Introduction

Our team set out with the goal of developing an integrated system to better combat the fatal disease of Malaria. We initially started the brainstorming process with the intention of developing a therapeutic drug that would help combat the growing problem of drug resistance in Plasmodium parasites against currently used antimalarials. While coming up with possible strategies to accomplish this, our team was also independently researching previous iGEM teams and looking for ways to expand upon their work.

Our System

We decided to work with E. coli, as it has been very well studied. It is a fast growing and easy to work with organism, and biobricks for cyclotide circularization already existed for the system. The vector we chose to work with for the expression of proteins was Pet28-a (+), as it is an inducible expression construct, and thus allows for controlled protein production.

Why Cyclotides

Kalata B1

Cyclotides are globular microproteins with a unique head-to-tail cyclized backbone, stabilized by three disulfide bonds forming a cystine knot. On further research, we realised that they would be extremely advantageous to incorporate into our project. Cyclotides, which are plant derived circular peptides, can be made orally bioavailable.^[1][2] The knotted arrangement of the three disulfide bonds makes them exceptionally stable to chemical, thermal and biological degradation. Cyclotides can cross cellular membranes and are able to modulate intracellular protein-protein interactions both in vitro and in vivo.^[10] They are highly tolerant to sequence variability, making them amenable to the insertion of protein sequences, and thus our project evolved into a therapeutics based approach to Malaria.

On researching available literature, we decided that these peptides would then be engineered into loop 6 of the relatively well studied cyclotide Kalata B1, which was originally found in the leaves of a plant native to Congo in Africa. Kalata B1 has also previously been used as a scaffold for the development of drugs.^[3] The decision to edit the peptide into loop 6 was based on a literature study of the effectiveness of such a protein drug when the peptide was edited into different loops.^[4]

Cyclotide Assembly using Split-Inteins

For the in-vivo synthesis of kalata-B1 we had to find an efficient way for the circularisation of protein. After some literature reviews, we decided to use protein trans-splicing for the circularisation of the linear cyclotide precursor. We also came across the work of the Team Heidelberg 2014 , who had worked on developing a split-intein based method for generating cyclized proteins.

Protein trans-splicing is a post-translational modification similar to protein splicing, the difference being that the intein self-processing domain is split into N- (I_N) and C-intein (I_C) fragments.^[5] The first native split intein was identified in Synechocystis sp. PCC6803 (Ssp).

In these species, it was observed that the replicative polymerase is encoded by two distinct genes. The gene encoding the amino-terminal fragment of the polymerase is terminated with a sequence resembling the amino terminus of an intein (N intein or I_N), and the gene encoding the carboxyl-terminal fragment of the polymerase is initiated with a sequence resembling the carboxyl terminus of an intein (C intein or I_C). Co-expression of the two gene fragments in Escherichia coli results in the production of the full-length polymerase, suggesting that the two intein fragments associate in-vivo to function as a heterodimeric (or split) intein.^[6]

By fusing the I_N and I_C fragments to the C- and N-termini of the polypeptide for cyclization, the trans-splicing reaction yields a backbone-cyclized polypeptide. The naturally occurring Nostoc puntiforme (Npu) DnaE split-intein, which has high splicing yields,has shown high tolerance to the amino acid composition of the intein-extein junctions and is reported to have the highest rate of protein trans-splicing (𝜏_1/2 ≈ 60 s), was selected for our project.^[7]

The split-intein fragments are not active individually. However, they can bind to each other with high specificity under appropriate conditions to form an active protein splicing or intein domain in trans.^[5]

Protein splicing is a rapid process of four nucleophilic attacks that occurs after the formation of the active intein domain, mediated by three of the four conserved splice junction residues. In step 1, the splicing process begins with an N−O shift if the first intein residue is Ser, or N−S acyl shift, if the first intein residue is Cys. This forms a (thio)ester bond at the N-extein/intein junction. In step 2, the (thio)ester bond is attacked by the OH- or SH-group of the first residue in the C-extein (Cys, Ser, or Thr). This leads to a transesterification, which transfers the N-extein to the side-chain of the first residue of the C-extein. In step 3, the cyclization of the conserved Asn residue at the C-terminus of the intein releases the intein and links the exteins by a (thio)ester bond. Finally, step 4 is a rearrangement of the (thio)ester bond to a peptide bond by a spontaneous S−N or O−N acyl shift.^[8]

Circular ligation mechanism. The expressed fusion protein (F) folds to form an active protein ligase. The enzyme catalyzes an N-to-S acyl shift at the target–I_N junction to produce a thioester intermediate (T), which undergoes transesterification with a side-chain nucleophile (S) at the I_C–target junction to form a lariat intermediate (R). Asparagine side-chain cyclization liberates the cyclic product as a lactone, and an S-to-N acyl shift generates the thermodynamically favored, lactam product (O) in vivo.^[2]

Our Parts

For the purpose of illustration of our parts, we will consider one of the interactions our team chose to work on, the interaction between the ClDRa domain of the MCvar1 PfEMP1 protein and the CD36 protein. For each of our interactions, similar parts exist.

The first part consists of all the components required to express the host protein in an E. coli cell, including requisite promoters. The expression of this protein is necessary to confirm the activity of our cyclotide as an inhibitory protein drug. The Strep tag helps in the purification of the protein.

The second part consists of all the components required to express the Plasmodium protein in an E. coli cell, including requisite promoters. The expression of this protein is necessary to confirm the activity of our cyclotide as an inhibitory protein drug. The 6xHis tag helps in the purification of the protein. This tag will also prove useful in a novel assay we are developing to measure the inhibitory activity of the peptide drug.

The third part consists of all the components required to express and circularise Kalata B1 in E. coli. The cyclotide gene itself will contain a Strep tag for purification in Loop 3 of the cyclotide and the sequence for the engineered peptide inserted in Loop 6 of the cyclotide. The N-intein and C-intein sequences come from pre-existing biobricks and are responsible for the circularisation of the cyclotide.

inhibitory sequence grafted in cyclotide kalata B1 (BBa_K3582005) in the plasmid backbone pet28a+ (BBaK3582500)

In-Silico Modelling of the project

Protein interactions between the human host and the malarial parasite Plasmodium falciparum were selected as drug targets for our project. Peptide inhibitors were designed against the parasitic proteins in these interactions, using the human protein components. ^[9]

Invasion of the red blood cell by Plasmodium merozoites is essential for parasite survival and proliferation, thus representing an attractive target for therapeutic development. Red blood cell invasion requires a coordinated series of protein/protein interactions, protease cleavage events, intracellular signals, organelle release and engagement of an actin-myosin motor, which provide many potential targets for drug development. As these steps occur in the bloodstream, they are directly susceptible and exposed to drugs.^[9]

Hence most of our interactions were from the blood-stage of the malarial parasite. The peptide inhibitors were modelled in-silico and the process can be summarised as follows:

RCSB Data mining for finding suitable interactions
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Preprocessing of the PDB files
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Identifying the interacting regions
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Computational Saturated Mutagenesis
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Scoring and Analysis
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Molecular Dynamics Simulations
↓
Grafting the peptide onto the cyclotide backbone

Further details about each step could be found under the Modelling Subsection.

Designing Novel Wet Lab Experiments

For our project, we had to design a novel assay that would measure the binding between the human and Plasmodium proteins and experimentally verify that our engineered cyclotide would inhibit this binding. We designed an assay that would exploit the His tag used to purify the Plasmodium protein. Due to this His tag on the protein, it would bind and be immobilized to Ni-NTA coated agarose beads. We then measure the amount of host protein that will bind to this immobilized parasite protein. On the addition of our cyclotide containing the designed inhibitory peptide, a reduction in the amount of host protein that will bind to the parasite protein would serve as experimental verification that our cyclotide has effectively inhibited the interaction. For more information refer to the Ni-NTA Assay on the Experiments page.

References

1: Sancheti, H., & Camarero, J. A. (2009). “Splicing up” drug discovery. Cell-based expression and screening of genetically-encoded libraries of backbone-cyclized polypeptides. Advanced Drug Delivery Reviews, 61(11), 908–917.
DOI: 10.1016/j.addr.2009.07.003

2: Gould, A., & Camarero, J. A. (2017). Cyclotides: Overview and Biotechnological ApplicationChemBioChem, 18(14), 1350–1363.
DOI: 10.1002/cbic.201700153

3: Wurch, T., Pierré, A., & Depil, S. (2012). Novel protein scaffolds as emerging therapeutic proteins: from discovery to clinical proof-of-concept. Trends in Biotechnology, 30(11), 575–582.
DOI: 10.1016/j.tibtech.2012.07.006

4: Wang, C. K., Gruber, C. W., Cemazar, M. š., Siatskas, C., Tagore, P., Payne, N., … Craik, D. J. (2013). Molecular Grafting onto a Stable Framework Yields Novel Cyclic Peptides for the Treatment of Multiple Sclerosis. ACS Chemical Biology, 9(1), 156–163.
DOI: 10.1021/cb400548s

5: Recombinant expression of cyclotides using split inteins, Krishnappa Jagadish and Julio A. Camarero; 2018;
DOI: 10.1007/978-1-4939-6451-2_4

6: Production of cyclic peptides and proteins in vivo; Charles P. Scott, Ernesto Abel-Santos, Mark Wall, Daphne C. Wahnon, and Stephen J. Benkovic, October 7, 1999;
DOI: 10.1073/pnas.96.24.13638

7: The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction. Zettler J, Schütz V, Mootz HD; 2009 Mar 4; 583(5):909-14.
DOI: 10.1016/j.febslet.2009.02.0

8: Inteins, valuable genetic elements in molecular biology and biotechnology; Skander, Elleuche, Stefanie Pöggeler. 2010;
DOI: 10.1007/s00253-010-2628-x

9: Targeting malaria parasite invasion of red blood cells as an antimalarial strategy; Amy L Burns, Madeline G Dans, Juan M Balbin, Tania F de Koning-Ward, Paul R Gilson, James G Beeson, Michelle J Boyle, and Danny W Wilson; 2019 May;
DOI: 10.1093/femsre/fuz005

10: Craik, D. J., Swedberg, J. E., Mylne, J. S., & Cemazar, M. (2012). Cyclotides as a basis for drug design. Expert Opinion on Drug Discovery, 7(3), 179–194. 10.1517/17460441.2012.661554

Team:IISER-Pune-India/Design