University of Otago Otago Innovation Ltd

Membrane Protein Expression System

Summary

Superior Membrane Protein Expression for drug discovery.

Fungal infections in humans are opportunistic, often difficult to treat and sometimes fatal. There is rising resistence in many fungal pathogens to the limited number of antifungal drugs. This lead to the present opportunity of a superior Membrane Protein Expression system to investigate and overcome drug resistance in fungal pathogens. In addition, the system has been further developed to aid in the discovery of new therapeutics targeting human membrane proteins.

Technology

Otago researchers developed a Membrane Protein Expression system that expresses any membrane protein required by research groups and companies. The sytem uses a Saccharomyces cerevisiae host (baker's yeast). Importantly, the expressed membrane protein is not species dependent and we have expressed a variety of fungal pathogen and human membrane proteins. Our system expresses the "foreign" membrane protein so that it is functional and located on the membrane as if it were expressed naturally. Our system consistently "over-expresses" the target at a high level and competing "background noise" of other unwanted membrane protein expression is minimised - improving protein crystallisation for indentification and functional analysis. The Membrane Protein Expression system can also build the scaffolding for appropriate drug discovery screening models as it amplifies the feature of interest and indentifies any interference with the expressed drug target.

We have a number of granted and filed patents for the system.

Application

By 2012, blockbuster drugs with revenue of $65 billion come off patent. Pharmaceutical companies face a lack of new drugs in their pipelines and are therefore seeking new drugs, drug classes and drug targets. Importantly for our system, more than 70% of all drug targets are membrane proteins, but only 0.3% of indentified protein structures are membrane proteins. This lack of structural knowledge and the trend towards "structure directed drug design" provides a tremendous opportunity for our system.

We have multiple systems that express efflux pump membrane proteins. The pumps we have expressed transport specific compounds out of the cell, for example cancer therapeutics and antifungal treatments. The cancer drug pump is a human membrane protein, whereas the antifungal drug pump is a fungus specific drug pump. Finding drug pump inhibiting compounds leads to new combination therapies that address resistance. Using the system to undertake initial screening activities, in collaboration with external parties, we have identified a novel pump inhibitor for potential use with anti-fungals. This candidate is now under further investigation.

We are currently further developing our system to improve human membrane protein expression to develop drug targets for many major human diseases.

Partnership

The existing Membrane Protein Expression systems are available for licensing.

We also offer the development (and licensing) of customised systems to express the membrane protein drug targets of interest and for its use in drug discovery screening models.

Potential applications in malaria, crop protection, and development of a research kit for academia provide additional partnering opportunities.

In the near future, we will seek investment for a company formation to undertake drug discovery and to extend uses of the Membrane Protein Expression system.

Contact

Dr Alexandra Tickle
Otago Innovation Limited
PO Box 56
Dunedin. New Zealand
Tel 64 3 479 4145
Email alexandra.tickle@otagoinnovation.com

Intellectual Property

Yeast membrane protein expression system and its application in drug screening.

International Patent Application No. PCT/NZ02/00163 (Priority Date 24 August 2001)

Granted:

Pending:

Publications

Identification of Nile red as a fluorescent substrate of the Candida albicans ATP-binding cassette transporters Cdr1p and Cdr2p and the major facilitator superfamily transporter Mdr1p.
Ivnitski-Steele I, Holmes AR, Lamping E, Monk BC, Cannon RD, Sklar LA.
Anal Biochem. 2009 Nov 1;394(1):87-91. Epub 2009 Jul 3.

Efflux-mediated antifungal drug resistance.
Cannon RD, Lamping E, Holmes AR, Niimi K, Baret PV, Keniya MV, Tanabe K, Niimi M, Goffeau A, Monk BC.
Clin Microbiol Rev. 2009 Apr;22(2):291-321, Table of Contents. Review.

Abc1p is a multidrug efflux transporter that tips the balance in favor of innate azole resistance in Candida krusei.
Lamping E, Ranchod A, Nakamura K, Tyndall JD, Niimi K, Holmes AR, Niimi M, Cannon RD.
Antimicrob Agents Chemother. 2009 Feb;53(2):354-69. Epub 2008 Nov 17.

ABC transporter Cdr1p contributes more than Cdr2p does to fluconazole efflux in fluconazole-resistant Candida albicans clinical isolates.
Holmes AR, Lin YH, Niimi K, Lamping E, Keniya M, Niimi M, Tanabe K, Monk BC, Cannon RD.
Antimicrob Agents Chemother. 2008 Nov;52(11):3851-62. Epub 2008 Aug 18.

Characterization of three classes of membrane proteins involved in fungal azole resistance by functional hyperexpression in Saccharomyces cerevisiae.
Lamping E, Monk BC, Niimi K, Holmes AR, Tsao S, Tanabe K, Niimi M, Uehara Y, Cannon RD.
Eukaryot Cell. 2007 Jul;6(7):1150-65. Epub 2007 May 18.

Amino acid residues affecting drug pump function in Candida albicans--C. albicans drug pump function.
Holmes AR, Tsao S, Lamping E, Niimi K, Monk BC, Tanabe K, Niimi M, Cannon RD.
Nippon Ishinkin Gakkai Zasshi. 2006;47(4):275-81. Review.

Heterozygosity and functional allelic variation in the Candida albicans efflux pump genes CDR1 and CDR2.
Holmes AR, Tsao S, Ong SW, Lamping E, Niimi K, Monk BC, Niimi M, Kaneko A, Holland BR, Schmid J, Cannon RD.
Mol Microbiol. 2006 Oct;62(1):170-86. Epub 2006 Aug 30.

Overexpression of Candida albicans CDR1, CDR2, or MDR1 does not produce significant changes in echinocandin susceptibility.
Niimi K, Maki K, Ikeda F, Holmes AR, Lamping E, Niimi M, Monk BC, Cannon RD.
Antimicrob Agents Chemother. 2006 Apr;50(4):1148-55.

[ABC transporters of pathogenic fungi: recent advances in functional analyses]
Niimi M, Tanabe K, Wada S, Yamazaki A, Uehara Y, Niimi K, Lamping E, Holmes AR, Monk BC, Cannon RD.
Nippon Ishinkin Gakkai Zasshi. 2005;46(4):249-60. Review. Japanese.

Characterization of the Saccharomyces cerevisiae sec6-4 mutation and tools to create S. cerevisiae strains containing the sec6-4 allele.
Lamping E, Tanabe K, Niimi M, Uehara Y, Monk BC, Cannon RD.
Gene. 2005 Nov 21;361:57-66. Epub 2005 Sep 26.

Functional analysis of fungal drug efflux transporters by heterologous expression in Saccharomyces cerevisiae.
Niimi M, Wada S, Tanabe K, Kaneko A, Takano Y, Umeyama T, Hanaoka N, Uehara Y, Lamping E, Niimi K, Tsao S, Holmes AR, Monk BC, Cannon RD.
Jpn J Infect Dis. 2005 Feb;58(1):1-7. Review.

Phosphorylation of candida glabrata ATP-binding cassette transporter Cdr1p regulates drug efflux activity and ATPase stability.
Wada S, Tanabe K, Yamazaki A, Niimi M, Uehara Y, Niimi K, Lamping E, Cannon RD, Monk BC.
J Biol Chem. 2005 Jan 7;280(1):94-103. Epub 2004 Oct 21.

Regulated overexpression of CDR1 in Candida albicans confers multidrug resistance.
Niimi M, Niimi K, Takano Y, Holmes AR, Fischer FJ, Uehara Y, Cannon RD.
J Antimicrob Chemother. 2004 Dec;54(6):999-1006. Epub 2004 Oct 14.

Chemosensitization of fluconazole resistance in Saccharomyces cerevisiae and pathogenic fungi by a D-octapeptide derivative.
Niimi K, Harding DR, Parshot R, King A, Lun DJ, Decottignies A, Niimi M, Lin S, Cannon RD, Goffeau A, Monk BC.
Antimicrob Agents Chemother. 2004 Apr;48(4):1256-71.

Identification of two proteins induced by exposure of the pathogenic fungus Candida glabrata to fluconazole.
Niimi M, Nagai Y, Niimi K, Wada S, Cannon RD, Uehara Y, Monk BC.
J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Dec 25;782(1-2):245-52.

Candida glabrata ATP-binding cassette transporters Cdr1p and Pdh1p expressed in a Saccharomyces cerevisiae strain deficient in membrane transporters show phosphorylation-dependent pumping properties.
Wada S, Niimi M, Niimi K, Holmes AR, Monk BC, Cannon RD, Uehara Y.
J Biol Chem. 2002 Nov 29;277(48):46809-21. Epub 2002 Sep 19.

Functional expression of Candida albicans drug efflux pump Cdr1p in a Saccharomyces cerevisiae strain deficient in membrane transporters.
Nakamura K, Niimi M, Niimi K, Holmes AR, Yates JE, Decottignies A, Monk BC, Goffeau A, Cannon RD.
Antimicrob Agents Chemother. 2001 Dec;45(12):3366-74.