Neuroactive venom compounds obtained from <i>Phlogiellus bundokalbo</i> as potential leads for neurodegenerative diseases: insights on their acetylcholinesterase and beta-secretase inhibitory activities <i>in vitro</i>

Lopez, Simon Miguel M.; Aguilar, Jeremey S.; Fernandez, Jerene Bashia B.; Lao, Angelic Gayle J.; Estrella, Mitzi Rain R.; Devanadera, Mark Kevin P.; Ramones, Cydee Marie V.; Villaraza, Aaron Joseph L.; Guevarra Jr., Leonardo A.; Santiago-Bautista, Myla R.; Santiago, Librado A.

doi:10.1590/1678-9199-JVATITD-2021-0009

Simon Miguel M. Lopez

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Institute of Chemistry, College of Science, University of the Philippines Diliman, Quezon City, Philippines, 1101.

http://orcid.org/0000-0001-7353-7376

Jeremey S. Aguilar

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Jerene Bashia B. Fernandez

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Angelic Gayle J. Lao

Mitzi Rain R. Estrella

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Mark Kevin P. Devanadera

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Research Center for Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines, 1015.

The Graduate School, University of Santo Tomas, Manila, Philippines, 1015.

http://orcid.org/0000-0002-0173-6955

Cydee Marie V. Ramones

Institute of Chemistry, College of Science, University of the Philippines Diliman, Quezon City, Philippines, 1101.

http://orcid.org/0000-0001-9376-5034

Aaron Joseph L. Villaraza

Institute of Chemistry, College of Science, University of the Philippines Diliman, Quezon City, Philippines, 1101.

Leonardo A. Guevarra Jr.

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Research Center for Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines, 1015.

http://orcid.org/0000-0002-2031-0300

Myla R. Santiago-Bautista ^**Correspondence: mrsantiago@ust.edu.ph

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Research Center for Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines, 1015.

The Graduate School, University of Santo Tomas, Manila, Philippines, 1015.

http://orcid.org/0000-0001-9173-7320

Librado A. Santiago

Department of Biochemistry, Faculty of Pharmacy, University of Santo Tomas, Manila, Philippines, 1008.

Research Center for Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines, 1015.

The Graduate School, University of Santo Tomas, Manila, Philippines, 1015.

http://orcid.org/0000-0002-4750-0729

*Correspondence: mrsantiago@ust.edu.ph

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

SMML, JSA, JBBF, AGJL, MRRE, and MKPD designed and performed the enzymatic and worm experiments, analyzed and interpreted the data, and wrote the paper and participated in the revisions of it. They were supervised by LAS that conceived the research and participated in the revision of the manuscript. LAG conducted the venom extraction, optimization of RP-HPLC conditions and helped in the interpretation of the results and revision of the manuscript. CMVR performed MALDI-TOF-MS on spider venom fractions which was supervised by AJLV. SMML and MRSB helped in the mass analysis of peptides in samples as well as participated in the revision of the manuscript. MRSB is the principal investigator of the CHED RDE-GIA program, “Screening and Characterization of Peptides with In vitro Bioactivity from Venoms Isolated from Theraphosidae and Lycosidae of Philippine Spiders for Therapeutic Leads” from where the some of the funds of this research was obtained. She also helped in the revision of the manuscript. All authors read and approved the final manuscript

Sample	K_M (mM)	V_max (mM/min)	Type of inhibition
Untreated AChE Reaction	1.15 x 10^-3 ± 1.5 x 10^-3	1.61 x 10^-2 ± 3.3 x 10^-5	-
Donepezil	4.24 x 10^-3 ± 2.1 x 10^-3	1.59 x 10^-2 ± 5.6 x 10^-5	Competitive
Whole venom	4.20 x 10^-3 ± 4.2 x 10^-3	1.58 x 10^-2 ± 5.1 x 10^-5	Competitive
Fraction 1	7.50 x 10^-4 ± 5.0 x 10^-4	1.58 x 10^-2 ± 5.4 x 10^-6	Uncompetitive
Fraction 3	4.16 x 10^-3 ± 2.4 x 10^-3	1.59 x 10^-2 ± 4.3 x 10^-5	Competitive
Fraction 16	5.60 x 10^-3 ± 9.0 x 10^-5	1.58 x 10^-2 ± 7.2 x 10^-6	Uncompetitive

Fractions	RT (min)	+ [M+H] (Da)	Matched Peptide	+ [M+H] (Da)	Species	Activity	References
6	37.72	1028.33	U -ctenitoxin-Pn1c 29	1027.57	Phoneutria nigriventer	Contracts smooth muscle of guinea pig ileum	Pimenta et al. [57]
7	40.555	3406.14	U -scytotoxin-Sth1a 1	3406.36	Scytodes thoracica	Unknown	Zobel-Thropp et al. [5858. Zobel-Thropp PA, Correa SM, Garb JE, Binford GJ. Spit and venom from scytodes spiders: a diverse and distinct cocktail. J Proteome Res. 2014;13(2):817-35. doi:10.1021/pr400875s https://doi.org/10.1021/pr400875s... ]
		3410.14	κ-sparatoxin-Hv1b	3410.34	Heteropoda venatoria	Blocks vertebrate Kv4.x and transient outward VGKC in rat ventricular myocytes; weak blocker of calcium channels in rat cerebellar granule cells	Sanguinetti et al. [5959. Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropadatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997;51(3):491-8.]
		3428.07	M-zodatoxin-Lt5a	3427.94	Lachesana tarabaevi	Antimicrobial activity against gram-negative and positive bacteria, and yeasts. It has hemolytic activity against rabbit erythrocytes and causes non- lethal paralysis to Musca domestica larvae	Kozlov et al. [6060. Kozlov SA, Vassilevski AA, Feofanov AV, Surovoy AY, Karpunin DV, Grishin EV. Latarcins, antimicrobial and cytolytic peptides from the venom of the spider Lachesana tarabaevi (Zodariidae) that exemplify biomolecular diversity. J Biol Chem. 2006;281(30):20983-92. doi:10.1074/jbc.M602168200 https://doi.org/10.1074/jbc.M602168200... ]
		4356.94 4357.98	U -theraphotoxin-Cg1b 3	4357	Chilobrachys guagxiensis	Unknown	Chen et al. [6161. Chen J, Deng M, He Q, Meng E, Jiang L, Liao Z, Rong M, Liang S. Molecular diversity and evolution of cystine knot toxins of the tarantula Chilobrachys jingzhao. Cell Mol Life Sci. 2008;65(15):2431-44. doi: 10.1007/s00018-008-8135-x https://doi.org/10.1007/s00018-008-8135-... ]
		4360	ω-hexatoxin-Hv2o	4360.05	Hadronyche versuta	Insecticidal toxin that blocks insect VGCC	King and Wang [6262. King GF, Wang X, inventors; University of Connecticut, applicant. Insecticidal compounds and methods for selection thereof. Australia patent AU2001238152. May 10, 2011.]
		4362.04	ω-hexatoxin-Hv2i	4362.03	Hadronyche versuta	Insecticidal toxin that blocks insect VGCC	King and Wang [6262. King GF, Wang X, inventors; University of Connecticut, applicant. Insecticidal compounds and methods for selection thereof. Australia patent AU2001238152. May 10, 2011.]
			ω-theraphotoxin-Bs1b	4362.23	Brachypelma smithi	Unknown	Escoubas et al. [6363. Escoubas P, Célérier M, Nakajima T. High-performance liquid chromatography matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide fingerprinting of tarantula venoms in the genus Brachypelma: chemotaxonomic and biochemical applications. Rapid Commun Mass Spectrom. 1997;11(17):1891-9. doi: 10.1002/(SICI)1097-0231(199711)11:17<1891::AID-RCM94>3.0.CO;2-X.[64. https://doi.org/10.1002/(SICI)1097-0231(... ]
		4380.98	ω-theraphotoxin-Asp1a	4380.2	Aphonopelma sp.	Inhibits rat cerebellar granule VGCC	Nason et al. [6464. Nason DM, Phillips D, Saccomano NA, Volkmann RA, inventors; Pfizer Inc. NPS Pharmaceuticals Inc., applicants. Calcium channel blocking polypeptides from Theraphosidae Aphonopelma. International patent WO 94/10196. May 11, 1994. Available from: https://patentimages.storage.googleapis.com/30/b7/58/042d09da06b2d6/WO1994010196A1.pdf. https://patentimages.storage.googleapis.... ]
8	43.324	3792.65	U -agatoxin-Ao1d 2	3792.73	Agelena orientalis	Unknown	Kozlov et al. [6565. Kozlov S, Malyabka A, McCutchen B, Lu A, Schepers E, Herrmann R, Grishin E. A novel strategy for the identification of toxinlike structures in spider venom. Proteins. 2005;59(1):131-40. doi:10.1002/prot.20390 https://doi.org/10.1002/prot.20390... ]
		3901.81	U -agatoxin-Ao1l 2	3901.76	Agelena orientalis	Unknown	Kozlov et al. [6565. Kozlov S, Malyabka A, McCutchen B, Lu A, Schepers E, Herrmann R, Grishin E. A novel strategy for the identification of toxinlike structures in spider venom. Proteins. 2005;59(1):131-40. doi:10.1002/prot.20390 https://doi.org/10.1002/prot.20390... ]
9	44.143	3788.53	U -agatoxin-Ao1j 2	3788.7	Agelena orientalis	Unknown	Kozlov et al. [6565. Kozlov S, Malyabka A, McCutchen B, Lu A, Schepers E, Herrmann R, Grishin E. A novel strategy for the identification of toxinlike structures in spider venom. Proteins. 2005;59(1):131-40. doi:10.1002/prot.20390 https://doi.org/10.1002/prot.20390... ]
		3792.56	U -agatoxin-Ao1d 2	3792.73	Agelena orientalis	Unknown	Kozlov et al. [6565. Kozlov S, Malyabka A, McCutchen B, Lu A, Schepers E, Herrmann R, Grishin E. A novel strategy for the identification of toxinlike structures in spider venom. Proteins. 2005;59(1):131-40. doi:10.1002/prot.20390 https://doi.org/10.1002/prot.20390... ]
		3793.64	ω-hexatoxin-Ar1b	3793.51	Atrax robustus	Blocks insect VGCC	King et al. [6666. King GF, Mukherjee AK, Wikel SK, McFarland BS, inventors. University of Connecticut, assignee. Acaricidal compostions and methods of use therof. United States patent US7,575,758B2. August 18, 2009. Available from: https://patentimages.storage.googleapis.com/d3/dc/16/1cd5c910cbb80f/US7575758.pdf. https://patentimages.storage.googleapis.... ]
		3805.59	U -theraphotoxin-Hs1b 20	3805.69	Haplopelma schimidti	Unknown	Zhang et al. [6767. Zhang Y, Huang Y, He Q, Liu J, Luo J, Zhu L, Lu S, Huang P, Chen X, Zeng X, Liang S. Toxin diversity revealed by a transcriptomic study of Ornithoctonus huwena. PloS ONE. 2014;9(6). doi:10.1371/journal.pone.0100682 https://doi.org/10.1371/journal.pone.010... ]
		3808.75	U -theraphotoxin-Cg1a 15	3808.73	Chilobrachys guagxiensis	Unknown	Chen et al. [6868. Huang RH, Liu ZH, Liang SP. Purification and characterization of a neurotoxic peptide huxentoxin-III and a natural inactive mutant from the veom of the spider Selenocosmia huwena Wang (Ornithoctonus huwena Wang). Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 Nov;35(11):976-80.]
10	45.768	3849.67	μ-theraphotoxin-Hs1a	3849.66	Haplopelma schimidti	Blocks insect sodium channels in cockroach DUM neurons; reversibly paralyzes cockroaches and enhances the muscular contractions of rat vas deferens	Huang et al. [6868. Huang RH, Liu ZH, Liang SP. Purification and characterization of a neurotoxic peptide huxentoxin-III and a natural inactive mutant from the veom of the spider Selenocosmia huwena Wang (Ornithoctonus huwena Wang). Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 Nov;35(11):976-80.]
		3850.49	U -theraphotoxin-Hhn1q 3	3850.61	Haplopelma hainanum	Unknown	Tang et al. [6969. Tang, X, Zhang, Y, Hu, W, Xu D, Tao H, Yang X, Li Y, Jiang L, Liang S. Molecular diversification of peptide toxins from the tarantula Haplopelma hainanum (Ornithoctonus hainana) venom based on transcriptomic, peptidomic, and genomic analyses. J Proteomeo Res. 2010;9(5):2550-64. doi: 10.1021/pr1000016 https://doi.org/10.1021/pr1000016... ]
		3850.97	U -theraphotoxin-Hhn1e 3	3850.61	Haplopelma hainanum	Unknown	Tang et al. [6969. Tang, X, Zhang, Y, Hu, W, Xu D, Tao H, Yang X, Li Y, Jiang L, Liang S. Molecular diversification of peptide toxins from the tarantula Haplopelma hainanum (Ornithoctonus hainana) venom based on transcriptomic, peptidomic, and genomic analyses. J Proteomeo Res. 2010;9(5):2550-64. doi: 10.1021/pr1000016 https://doi.org/10.1021/pr1000016... ]
		3862.69	U -agatoxin-Ao1s 2	3862.86	Agelena orientalis	Unknown	Kozlov et al. [6565. Kozlov S, Malyabka A, McCutchen B, Lu A, Schepers E, Herrmann R, Grishin E. A novel strategy for the identification of toxinlike structures in spider venom. Proteins. 2005;59(1):131-40. doi:10.1002/prot.20390 https://doi.org/10.1002/prot.20390... ]
11	47.819	3404.92	U -theraphotoxin-Cv1a 1	3404.43	Coremiocnemis valida	Unknown	Balaji et al. [7070. Balaji RA, Sasaki T, Gopalakrishnakone P, Sato K, Kini RM, Bay BH. Purification, structure determination and synthesis of covalitoxin-II, a short insect-specific neurotoxic peptide from the venom of Coremiocnemis Validus (Singapore tarantula). FEBS Lett. 2000;474(2-3):208-12. doi: 10.1016/s0014- 5793(00)01593-3 https://doi.org/10.1016/s0014- 5793(00)0... ]
Whole venom	-	3595.56	κ-sparatoxin-Hv1c	3596.46	Heteropoda venatoria	Blocks vertebrate Kv4.x and transient outward VGKC in rat ventricular myocytes; weak blocker of calcium channels in rat cerebellar granule cells	Sanguinetti et al. [5959. Sanguinetti MC, Johnson JH, Hammerland LG, Kelbaugh PR, Volkmann RA, Saccomano NA, Mueller AL. Heteropadatoxins: peptides isolated from spider venom that block Kv4.2 potassium channels. Mol Pharmacol. 1997;51(3):491-8.]
		3788.46	U -agatoxin-Ao1j 2	3788.7	Agelena orientalis	Unknown	Kozlov et al. [6565. Kozlov S, Malyabka A, McCutchen B, Lu A, Schepers E, Herrmann R, Grishin E. A novel strategy for the identification of toxinlike structures in spider venom. Proteins. 2005;59(1):131-40. doi:10.1002/prot.20390 https://doi.org/10.1002/prot.20390... ]
			κ-theraphotoxin-Sc1a	3788.57	Stromatopelma calceatum	Blocks Kv4.2, Kv2.2 and Kv2.1 in vertebrates	Shiau et al. [7171. Shiau YS, Huang PT, Liou HH, Liaw YC, Shiau YY, Lou KL. Structural basis of binding and inhibition of novel tarantula toxins in mammalian voltage-dependent potassium channels. Chem Res Toxicol. 2003;16(10): 1217-25. doi: 10.1021/tx0341097. https://doi.org/10.1021/tx0341097.... ]
		4051.5	M-theraphotoxin-Gr1d	4051.93	Grammostola rosea	Antimicrobial and atrial fibrillation activity by blocking KvAP; blocks mechanosensitive ion channels	Kimura et al. [7272. Kimura T, Ono S, Kubo T. Molecular cloning and sequence analysis of the cDNAs encoding toxin like peptides from the venom glands of tarantula Grammostola rosea. Int J Peptides. 2012;1-12. doi: 10.1155/2012/731293 https://doi.org/10.1155/2012/731293... ]

[1] *Correspondence: mrsantiago@ust.edu.ph

Brasil

Brasil

Neuroactive venom compounds obtained from Phlogiellus bundokalbo as potential leads for neurodegenerative diseases: insights on their acetylcholinesterase and beta-secretase inhibitory activities in vitro

Abstract

Background

Methods

Results

Conclusion