Development of synthetic strategies for lasso peptides with anticancer activity

Abstract

Nowadays, the discovery and development of novel constrained peptides which are likely to combine the advantages of therapeutic proteins with those of small molecules is of special interest. This has partially prompted the re-emergence of peptides as therapeutics. Thus, potentially, these peptides provide both the selectivity and potency of larger protein biologics but with zero or low immunogenicity, and the stability and bioavailability of small molecules. Furthermore, they are smaller than biologics, more accessible and cheaper to manufacture using chemical methods, thus presumably combining the advantages of the two therapeutic approaches.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified natural products with a unique three-dimensional structure, in which the C-terminus threads through an N-terminal macrolactam ring in a right-handed conformation. These peptides consist of 15–26 proteinogenic amino acids and share an N-terminal 7- to 9- residue macrolactam ring where the N-terminal amino acid is always glycine or cysteine and the amino acid that closes the ring is aspartic or glutamic acid. The lasso topology is predominantly stabilized by steric interactions, in the case of class II lasso peptides, but sometimes is assisted by the presence of disulfide bridges; two in the case of class I or one in class III lasso peptides. Currently, a total of 43 lasso peptides have been described; 3 belong to class I, 39 to class II and 1 to class III.1 Prior to 2008, most of these lasso peptides were discovered by isolation from bacteria; however, capistruin, the first lasso peptide isolated by a genome mining approach, changed this scenario.2

The diverse functionality of lasso peptides makes these molecules attractive candidates for drug discovery. In addition, given their extraordinary stability against chemical, thermal and proteolytic degradation1 and reduced flexibility, these peptides are suitable scaffolds for drug design and epitope grafting approaches.3,4 Considering this, it is possible to use a rational approach to further improve and optimize such a scaffold toward the generation of more potent and more selective bioactive compounds.

Currently, all research into new peptide drugs pursues two main common objectives: development of new compounds resistant to enzymatic degradation and the modulation of peptide topology, since the properties are highly related to the shape.5 In this regard, most lasso peptide synthetic strategies are based on the imitation of the interlocked structure of rotaxanes and catenanes.6,7,8,9 Furthermore, lasso peptide-like bicyclic peptides is also a suitable chemical approach, in which the loop sequence is tied with a covalent bond.10

Sungsanpin is a class II lasso peptide isolated from a Streptomyces sp. strain collected in Korea in 2012.11 It shows an inhibitory effect on the invasion of human non-small cell lung cancer (NSCLC), an effect that has been reported with the A549 cell line. Regarding the previously mentioned, the aim of this project is the synthesis of sungsanpin and analogs with linkages able to maintain the threaded lasso structure.

Several characterization techniques have been established for lasso peptides identification. A representative and recent technique that allows rapid structural detection and dynamical features is ion-mobility mass spectrometry (IM-MS). It is a complementary approach to MS/MS experiments that provides information on the global shape of molecules,12 and has proven useful for the structural characterization of many lasso peptides.13,14

To date, no synthetic access to lasso peptides is available due to the difficulty in building and maintaining the threaded lasso structure. The ability to generate lasso peptides synthetically remains a challenging endeavor and it would open the door to the production of lasso peptide analog with unnatural amino acids or other nonproteinogenic building blocks. From a therapeutic point of view, these small and constrained structures would represent a new paradigm in drug discovery.

(1) Hegemann, J. D.; Zimmermann, M.; Xie, X.; Marahiel, M. A. Acc. Chem. Res. 2015, 48 (7), 1909. (2) Knappe, T. a.; Linne, U.; Zirah, S.; Rebuffat, S.; Xie, X.; Marahiel, M. a. J. Am. Chem. Soc. 2008, 130 (17), 11446. (3) Knappe, T. A.; Manzenrieder, F.; Mas-Moruno, C.; Linne, U.; Sasse, F.; Kessler, H.; Xie, X.; Marahiel, M. A. Angew. Chemie - Int. Ed. 2011, 50 (37), 8714. (4) Hegemann, J. D.; De Simone, M.; Zimmermann, M.; Knappe, T. A.; Xie, X.; Di Leva, F. S.; Marinelli, L.; Novellino, E.; Zahler, S.; Kessler, H.; Marahiel, M. A. J. Med. Chem. 2014, 57 (13), 5829. (5) Clavel, C.; Fournel-Marotte, K.; Coutrot, F. Molecules 2013, 18 (9), 11553. (6) Mohr, B.; Weck, M.; Sauvage, J.-P.; Grubbs, R. H. Angew. Chem. Int. Ed. Engl. 1997, 36 (12), 1308.

(7) Hogg, L.; Leigh, D. A.; Lusby, P. J.; Morelli, A.; Parsons, S.; Wong, J. K. Y. Angew. Chemie - Int. Ed. 2004, 43 (10), 1218. (8) Hänni, K. D.; Leigh, D. A. Chem. Soc. Rev. 2010, 39 (4), 1240. (9) Yan, L. Z.; Dawson, P. E. Angew. Chemie Int. Ed. 2001, 40 (19), 3625. (10) Soudy, R.; Wang, L.; Kaur, K. Bioorganic Med. Chem. 2012, 20 (5), 1794. (11) Um, S.; Kim, Y.-J. J.; Kwon, H. H. C.; Wen, H.; Kim, S.-H. H.; Kwon, H. H. C.; Park, S.; Shin, J.; Oh, D.-C. C. J. Nat. Prod. 2013, 76 (5), 873. (12) Clemmer, D. E.; Jarrold, M. F. J. Mass Spectrom. 1997, 32 (6), 577. (13) Jeanne Dit Fouque, K.; Afonso, C.; Zirah, S.; Hegemann, J. D.; Zimmermann, M.; Marahiel, M. A.; Rebuffat, S.; Lavanant, H. Anal. Chem. 2015, 87 (2), 1166. (14) Fouque, K. J. D.; Lavanant, H.; Zirah, S.; Hegemann, J. D.; Zimmermann, M.; Marahiel, M. A.; Rebuffat, S.; Afonso, C. J. Am. Soc. Mass Spectrom. 2016.

Els pèptids llaç són una classe de productes naturals sintetitzats al ribosoma i modificats després de la translació amb una estructura tridimensional única, en la qual el C-terminal travessa l’anell de macrolactama N-terminal en una conformació de mà dreta. Aquests pèptids consisteixen en 15-26 aminoàcids proteïnògens i comparteixen un anell de macrolactama N-terminal de 7 - 9 residus on l'aminoàcid N- terminal és sempre glicina o cisteïna i l'aminoàcid que tanca l'anell és àcid aspàrtic o glutàmic. La topologia de llaç està predominantment estabilitzada per interaccions estèriques, en el cas dels pèptids de la classe II, però de vegades és assistida per la presència de ponts disulfurs; dos en el cas dels pèptids de la classe I o un, en la classe III.

Tenint en compte la diversa funcionalitat dels pèptids llaç i la seva extraordinària estabilitat davant la degradació química, tèrmica i proteolítica, aquests pèptids són bastiments proteínics adequats pel disseny de fàrmacs i les aproximacions d’empelt d’epítops.1,2 Tenint en compte això, és possible utilitzar un enfocament racional per millorar i optimitzar encara més aquests bastiments proteínics cap a la generació de compostos bioactius més potents i selectius.

Sungsanpin, és un pèptid llaç de classe II, aïllat d'una soca de Streptomyces sp. recollida a Korea al 2012.3 Sungsanpin mostra un efecte inhibitori sobre la invasió del càncer de pulmó humà no microcític (NSCLC), un efecte que s'ha estudiat amb la línia cel·lular A549. L'objectiu d'aquest projecte és la síntesi del sungsanpin i anàlegs amb enllaços capaços de mantenir l'estructura roscada de llaç. Tanmateix, fins avui, no hi ha accés sintètic als pèptids llaç a causa de la dificultat a l'hora de construir i mantenir l'estructura roscada. La capacitat de generar aquests tipus de pèptids sintèticament continua sent un repte desafiant i obriria la porta a la producció d’anàlegs de pèptids llaç amb aminoàcids no naturals o altres blocs de construcció no proteïnògens. Des d'un punt de vista terapèutic, aquestes estructures petites i restringides representarien un nou paradigma en el descobriment de fàrmacs.

(1) Knappe, T. A.; Manzenrieder, F.; Mas-Moruno, C.; Linne, U.; Sasse, F.; Kessler, H.; Xie, X.; Marahiel, M. A. Angew. Chemie - Int. Ed. 2011, 50 (37), 8714. (2) Hegemann, J. D.; De Simone, M.; Zimmermann, M.; Knappe, T. A.; Xie, X.; Di Leva, F.

S.; Marinelli, L.; Novellino, E.; Zahler, S.; Kessler, H.; Marahiel, M. A. J. Med. Chem. 2014, 57 (13), 5829. (3) Um, S.; Kim, Y.-J. J.; Kwon, H. H. C.; Wen, H.; Kim, S.-H. H.; Kwon, H. H. C.; Park, S.; Shin, J.; Oh, D.-C. C. J. Nat. Prod. 2013, 76 (5), 873.