Design and synthesis of sphingosine-1-phosphate lyase inhibitors and fluorogenic probes for the development of HTS assays

Abstract

[eng] Sphingolipids (SLs) are essential structural and signaling molecules of eukaryotic cells. An important group of SLs metabolites are those showing a phosphate group at the C1‒ OH. Among them, sphingosine‒1‒phosphate (S1P) is a well‒recognized signaling molecule that can act both as an intracellular second messenger and as a ligand of specific G‒protein coupled receptors (S1P1‒5), giving rise to a series of downstream signaling pathways involved in vascular development, control of cardiac rhythm, and immunity responses, among others. Sphingosine‒1‒phosphate lyase (S1PL) is a pyridoxal 5’‒phosphate (PLP) dependent enzyme that catalyzes the irreversible degradation of S1P into ethanolamine phosphate and trans‒2‒hexadecenal in the endoplasmic reticulum. Together with S1P phosphatase, and sphingosine kinase, S1PL regulates the intracellular levels of S1P and contributes to the so‒called ‘sphingolipid rheostat’, a system that controls cell fate based on the ratio of intracellular proliferative S1P and the apoptogenic sphingosine and ceramide. Notably, S1PL plays an important role in regulating the immune system, since its inhibition disrupts the S1P gradient that promotes T‒cell egress from lymphoid tissues. In this context, S1PL has been validated as therapeutic target for the treatment of some autoimmune diseases, such as multiple sclerosis or rheumatoid arthritis. In light of the therapeutic potential associated to the modulation of S1PL activity, we undertook the design of new S1PL inhibitors based on two different approaches. Firstly, a structure‒based drug design of S1PL inhibitors was performed using the crystal structures of the bacterial (StS1PL) and human (hS1PL) enzymes, which share a high level of sequence and structural similarities. Taking into account the common structural features of a series of hits, identified on a preliminary screening of potential S1PL inhibitors, and based on the results arising from docking studies using the hS1PL and StS1PL X‒ray structures, a small library of putative S1PL inhibitors, derived from a common scaffold, were designed and synthesized. Compound RBM13, a previously reported fluorogenic S1PL substrate, was used in the development of an ‘on‒plate’ assay for the S1PL activity determination using recombinant StS1PL and hS1PL as enzyme sources. Unfortunately, the rational design of new S1PL inhibitors was unsuccessful, as evidenced by the modest activities found for the designed inhibitors under our optimized assay conditions. In addition, although comparable kinetic parameters were determined for RBM13 against the two enzymes, a reference hS1PL inhibitor did not show any activity on the bacterial enzyme. In a second approach, two families of S1PL inhibitors were designed based on S1PL mechanistic considerations. In this sense, a small family of non‒reactive analogs of some key enzyme reaction intermediates, as well as a series of stereodefined azide analogs of the natural S1P, were synthesized and tested against human and bacterial S1PL. Although compounds mimicking the intermediates of the catalytic process were weak inhibitors, all the azido phosphates behaved as competitive inhibitors in the low µM range on the two S1PL isozymes. These results suggested that StS1PL can be a reliable model for the design of hS1PL inhibitors that bind into the active site. However, the usefulness of this model protein is more limited if one wants to design compounds that target the active site access channel. Finally, two new coumarin‒containing probes with potential applicability in HTS assays were designed and synthesized. Structurally, both compounds are formally derived from RBM13 by intercalation of a vinyl group between the amino alcohol phosphate moiety and the ether‒linked coumarin group of the parent compound. Gratifyingly, both probes were validated as hS1PL substrates with better kinetic parameters than those determined for RBM13.

[cat] Els esfingolípids (SLs) són components estructurals essencials de les membranes cel·lulars. Alguns metabòlits dels SLs actuen, a més, com a molècules de senyalització en cèl·lules eucariotes. L’esfingosina-1-fosfat (S1P) és un esfingolípid bioactiu, el qual està involucrat en processos biològics rellevants tals com el desenvolupament vascular, el control del ritme cardíac o la resposta immunitària, entre d’altres. L’esfingosina‒1‒fosfat liasa (S1PL), és un enzim depenent de piridoxal 5’‒fosfat (PLP) situat al reticle endoplasmàtic, el qual catalitza la degradació irreversible de la S1P, per donar fosfat d’etanolamina i 2‒hexacedenal. La S1PL ha estat recentment validada com a diana terapèutica pel tractament de certes malalties autoimmunes. Degut al potencial terapèutic associat a la modulació de l’activitat S1PL, en la present tesi doctoral s’ha abordat el disseny de nous inhibidors de la S1PL des de dues aproximacions diferents. En primer lloc es va duu a terme un disseny basat en la estructura de nous inhibidors de la S1PL emprant les estructures cristal·lines de la S1PL humana (hS1PL) i la de Symbiobacterium thermophilum (StS1PL). No obstant, el còmput de molècules seleccionades com a potencials inhibidors, varen presentar activitats moderades en un nou assaig fluorogènic, el qual va ser desenvolupat per a la determinació de l’activitat S1PL emprant enzims purificats. En una segona aproximació, basant-se en consideracions mecanístiques del cicle catalític de l’enzim, es varen dissenyar i sintetitzar dues famílies d’inhibidors de la S1PL, les quals varen ser testades contra la hS1PL i la StS1PL. D’entre els compostos sintetitzats, una sèrie d’azido- derivats del substrat natural de l’enzim van resultar ser inhibidors competitius en el rang µM baix en els dos isoenzims. Finalment, dues noves sondes fluorogèniques per a la determinació de l’activitat S1PL amb potencial aplicabilitat ens assaigs HTS van ser dissenyades i sintetitzades. Estructuralment, ambdues sondes corresponen a dos vinílegs d’un substrat no natural de la S1PL prèviament reportat. Els dos nous compostos varen ser validats com a substrats de la hS1PL, presentant, en ambdós casos, millors paràmetres cinètics que el compost de referència.