IISER-BPR IGEM

Implementation

FRaPPe : A RESOURCE FOR RESEARCHERS

The implementation of our reporter system, FRaPPe will find applications in many branches of study.

Researchers working in the area of protein-protein interactions

Researchers working in the area of host viral interactions

Researchers working in the area of viral diseases

Drug discovery, Pharmaceutical Research and Development

Researchers working at the interface of chemistry and biology

...and many more!

We hope to make this tool a valuable addition to the arsenal of resources for researchers not only in molecular, cellular and infectious disease biology but also, chemistry and interdisciplinary fields. A new discovery takes several years to be refined and matured into a viable product. For viruses, in particular, the problems are many-fold, considering their diversity and mechanisms of hijacking host machinery. Investing a huge deal of money and resources to study every unique virus stretches out the time needed for antiviral development immensely. Using this tool, the initial stages of studying the basic biology of the disease can be accelerated, combined with preliminary drug screening too.

FEATURES

The utility of FRaPPe is reflected in its various characteristics:

It helps in understanding how any two proteins in an important pathway come together and help in cellular function.

It helps in modulating the strength of the above interaction through chemical biology.

It can be used as a High Throughput Screening (HTS) system, to assay for chemical and biological modulators of the above protein-protein interaction. And thus, can be used for drug discovery and development.

FRaPPe will thus facilitate the development of an efficient reporter to study host-pathogen protein interactions and enable the screening of potential drugs such as peptide inhibitors.

Figure 1: The Transition of FRaPPe from the research lab to the clinic, summarising it's feasibility, safety considerations and challenges to be addressed.

CHARTING OUT THE FUTURE PLAN

The proposed end-use of our tool is an HTS system in the drug discovery field. However, there are multiple stages to tackle before this can be achieved. Hence, we decided to proceed strategically in this aspect. Before pitching our idea for large-scale usage, we wanted to assess the feasibility of its usage. Discussing with the stakeholders of the pharmaceutical companies to propose this HTS system for drug discovery was our first step. We had a meeting with Hardik V Sheth, Area Sales Manager for PerkinElmer (India) Pvt Ltd. and his technical team from drug designing to review the current status of the field. While there are several sophisticated instruments to screen drugs, cost, time and labour are major constraints. Our system, on the other hand, is simple and easy to use, requiring basic molecular cloning and cell culture procedures. Owing to the widespread usage of fluorescence tools in cell biology in modern research, these instruments and procedures are also accessible in most labs. The biggest boon that our system confers compared to more conventional immunoprecipitation based methods for studying protein complexes, is the obtainment of a rapid readout. We would like to improve and strengthen our quantification procedures using fluorescent tools to address any issues that may arise during its implementation. We will also collaborate with researchers as discussed with NCBS, to take this forward in a research setting. In our meeting with Prof. Sudhir Krishna and his research group, we realised that if our system works, there will be sufficient demand for it given the race to find cures for viral diseases. (Visit our Human Practices section for more details.) Troubleshooting and optimising are thus going to be our next order of business once we can get our wet lab completed.

SAFETY

Precautions have been observed during our preliminary optimisation experiments. Since the end-users of our project are researchers in universities as well as pharmaceutical companies, the organisms we build do not possess a risk to the general public. 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. They are considered to belong to risk group 1. They have limited ability to grow in environments other than laboratory culture conditions, and hence there is minimal risk associated with these bacterial cells. The reporter system that will be cloned in the above chassis, will consist of ORFs of human origin or dengue viral origin. Since these are cloned under different promoters, they will not retain any infectivity. Also, the cloning related bacterial culture work will be performed in a designated BSL1 facility.

HEK293 cells are classified as risk group 2. They have a portion of human adenovirus 5 integrated into their genome, however since this is not the entire viral genome, it is considered incapable of resulting in any viral infections. Generalized infections can occur in immunocompromised individuals. Transient transfection of the reporter systems will be done in this chassis. Because of this reason, there will be no integration of the DNA and thus no genetically modified lines will be created. All cell culture work will be performed in designated BSL2 facilities.

Lentiviruses belong to the class of biosafety level 2 material. Due to its modified features (deletion of virulence genes), it is unable to produce viral materials after infecting the host cells. To minimize the environmental impacts replication-competent virus testing will be done. Based on the IHP, we will also be cloning FRaPPe in lentiviral construct. 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 in front of us. Vaccine development for DENV is difficult as inducing protection against four different serotypes presents a significant challenge. Incomplete vaccine-induced immunity against any single serotype could predispose an individual to severe disease during subsequent natural infection.

Although the efforts into developing suitable therapeutics have been low, the research landscape in this field is gradually changing with increasing awareness. A suitable therapeutic should meet several criteria including target populations (adults and children), dosage and pill burden, pharmacokinetics, safety, stability and cost. A DENV antiviral in particular, must be fast-acting and will be more effective when used with rapid diagnostics of early DENV infection (such as the NS1 test). (Low et al., 2017)

Notably, the current rationale for a dengue antiviral is to rapidly reduce viremia by >10-fold during the early phase of DENV infection; as this would translate into clinical benefits and prevent the development of DHF/DSS (Khanna et al., 2016). Prevention of the infected patients from development into severe diseases (DHF/DSS) should be used as the ultimate Proof-of-Concept criteria in designing DENV antivirals. However, testing this would require more than several thousand patients in order to draw a statistically sound conclusion. While such preclinical and clinical studies are beyond the scope of our particular project, it may be taken up as a future initiative. Consulting with appropriate medical and public health officials can help us understand the logistics of commercialisation.

Our inhibitors of choice, namely, interference peptides also pose a few challenges. The need for intravenous administration and low-shelf life could limit their use in clinics. Given that dengue is prevalent mostly in developing countries, it will be challenging to make this class of inhibitors accessible to poor patients living in rural and low-resource regions. Delivery into the recipient requires stability, however, we have tried to address this by making relevant chemical modifications to the drugs. (Refer to our Modelling Section.)

References:

Low, J. G., Ooi, E. E., & Vasudevan, S. G. (2017). Current Status of Dengue Therapeutics Research and Development. The Journal of infectious diseases, 215(suppl_2), S96–S102. https://doi.org/10.1093/infdis/jiw423

Niyati Khetarpal, Ira Khanna, "Dengue Fever: Causes, Complications, and Vaccine Strategies", Journal of Immunology Research, vol. 2016, Article ID 6803098, 14pages, 2016.https://doi.org/10.1155/2016/6803098