This is a translational study in which, starting from the molecular mechanisms involved in the early steps of autophagy, we will investigate the role of the autophagic process in the pathogenesis of Rheumatoid Arthritis (RA), implementing a rational drug design approach to discover and develop new drug compounds.
Autophagy is a catabolic process aimed at engulfing cellular components and damaged organelles in internal vesicles (autophagosomes) that then fuse with lysosomes. This process occurs at a basal level in most tissues and contributes to the steady-state turnover of cytoplasmic components. Autophagosomes may originate from different membrane sites, mainly Mitochondria-associated membranes (MAMs) where raft-like microdomains are enriched.
Autophagy may represent a functional processing event creating a substrate for autoreactivity. Recently, we demonstrated that autophagy activate peptidyl arginine deiminase, generating citrullinated peptides in fibroblasts and synoviocytes, with consequent triggering for anti-citrullinated peptide antibodies, the serological marker of RA. Thus, this research program is divided into 5 main objectives:
1.Analysis of the role of "raft-like microdomains" in the regulation of the signals involved in autophagy;
2.Analysis of a possible regulation of the autophagy at the level of MAMs;
3.Proteomic analysis of MAMs in the absence or following autophagic stimulus;
4.Analysis of the role of autophagy on post-translational modifications of proteins: a possible trigger for anti-citrullinated and anti-carbamylated peptide antibodies:
5.Synthesis and evaluation of the effect of heparanase inhibitors on autophagy modulation. All the compounds will be previously tested for selectivity, efficacy and safety.
Research in this field may contribute to clarify whether pharmacological regulation of autophagy might modify the autoimmune response and progression of RA, thus disclosing new potential therapeutic targets for autoimmune diseases.
The combined data from the multidisciplinary approaches described above will enlighten the molecular mechanisms controlling the upstream subtle regulation of autophagy, key process in cell metabolism and stress response and their involvement in RA or other autoimmune diseases. The targets and compounds identified will create the basis for the development of novel types of drugs targeting cellular catabolism.
Syntheses will be performed applying the most modern organic and pharmaceutical chemistry approaches to deliver compounds with maximized purity, yields and throughput. Techniques will include multicomponent reactions, liquid-phase parallel synthesis, microwave and flow chemistry techniques, under pressure and inert reactions, applying selective protective groups and stereoselective synthesis. Series of congeners will be obtained by means of a liquid-phase parallel synthetic approach that provides high output, optimizing the time needed to produce the series. In particular, flow chemistry in which reactive components flow down a temperature controlled pipe or tube will provide faster reactions, purer products, safer reactions, rapid reaction optimization, easy scale-up, and the integration of classically separate processes (such as synthesis, work-up and analysis). All compounds will be produced in a sufficient quantity based on the needs of biological assays. The structure and purity (>95%) of the molecules will be confirmed by HPLC, LC-MS and NMR spectroscopy.
Moreover, we expect to identify novel interactors of the given molecules, their targets and find post-translational modifications turning out to be required in the ontogenesis or progression of other diseases, such as neurodegeneration or cancer.
In addition, taking advantage of the deeper knowledge of some pathogenic aspects of RA and on the recently discovered cell processes (such as citrullination, autophagy and NETosis) might represent an innovative tool for the discovery of new biomarkers, e.g. characteristics of germs, their enzymatic machinery, their interaction with host immune response and the induction of cellular response affecting survival. Expected results might rapidly have applicative implications aiming to evaluate their weight in diagnostic procedures and, even more, prognosis and response to treatment.
Our final goal is to validate the identified biomarkers by larger clinical studies accordingly to specific therapeutic protocols based on the use of new strategies of care (standard DMARDs) or new drugs (biological drugs). All this could have great scientific value (for the progress of knowledge), great importance for clinical, preventive and therapeutic aspects (to better answer the patients needs), but also great economic relevance (to allow a rationalization of the health care costs). As for this last point, it is noteworthy to mention that the new and effective treatments of RA, with the employment of biologic drugs, suffer from the huge costs and the weight of severe side effects. Moreover, these therapies are effective only in a limited percentage of patients (about 30%), with response to drugs being sometimes ambiguous and unpredictable. This scenario imposes the need of identifying effective and selective markers able to drive therapeutic choices. We expect that the achievements of the aims of this project will have positive consequences in terms of economic costs as well as for the life quality of patients suffering from RA.