Characterization of in vitro and in vivo properties of pharmacologically active compounds in a GLP-Like laboratory

The first chapter of my PhD thesis is part of TUSCAVIR.NET project funded on November 23rd, 2018, by the Tuscany Region in the public call for health research. This work was developed to establish and validate standard operating procedures (SOPs) and protocols for the study of ADME parameters and in vivo PK parameters of new potential antiviral compounds. To do this, MAS584 and EIDD-1931, which were found to be particularly active on several viruses, were chosen from an internal library of compounds and the literature and then used as standards for further studies. MAS584 is a thiobarbituric derivative that has shown interesting antiviral activity on enveloped viruses. EIDD-1931, on the other hand, is a nucleoside analogue capable of inhibiting viral RNA-polymerase as a chain terminator and has shown antiviral activity on a wide library of viruses but is characterized by poor oral bioavailability and reduced lipophilicity. Once we developed and validated SOPs and protocols for the ADME and pharmacokinetics studies, the second part of the project saw the application of these on pharmacologically active compounds, which led us to several scientific papers with relevant results, and here are reported three of them as examples. This made it possible to provide the Regional Health System with a qualified tool to support the research and development process. The second chapter of my PhD thesis deals with the development of a sensitive and reproducible LC-MS/MS method that uses O-BHA (O-benzylhydroxylamine) as a derivatizing agent for quantitative derivatization of the major SCFAs (acetic acid, propionic acid, butyric acid, and isobutyric acid) for quantification their plasmatic levels in human, rat, and mouse samples. The development of an analytical technique to measure these molecules is crucial to understanding how SCFAs affect behavior and cognitive function and how they influence metabolism and signaling in different disease states. It was, therefore, necessary to develop a chromatographic method for quantification: first by optimizing the instrumental parameters of the mass spectrometer, then by searching for the best chromatographic conditions. To verify that the chosen analytical conditions had good linearity and excellent sensitivity, methanolic solutions, with scaled-up content, of the four previously mentioned acids were analyzed, after derivatizing and characterizing them. Subsequently, the ideal conditions for derivatization and extraction from plasma were refined. Aliquots of human, rat and mouse plasma were used for a series of determinations to validate the overall procedure (derivatization, extraction, and LC-MS/MS determination). Once the method was validated and its applicability to different biological matrices established, major SCFAs were quantified in adult and aged mice, germ-free (GF) mice and GF recipient mice undergoing fecal microbiota transplantation (FMT) from adult (FMT-adult) and aged (FMT-aged) donors. In the third and last chapter, we tried to find new derivatives of 2,3-dihydrophthalazine-1,4-dione that could have higher affinity and selectivity on PARP10, an ADP-ribosyltransferase capable of attaching an ADP-ribose unit post-translationally on various amino acid residues of target proteins. This protein plays an important role in apoptosis, DNA repair, and transcription and several studies showed that this protein and the other enzymes like this are overexpressed in some tumors. For this reason, PARP10 represents a potential drug target for small molecule inhibitors and to validate a possible approach to cancer treatment. We performed several activity assays to establish which compounds could be most promising as PARP10 inhibitors, and from the preliminary data obtained, OULL-0312 resulted in the best one. OULL-0312 also profiled with other PARP family enzymes was characterized by a selective action on PARP10. This compound was then chosen to study the interaction and bond with the PARP10 protein co-crystallizing them. These studies with PARP10 did not yield good results, highlighting the need for further studies to optimize crystallization conditions. We tried then to co-crystallize the OULL-0312 with the PARP15, used in structural studies with several PARP inhibitors because we already set up the best crystallization condition. We obtained good crystals, capable of diffracting but the structural studies are still ongoing.