an improvement over existing technology in several ways. Through the use of different nucleic acid substrates, the assays can be adapted to screen for inhibitors of numerous different classes of topoisomerase enzymes and assay multiple different topoisomerase enzymes in a single reaction, thus enhancing throughput. The assays can be run in a parallel fashion such that multiple different topoisomerase enzymes and/or modulators are assayed simultaneously. The assays can be performed in the liquid or solid phase and each of the formats is readily amenable for automation and high throughput screening. Further, the assays are are extremely sensitive relative to previous assay formats and only minimal quantities of reagents are required..

References
Nucleic Acids Res 28(24):4884-92 (December 2000)

Patent Status:

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Alpha-Complementation Viral Fusion Assay (S02016A.pdf)

Inventors
Carsten Muenk, Anne Holland and Nathaniel Landau

Applications
Antiviral Drug Discovery, Infection
A rapid and quantitative assay to detect HIV and other viral mediated fusion to screen for inhibitors

Entry is an attractive and promising step in HIV-1 replication to target with small molecules. Unlike current drugs that act mainly at reverse transcription or virus maturation, entry inhibitors protect cells from becoming infected and do not need to penetrate the cell. HIV-1 entry is mediated by the interaction of the viral glycoprotein with CD4 and either of the CC chemokine receptors, CCR5 or CXCR4. Current methods for high throughput screening for coreceptor antagonists have advantages and disadvantages. These assays include ligand-receptor competition assays, or cell based assays for cell-cell fusion, virus entry, or single cycle reporter virus infection. None of the assays directly measures the fusion event , but rather measure a secondary result of fusion. As a result, they pick up numerous false positives that act on irrelevant targets. The alpha complementation fusion assay described in the invention provides a means to directly detect envelope glycoprotein-mediated fusion. Moreover, the assay is much simpler and more rapid than any other method. Also, importantly, the assay does not use live virus and therefore can be performed without biohazard containment. The assay is done in 96 well microtiter plates and is easily adaptable to 384 or 1024 well format. The assay is scalable and easily adapted to high throughput screening. In addition, the assay can be adapted to any enveloped virus..

References
Virology 319(2):343-352 (February 2004)

Patent Status:
U.S. Patent Application Serial No. 10/729,069 filed December 4, 2003

License Terms:
Non-exclusive licenses available

Reference_Number: S02016A
Contact: Mike White, Ph.D., CLP o Director, OTM o 858.453.4100 x1703 o mwhite@salk.edu

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Sequence and Method for Increasing Protein Expression in Cellular Expression Systems (S99046.pdf)

Inventors
Martin Latterich and Kendall Powell

Applications
Infection, Protein Production, Antibiotics, Drug Discovery
A bacterial expression system to increase biomass and cellular protein production

There is a continuing need for improved cellular expression systems that produce higher yields of heterologous proteins and can be subjected to relatively easy production and purification techniques. The invention relates to a bacterial expression system which includes a novel protein, Vff2p (vesicular fusion factor 2), that, when expressed in a host cell, increases cellular biomass and cellular protein production and secretion. The invention also relates to a polynucleotide, VFF2, encoding the novel protein, methods for using the polynucleotide and encoded protein to increase cellular biomass and cellular protein production and secretion, methods of screening for antibiotics, as well as a genetically altered mutant cell strain with enhanced production and secretion of cellular and heterologous proteins..

References
No publications to date

Patent Status:

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Modulation of Mevalonate-Independent Isoprenoid Biosynthetic Pathway (S00010B.pdf)

Inventors
Joseph Noel, Marianne Bowman and Stephane Richard

Applications
Infection, Drug Discovery, Natural Products, Agriculture, Plant Biology
Templates for the design of novel antibacterial and antiparasitic drugs

The invention provides the three dimensional structure of the enzyme 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthase, a member of the cytidyltransferase family of enzymes. CDP-ME is a critical intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis in a number of prokaryotic organisms, in algae, in the plastids of plants and in the malaria parasite. Since vertebrates synthesize isoprenoid precursors using a mevalonate pathway, CDP-ME synthase and other enzymes of the mevalonate-independent pathway for isoprenoid production represent attractive targets for the structure-based design of selective antibacterial, herbicidal and antimalarial drugs. The invention provides methods for screening for compounds that inhibit enzymes of the mevalonate-independent pathway and pharmaceutical compositions and antibacterial formulations thereof. Further provided are methods of inhibiting the enzymes of the pathway and bacterial terpenoid synthesis and methods for treating a subject suffering from a bacterial infection..

References
Nat Struct Biol 2001 Jul: 8(7):641-8
Published PCT Application WO01/083769

Patent Status:
U.S. Application pending

License Terms:
Exclusive or Nonexclusive licenses available

Reference_Number: S00010B
Contact: Mike White, Ph.D., CLP o Director, OTM o 858.453.4100 x1703 o mwhite@salk.edu

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Methods of Inhibiting Poxvirus Growth (S05006.pdf)

Inventors
Bart Sefton, Roberta Schulte

Applications
Infection, Drug Discovery and Development, Poxvirus treatments
Targets for the development of anti-smallpox drugs

A drug that blocked poxvirus growth would be valuable if smallpox virus were to be used as a weapon. We have tested the ability of inhibitors of poxvirus protein kinases to inhibit poxvirus growth. Poxviruses encode two essential protein kinases, B1 and F10. We identified a number of small molecules that inhibited these protein kinases in vitro. One of these compounds inhibited the growth of vaccinia virus strongly. The drug reduced virus growth by 99% at a concentration of approximately 35 µM. This effect is apparently specific. This compound had no effect on the growth of the unrelated vesicular stomatitis virus or on the growth of human cells at a concentration that inhibited vaccinia growth by more than 90%. This compound shows promise for further development into an effective anti-small pox drug. In addition, the two essential viral protein kinases represent useful targets for the development of additional anti-poxvirus drugs..

References
No publications to date

Patent Status:
U.S. Patent Application filed October 10, 2005

License Terms:
Exclusive or Non-Exclusive Licenses Available

Reference_Number: S05006
Contact: Dave Odelson, Ph.D. o Senior Licensing Executive o 858.453.4100 x1223 o dodelson@salk.edu

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A Novel Antitoxin and Vaccine Platform Based on Nodavirus VLPS (S07001.pdf)

Inventors
John Young, Anette Schneemann, Marianne Manchester, Kelly Dryden, John Harlett, Darly Manayani, Godfrey Rainey, Vilay Reddy, Marc Saladi, Heather Scobie, Diane Thomas, Mark Yeager

Applications
Infection, Vaccine, Drug Discovery and Development
A novel, chimeric virus-like particle (VLP) capable of multivalent display of PA shows significant advantages over existing monovalent PA immunogens for anthrax toxin. Studies indicate VLP-PA complex may eliminate the need for lengthy immunization schedules by providing immunity after a single injection, and has potential to generate effective vaccines against other pathogens, including combination vaccines immunizing against multiple toxins.

The recent use of Bacillus anthracis as a bioweapon has stimulated the search for novel anthrax antitoxins and vaccines that act rapidly with minimal adverse effects. B. anthracis produces a toxin composed of the receptor-binding moiety protective antigen (PA) and the enzymatic moieties edema factor and lethal factor. Current anthrax-vaccine strategies are based on targeting PA as the critical immunogen; however, these vaccines are molecularly ill-defined, can cause adverse reactions, and are administered in a lengthy immunization schedule (6 doses over 18 months). The development of a well-characterized vaccine that induces rapid immunity after a single injection remains an important goal. We have developed a novel, chimeric virus-like particle (VLP) capable of multivalent display of PA. In animal studies, rats (5/group) were immunized once with VLP-PA complex, PA alone, or VLP alone as a control. After four weeks the animals were challenged with anthrax lethal toxin. All rats that were immunized with the VLP-PA complex survived, whereas all other animals died. These results illustrate that this recombinant VLP platform, capable of multivalent display of PA, yields a significant advantage over monovalent, soluble PA as an immunogen for anthrax toxin, and represents a novel and highly effective reagent for protection against anthrax. An additional advantage of this VLP platform is its potential to be used to generate effective vaccines against pathogens other than anthrax, through the use of fusion protein technology to display other immunogens on the VLP. These pathogens include category A, B, and C agents, some of which represent major bioterrorism threats, such as ricin and botulinum neurotoxins. Further, by taking advantage of the multivalent display capability of the VLPs, it is possible that this approach could be used to generate combination vaccines, which could simultaneously immunize against anthrax, ricin, and botulinum toxins while circumventing the need for multiple dosings of immunogens..

References
No publications to date

Patent Status:
U.S. Patent Application Pending

License Terms:
Non-exclusive and Exclusive by Field of Use Licenses Negotiable

Reference_Number: S07001
Contact: Dave Odelson, Ph.D. o Senior Licensing Executive o 858.453.4100 x1223 o dodelson@salk.edu

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Non-Nucleoside Reverse Transcriptase Inhibitors (S07014.pdf)

Inventors
Daniel Elleder, John Young, Thomas Baiga, Joseph Noel

Applications
HIV, Infection, Virus, Drug Development
Novel NNRTIs, compounds and pharmaceutical compositions for reducing HIV infection and replication.

Resistance of the human immunodeficiency virus (HIV) to HIV drugs has always been a major cause of treatment failure, leading to the use of combination therapy employing multiple anti-HIV agents, each usually having a different activity profile. The introduction of HAART therapy (Highly Active Anti-Retroviral Therapy), for example, has resulted in a significant reduction of morbidity and mortality in those HIV patients receiving it. HAART involves various combinations of nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs). Current guidelines for antiretroviral therapy now recommend triple combination therapy regimens for initial treatment. Unfortunately, multi-drug therapies do not completely eliminate HIV, and long-term treatment usually results in multi-drug resistance. Half of the patients receiving anti-HIV combination therapy fail to respond fully, mainly because of resistance of the virus to one or more drugs used. The resistant virus is also carried over to newly infected individuals, who as a result have severely limited therapy options. Recently Salk Institute scientists identified a novel lead compound: a non-nucleoside reverse transcriptase inhibitor (NNTRI) that substantially reduces the activity of HIV-1 reverse transcriptase, thereby reducing HIV infection. This NNTRI also acts synergistically with one particular NRTI, zidovudine (or AZT), the first drug approved for the treatment of HIV. Structural Activity Relationship (SAR) analysis has identified additional, more potent derivatives of this compound, and experiments have confirmed their efficacy against wild-type and existing drug-resistant HIV-1 variants isolated from patients. The technology disclosed in the pending patent includes novel NNRTIs and compounds that can substantially inhibit HIV infection and replication and can be used in combination with other retriviral inhibitors. Also described are pharmaceutical compositions, prodrugs of the compounds, and methods for their creation, preparation and use. .

References
None to date

Patent Status:
U.S. Patent Application Filed 11/09/2007

License Terms:
Exclusive and Non-Exclusive Licenses Available

Reference_Number: S07014
Contact: Dave Odelson, Ph.D. o Senior Licensing Executive o 858.453.4100 x1223 o dodelson@salk.edu