Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom

Journal Pre-proofs Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom Peter V. Dubovskii, Anastasia A. Ignatova, Alexey V. Feofanov, Yuri N. Utkin, Roman G. Efremov PII: DOI: Reference:

S0960-894X(19)30868-6 https://doi.org/10.1016/j.bmcl.2019.126890 BMCL 126890

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Bioorganic & Medicinal Chemistry Letters

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28 October 2019 3 December 2019 4 December 2019

Please cite this article as: Dubovskii, P.V., Ignatova, A.A., Feofanov, A.V., Utkin, Y.N., Efremov, R.G., Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126890

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Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom

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Peter V. Dubovskii, Anastasia A. Ignatova, Yuri N. Utkin, Alexey V. Feofanov, Roman G. Efremov

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com

Antibacterial activity of cardiotoxin-like basic polypeptide from cobra venom Peter V. Dubovskiia* , Anastasia A. Ignatovaa,b , Alexey V. Feofanova,b, Yuri N. Utkina, Roman G. Efremova,c,d Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia M.V.Lomonosov Moscow State University, Biological Faculty, ul. Leninsky Gory 1, 119234 Moscow, Russia cHigher School of Economics, ul. Myasnitskaya 20, 101000 Moscow, Russia dMoscow Institute of Physics and Technology (State University), 9 Institutskiy per., Dolgoprudny, Moscow Region, 141700 Russia a b

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ABSTRACT

Article history: Received Revised Accepted Available online

Antibacterial activity of the three-finger toxins from cobra venom, including cytotoxin 3 from N. kaouthia, cardiotoxin-like basic polypeptide A5 from N. naja (CLBP), and alpha-neurotoxin from N. oxiana venom, was investigated. All toxins failed to influence Gram-negative bacteria. The most pronounced activity against Bacillus subtilis was demonstrated by CLBP. The latter is ascribed to the presence of additional Lys-residues within the membrane-binding motif of this toxin.

Keywords: Cobra venom Three-finger toxins Antibacterial activity Bacillus subtilis Electric charge distribution

2019 Elsevier Ltd. All rights reserved.

Resistance of continually increasing number of bacterial strains to conventional antibiotics urges for development of novel antibacterial substances1. Membrane-active polypeptides (MAP) are the promising prototypes for design of such molecules2. MAP can be classified into five groups: (a) -helical peptides, (b) rich in cysteine residues and disulfide bonds, (c) -sheet forming, (d) rich in particular regular amino acids, e.g. arginine, proline etc. and (e) composed of rare and/or modified amino acids3. The plasma membrane of bacteria is supposed to be their target. In this case, resistance in bacteria cannot develop easily. The most widely spread MAP are linear peptides, i.e. those lacking disulfide bonds4. As a rule, they are unstructured in aqueous solution. When bound to lipid membranes they often form amphiphilic helices. However, considering only this class of molecules, it is difficult to establish relationships between spatial structure and antibacterial effect. This is because the spatial organization of these molecules in the peptidoglycan layer, or in the lipopolysaccharide envelope is of importance5. However, this often remains unknown for linear peptides. To illustrate the importance of this issue we appealed to cytotoxins (or cardiotoxins, CT) from cobra venom6. CT belong to the family of three-finger toxins (TFT). These are disulfide-rich polypeptides, adopting a compact beta-sheet fold, which is conserved in a variety of environments7. Only local structural changes within CT are observed during their interactions with model lipid membranes8-10. Recently, using a set of CT, distinct in their net electric charge and membrane activity, we demonstrated that a correlation exists between minimum inhibitory concentrations of CT towards Gram-positive bacteria and favorable distribution of positively and negatively charged amino acid residues over the molecular surface11. The goal of the present work is to elucidate whether these observations hold for a broader range of TFT. Cobra venom is a cocktail of small amount of low molecular substances and predominately polypeptides, among which TFT represent a significant part12. Its largest portion is represented by CT, the 59-61 residues long MAP. Cardiotoxin-like basic polypeptides (CLBP) is an additional group of MAP (61-62-residue long). CLBP are weaker MAP, compared to CT. Probably, they target not only lipid membranes but exhibit affinity to proteins, such as integrins13. Short neurotoxins (NT) are 60-62-residue-long polypeptides, lacking any membrane affinity, and targeting nicotinic acetylcholine receptors14. In the present work we investigated antibacterial properties of the toxins from the above three families. TFT can be purified from cobra venom or obtained either via recombinant bacterial production15, or chemical synthesis16. For testing biological properties of TFT, their extraction and purification from venom can be used11. In this work, cytotoxin 3 (CT3Nk), cardiotoxin-like basic polypeptide A5 (CLBP A5), and neurotoxin 2 (NT2) were purified from N. kaouthia, N. naja, and N. oxiana cobra venoms, respectively17. CT3Nk contained an admixture of cytotoxin 2 (CT2Nk), featuring a single amino acid substitution

Ala41/Val41. These two homologues were inseparable chromatographically. The analysis of the mixture was performed with NMRspectroscopy18. The amino acid sequences of the TFT investigated in the present work are shown in Fig. 1.

Fig. 1. Amino acid sequences of TFT. The rows (from upper to lower) correspond to the amino acid sequences in one-letter abbreviation of cytotoxin 3 from N. kaouthia (CT3Nk), CLBP from N. atra (A5), and short-chain neurotoxin 2 (NT2) from N. oxiana venom. Charged amino acid residues are marked bold. The cysteine residues (shown in an enlarged font) forming disulfide bonds, possess coinciding indices. The putative membrane binding residues of CT3Nk and A5 are enclosed into boxes. The numbering of the residues is shown above the upper row. The amino acid sequences were aligned manually.

Antibacterial activity of the TFT was studied, as described19. Cytotoxicity of the toxins against A549 cells was evaluated20. The results are presented in Table 1. Table 1. Antibacterial, cytotoxic and electric properties of TFT. Toxin

CT3Nk3 A5 NT2

Electric charge (at pH 7) +9 +10 +6

Antibacterial activity, MIC1, M B. subtilis B-501

1.6±0.4 0.4±0.1 >40

S. aureus 209-P

E. coli C-600

>40 >40 >40

>40 >40 >40

Cytotoxicity2, ED50, M 2.6±0.24 >200 >200

MIC, Minimum Inhibitory Concentration; For A549 human lung adenocarcinoma cells, ED50 – concentration causing death of 50% of the cells; 3In fact, the antibacterial effect was determined for a mixture of CT3Nk/CT2Nk toxins, differing by a single amino acid substitution A41/V41. Taking into account our structure-functional study of CT11, this substitution should not affect the antibacterial activity. Thus, we assume that CT3Nk and CT2Nk feature identical antibacterial activity; 4According to the previously published data20. 1 2

All the TFTs were inactive against E.coli (Gram-negative bacteria), and S. aureus (Gram-positive one). Two of them exhibited activity against B. subtilis (Gram-positive bacteria). Interestingly, the strongest effect (the lowest MIC) was found for CLBP A5 (Table 1). To the best of our knowledge, this rather high activity was not previously reported for the toxins of this group. Taking into account the spatial organization of the TFT investigated in this work (Fig. 2) and our previous investigations of antibacterial properties of CT11, we propose the following interpretation of the current findings.

Fig. 2. Spatial organization and membrane binding modes of TFT. Spatial structure of CT3Nk (a), A5 (b), and NT2 (c) is shown. The PDB-codes used to depict the structures are indicated below each panel. The backbone of all molecules is shown in ribbon representation. Only side-chains of negatively charged (Asp, Glu) and positively charged (Arg, Lys, His) residues are shown in red and blue colors, respectively. The tentative membrane-water interface is shown for the

membrane active toxins with solid horizontal line (panels a, b), according to the experimental21 and computer modeling22 data. The fingers (roman numerals) are numbered, and the N- and C-termini are marked. The program MOLMOL23 was used to draw this figure.

CT3Nk, containing a single Pro-residue within the extremity of the loop-1, exhibits conformational equilibrium between the two forms, like other CT24. The major form of this toxin features trans-configuration of the Val7-Pro8 peptide bond and is favorable for lipid binding8,9. The structure of the representative conformer from the NMR-determined set is shown in Fig. 2, a. This molecule features the net positive charge of +9 (Table 1), which is smoothly distributed over the protein surface. Thus, one can speculate that this is the reason, why the molecule is not capable of passing through the lipopolysaccharide layer of Gram-negative bacteria11. Alternatively, high density of positive charge within spider venom toxins is favorable for that25. CT are known to interact with lipid membranes, containing acidic lipids26. It is not surprisingly that the plasma membrane of these bacteria, composed mostly of anionic phospholipids27, can be damaged by CT. It is likely that the extremities of all the loops of CT3Nk are involved in binding to lipid membranes of this kind (Fig. 2, a)9,18. When toxin concentration in the plasma membrane becomes higher than a threshold value, the membrane bilayer is disrupted, causing death of bacteria28. The respective MIC values fall in the micromolar range11. The spatial organization of CLBP A5 is shown in Fig. 2, b. Irrespective the fact that the amino acid sequence of this toxin is by 2 residues longer than that of CT3Nk, there is much similarity in their spatial structures (Fig. 2 a, b). Importantly, the tip of the loop-2 of A5 features also -like shape, like in CT24,29. As suggested earlier, such shape of the loop-2 entails into a single membrane-binding motif the tips of all three loops (Fig. 2, a, b). Numerous experimental and computer modeling data evidence that A5, similarly to CT, interacts with detergent micelles and lipid membranes via the tips of all three loops (Fig. 2, b)22,30. The lower MIC of A5, compared to CT3Nk (Table 1) permits a proposal that the former toxin is more perturbing to the plasma membrane of B. subtilis. This effect might be ascribed to the presence of transmembrane potential across a bacterial plasma membrane with the cytoplasmic side being more negative4. Thus, the positive charge of the toxin molecule would promote its translocation toward the membrane interior. Due to strong dielectric screening effects in water-lipid milieu, the membrane does not sense the total positive charge of the toxin molecule31,32. Only charged amino acid residues, positioned close to the membrane-water interface, contribute to this “effective” charge32. Indeed, only CT bearing a single Lys (or His) residue within the tip of the loop-2 demonstrated maximal antibacterial activity11. However, A5 bears a pair of additional Lys-residues within the tip of the loop-2, compared to CT3Nk (Fig. 1, second raw, the fragment, enclosed into middle box; Fig. 2, a, b). Taken this into account, we assume that the “effective” charge of this toxin sensed by the membrane is higher than that of CT3Nk. As a result, the membrane perturbing activity of this toxin outweighs that of CT. NT2 does not show the antibacterial activity (Table 1). Of note, CT with a positive charge below +9 did not show antibacterial effect on Gram-positive bacteria, too11. This was ascribed to failure of these toxins for passing through the bacterial peptidoglycan layer. Probably, this holds true for NT2. This toxin is not membrane-active one due to its low hydrophobicity within the tips of the loops (Fig. 1). In molecular dynamics study of the interaction of NT2 with lipid membranes these residues were found to avoid contacts with the lipid bilayer33. Thus, it is likely that due to both the low net overall positive charge and low hydrophobicity NT2 lacks antibacterial activity. Finally, let us concern cytotoxic properties of TFT. NT2 exhibits very low cytotoxicity, like CLBP A5. CT3Nk acts in low micromolar range of concentrations against A549 cells (Table 1) and PC12 cell line34. Earlier, it was noted that cardiotoxin-like basic polypeptides possess at least one order of magnitude lower cytotoxicity than CT toward FL cells35. Therefore, we assume that A5 is less cytotoxic toward mammalian cells than CT. Concluding, we investigated antibacterial properties of several three-finger toxins, including CLBP A5. Taking account the present data and previous results11, we hypothesize that both the overall protein positive charge and its higher density within the membranebinding motif promote the antibacterial effect, while keeping at low level toxicity against mammalian cells. Interestingly, similar effect was observed for -helical antimicrobial peptides, derived from melittin36. Peptides with substitution of a hydrophobic residue on the nonpolar face of the helix to lysine showed only slightly reduced antimicrobial activity towards B. subtilis, but had much lower haemolytic activity and cytotoxicity against mammalian cells.

Acknowledgments The work was supported by the Russian Foundation for Basic Research (grant #19-04-00350), the Basic Research Program at the National Research University Higher School of Economics and the Russian Academic Excellence Project ‘5-100’. Microbiology branch of Biological faculty of Moscow State University is thanked for providing bacterial strains. References and notes 1. Czaplewski, L.; Bax, R.; Clokie, M.; Dawson, M.; Fairhead, H.; Fischetti, V. A.; Foster, S.; Gilmore, B. F.; Hancock, R. E. W.; Harper, D.; Henderson, I. R.; Hilpert, K.; Jones, B. V.; Kadioglu, A.; Knowles, D.; Olafsdottir, S.; Payne, D.; Projan, S.; Shaunak, S.; Silverman, J.; Thomas, C. M.; Trust, T. J.; Warn, P.; Rex, J. H. Lancet Infectious Diseases 2016, 16, 239-251. 2. Li, J. G.; Koh, J. J.; Liu, S. P.; Lakshminarayanan, R.; Verma, C. S.; Beuerman, R. W. Front Neurosci 2017, 11:73. 3. Reddy, K. V. R.; Yedery, R. D.; Aranha, C. Int J Antimicrob Agents 2004, 24, 536-547. 4. Dubovskii, P. V.; Vassilevski, A. A.; Kozlov, S. A.; Feofanov, A. V.; Grishin, E. V.; Efremov, R. G. Cell Mol Life Sci 2015, 72, 4501-4522. 5. Dubovskii, P. V.; Efremov, R. G. Expert Rev Proteomics 2018, 15, 873-886. 6. Dubovskii, P. V.; Utkin, Y. N. Acta Naturae 2014, 6, 11-18. 7. Lo, C. C.; Hsu, J. H.; Sheu, Y. C.; Chiang, C. M.; Wu, W. g.; Fann, W.; Tsao, P. H. Biophys J 1998, 75, 2382-2388. 8. Dubovskii, P. V.; Dementieva, D. V.; Bocharov, E. V.; Utkin, Y. N.; Arseniev, A. S. J Mol Biol 2001, 305, 137-149.

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Highlights >Carditoxin-like-basic-polypeptide A5 is a three-finger toxin (TFT) > CLBP A5 possesses high antibacterial activity against Bacillus subtilis >Cardiotoxins (CT) and neurotoxins (NT) also belong to TFT > CT and NT exhibit weaker antibacterial activity, compared to A5 > This is ascribed to the presence of additional Lysresidues within the membrane-binding motif of A5.