• Slow Drug Development pipeline- To understand the predicament faced by humanity in developing effective therapeutics against deadly diseases, one need not look further than the COVID-19 pandemic waging around us right now. One might have often heard that it takes at least 10 years for a drug to be discovered and commercialised. However the actual journey might even take three times this time period! Our project arose out of this need for more effective techniques to speed up drug development. We realised that in vitro systems for testing drugs is of paramount importance.
• Viruses, a potent enemy- For viruses, in particular, the problems for drug development are many-fold. The arsenal is much more limited when compared to other infectious agents such as bacteria. Moreover, any drug that targets a part of the viral life cycle, could end up harming the host in the process since viruses hijack the cells of our own body to feed their replication machinery. And then comes the issue of diversity among viruses. Investing a huge deal of money and resources to study every unique virus stretches out the time needed immensely.
• Dengue- a socio-economic issue in the tropics and sub-tropics- Dengue is a critical disease that has now reached 129 countries and has put nearly half the world population at risk. Moreover this virus shows a diverse clinical picture making accurate diagnosis difficult. The existence of four different DENV serotypes makes it even more difficult to produce an effective, commercialised antiviral drug or vaccine. Yet there remains very little awareness about the mechanisms of its spread among the common people. Keeping these points in mind, we decided to focus on dengue as a proof-of-concept for our main project.

What is the problem your team is working to solve? How does it affect the world?

We took an approach of ‘Imagine each step -> Ask Questions: Why? How? -> Modelling to answer the questions -> Integrate the answers into the project -> Proceed ahead’ for the designing our overall project.
WHY DENGUE? → Epidemiological Studies
WHICH ONE TO TARGET: NON-STRUCTURAL OR STRUCTURAL PROTEINS? → Mutational Analysis

Figure Legend: Mutation Profile for DENV1

Conclusion: Evolutionarily speaking, targeting NSPs for iPEP designing is better than targeting SPs as the latter is more prone to mutations.

WHICH PPI TO TARGET? → Protein-Protein Interaction Studies (i) Aim 1: To select a particular PPI as the target.

Figure Legend: Representative Interactome of DENV NS5

Conclusion: Interaction between DENV NS5 and hSTAT2 was selected as target PPI.

(ii) Aim 2: Building structures for DENV NS5- hSTAT2 complex.

Figure Legend: Model of DENV NS5- hSTAT2 complex (Colour Codes: DENV NS5- Blue, hSTAT2- Green) obtained through Docking


Figure Legend: Model of DENV NS5 - hSTAT2 complex (Individual Structures were first obtained through Homology Modelling and then were docked. The insights from this model were helpful in FRaPPe plasmid designing) (Colour Codes: DENV NS5- Blue, hSTAT2-Green, White Sphere- N-terminal residues, Red Spheres- C-terminal residues)

HOW TO INHIBIT TARGET PPI? → Peptide Inhibitor Designing
Peptides binding to DENV NS5 and inhibiting DENV NS5 - hSTAT2 interactions were designed in-silico and were characterised in-silico for various physicochemical properties and their ability to bind to the target site.

[Figure Legend: iPEP library generated in-silico to target DENV NS5- hSTAT2 interaction]

HOW THE TARGET PROTEINS INTERACT? → MDS Studies

Molecular Dynamics Simulations were performed to study the interaction between DENV NS5 and hSTAT2.

[Figure Legend: Plot showing distance between the centre of mass of DENV NS5 and hSTAT2]

Contributions
1. Complex Models

• Docked Model of DENV NS5 and hSTAT2 structures (not the whole protein) obtained from PDB
• Docked Model of DENV NS5 and hSTAT2 structures (whole protein) obtained from homology modelling.

2. iPEP library containing potential peptide inhibitors targeting DENV NS5 - hSTAT2 interaction

3. Tips and Tricks for GROMACS troubleshooting while performing MDS on PPIs.

Owing to the pandemic, we did not have access to lab facilities during the lockdown. We have started our preliminary optimization experiments recently.

A FRET reporter, pCKI-PYRATES (Bulusu et al., 2017), that was available in the Department of Biological Sciences (DBS), IISER Berhampur was used to carry out restriction digestion with three sets of enzymes and validate it. We also performed in silico digests with the circular pCKI sequence. Since it contains coding sequences of the CFP and YFP variants, we decided to excise these fragments out for additional FRaPPe constructs. We aim to use these variants as they are more stable forms than conventional CFP and YFP to generate enhanced FRET sensors. We have also planned how we would be conducting the experiments if the opportunity came up.


Schematic for Optimization Experiments

Map of pCKI Optimization Plasmid features with enhanced FRET pair (mTurquoise ; cp173Venus) sequences
In silico digests to release fluorescent reporters

Restriction digestion of pCKI with EcoRI and HindIII, 0.8% agarose gel, 1X TBE

CODE	CONTROL CONSTRUCTS	CODE	TEST CONSTRUCTS
FRaPPe C1	FKBP-CFP	FRaPPe T1	FKBP C-hSTAT2(FL)-N CFP
FRaPPe C2	FRB-YFP	FRaPPe T2	FRB C-NS-5(FL)-N YFP
FRaPPe C3	C-hSTAT2(FL)-N CFP	FRaPPe T3	CFP FKBP N-hSTAT2(FL)-C
FRaPPe C4	C-NS-5(FL)-N YFP	FRaPPe T4	YFP FRB N-NS-5(FL)-C
FRaPPe C5	FKBP-N-hSTAT2(FL)-C
FRaPPe C6	FRB-N-NS-5(FL)-C

Table of constructs to be prepared. Cloning for NS-5 (GenBank : KR919821.1, DENV TSV08 Serotype 1) into Addgene plasmid # 20148 using HindIII at 5’ and SalI at 3’ and for hSTAT2 (GenBank : U18671.1) into Addgene plasmid # 20160 using XhoI at 5’ and EcoRI at 3’ to create test constructs.

Overview of the Reporter System showing the overall test procedure for experimentation and characterization of its working.


Dose dependent Rapamycin assay and corresponding FRET measurement to choose an optimum Rapa concentration. FRaPPe T2 and T3 co-transfected cells will be treated with the inhibitor and the efficiency of each peptide inhibitor in inhibiting the PPI will be quantified based on the FRET readout with respect to different concentrations of the iPEPs.




Parts



The proposed constructs that will be generated as a part of project FRaPPe.The designed constructs T2 and T3 have been registered with the standard registry of biological parts.

T2: BBa_K3646010 Is a composite project part composed of basic parts BBa_I712004 (CMV promoter), BBa_K3646002 (eYFP), BBa_K3646000 (DENV NS-5) and BBa_K3646004 (FRB)
T3: BBa_K3646009 Is a composite project part composed of basic parts BBa_I712004 (CMV promoter), BBa_K3646007 (eCFP), BBa_K3646008 (FKBP) and BBa_K3646006 (hSTAT2)


Plasmid Maps for FRaPPe project parts showing the tentative design for test and control constructs. Created using SnapGene.

Frappe, goes to Clinic

The proposed end-use of our tool is an HTS system in the drug discovery field. Compared to several sophisticated instruments, it's simple and easy to use, requiring basic molecular cloning and cell culture procedures.

Initial stages of studying the basic biology of the disease can be accelerated, combined with preliminary drug screening.

We will collaborate with researchers as discussed with NCBS, to take this forward in a research setting.

Troubleshooting and optimising the tool, is our next focus before making it available for researchers.





Safety

The FraPPe screening system is made with the HEK293 cell lines. It should be only used by researchers with at least BSL-2 infrastructure.

E.coli K12 derived DH5alpha; the bacterial strains we are using for cloning, are considered to be non-colonizing and non-pathogenic to humans or animals.

Based on the IHP, we will also be cloning FRaPPe in lentiviral construct.Lentiviruses belong to the class of biosafety level 2 material.
This work will be done in our BSL2+ facility and all safety guidelines relevant to cell cultures will be followed.


Challenges

The transition from the research lab to the clinic is by far the biggest challenge. A suitable therapeutic should meet several criteria including target populations (adults and children), dosage and pill burden, pharmacokinetics, safety, stability and cost.

Testing Antivirals would require more than several thousand patients in order to draw a statistically sound conclusion which may be taken up as a future initiative.

Interviews

We got in touch with experts from different fields including researchers, medical professionals, and government authorities from whom we got valuable insights into our project design and outreach activities. The takeaways from these interviews were implemented into our project lifecycle which helped in improving our results.

Outreach

We conducted a crowdfunding campaign to provide mosquito nets, masks, and information brochures to people living in low-resource areas. Fortunately, we were able to collect around 800 USD for this cause through which we distributed amenities to 70 families in Berhampur. At another level, we spread awareness about Dengue through our social media handles in the form of posters and threads.

Education

In collaboration with team iGEM IISER Pune, we conducted an interactive online workshop for middle school students on the prevention of Mosquito-borne diseases. We contributed several articles spanning domains of Synthetic Biology to different established blogs, designed pamphlets, posters, and comic strips. We also created an online forum named 'Syntillate', engaging writers from different disciplines of our home institute.

Splash

By creating this colouring book, called ‘Splash’, we tried to make an impact on primary class students by introducing to them some concepts of biology that they were able to learn in a fun way. To kindle their curiosity, we invited questions from them and tried to answer those in our Q&A forum. We received an enthusiastic response in this venture.