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Tesi etd-03202014-130416

Thesis type
Tesi di laurea specialistica LC5
Design, synthesis and biological evaluation of new heterocyclic derivatives as serine palmitoyltransferase inhibitors
Corso di studi
relatore Dott. Saccomanni, Giuseppe
relatore Dott.ssa Del Carlo, Sara
Parole chiave
  • Synthesis
  • SPT
  • inhibitors
Data inizio appello
Riassunto analitico
Serine palmitoyltransferarase (SPT) is a PLP dependent enzyme which catalyzes the first step of the de novo ceramide biosynthesis condensing serine and palmitoyl-CoA and producing 3-ketodihydrosphingosine. This step is conserved among different species (conversely to other steps involved in the SL biosynthesis). SPT has significant similarities to members of a subfamily of PLP-dependent enzymes which catalyze condensations of amino acids and carboxylic acid CoA thioesters to produce alpha-oxoamines (AOS). Among the members of this family identified to date, only eukaryotic (but not prokaryotic) SPT is a membrane-bound heterodimer enzyme, while all other members are soluble homodimer enzymes.

Eukaryotic SPT is present in the ER and is constituted by LCB1–LCB2 heterodimer. The PLP containing LCB2 subunit carries out the condensation reaction and, although the LCB1 subunit appears to lack the residues that bind PLP, it is still required for activity. A second isoform of LCB2 named LCB3, is expressed only in certain tissues. Recently two novel small subunits named ssSPTa and ssSPTb were discovered. These subunits can enhance activity >10 fold when bound to LCB1-LCB2 heterodimer. Moreover its seems that orosomucoid-like (ORMDL) proteins are able to interact with the LCB1-LCB2 heterodimer.
An abridged version of the SPT enzymatic reaction indicates three important intermediates:
(I) the holo-form or internal aldimine of the enzyme with PLP cofactor bound to the active-site Lys265 via a Schiff base;
II) the external aldimine with L-serine following transamination; and
(III) the quinonoid intermediate (carbanion equivalent) following deprotonation at Cα that attacks the incoming thioester substrate.
Sphingolipid metabolites have crucial roles in cell pathway like apoptosis, proliferation and differentiation. Inhibition of late steps of sphingolipid biosynthesis causes accumulation of intermediate products which are death effectors. Conversely SPT inhibition prevents accumulation of toxic intermediates.
Recently different studies have demonstrated that some neurodegenerative disease are caused by alteration of SPT activity. Mutation of the gene encoding for LCB1 subunit causes a shift in substrate specificity leading to the production of atypical deoxysphingoid bases. These intermediates are not metabolized and they are accumulated in cells producing neurotoxic effects. Moreover higher levels of de novo synthesized ceramide has been related to photoreceptor death in retinitis pigmentosa and inhibition of SPT causes a reduction of ceramide levels reducing apoptotic events.2 Moreover inhibition of SPT could be a safe therapeutic strategy to ameliorate the Alzheimer’s disease.3 Thereby inhibitors of SPT could exert neuroprotective activity.
Few inhibitors of SPT are known. Several natural product have been identified as potent SPT inhibitors (such as myriocin and cycloserine) but few complete investigations into the exact mechanism of inhibition by each have been undertaken.
The only data reported in literature about aromatic SPT inhibitors are represented by a patent of Pfizer. In this patent a lot of compound are reported whose general structure is mainly represented by 2-oxo-benzimidazole.
In the research lab where I performed my thesis, new aromatic SPT inhibitors has been designed and synthesized and a new hit compound has been identified. During my thesis, I have synthesized phenyl and heterocyclic derivatives whose structure was designed by modification of hit compound central core and substituents with the aim to enhance the inhibitory activity.

1 Lowther J., Naismith J. et al., Biochem. Soc. Trans., 2012, 40, 547–554.
2 Strettoi E., Gargini C., et al.; PNAS, 2010, 107, 18706-18711.
3 Geekiyanage H, Upadhye A, et al., Neurobiol Aging. 2013, 34, 2037-2051.