Novel Antimocrobial Drug Targets:  Genetic Screens for Intein Function

Our invention provides a novel approach to drug screening by adopting inteins as a drug target and testing the efficacy of agents which inhibit intein function in microbial pathogens. Inteins are naturally occurring protein elements that catalyze their own excision from within a larger protein together with the ligation of their flanking “extein” sequences which allow normal protein processing to occur. If the inteins are not removed, protein function will be interrupted and the viability of the cell may be compromised. Inteins are widespread, occurring in microbial pathogens such as Mycobacterium and Cryptococcus. This invention is directed at screening novel drugs to determine which have application for administration as intein-blockers or intein-inhibitors.

Our system of drug targeting against intein function provides a new approach to prevent the growth of organisms in critical genes by inhibiting inteins. Of additional benefit is that, while inteins are widespread, >400 inteins have been found in >35 different types of proteins, they are absent in mammalian genes. This is a positive indication for toxicity concerns which surround the development, approval and use of drugs. Our method provides a simple and targeted drug screening application for a new line of antimicrobial drugs. Crucial to pathogen survival, yet absent from all human proteins, the intein is a promising new drug target to overcome the drug resistance problem.

Applications

  • Identification of drugs that inhibit intein function to be useful as antimicrobial agents for a broad spectrum of pathogens
  • Testing & monitoring the efficacy of antimicrobial agents that inhibit intein function
  • A novel method of monitoring of intein function of Mycobacterium tuberculosis

Advantages

  • Unique ability to interrupt genetic sequences containing inteins and retard the growth of the organisms
  • Overcoming drug resistance of traditional antimicrobial drugs via intein-blocking
  • Ability to screen drugs against novel molecular targets within microbial pathogens

State of Development

Empirical assay for screening chemical agents available for use

Patents

U.S. Patent no. 5,795,731

Licensing Potential

Available for licensing.

Inventor

Marlene Belfort, Ph.D.Marlene Belfort, Ph.D.
Dr. Marlene Belfort has been actively engaged as a collaborator, mentor and reviewer in the field of inteins for over 15 years. She has been fascinated by their evolution and convinced of their utility and states that, “understanding their mechanism is the key to understanding the role of exteins in cellular evolution for biotechnological advancement”. Using a combination of genetics, biochemistry, structural biology and quantum mechanical modeling, her group has key residues that have aided in identifying acid and base catalysts that coordinate activities at splice sites which have shown the importance of allosteric effects for splicing. Her work has demonstrated the dramatic effects of the flanking extein sequences in splicing sequences in splicing and we designed an extein-based redox trap that no only promises further mechanistic insight, but also have implications for the persistence of inteins within cells. Her theory is that some inteins not only adapt to their extein, but regulate splicing in a manner consistent with cellular environment providing benefit to the cell. This challenges the “selfish intein” hypothesis.

Marlene holds two patents for intein technology and recently applied for a third. She co-developed an intein-based protein purification system for research, industrial-scale and microfluidic separation (Wood et al., 1999; Wood et al., 2000; Wu et al., 2002; Miao et al., 2005), several functional assays, and a protease assay (Callahan et al., In press) that forms the basis of an intein-based botulinum sensor. Additionally, she co-discovered an anti-mycobacterial lead compound, the antineoplastic agent cisplatin.

Awards & Honors
Distinguished Scientist, Wadsworth Center, Molecular Genetics
Fellow, American Association for Advancement of Science
Doctor Philosophiae Honoris Causa, Hebrew University
Distinguished Professor, School of Public Health, Biomedical Sciences
Alice Evans Award, American Society for Microbiology
MERIT Award NIH
Fellow, American Academy of Microbiology
Member, National Academy of Sciences (NAS)
Fellow, American Academy of Arts and Sciences
Ph.D., University of California at Irvine (1972)
Postdoctoral training, Hebrew University, Jerusalem

Publications

Peer-reviewed Publications (of ~170)
Most relevant to current invention

Derbyshire, V., Wood, D.W., Wu, W., Dansereau, J.T., Dalgaard, J.Z. and Belfort, M. Genetic definition of a protein-splicing domain: functional mini-inteins support structure predictions and a model for intein evolution. Proc. Natl. Acad. Sci. USA (1997) 94, 11466-11471.

Derbyshire, V. and Belfort, M. Lightning strikes twice: intron-intein coincidence. Proc. Natl. Acad. Sci. USA (1998) 95, 1356-1357.

Wood, D., Wu, W., Belfort, G., Derbyshire, V. and Belfort, M. A genetic system yields self-cleaving element for bioseparations. Nature Biotechnol. (1999) 17, 889-892.

Wood, D.W., Derbyshire, V., Wu, W., Cartrain, M., Belfort, M., and Belfort, G. Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor. Biotechnol. Prog. (2000) 16, 1055-1063.

Wu, W., Wood, D.W., Belfort, G., Derbyshire, V. and Belfort, M. Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing. Nucleic Acids Res. (2002) 30, 4864-4871.

Miao, J., Wu, W., Spielmann, T., Belfort, M., Derbyshire, V., and Belfort, G. Single-step affinity purification of toxic and non-toxic proteins on a fluidics platform. Lab on a Chip (2005) 5, 248-253.

Hiraga, K., Derbyshire, V., Dansereau, J. T., Van Roey, P. and Belfort, M. Minimization and stabilization of the Mycobacterium tuberculosis recA intein. J. Mol. Biol. (2005) 354, 916-926.

Van Roey, P., Pereira, B., Li, Zh., Hiraga, K., Belfort, M., and Derbyshire, V. Crystallographic and mutational studies of Mycobacterium tuberculosis recA mini-inteins suggest a pivotal role for a highly conserved aspartate residue. J. Mol. Biol. (2007) 367, 162-173.

Amitai, G., Callahan, B.P., Stanger, M.J., Belfort, G. and Belfort, M. Modulation of intein activity by its neighboring extein substrates. Proc. Natl. Acad. Sci. USA (2009) 106, 11005-11010.

Du, Z., Shemella, P.T., Liu, Y., McCallum, S.A., Pereira, B., Nayak, S.K., Belfort, G., Belfort, M. and Wang, C. Highly conserved histidine plays a dual catalytic role in protein splicing: a pKa shift mechanism. J. Am. Chem. Soc. (2009) 131, 11581-11589.

Hiraga, K., Soga, I., Dansereau, J.T., Pereira, B., Derbyshire, V., Du, Z., Wang, C., Van Roey, P., Belfort, G. and Belfort, M. Selection and structure of hyperactive inteins: peripheral changes relayed to the catalytic center. J. Mol. Biol. (2009) 393, 1106-1117.

Du, Z., Liu, Y., Ban, D., Lopez, M.M., Belfort, M. and Wang, C. Backbone dynamics and global effects of an activating mutation in minimized Mtu RecA inteins. J. Mol. Biol. (2010) 400, 755-767.

Callahan, B.P., Stanger, M.J. and Belfort, M. Cut and Glow: Protease activation of split green fluorescent protein ChemBiochem (2010). In press.

Additional Recent Publications

Coros, C.J., Piazza, C. L., Chalamcharla, V.R., Smith, D. and Belfort, M. Global regulators orchestrate group II intron retromobility. Mol. Cell (2009) 34, 250-256.

DiChiara, J.M., Contreras-Martinez, L.M., Livny, J., Smith, D., McDonough, K.A. and Belfort, M. Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in M. tuberculosis and M. smegmatis. Nucleic Acids Res. (2010) 38, 4067-4078.

Chalamcharla, V.R., Curcio, M.J. and Belfort, M. Nuclear expression of a group II intron is consistent with spliceosomal intron ancestry. Genes Dev. (2010) 24, 827-836.

Contact

Diane L. Borghoff, B.S., M.S.
Marketing & Licensing Associate – Intellectual Property
Health Research, Inc. – 150 Broadway – Suite 560, Menands, New York 12204-2719 U.S.A.
Phone 518-431-1213  Fax 518-431-1234
E-mail:  diane.borghoff@healthresearch.org  Website:  www.healthresearch.org