macrocyclic peptide synthesis and novel approaches to ... · aminoacyl-trna synthetases in biology...

ShaopeiwenLuoSuga’sLab

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Macrocyclic peptide synthesis and novel approaches to peptide

selection Shaopeiwen Luo

Hiroaki Suga’s lab, Department of Chemistry, University of Tokyo Principle Investigator: Prof. Hiroaki Suga

Supervisor: Dr. Toby Passioura

Abstract Cyclic peptides are of good use of drug and have various clinical merits. To develop drug-like macrocyclic peptides that target on TAB2 protein, flexible in vitro translation (FIT) system combining random nonstandard peptide integrated display (RaPID) selection were done. By calculating positive and negative percentage recovery rate, the purity of desired peptide library was determined.


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1 Introduction Many current tentative drugs are of insufficient efficacy and of excessive risks of unpredictable immunological response and thus alternatives are needed. Despite of some limitations, peptides have been found as good alternatives to small synthetic molecules because they get degraded rapidly into amino acids and thus are less harmful after acting on target molecules. Also, peptides can work on targets very selectively because of the specific interaction with the targets. Cyclic peptides could make even better peptide drugs besides the advantages of peptides as drug molecules. Usually cyclic peptides show better biological activity compared to their linear counterparts because of the conformational rigidity: cyclization reduces conformational entropy, increasing rigidity and therefore binding affinity and receptor selectivity for the target protein. Cyclic peptides are also resistant to hydrolysis by exopeptidases, because they lack both amino and carboxyl termini. Several cyclic peptides found in nature have already been used in clinic for various activities: gramicidin and tyrocidine are used for bactericidal activity; cyclosporine A is used for immunosuppressive activity. (S. Joo, 2012) As cyclic peptides have several merits for drug development, our lab has made efforts to develop cyclic peptide compounds in vitro. The majority of cyclic peptides has noncanonical structures, such as atypical amino acid side chains or unnatural stereochemistries, and is synthesized by nonribosomal peptide synthases (NRPSs). (T. Passioura & H. Suga, 2013) To artificially cyclize peptides, we use flexizyme to charge tRNA with unnatural amino acids in our lab. Combined with RaPID selection (random nonstandard peptide integrated display) and FIT system (flexible in vitro translation), we could select artificial cyclic peptides to our target protein. In my research project, the target protein was called TAB2, and both D and L peptide libraries were created for selection.

2 Theoretical Background 2.1 Flexizyme and Genetic Code Reprograming Genetic preparation of peptide is usually limited to ribosomal 20 natural amino acids, and the resulting compounds are linear peptides. Though nonribosomal peptide synthases (NRPSs) can be used to synthesize cyclic peptides and some success has been made in engineering NRPSs with novel function, the complexity and mRNA-independence of NRPSs has impeded the alternation of cyclic peptide products’ structures. (T. Passioura & H. Suga, 2013) However, as the fact that the fidelity of polypeptide synthesis is dependent upon the fidelity of tRNA aminoacylation, the intentional misacylation of tRNAs with noncannonical amino acids could lead to cyclic peptides. (T. Passioura & H. Suga,


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2013) To misacylate tRNAs, flexizyme is used for charging tRNAs with noncanonical amino acids. Flexizymes are small (~45 nucleotide) ribozyme aminoacyl-tRNA synthetase (ARS) that are highly flexible and can charge various noncanonical amino acids to tRNAs. In the process of acylation of any tRNA that has a C-terminal CCA motif (figure 11), flexizymes can catalyze the reaction by [using an amino acid substrate activated by a leaving group conjugated through an ester linkage] (figure 2). (T. Passioura & H. Suga, 2013)

Figure 1. Structures of flexizyme and tRNA with C-terminal CCA motif. Structures of tRNA and flexizymes with similar structures are shown. The C-terminal CCA motif is highlighted in red.

Figure 2. Misacylation using flexizyme. Flexizymes (Fx) catalyze the reaction by [using an amino acid substrate activated by a leaving group conjugated through an ester linkage].

There are three kinds of flexizymes that have been identified. One kind is called enhanced flexizyme (eFx). eFx flexizyme can recognize aromatic part in the side chains of amino acids, such as phenylalanine and tyrosine. Though it is not critical for the structure of leaving group in the acylation process, cyanomethyl ester (CME) has

1 Passioura T., Suga H. (2013) Flexizymes, Their Evolutionary History and Diverse Utilities. In: Kim S. (eds)

Aminoacyl-tRNA Synthetases in Biology and Medicine. Topics in Current Chemistry, vol 344. Springer,

Dordrecht


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commonly been used because of the historical reasons. (T. Passioura & H. Suga, 2013) In my research, CME attached noncanonical amino acid was used. 2.2 Cyclization of peptides The introduction of chloroacetylated noncanonical residues allows the automatic post-translational cyclization. The cyclization occurs between the noncanonical amino acids and cysteine side chains (figure 3). However, the attachment only occurs to the first cysteine, even when more than one cysteine exist. (T. Passioura & H. Suga, 2013) The peptide cyclization reduces entropy due to conformational change that associates with binding pocket, and thus increases rigidity and binding affinity to the target protein.

Figure 3. Automatic post-translational cyclization. Species with noncanonical amino acids would automatically cyclize by losing a hydrogen from the first cysteine and a chloride from chloroacetyl tyrosine.

2.3 FIT system Forster and co-workers have reprogrammed the translation system that contains purified E.coli ribosomes and other recombinant proteins and co-factors. In this system, desired canonical amino acid and its corresponding aminoacyl-tRNA synthetase (ARS) can be omitted, and its space can be taken by the introduced noncanonical amino acids. (T. Passioura & H. Suga, 2013) In my research, flexible in vitro translation (FIT) system was used. FIT system combined reprogramed translation system similar as Forster’s and flexizyme technology, which then allowed intentional noncanonical amino acids to involve in peptide translation. Consequently, cyclic peptide could be produced.

3 Method The acylation step was done first to charge tRNA with L or D-chloroacetyl tyrosine and other 19 natural amino acids without methionine. The protein TAB2 was optimized on beads by treating variable concentrations of beads to fixed concentration of TAB2 before the selection step. To select desired high-affinity protein binding cyclic peptides, RaPID selection (random nonstandard peptide integrated display) involving FIT system (flexible in vitro translation) was used. 5 rounds of selections


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were performed to select macrocyclic peptides with high affinity to TAB2 protein. The RaPID selection involved 6 steps as shown in figure 42.

Figure 4. RaPID selection process. Part of mRNA library was ligated with puromycin linker (the purple balls). Translation of the library using FIT system resulted in cyclic peptide library that attached to the puromycin linker. Reverse transcription of the mRNA that covalently bound to the puromycin linker leaded to DAN-RNA duplex. Cyclic peptides that had high affinity to the target molecule were isolated in selection process and the impurities were removed by wash. Desired peptides left and their corresponding DNA were amplified by PCR.

3.1 In vitro transcription DNA libraries were translated into mRNA libraries at a 20µl scale. 3.2 Puromycin linker reaction: To display the activity of peptide libraries in later steps, translation-terminating molecule puromycin (figure 53), which would form a covalent bond with the nascent mRNA at 3’end, was used. (Wilson et al., 2001)

Figure 5. Puromycin structure

2 Passioura, T. and Suga, H., (2013). Flexizyme-Mediated Genetic Reprogramming As a Tool for Noncanonical Peptide Synthesis and Drug Discovery. Chemistry – A European Journal, vol. 19, no. 21, pp. 6530-6536 ISSN 0947-6539. DOI 10.1002/chem.2013002473 Compound Summary for CID 439530, PubChem, Retrieved on September 3rd, from https://pubchem.ncbi.nlm.nih.gov/compound/puromycin#section=Top


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The puromycin would also stalk the polymerase and thus stopped chain elongation. (Wilson et al., 2001) The mRNA molecules attached on the puromycin could later be displayed after selection with beads. However, the ligation was not 100% proficiency. Any mRNA molecules lacking puromycin would not be able to covalently attach to corresponding peptide sequences and would not be enriched through PCR in the later step. (Barendt et al., 2013) 3.3 FIT translation reaction: The flexible in vitro translation (FIT) system was used to create peptide libraries with L or D-chloroacetyl tyrosine charged initiator tRNA and other 19 natural amino acids without methionine at 10µl scale. (Yu et al., 2017) The peptides involving chloroacetyl tyrosine should self-cyclize.

3.4 Reverse transcription (RT): The mRNA libraries were reverse transcribed into DNA libraries for DNA recovery by PCR in later steps. 3.5 Negative and positive selection: Two types of selection were performed. For negative selection, peptide libraries were treated to beads that were not coated by TAB2 to remove impurities. As a result, impurities would bind to the beads with the rest remained in the supernatant. For positive selection, beads were first coated with target proteins, which was TAB2. Then the supernatant from negative selection was treated to positive beads. In this way, desired peptides would bind to the positive beads and were selected.

3.6 Real time PCR and PCR: Desired peptides had corresponding DNA sequences attached and were amplified by PCR.

4 Data & Result 4.1 Acylation: Automatic self-cyclization check The acylation step was checked by MALDI-MS method. MALDI-MS could analyze molar mass of targets. Because species with noncanonical amino acids would automatically cyclize by losing a hydrogen from the first cysteine and a chloride from chloroacetyl tyrosine, peaks around 36, which was the molar mass of HCl should be seen if the acylation was successful. The figure (figure 6) showed that acylation was successful.


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Figure 6. MALDI-MS result. The MALDI-MS spectrum showed molar mass differences at around 36.

4.2 Optimization protein on beads The lightest band of supernatant occurred in the column corresponding to 2.5µm (figure 7) and thus the optimal concentration of beads was 2.5µm. The lightest band of supernatant meant most protein bound to the beads and thus almost nothing was left in the supernatant. The column that had nothing occur in the supernatant was not chosen, because it could not be told if the beads were saturated or not.

Figure 7. Optimization protein on beads result. The protein TAB2 was optimized on beads by treating variable concentrations of beads to fixed concentration of TAB2 before the selection step.

4.3 Puromycin linker reaction check 8% Urea/acrylamide gel was used to check puromycin-linker reaction. This method separated protein according to mass. Peptides with smaller mass would go further. The figure (figure 8) showed, in each lane, protein traveled to different distances, meaning puromycin was linked to peptides.


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Figure 8. Puromycin linker reaction result. In each lane, protein traveled to different distances, meaning puromycin was linked to peptides.

4.4 Library Check after each selection round 3% agarose gel was used at the end of each round selection to check if only DNAs that could produce desired macrocyclic peptides were selected. From the figure, only one band occurred, indicating only selected DNAs that had similar features existed.

Figure 9. DNA result after each selection round. Only one band occurred in both D and L lanes, meaning desired DNAs were selected.

4.5 Selection Result To measure selection, percentage recovery rate was calculated. The percentage recovery rate was defined such that recovery rate % = (positive/negative concentration * 2) / (input concentration * 1000) * 100%. The input solutions were the diluted L or D DNA library solutions. Five rounds of RaPID selection were performed as the time was limited.

TAB2 Positive % Recovery Negative % Recovery

Round D positive D negative Round L positive L negative 1 0.094 0.232 1 0.082 0.486 2 0.084 0.062 2 0.085 0.053 3 0.0024 0.054 3 0.0028 0.049 4 0.031 0.83 4 0.323 2.48 5 0.03 0.034 5 0.011 0.079

Table 1. Percentage recovery rate of D and L libraries

*The data highlighted with red color was rare and suggested the appearance of error.


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Figure 10. Comparison of positive/ negative recovery rate % for TAB2 D library. Blue represented positive percentage recovery rate, and red represented negative percentage recovery rate.

Figure 11. Comparison of positive/ negative recovery rate % for TAB2 L library. Blue represented positive percentage recovery rate, and red represented negative percentage recovery rate.

5 Discussion and future developments For both D and L libraries, the positive percentage recovery rate went down in the first three rounds and went up in the 4th round. This trend occurred because at first most peptides were “impurities” to the target protein. Though washed by negative beads, some impurities would bind to the positive beads accidentally as the positive beads were positive charged and DNA molecules were negative charged. In further selection rounds, more impurities were removed. Only peptides that had high affinity to TAB2 could bind and thus positive percentage recovery rate went down. When most peptides left were the desired peptides, the positive selection rates would then went up. However, the 4th L negative percentage recovery rate did not look fine. The rate went up largely and was above 1.0. This rare data suggested some machinery or human errors might have occurred. In 5th round, the positive and negative percentage recovery rate were pretty close, meaning the desired peptides were not pure enough, and there were still many impurities left in the sample. To run effective sequencing, the recovery rate of positive selection must be much higher than that of negative selection, meaning most desired peptides were left, and most impurities were removed. Consequently, further selection rounds would be needed in the future for developing drug-like molecules for TAB2 protein.

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0.5

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1 2 3 4 5

TAB2D

Dpositive Dnegative

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1 2 3 4 5

TAB2L

Lpositive Lnegative


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Besides developing drug-like molecules for TAB2 protein, the techniques also allowed synthesis of other natural-product-like macrocyclic compounds and could facilitate further clinical therapeutics.


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Acknowledgement First, I want to thank to the UTRIP committee and ILO, for choosing me as a participant to the research program at University of Tokyo and planning all the events, and Ito Foundation U.S.A. and Friends of UTokyo, Inc., for selecting me as a FUTI scholar and providing me with financial support. I would also like to thank professor Hiroaki Suga for providing me with opportunities to get in touch with frontier science, and Dr. Toby Passioura for guidance and help within my project. In addition, I would like to thank my supporter Manuel E. Otero Ramirez for supporting me and Ms. Kawai for planning the welcome party to the lab and the University. It is my great honor to have the experience this summer.


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References [1] Joo, S. H. (2012). Cyclic Peptides as Therapeutic Agents and Biochemical

Tools. Biomolecules & Therapeutics, 20(1), 19–26. http://doi.org/10.4062/biomolther.2012.20.1.019

[2] Passioura, T. and Suga, H., (2013). Flexizyme-Mediated Genetic

Reprogramming As a Tool for Noncanonical Peptide Synthesis and Drug Discovery. Chemistry – A European Journal, vol. 19, no. 21, pp. 6530-6536 ISSN 0947-6539. DOI 10.1002/chem.201300247.

[3] Wilson, D.S., Keefe, A.D., & Szostak J.W. (2001). The use of mRNA display to

select high affinity protein-binding peptides. PNAS, vol.98, no.7, pp. 3750-3755. DOI 10.1073/pnas.061028198

[4] Barendt, P. A., Ng, D. T. W., McQuade, C. N., & Sarkar, C. A. (2013). Streamlined Protocol for mRNA Display. ACS Combinatorial Science, 15(2), 77–81. http://doi.org/10.1021/co300135r

[5] Yu, H., Dranchak, P., Li, Z., MacArthur, R., Munson, M. S., Mehzabeen, N., … Inglese, J. (2017). Macrocycle peptides delineate locked-open inhibition mechanism for microorganism phosphoglycerate mutases. Nature Communications, 8, 14932. http://doi.org/10.1038/ncomms14932

[6] Compound Summary for CID 439530, PubChem, Retrieved on September 3rd,

from https://pubchem.ncbi.nlm.nih.gov/compound/puromycin#section=Top [7] Passioura T., Suga H. (2013) Flexizymes, Their Evolutionary History and

Diverse Utilities. In: Kim S. (eds) Aminoacyl-tRNA Synthetases in Biology and Medicine. Topics in Current Chemistry, vol 344. Springer, Dordrecht

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