Revealing the MoA of the innovative antileishmanial agent H80, leveraging MS omics tools combined with ADME/Tox/Ecotox Chemoinformatic

 Motivation and Workplan Summary 

I am presently at my III year (last) of my PhD course in Clinical and Experimental Medicine (CEM), Nanosciences and Pharmaceutical Sciences Curriculum, at the University of Modena and Reggio Emilia (Dept of Life Sciences) within the laboratory coordinated by Professor Maria Paola Costi (Drug Discovery and Biotech Lab). 

The primary focus of my research lies in the development of innovative techniques to characterize the mechanism of action of novel inhibitors targeting the Transcriptional Enhancer Associated Domain 4 (TEAD4), a key protein in tumour growth a metastases development. 

However, during my three years of PhD (Nov 2021-Ongoing), and two years of junior research assistant and contracts (Sept 2019-Oct 2020), I have developed a keen interest in parasite diseases and their connection to environmental changes in both animals and humans, thanks to the guidance and involvement of Professor Maria Paola Costi and my participation in the COST Action CA21111 meetings and workshops. During these years, I have worked on Leishmaniases and Trypanosomiases treatments with innovative inhibitors of Pteridine reductase 1 (PTR1) and Dihydrofolate reductase (DHFR-TS). Also, I have performed different MS Proteomics and Chemoproteomics experiments to study the molecular bases of drug resistance and therapeutic failure mechanisms of these parasites, which resulted in two research articles on the ACS Inf Dis. 

Since I am particularly intrigued by the notion of One Health, which explores the interconnectedness between humans and animals, during a recent research pipeline of the Drug Discovery Lab, I have investigated the Guest-Host interplay in L. infantum amastigotes infecting THP-1 cells under the one health umbrella through novel Ecotoxicological tools. The collaboration with different members of the COST Action all over the World, encompassing diverse skills of research (from molecular biology to ecotoxicology and environmental sciences) has fascinated me and reinforced my idea that these concepts must fit any stage of the drug discovery pipeline. Indeed, from my point of view, a good Medicinal Chemistry programme should encompass ecotoxicology screening and One Health applications at any level, not just at the end of it i.e. when the lead compound has already emerged. This approach would also help obtain compounds whose activity is species-specific, avoiding off target effects and thus cross resistance mechanisms arousal. 

Parallelly, I have contributed to different research lines that deal with Drug Discovery and development of new antiparasitic agents, in particular anti trypanosoma and anti-leishmania agents, with the aim to limit the emerging problem of drug resistance to common available drugs. 

The main force that had me passionate for Medicinal Chemistry of NTDs, and the main issue that I think should drive people to invest time and money in this field, is the actual diminished level of interest exhibited by the pharmaceutical industry towards the exploration and innovation of new therapeutic interventions. 

Overall, vector borne NTDs is an umbrella term to point out a heterogeneous group of 20 diseases, affecting not only the low-income countries like sub-Saharan Africa, but also the Mediterranean cost and some areas in the southern and central America. 

Trypanosomatids are an order of unicellular protozoan parasites that are causative agents of several diseases, such as Leishmaniasis, Chagas disease, and human African trypanosomiasis (HAT), all of which are classified as NTDs. Leishmania infantum is the aetiological agent of visceral leishmaniases (VLs) in south Europe and Africa, and strictly related to L. chagasi, endemic in south America, and L. donovani, which share large part of the genome and protein similarity. 

Due to my increasing interest in this topic presented by the Cost Action CA21111 initiative for young researcher innovators (YRI), I have made the decision to submit my project proposal for the Short-Term Scientific Mission (STSM) in partnership with Professor T Katsila from the National Hellenic Research Foundation (NHRF), Athens. 

The decision to collaborate with Dr. Katsila in this Short-Term Scientific Mission (STSM) project is highly advantageous for my professional advancement and the acquisition of innovative skills in MS metabolomics and bioinformatic in the drug discovery field. 

Dr. Katsila primary research focus includes the study of the mechanism of action of anticancer and antiproliferative agents through innovative techniques like MS metabolomics and proteomics, and the exploitation of these tools in the field of translational medicine and biomarker research. Dr. Katsila’s laboratory has a strong background in a. mass spectrometry-based multi-omics and exosomal profiling, 3D cell models and cheminformatics, ADME-Tox in 3D and computational statistics to disrupt healthcare solutions and empower biomedical innovation. This toolbox at hand maps interindividual variability upon xenobiotics profiling and performs state-of-the-art drug repurposing. The extensive knowledge and proficiency possessed by Dr. Katsila in the areas of MS metabolomics and chemoinformatic predictive models present a valuable prospect for me to enhance my own understanding of these methodologies and employ them in the context of neglected tropical diseases (NTDs). 

In the context of this Short-Term Scientific Mission (STSM), we initiated our investigation by analysing the preliminary data derived from a previous study that employed mass spectrometry-based, label-free proteomics combined with cell fluorescence assays to describe the Mechanism of action of the Novel antileishmanial agent H80, a flavonoid-like derivative identified by a phenotypic screening. Preliminary results suggest its good cellular activity (EC 50 = 1.88 ± 0.32 μM in L. infantum promastigotes) with negligible cytotoxicity, and fluorescence and proteomics assays indicate its main mechanisms of action in the cytosolic fraction, with particular attention to Kreb’s energetic metabolism. However, the exact target of H80 is still unknown, along with its cellular pharmacokinetics and ADME/Tox. 

During my STSM, I would investigate- through MS metabolomics (untargeted, then targeted), the effects of H80 to parasites, and the metabolic modifications induced with 

respect to Miltefosine, its reference control. These data will be integrated with MS proteomics experiment, to obtain a multi-Omic panel of information. Also, predictive tools for the determination of ADME/Tox will be employed to characterize the PK of H80. This will help in the Drug Discovery Pipeline, to obtain more selective and less toxic derivative. 

Ultimately, the outcomes of this initiative have the potential to facilitate new partnerships with other research groups affiliated with the CA21111 network, as well as with Dr. Katsila.