Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines Inhibit Mammalian and Protozoan DYRK & CLK Kinases

A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological leads for the treatment of several diseases, including Alzheimer’s disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.


HABITAT AND DISTRIBUTION
Leucetta microraphis is a common species in the Indo-Pacific, where it can be found in exposed and light-protected areas, such as crevices and steep walls (Wörheide & Hooper, 1999). The distribution of L. microraphis has already been considered cosmopolitan, including the Indo-Pacific, Atlantic, Mediterranean, Red Sea and Antarctica (see Wörheide & Hooper, 1999). However, in 2009, Valderrama et al. (2009 showed that what had been called L. microraphis in the Atlantic was in fact L. floridana. Presently, L. microraphis is considered to be present only in the Indo-Pacific, but even this more restricted distribution may still be hiding other species. Its currently accepted distribution is Australia, New Zealand, New Caledonia, French Polynesia and Madagascar. Although Haeckel (1872) had not made clear where the type locality of Leucetta primigenia var. microraphis is, since Lendenfeld (1885) elevated it to the species category, Australia is considered as its type locality.

HABITAT AND DISTRIBUTION
Leucetta chagosensis is a very abundant sponge both in protected and in illuminated areas. It was already reported to the Indian Ocean (Chagos Archipelago -type locality; Madagascar, Northern Red Sea, Japan (Okinawa), Indonesia, Australia (Queensland and Fremantle), New Caledonia and French Polynesia (Dendy, 1913;Wörheide & Hooper, 1999;Borojevic & Klautau, 2000). Phylogeographical studies showed, however, that populations of this species are highly structured and this wide distribution may be concealing cryptic species (Wörheide et al., 2002;.

Clathrina Minchin, 1900
Clathrina is the most diverse genus of the family Clathrinidae Minchin, 1900. It comprises delicate clathroid species whose skeleton is composed only of triactines (Rossi et al., 2011;Klautau et al., 2013). It is a widespread genus, present in all oceans and latitudes, however, most of its species have a restricted distribution.

HABITAT AND DISTRIBUTION
Clathrina clathrus is a very delicate species that lives in light protected habitats, such as caves and crevices. It has already been considered as a cosmopolitan species, but currently its accepted distribution is restricted to the whole Mediterranean Sea and to the Northwestern coast of the Atlantic. In the past, all yellow clathrinas were identified as C. clathrus, however, a genetic study showed that Mediterranean and Southwestern Atlantic populations of yellow clathrinas were not conspecific (Solé-Cava et al., 1991). Since then, molecular and morphological studies have proved the existence of several different species of yellow clathrinas in the world (Wörheide & Hooper, 1999;Borojevic & Klautau, 2000). However, all of them can be grouped in the same monophyletic clade (Rossi et al., 2011;Klautau et al., 2013).

Remarks
Clathrina clathrus, L. microraphis and L. chagosensis group within the subclass Calcinea, but the phylogenetic tree of Calcinea shows three main groups, one that reunites sponges without tetractines (Clathrina) and two of sponges with tetractines (all the other genera) (Rossi et al., 2011;Klautau et al., 2013) ( Figure S5). Clathrina clathrus groups in the clade of Clathrina, more specifically in a clade of yellow clathrinas only. It is a sister group of C. aurea from the Western Atlantic (Brazil). Leucetta microraphis and L. chagosensis group in the clade of Leucetta, but separately from each other. Leucetta microraphis is more related to L. potiguar and L. floridana from the Western Atlantic, while L. chagosensis is more related to L. pyriformis and L. antarctica, both from Antarctica.

DNA sequencing, alignment and phylogenetic analyses
The analysed region was the internal transcribed spacer (ITS), including 18S partial, ITS1, 5.8S, ITS2 and 28S partial. All sequences were obtained from the GenBank (Table S1). Sequences were aligned with the Q option of the MAFFT program (Katoh & Standley, 2013), which takes into consideration the secondary structure of the ITS. Scoring matrix was PAM/k1/42, gap penalty 1.53 and offser value 1/40. As an appropriate outgroup is not available, we root the tree with the mid-point rooting method. Final alignment contained 1,096 bp.