Background

Malaria is a mosquito-borne disease caused by the Plasmodium parasite, and is one of the toughest global health crises we face today.

In 2018 alone, it was responsible for 228 million infections and 405,000 deaths worldwide, with children alone accounting for 67%.^[1]

The Plasmodium parasite completes its life cycle through two different hosts: human beings and female Anopheles mosquitoes (as illustrated below). Female Anopheles mosquitoes act as the vectors for this disease by carrying the malarial sporozoite in their salivary glands. Out of the 5 species of these parasites that cause malaria in humans, Plasmodium falciparum is the deadliest.^[2]

Over the past few decades, Plasmodium falciparum has gained resistance to drugs commonly used to treat Malaria, such as chloroquine, sulfadoxine, quinine, and mefloquine, especially in the South-Eastern parts of Asia. As of today, Artemisinin is the most potent anti-malarial drug. There is, however, growing evidence for malarial parasites attaining Artemisinin resistance worldwide. ^[3] Our last line of defense is Artemisinin Combination Therapy (ACT)*, and it’s failing fast, potentially making malaria untreatable in the years to come.

*ACT uses an artemisinin derivative with a partner drug. Artemisinin has a short lifespan in the human body, reducing the number of parasites during the first 3 days of treatment, while the partner drug stays in the human body for longer, eliminating the remaining parasites.

Therapeutics

We are designing a novel class of orally administrable drugs to treat malaria and overcome the antimicrobial resistance of the pathogen.

The drugs consist of two parts: an inhibitory peptide and a cyclotide backbone.

We aim to create a library of inhibitory peptide molecules, which target multiple, crucial host-pathogen interactions in the blood stage of Plasmodium falciparum malaria, effectively preventing the parasites from infecting human red blood cells (RBCs).

The peptide molecules are then grafted into a cyclotide backbone, which acts as the drug scaffold, in this case, the cyclotide Kalata B1.

Cyclotides are plant-derived circular peptides and have been previously used to design drugs. Their unique circular backbone topology and knotted arrangement of three disulfide bonds make them exceptionally stable to degradation compared to other peptides of similar size. They are also highly tolerant of sequence variability and are orally bioavailable.

Apart from this, cyclotides are robust, efficient, and cost-effective making them appealing candidates for drug design.^[5-9]

Using in-silico modeling, we have created inhibitory sequences for particular human - parasite protein interactions, which will then be grafted into Kalata B1 for expression.

We hypothesize that the drug will essentially outcompete the host epitope, thus preventing pathological infections.

Creating a library of peptide molecules ensures that the treatment of Malaria is not affected by the development of resistance to drugs by Plasmodium falciparum. Once it develops resistance to a particular drug, a different one can be picked from the library to treat Malaria, effectively circumventing the problem of drug resistance.

*A schematic depicting the development of potential inhibitory peptides against Malaria*

Diagnostics

Successfully eradicating malaria requires us to address the complex interaction of the various factors that cause the prevalence of the disease. Keeping this in mind, we wanted to create a project that would address all the different aspects of the disease.

Through our research and on talking to various experts, we realised that a lack of infrastructure in remote areas was a major hindrance in eliminating the disease.

This means that it is not only harder to study the genetic variations in malarial parasites in remote areas, but also to diagnose patients due to the lack of testing facilities.

The diagnosis of malaria is currently done by testing a blood sample in the lab. The current gold standard for the diagnosis of malaria involves microscopy with visualization of Giemsa-stained parasites in a blood sample.^[10,11] The species determination is done based on the morphological characteristics of human malaria parasites and the infected red blood cells. This requires access to a lab with the technical expertise to identify malarial parasites and sophisticated microscopes.^[12]

However, in isolated and remote areas, this poses a huge challenge in accurately diagnosing malaria, which affects the treatment patients can avail. In order to bridge this gap, we aim to create a portable diagnostics kit that will effectively enable healthcare workers to increase their testing capabilities. All they have to do is capture an image of a blood smear using a smartphone and test for the presence of malaria parasites using a Web API and voila, you have a diagnosis of whether a patient has malaria!

COVID-19

Covid-19 significantly impacted various aspects of our project. Our college shut down all operations in the month of March.

Since all team members were at home in different cities due to quarantine, physical meetings were affected and we had to come up with an efficient way to work remotely and digitally. Network issues, time constraints, remote human practices, and online meetings posed unique challenges during the course of our project; real-time online collaboration, keeping track of work being done across the team, formulating human practices strategies that could be conducted primarily online, and so on were some of the issues we faced. We powered through, however, and used various digital platforms, like zoom, trello and slack for efficient collaborations.

Due to the pandemic, our institute was fully closed and non-operational till August 2020, after which a few students returned in a staggered manner according to a priority order. Obtaining the necessary space and permissions, two members were able to work in the lab starting from the 20th of September, months later than originally planned, greatly affecting the scope of experimental verification in our project.

Nevertheless, we have wholeheartedly worked to the best of our abilities to develop this project as much as possible and have documented the same throughout this wiki.

Project Inspiration

While searching for a project idea, all of us had a common goal in mind: we wanted to contribute to solving a real-world problem that was affecting the communities around us.

Mosquitoes are quite an issue on our college campus and one day while lamenting over our annoyances at being bitten by them, we thought of looking up mosquito-related diseases, just out of curiosity. The statistics that we found on malaria shook us, to say the least. We knew that malaria was a problem, but that big and pervasive a problem? We didn’t have the slightest idea.

As we progressed through our research on the impact of malaria on the world, the idea of working on it was increasingly cemented in our heads. But we didn’t know how we could make an impact through synthetic biology. We thought of creating drugs but there already existed so many that it didn’t really seem groundbreaking. That is until we came across reports of increasing drug resistance.

What if we could create a class of drugs that could bypass resistance? This would allow us to have the evolutionary upper hand in some sense. The idea was an exciting possibility. But how would we create such a class of drugs? We knew the basic epidemiology of malaria, and we knew that it infected RBCs. It had to do that by entering them, and that would potentially be mediated by some sort of interactions between the RBCs and the parasite. And that’s when we had our light bulb moment, what if we could inhibit it from infecting the RBCs by blocking those interactions? And if you blocked multiple interactions, you could create multiple inhibitors, which would bypass resistance, as and when the parasite developed resistance to a particular inhibitor. We were absolutely thrilled at having made that leap. Just one problem remained. How would we deliver said inhibitors?

And that’s when we hit the jackpot. Cyclotides! A team member came across the work of Team Heidelberg 2014 that had worked on the synthesis of cyclized proteins. And on reading more about it, we realised that cyclotides could be grafted with a peptide of choice to give them more stability.

And that was it. We had a cyclotide based drug! Now all that was left was the herculean task of actually coming up with the system. Browse through our wiki to find out how!

References

1: World Health Organisation; The “World malaria report 2019” at a glance (December 4, 2019)
Link

2: Ultrastructure of the Asexual Blood Stages of Plasmodium falciparum, Eric Hanssen, Kenneth N.Goldie, LeannTilley (2010).
DOI: 10.1016/S0091-679X(10)96005-6

3: Artemisinin-Resistant Plasmodium falciparum Malaria, Rick M. Fairhurst, Arjen M. Dondorp (2016).
DOI: 10.1128/microbiolspec.EI10-0013-2016

4: World Health Organisation, Q&A on artemisinin resistance (August 2020)
Link

5: Spider-venom peptides that target voltage-gated sodium channels: Pharmacological tools and potential therapeutic leads. Julie K.Klint, SebastianSenff, Darshani B.Rupasinghe, Sing YanEr, VolkerHerzig, Graham M.Nicholson, Glenn F. King (2012).
DOI: 10.1016/j.toxicon.2012.04.337

6: Novel protein scaffolds as emerging therapeutic proteins: from discovery to clinical proof-of-concept. Thierry Wurch, Alain Pierré, Stéphane Depilst 2012.
DOI: 10.1016/j.tibtech.2012.07.006

7: Discovery and development of the χ-conopeptide class of analgesic Richard J.Lewis (2011).
DOI: 10.1016/j.toxicon.2011.07.012

8: “Splicing up” drug discovery. Cell-Based Expression and Screening of Genetically-Encoded Libraries of Backbone Cyclized Polypeptides, Harshkumar Sancheti and Julio A. Camarero (2009).
DOI: 10.1016/j.addr.2009.07.003

9: FN3: a new protein scaffold reaches the clinic. LairdBloom, Valerie Calabro (2009).
DOI: 10.1016/j.drudis.2009.06.007

10: Malaria Diagnosis Using a Mobile Phone Polarized Microscope. Casey W. Pirnstill, Gerard L. Coté. Sci Rep 5, 13368 (2015).
DOI: 10.1038/srep13368

11:Imaging and identification of malaria parasites using cellphone microscope with a ball lens. Temitope E. Agbana, Jan-Carel Diehl, Fiona van Pul, Shahid M. Khan, Vsevolod Patlan, Michel Verhaegen, and Gleb Vdovin.
DOI: 10.1371/journal.pone.0205020