Attività enzimatica e proprietà ADME-Tox di nuovi inibitori del hDHODH

Dihydroorotate Dehydrogenase (DHODH) is the fourth and rate-limiting enzyme of de novo pyrimidines biosynthetic pathway. This synthetic route is indispensable in proliferating cells, in order to meet the increased demand for nucleic acid precursors and other cellular components. DHODH, which converts dihydroorotic acid (DHO) into orotic acid (ORO) via a ping-pong mechanism played with ubiquinone (CoQ10), is usually overexpressed in most of tumors and viral infected cells, and that’s why the inhibition of human DHODH (hDHODH) has shown interesting results in terms of therapeutic effects against several cancer lines and several viral infections as well. During my thesis I worked in MeDSynth group, which is specialized in the production of hDHODH inhibitors by exploiting the bioisosterism concept; this tool allowed to produce active compounds structurally related to the two most historically studied hDHODH inhibitors, Brequinar and Leflunomide. Our lead compound, MEDS433, exhibited an excellent activity in the low nanomolar range (IC50=1.2 nM) at enzymatic level, even though resulted poorly water soluble. Thus, several analogues to this molecule have been developed, and the aim of my project was to determine the activity and the ADME-Tox properties of such of them. Specifically, I firstly studied activity and solubility of one Class of MEDS433’s analogues characterized by a phenoxy moiety instead of the original biphenyl group presents in the lead compound: this modification was done in order to improve the solubility by the presence of one more polar atom and, theoretically, to improve the enzymatic activity too by exploiting the better flexibility of the phenoxy ring. Unfortunately, in this case the analyses revealed that the inserted functional groups didn’t increase either the general enzymatic activity nor the solubility. In the meantime, I studied another Class of MEDS433’s analogues: in this case, these molecules were functionalised on the second ring of the original biphenylic group of the MEDS433’s scaffold, in order to improve the enzymatic activity. The most active one of the Class, MEDS613, was tested in order to assess its in vitro metabolic stability, to evaluate wether it could be a suitable back-up of MEDS433: the compound resulted to be fastly hydroxylated (T1/2 <15 min), therefore, several structurally related compounds will be produced, consisting of the same MEDS613’s scaffold but further functionalised to protect its metabolically liable site. T . During my thesis I also took part in the identification of the proper vehicle to administrate MEDS433 to mice for its acute and subacute toxicity tests, fundamental parameter that needs to be evaluated before approaching the clinical trials. The poor MEDS433’s solubility pushed us to use, as first attempt, a Self MicroEmulsifying Drug Delivery System (SMEDDS) made of corn oil, PEG400 and Cremophor®: since this despersant medium exhibited a significant intrinsic toxicity, it was further replaced by a less harmful sugar-based one, named MediGel Sucralose®, on which the MEDS433, previously dissolved in DMSO, was dispersed. Before the administration, the homogeneity of the dispersion and the time-related stability of the molecule in the system were determined, both via a UHPLC-UV system and via electronic microscopy. Since MediGel Sucralose® resulted to be a suitable vehicle for per os administration, a 14 days long experiment was conduced on mice. During the in vivo experiment, the hematological parameters have been analyzed.