Some examples are Bg 16.42 (1517.7 Da) and Bcg 16.00–17.00 (1517.6 Da), Bg 25.63 (3059.3 Da) and Sh 25.79 (3059.9 Da), Bg 20.79 (3932.7 Da) and ATM/ATR inhibitor drugs Bcg 20.64 (3933.5 Da), Bg 30.00 (4370.6 Da) and Bcg 31.16 (4371.1 Da), Bg 28.95 (4669.2 Da) and Bcg 28.78 (4669.1 Da), Bg 22.66 (4700.8 Da) and Sh 22.05 (4699.6 Da), Bg 27.35 (5071.9 Da) and Sh 26.77 (5072.2 Da).

Considering the diversity of peptides with the mass range of 4000–5000 Da in the final portion of the RPC18 chromatogram of B. granulifera neurotoxic fraction (Bg-3-4), and the higher abundance of mass signals in this species, we decided to focus our transcriptome analysis on these proteins. Transcriptome profiling with cDNA new generation sequencing technology was used to identify some of the expressed genes of B. granulifera. The mRNA was isolated for the preparation of a library and subsequent pyrosequencing analysis. The total number of tags per library was approximately 59,000, with average read length of about 292 bp, which assembled 1.603 contigs. The contigs were mapped

RO4929097 to the NCBI non-redundant databases. A preliminary data mining could reveal five matches with annotated genes encoding novel peptide toxins from the sea anemone B. granulifera, having from 317 to 524 bp. The full coding sequences (CDS) were obtained for four out of the five matches, including the complete translated sequences of the precursors and mature regions for neurotoxins within the mass range of 4–5 kDa (mature Bay 11-7085 products), as shown in Fig. 4A and B. Translation of the nucleotides retrieved

could reveal sequence similarity to other known sea anemone toxins. A sequence similarity search (http://www.ebi.ac.uk/Tools/sss/fasta/) indicated that these peptides share homology with type 3 potassium channel toxins APETx1 [24], BDS-I and BDS-II [26], APETx2, an ASICs inhibitor [23] and the APETx-like toxins Bcg 25.52, Bcg 28.78, Bcg 29.21, Bcg 31.16 [85], BcIV [64] and BcV (accession number P86470). The highest sequence identity (57–65%) of the new toxins was observed in relation to APETx1 or APETx-like peptides. Moreover, multiple sequence alignment (http://www.ebi.ac.uk/Tools/msa/clustalw2/) showed that these new toxins are structurally close to each other (Fig. 4A), and therefore can be considered as new members of the APETx-like peptide group [64] and [85]. Given than their molecular targets are still unknown, these peptides (mature region, Fig. 4A) were named as U-AITX-Bg1a, U-AITX-Bg1b, U-AITX-Bg1c, U-AITX-Bg1d, and U-AITX-Bg1e (nucleotide sequences deposited at the EMBL Nucleotide Sequence Database having the following accession numbers assigned: HE577144, HE577145, HE577146, HE577147 and HE577148, respectively) according to the nomenclature system proposed by King et al. [44]. Their theoretical molecular masses are 4586.3 Da (U-AITX-Bg1b), 4921.6 Da (U-AITX-Bg1c), 4684.4 Da (U-AITX-Bg1d), and 4142.