De Novo Sequencing and Dereplication of Cyclic Nonribosomal Peptides

Researchers from UC San Diego have developed automated, rapid, cost-effective mass-spectrometry based approaches to perform de novo sequencing of cyclic nonribosomal peptides(NRPs) without knowing amino acid masses and the dereplication of known NRPs for compound identification.

Background: Nonribosomal peptides (NRPs) are natural products with cyclic structure and relative short sequences of up to fifty amino acids. They often are involved in the host’s chemical defense and communication and represent a rich reservoir of potential novel drugs. Nine out of the top twenty best-selling drugs were directly or indirectly derived from natural NRPs. Determining the structure of an unknown NRP structure is a slow and expensive part of drug screening and development, because the usual peptide sequencing tools do not work. The cyclic structures of NRPs, the prevalence of non-standard amino acids, and the lack of structural information directly inscribed in the genomic DNA due to the nonribosomal nature of the peptides all contribute to the difficulty. Researchers have had to rely on slow, manual, expensive, and not always reliable approaches to decipher the structure of NRPs.

Technology Description: UC San Diego researchers have devised novel methods to achieve both de novo sequencing and dereplication of NRPs, without the need to have the knowledge of the structure of the original compound. The approach uses multistage mass spectrometry (MS) to break NRP rings apart and then break the resulting peptide strings into smaller and smaller subunits of the original ring using multiple passes with a mass spectrometer. It allows the collection of data on the weights of ring fragments as these fragments get progressively shorter and more numerous with each pass of the mass spectrometer.

The de novo sequence of the NPR is achieved by applying novel algorithms that both decipher the mass of each fragment pass and determine the order of the fragments within the ring. The sequencing data provides crucial information for uncovering both the structure of the molecule and its pharmacological activities. A complimentary dereplication process applies the above approach in a reverse order; it takes the chemical structures of known NRPs and other related information and determines what the data signature would look like if a multistage MS had been applied.

By using these two approaches, researchers can both characterize the compound they have isolated and check to see if it, or something similar, has been previously described. With dereplication, researchers can leverage known information and circumvent the necessity to do extra sequencing each time a new compound needs to be identified.

Applications:

  • High throughput screening of natural products to identify new therapeutics for infectious disease, cancer and other disorders.
  • Facilitating biosynthetic engineering of E. coli to produce NRPs.

Related Materials: See references below for more information.
1. Ng, J., Bandeira, N., Liu, W.T., Ghassemian, M., Simmons,T.L., Gerwick,W., Linington,R., Dorrestein,P., and Pevzner, P.. Dereplication and De Novo Sequencing of Nonribosomal Peptides. Nature Methods online issue, July 13, 2009
2. Liu WT, Ng J, Meluzzi D, Bandeira N, Gutierrez M, Simmons TL, Schultz AW, Linington RG, Moore BS, Gerwick WH, Pevzner PA, Dorrestein PC. Interpretation of Tandem Mass Spectra Obtained from Cyclic Nonribosomal Peptides. Anal Chem. 2009 Jun 1;81(11):4200-9.
3. Bandeiray, N., Ng, J., Meluzziz, D, Liningtonk, R.G., Dorresteinz, P., Pevznery, P.A.. De Novo Sequencing of Nonribosomal Peptides. Lecture Notes in Computer Science 2008. 4955(Research in Computational Molecular Biology):181-195.

State of Development: Provisional patent application filed.

Keywords: nonribosomal peptides, NRP, cyclic peptides, mass spectrometry, MS, new drug discovery, natural compounds, de novo sequencing, dereplication, multistage mass spectrometry, bioinformatics

Case Number: SD2008-183


Inquiries To: invent@ucsd.edu