PhytoDenCov-3 – Fighting Dengue and SarsCov2 with plants

A Conservation Effort on Malaysian Annonaceae Plants and its Identification of Novel SarsCoV-2 and Dengue Antiviral Agents

PITCH

 

INTRODUCTION TO THE PROJECT

According to the World Health Organization, dengue fever is the most critical and the most rapidly spreading mosquito borne disease in the world, and no antiviral specific to DENV has been identified (just like CoV-2 SARS). Dengue virus (DENV) is the key vector-borne virus that causes hemorrhagic fever in humans. The number of cumulative reported cases affected 80,590 last year in Malaysia and is projected to rise by 2030.
With nearly 15,000 species of vascular plants, Malaysia is one of 12 mega biodiversity countries in the world. However, modernization and global warming are now threatening the highly valuable plants producing active agents against the virus. Hence, the aim of the PHYTODENCOV project is to identify dual inhibitors effective against SARS-CoV-2 and DENV from Malaysian medicinal plants while building living plant collections to support conservation. Our laboratory, in collaboration with ICSN-CNRS, and PIMIT has identified two Malaysian plant extracts (Desmos dumosus and Goniothalamus giganteus) from Annonaceae family with dual anti-DENV and anti-HCoV- 229-E activities.

Objectives : To have natural substances that are capable of acting in concert against DENV infection and SARS-CoV-2.

PROJECT IN ACTION

Hypothesis: The 20 extracts of Malaysian plants active against HCoV-229-E are also effective against SARS-CoV-2 since it is genetically close to beta-coronavirus and shares common mechanisms of replication. On the other hand, the two extracts (Desmos dumosus and Goniothalamus giganteus) with a dual activity against DENV and HCoV-229-E would allow us to envisage a multiple therapeutic approach (when symptoms are similar or in the case of potential comorbidities).

Task 1: Biodiversity conservation : To conduct collection and replanting the selected Malaysian Annonaceae plants as well as initiate the formation of the natural products extract library

Plant collection and conservation : The species biology is used to inform the collecting strategy. Each of the population has been separately managed. Multiple accessions will be collected and maintained over a few years. The selected Annonaceae plant species were and will be replanted in the Rimba ilmu, a tropical botanical garden, set up in the University of Malaya campus in Kuala Lumpur, Malaysia.

Task 2 : Phytochemistry :
– To isolate and characterise the structure of bioactive compounds through various chromatographic and spectroscopic methods
– To expedite elucidation analysis, training, and creating the natural products database and library

Purification and structural elucidation of the isolated compounds from the active fractions : 1) Isolation of bioactive molecules with > 90% purity. The purification of antiviral compounds used a variety of chromatographic methods: flash chromatography, HPLC, HPLC-MS, TLC. 2) The fractionation and isolation were guided by antiviral testing carried out on the HCOV-229-E and the DENV at PIMIT. 3) Structural characterization of active molecules and related non-active analogues, with a view to establishing structure-activity relationships (ICSN and UM). Once all the data in a text format (mzXML or mgf), the data was subjected to Spectral Networks, which includes MS-Clustering, followed by generating text files with attributes using MATLAB, which was imported into Cytoscape for visualization as a network.

Task 3 : Experimental virology : To perform antiviral assay against DENV infection and SARS-CoV-2 on the fractions and compounds isolated

1) Evaluation of the antiviral activity of selected extracts/compounds against a clinical isolate of SARS-CoV-2: A549 lung carcinoma cells expressing human ACE2 (SARS-CoV-2 receptor) was used as cell model. A549-ACE2 was infected with SARS-CoV-2- by presence of increasing concentration of extract to determine the median inhibitory concentration (IC50) corresponding to a 50% decrease in viral infection. Infection rates are measured by quantification of viral RNA in the infected cells by qRT-PCR as well as the measurement of infectious titers. 2) Evaluation of the antiviral activity of selected extracts/compounds against a clinical isolate of DENV: Human liver cells were used as cell model. Huh7 was infected with DENV (different serotype I, II and III which are currently circulating in Reunion Island) in the presence of different concentration of extract to determine the median inhibitory concentration (IC50) corresponding to a 50% decrease in viral infection. Infection rates are measured by quantification of viral titer by plaque forming assay (PFU).

NEXT STEPS

  • Publication of findings
  • Dissemination awareness towards local communities : through The Enviromental Education Programme (EEP) Scientific reports on research findings (Publication/Journal)
  • Built the foundation for the plant repository, starting with all the extracts of collected and studied plants, fractions and pure compounds
  • Further characterization of the identified biologically active compounds and potentially propose a medicinal chemistry program to synthesize active molecules and their analogues (hits to lead).
  • Impacts:
    1- Demonstrate the importance of rare plants as sources of a potential drug for treating microbial infections, such as COVID and dengue.
    2- The repository will be a point of reference for the biodiversity of Malaysian plants that could be developed further to be an international centre.

PhytoDenCov-3 ON THE WEB

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CONTACT

Project leader : Khalijah AWANG (khalijah@um.edu.my)

Associated researchers :
Muhamad Aqmal Othman (aqmal@um.edu.my)
Marc LITAUDON (marc.litaudon@cnrs.fr)
Chaker EL KALAMOUNI (Chaker.el-kalamouni@univ-reunion.fr)

Countries involved : Malaysia and France