Phd in LIFE SCIENCE

Thesis title: Design and Synthesis of New Indolyl Derivatives as SARS-CoV-2 Non-Structural Protein 13 Inhibitors

The severe and acute respiratory syndrome coronavirus 2 has caused to pandemic with considerably great socioeconomical impact worldwide. Throughout the global pandemic, vaccine development has been prioritized. Despite the outstanding job done in the development of vaccines, there are various downsides when it comes to lacking antiviral drugs active on SARS-CoV-2: first and foremost, the short-term vaccine-induced immunity, and obscurity of vaccines among new variants. Furthermore, the clinical efficacy of antivirals used throughout the pandemic also remains limited in spite of variety of the therapeutic agents. Taken together, these reasons rightfully highlight the pivotal importance of having broad spectrum antiviral inhibitors. Non-structural protein 13 was reported with 99.8% conservation ratio among variants with two druggable pockets the binding site occupied by AMP PNP, and 5′ end of the RNA substrate as well as potential allosteric hotspots. Targeting them with small molecule inhibitors could be advantageous considering the pace of evolution of variants. In this content, there are literature data suggesting the potential use indolyl diketo acids have as inhibitors of nsp13 for SARS-CoV-2. This class of compounds are particularly interesting since they were reported to have dual inhibitory activity, unwinding and ATPase activity with an allosteric mechanism of inhibition. Inspired by these outcomes, the main objective of the thesis is to focus at deepening the structure activity relationship of this novel class compounds with various strategies in addition to unveil a bioisosteric replacement analogue of indolyl diketo acids with pyrazole moiety placement. To state deeper, firstly the chemical space is assessed by systematically modifying the substituent patterns on the core scaffolds, which results in the design of new series of small molecules. The strategies that are employed within this include the removing of vinyl group that links the indolyl core to diketo acid moiety, the use of spacer with different chemical features in between indolyl core and phenyl ring as well as the placement of a Cl atom on position 5 of indolyl core and R-group substitutions of benzyl ring with a diverse set of functional groups with differing electronic, steric, and lipophilic properties. Bearing these strategies, the new analogues were designed, synthesized, characterised and their inhibitory profile were examined with in vitro studies asides from cellular antiviral activity assessment in addition to the assessment of inhibition type. The results indicate that almost all the newly obtained analogues were active in low micromolar range and displayed a non-covalent, reversible inhibition mechanism of action. Followingly, the binding pose was also examined. The results demonstrate a similar binding pattern with previously reported allosteric fragments in literature. Another strategy that was employed within the thesis is the bioisosteric replacement of diketo acid moiety. In order to improve the pharmacokinetic and pharmacodynamics profile, this is essential due to the ionized presence of diketo moiety at physiological pH, which could result in high metabolic turnover and poor cell membrane penetration. In this sense, the replacement of the DKA chain with a pyrazole ring – a Lewis base that could mimic the DKA functionality through number of heteroatoms and bonds was employed. Followingly, various analogues of this new class were also designed with a similar fashion with previously mentioned substituent patterns in a way that the comparison of diketo acid and pyrazole analogues can be done. Having synthesized and characterized these analogues, their inhibitory profile was assessed with in vitro studies. The results indicate that the pyrazole analogues are also active in the low micromolar range as it is with diketo acid analogues. Furthermore, the binding pose prediction was also done by in silico studies. There is a correlation with the binding modes of pyrazole analogues and diketo acids analogues as it would be expected. Lastly, the comparison of pyrazole and diketo acid analogues were assessed by SAR report option of Molecular Operating Environment to clearly illustrate the correlation between the analogues as well as assessment of contribution of each varying substitutes both individually and combination strategies. Taken together, lessons learned from the development of these analogues reveals promising insights and can accelerate the development of hit-to-led stage.