Evaluation of hepatic activity of immunomodulatory compounds and GSK3 inhibitors

Resumen

Thesis Title: Evaluation of hepatic activity of immunomodulatory compounds and GKS3 inhibitors Submitted in fulfillment of the requirements for the degree ofDoctor with international mention at the University of Granada by José Pérez del Palacio. February 2017, Granada, Spain Summary: Nitric-oxide synthase (NOS), the enzyme responsible for mammalian nitric oxide (NO) generation, and cytochrome P450 (CYP450), the main enzymes involved in drug metabolism, share striking similarities. First and foremost, both are heme-thiolate proteins, which employ the same prosthetic group to perform similar chemistry. Moreover, they share the same diflavoprotein reductase as redox partner. Therefore, it makes sense that cytochrome P450 drug-mediated biotransformations might play an important role in the pharmacological modulation of NOS. In this work, we have undertaken an integrated in vitro assessment of the hepatic metabolism and NO modulation of previously described dual inhibitors (imidazoles and macrolides) of these enzymes, in order assess the involvement of CYP450 activities in the production of NO. From the experience acquired during this aim, we developed a validated high-throughput screening (HTS) approach in 96-well plate format for the assessment and discovery of molecules with anti-inflammatory/immunomodulatory activity. The in vitro models were based on the quantitation of nitrite levels in lipopolysaccharide (LPS) stimulated RAW 264.7 murine macrophages and interleukin-8 (IL-8) in Caco-2 cells stimulated with interleukin 1β (IL-1β). In order to validate this new methodology, we used this platform in a pilot project to screen a subset of 5,976 non-cytotoxic crude microbial extracts from the MEDINA microbial natural product collection. As a result, we isolated a previously described molecule with potential activity against inflammation in the central nervous system (CSN). It is widely accepted that CSN inflammation conditions play a significant role in the progression of chronic neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, neurotropic viral infections, stroke, paraneoplastic disorders, traumatic brain injury and multiple sclerosis. Therefore, it seems reasonable to propose that the use of anti-inflammatory drugs might diminish the cumulative effects of inflammation. Indeed, some epidemiological studies suggest that the sustained use of anti-inflammatory drugs may prevent or delay the progression of neurodegenerative diseases. However, the anti-inflammatory drugs and biologics used clinically have the disadvantage of causing side effects and the high cost of treatment. Alternatively, natural products offer great potential for the identification and development of bioactive lead compounds into drugs for treating inflammatory diseases with an improved safety profile. To our knowledge, this is the first report on a high-throughput screening (HTS) of microbial natural product extracts for the discovery of immunomodulators. When assessing enzyme inhibition as is the case of the present work, aqueous solubility of test compounds is a key factor to be considered. This is really a hot topic within drug discovery research that has been explored in this work with the double objective of investigating how solubility is involved in the CYP450 and NOS interactions and in the other hand for improving the methodologies of drug metabolism evaluation at MEDINA drug discovery setting. Therefore, in a first approach we investigated role that compound aqueous kinetic solubility (KS) may play in this lack of correlation observed between different in vitro models for assessment of CYP450 inhibition. In terms of metabolism, drug-drug interactions mediated through CYP450 inhibition are a significant safety concern, and therefore the effect of new candidate drugs should be screened early on CYP450 activity. This is a common practice in drug discovery to screen new chemical entities in order to predict future drug-drug interactions. For this purpose, there are two main assay strategies, one based on human recombinant cytochrome P450 (hrCYP) enzymes and fluorescent detection, and the other one based on human liver microsomes (HLM) and liquid chromatography coupled to mass spectrometry (LC-MS). Many authors have reported a poor correlation between both technologies, giving rise to concerns about the usefulness of fluorometric methods for predicting these drug-drug interactions. To investigate the effect of aqueous solubility in CYP450 inhibition we used a set of drug discovery compounds within a project for the discovery of glycogen synthase kinase 3 (GSK3) Inhibitors along with well-known inhibitors of main isoforms of CYP450. As a result, we found that drug discovery compounds with unacceptable kinetic solubility tended to yield higher IC50 values in in vitro models based on HLM, whereas compounds with KS values higher than 50 µM showed very similar IC50 values in both in vitro models. Our results show that the KS assay is a useful tool to identify those discovery compounds that may require further investigation in order to avoid overlooking future drug-drug interactions. From these findings we conclude that the integration of solubility and activity (enzyme inhibition) of the tested products was of critical importance to achieve a rational ranking of drug candidate compounds. In the initial stages of drug discovery, when physicochemical properties such as aqueous solubility have not yet been optimized, there might be a large number of candidate compounds showing artificially low CYP450 inhibition, causing potential drug-drug interaction toxicity to be overlooked. For these reasons, within this work we set a new objective in order to develop and validate a novel in vitro approach to simultaneously assess CYP450 inhibition and KS solubility (NIVA-CYPI-KS). This new methodology is based on fluorogenic CYP450 activities and KS measurements for compound solubility and it provides a significant improvement in the use of resources and a better understanding of CYP450 inhibition data. We have used this approach, among others, to investigate the hepatic metabolism of imidazole derivatives and macrolides in the context of immunomodulation.