International Journal of Peptide Research and Therapeutics (2021) 27:385–395

https://doi.org/10.1007/s10989-020-10091-x

Isolation, Characterization and In‑Silico Study of Conotoxin Protein

from Conus loroisii and Its Anti‑cancer Activity
Anjali Kumari1 · Shijin Ameri2 · Najat Marraiki3 · Abdallah M. Elgorban3,4 · Vincent Aroulmoji5 ·
Kumar Ponnuchamy6 · Muthusamy Govarthanan7   · Thangaswamy Selvankumar1

Accepted: 16 June 2020 / Published online: 29 June 2020

© Springer Nature B.V. 2020

Abstract
The conopeptides are the conotoxin of Conus loroisii have wide applications in drug development. This work emphasizes the
isolation and identification of conopeptides form Conus loroisii and the peptides were characterized using LC–MS/MS ESI
fragmentation technique and showing 50 protein sequences with disulfide linkages of the molecular mass of 352–1263 m/z.
The conotoxin peptide having O1, O2, M, S, I1, J, A and T-superfamily. The gel electrophoresis results also confirmed the
protein bands above 20 kDa. The Fourier transform infrared (FT-IR) spectra reported the presence of halometabolites found
in marine-derived conopeptides. Moreover, the toxicity of conopeptide has been studied with brine shrimps, chorioallantoic
membrane, and breast cancer cell lines (MCF-7). The in-silico analysis of HPV protein databases reveals conotoxin protein
has strong interactions and the toxicity assay proved that the conotoxin of C. loroisii as a potential anti-cancer drug.

Keywords  Anticancer · Conotoxin · Conus loroisii · Human papillomavirus · Mass spectrometry

Introduction

* Muthusamy Govarthanan
gova.muthu@gmail.com
* Thangaswamy Selvankumar
selvankumar75@gmail.com
1
PG and Research Department of Biotechnology, Mahendra
Arts and Science College (Autonomous), Tamil Nadu,
Kalippatti, Namakkal 637501, India
2
Biodiversity Division, Central Marine Fisheries Research
Institute (ICAR), Kochi 682018, Kerala, India
3
Department of Botany and Microbiology, College of Science,
King Saud University, Riyadh 11451, Saudi Arabia
4
Centre of Excellence in Biotechnology Research, King Saud
University, Riyadh 11451, Saudi Arabia
5
Center for Research & Development, Mahendra Engineering
College (Autonomous), Tamil Nadu, Mallasamudram West,
Namakkal 637503, India
6
Department of Animal Health and Management, Alagappa
University, Karaikudi 630003, Tamil Nadu, India
7
Department of Environmental Engineering, Kyungpook
National University, Daegu, South Korea
The cone snails of the genus Conus loroisii are the group of
gastropods with predatory properties with their venomous
compounds as “conotoxin” (Robinson and Norton 2014).
The conotoxin shows an important source in pharmaceutics
for human health (Kalia et al. 2015). The conotoxins are
peptide molecules and identified through liquid chromatog-
raphy with electrospray ionization mass spectrometry tech-
nique (Davis et al. 2009; Asuma et al. 2015). Moreover, the
peptides have wide pharmacological properties such as neu-
rotransmitter transporters (Morales-González et al. 2015),
pain reliever and molecular probe in drug designing (Lewis
et al. 2012). Convulsive disorders, stroke, neuromuscular
block and cardioprotection (Thundimadathil 2012; Richard
and Michael 2006).
The earlier workers were reported on the conotoxin
“Conolysin-Mt” of Conus mustelinus showed therapeutic
mechanisms of membrane disruption (Biggs et al. 2007),
and conotoxin from Conus inscriptus having the anti-
cancer activity of cervical cancer cell lines (HeLa-HPV
associated) (Anjali et al. 2019), HPV associated anogenital
cancer (Barbara et al. 2019), anticancer activity (Kumari
et al. 2020) and pharmaceutical target of specific peptides
from Conus loroisii (Kohn 2001). The inhibitory effect of

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alpha-conotoxin TxID on lung cancer (Qian et al. 2019)
binding the membrane proteins channels and receptors (McI-
ntosh et al. 2000) and efficiently blocking the α3β4 nAChRs.
The study of conotoxin characterization includes extrac-
tion, purification, identification and assembly of biological
activity for peptides. This owns the prospective of mak-
ing efficient and selective pharmaceutical drug (Vetter and
Lewis 2012) from Conus venom. The latest classification of
conotoxin was based on characteristics such as gene super-
family, cysteine framework and disulfide linkages (Duque
et al. 2019). The simplicity of toxins from conus has placed
them on a valuable position in the research of neuroscience
and cancer that leads to novel drug development (Yao et al.
2008). Moreover, the peptide toxins display broadly, the
chemical disparity which bears an excellent efficiency to
bind with the target receptor. The protein diversity of cono-
toxins studied through different computational perspective
have been implemented for studying their structure, role and
their efficiency. Also, In-silico methods such as homology
modelling, molecular dynamics simulations and docking are
essentially used to understand the rapid screening, chemi-
cal configuration of conotoxins for applications (Lee et al.
2012).Although, the pharmaceutical and therapeutic applica-
tions of the conotoxin from Conus loroisii have not reported
the anticancer activity of this species.
Thus, the objectives of the current study were, Conus
loroisii (i) isolation and purification of conotoxin from
Conus loroisii,(ii) characterization of the conotoxin liquid-
chromatography mass spectrometry (LC–MS) analysis (iii)
structural characterization and functional groups identifica-
tion using Fourier transform infrared spectroscopy (FT-IR)
(iv) toxicity assay of Brine shrimp (Artemia salina) lethal-
ity test (BSLT) and chorioallantoic membrane assay on a
chicken embryo (v) The (vi) cytotoxicity assay of conotox-
ins on MCF-7 cancer cell lines (vii) In silico analysis of
GRAMM-X Protein–Protein Docking Web Server v.1.2.0
for MCF-7 cell line.
Materials and Methods
Collection of Specimen and Identification
Specimens of Conus loroisii were collected from the Bay of
Bengal, Thoothukudi, (Lat. 8.81°N, Lon. 78.14°E) (Fig. 1a
Tamil Nadu The Species identification was done by follow-
ing the standard keysets as reported earlier (Franklin et al.
2009).
Extraction of Venom
The C. loroisii shell was cracked by hammer and the soft
body was dissected for achieving venom duct. The venom
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International Journal of Peptide Research and Therapeutics (2021) 27:385–395
ducts were anatomized; blended in 2.0 ml of extraction
buffer (0.1 M NaCl, 0.05 M Tris–HCl and pH 8.8). The
extract was vortexed and centrifuged twice at 12,000×g for
10 min at 4 °C. The supernatant was collected and main-
tained at − 20 °C for further studies.
Protein Estimation and Molecular Weight
Determination
The total protein content from the isolated venom superna-
tant was estimated by Lowry’s methods (Lowry et al. 1951).
The Molecular weight of the venom proteins was estimated
by SDS-PAGE (Laemmli 1951) and Gel enzymatic digestion
of proteins was carried out according to Safavi et al. (2010).
The in-gel digestion was incubated overnight, and peptides
were extracted, lyophilized and stored at − 80 °C for further
studies (Wang et al. 2015).
Liquid Chromatography ESI‑Mass Spectrometer
and FT‑IR Analysis
The proteomics data were procured using mass spectrom-
eter fragmentation technique on instrument LC–ESI–MS
(Brukner Daltonis, Germany). The 5  µl of chemically
reduced and alkylated peptides were mixed with 2 µl of
acetic anhydride and volume was made up to 20 µl with
water. After 1 h of incubation at room temperature, the mix-
ture was exposed to LC–ESI–MS to determine the disulfide
linkages (Pundir et al. 2016). The peptides were separated
with analytical C18 column using Eksigent nano LC425 (AB
Sciex, Singapore, 5 µm particle size, 100 mm × 0.5 mm. The
sample 1 µl was loaded with a flow rate of 20 µl/min. The
one-dimensional solution B gradient was acquired from 0 to
100% in 90 min (TFA in ACN), solution A (TFA in water).
The data was achieved by Analyst software version 1.6. The
processing of data was with protein Pilot™ and Biopharma
view software (AB Sciex, Singapore). The IR spectra were
carried out using Bruker/OPUS 7.5.18, (India) with the lyo-
philized sample of C. loroisii venom peptides.
Brine Shrimp Lethality Assay (BSLA)
The toxicity test of the conopeptide involved brine-shrimp
lethality assay was studied based on the method of Nookala
et al. (2018). Accordingly, brine shrimp eggs were placed
in the artificially obtained seawater for hatching in a plastic
container for 2 days. After incubation, the mature nauplii
were used for lethality assay. The purified venom protein was
mixed with 24.9 ml of artificial seawater in the test tubes fol-
lowed by addition of 10 living mature nauplii into the tubes.
The experimental tubes were then allowed to stay at labora-
tory condition for 24 h. Next, the surviving nauplii were
counted of each test tube using magnifiers and data recorded.
International Journal of Peptide Research and Therapeutics (2021) 27:385–395 387

Fig. 1  a Conus loroisii samples

were collected from the sea-
shore of Thoothukudi district
of Tamil Nadu, which belongs
to Northeastern part of Indian
Ocean. b Image of Conus
loroisii and its venom gland, the
shell appears brownish black
and the venom glands were pale
white

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388
Fig. 2  SDS-PAGE, the electrophoretic observed bands of venom pro-
teins extracted from the Conus loroisii  the eraser algorithm. The grid box (20 Å × 20 Å × 20 Å) was
MTT Assay
Cytotoxicity and Apoptotic Staining
The cytotoxic property of the conopeptides was tested against
MCF-7 breast cancer cell lines. The MCF-7 breast cancer cell
lines were procured from National Center for Cell Sciences,
Pune and cultured as per their requirements (Kumar et al.
2014a). Further, the cytotoxicity of conotoxin peptides was
evaluated by MTT assay based on the methodology adopted
by Mosmann (1983). Further, the apoptotic features of cell
death were recorded by dual (acridine orange (AO) and Eth-
idium bromide (EtBr) and nuclear (Hoechst 33,344) staining
as reported by (Kumar et al. 2014b). Furthermore, the stained
cells were visualized under a fluorescence microscope (Accu-
scope—EXI-310, USA) at × 20 magnification.
Chorio Allantoic Membrane Assay
Freshly fertile nine-day-old chicken eggs were purchased
from nearby Poultry Breeding Farm, Mallasumdram, Tamil
Nadu. With the use of driller, a small hole was made and the
Fig. 3  LC–MS: total ion chromatogram, a mass spectrum of injected venom protein, isolated from Conus loroisii
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International Journal of Peptide Research and Therapeutics (2021) 27:385–395
air was aspirated to let the membrane down. The venom pep-
tide was mixed with dyes and injected into the embryonated
eggs at the linking of two blood vessels followed by closing
the hole by adhesion tape. The eggs were left at 37 °C for
incubation in BOD incubator for 72 h. After incubation, the
tape was opened and the development of blood vessels was
observed (Gupta et al. 2014).
Docking Studies
The structure of delta-Am2766 conotoxin from conus (PDB
ID:1YZ2) and the X-ray diffraction structure of E2 HPV
type 2 protein (PDB ID: 1A7G) were retrieved from protein
database RCSB protein data bank (www.rcsb.org/pdb/). The
water molecules were removed from both conotoxin and E2
HPV type 2 proteins before docking and the structures were
minimized by using Schrodinger suite (https​://www.schro​
dinger​ .com/). The active site pockets were identified by using
generated around receptor and the docking was conducted
by GRAMM-X tool.
Results and Discussion
In the present study, the crude toxin was extracted from the
ducts of C. loroisii (Fig. 1a, b). The total protein concentra-
tion estimated was 1 mg/ml. The SDS-PAGE determined
the molecular weight of the crude venom proteins, the gel
image is depicted in Fig. 2. The SDS-PAGE displayed above
20KD with different molecular weights (20, 29, 36, 45, 55,
63, 66, 97 and 166 KDa) of C. loroisii. this study also antici-
pated by Wang et. al. (2015) and showed the protein bands
corresponds to 20KD isolated from C. textile, C. lividus,
C. caracteristicus. The results also imply the proteome pro-
filing above 20KD showed the role in metabolism, folding
and synthesis of a protein with post-translational modifi-
cation. The results of high and low scads of protein were
chemically modified and exposed to LC–MS/MS is shown
in Fig. 3 and the sequence results are reported in Table 1.
The result confirms the presence of 50 proteins was identi-
fied and match with conoserver data for comparison (Kaas
et al. 2012). Earlier reports have shown their importance of
peptidomes analysis using LC–MS/MS (Franklin and Rajesh
 
International Journal of Peptide Research and Therapeutics (2021) 27:385–395 389

Table 1  Conoprotein sequence determination

S. no Description m/z value Super family Cysteine frame work Peptide sequence

1 CnIIIG conotoxin (Conus consors) 410.35 M III QKCCGKGMTCPRYFRDNFICGCC​

2 CnVIIA conotoxin (Conus consors) 605.01 O1 VI/VII CKGKGAOCTRL(Mox)YDCCHGSC-
SSSKGRC​
3 Ai6.1 conotoxin (Conus ammiralis) 437.01 O1 VI/VII WCKQSGEMCNLLDQNCCEGYCIV-
LVCT
4 St6.3 conotoxin (Conus Striolatus) 437 O1 VI/VII STTKVSKSTSCMKAGSYCVATTRICCG​
YCA​YFGKICIGYPKN
5 Pn6.9 conotoxin (Conus pennaceus) 448.7 O2 VI/VII GCSGTCHRREDGKCRGTCDCSGYSY-
CRCGDAHHFYRG​CTC​TC
6 Ca8a Conotoxin (Conus caracteristicus) 448.25 S VIII GCSGTCHRREDGKCRGTCDCSGYSY-
CRCGDAHHFYRG​CTC​TC
7 R11.5 Conotoxin (Conus radiatus) 407.7 I1 XI GAVPCGKDGRQCRNHADCCNCC-
PIGTCAPSTNWILPGCSTGQFMTR
8 MiK41 conotoxin (Conus miles) 407.15 O1 VI/VII TCRSSGRYCRSPYDCCR​RYC​RRIT-
DACV
9 CcoSc conotoxin (Conus consors) 979.23 S VIII GCTLVNNCRKRNGACNGDCHCKG-
KICKCSSSARPWKPGCA​CTC​RNA
10 Ac1.2 conotoxin (Conus Achatinus) 875.01 A I NGRCCHPACGGKYVKC
11 Fla6.17 conotoxin (Conus flavidus) 1263.59 Q VI/VII CTPCGPDLCCEPGTTCDTVLRHTHF-
GEPSCSY
12 G5.4 conotoxin (Conus geographus) 558.18 T V DCCEERWCCF
13 CnIA conotoxin (Conus consors) 535.24 A I GRCCHPACGKYYSC(nh2)
14 G6.7 conotoxin (Conus geographus) 774.9 O1 VI/VII PCANLGRACDTVPCCLGVRCFESRT-
PTCLLKQRGV
15 Vc6.14 conotoxin (Conus victoriae) 650.29 O2 VI/VII TNAESWWEGECLGWSNGCTQPSDC-
CSNNCKGRNCDIW
16 G6.3 conotoxin (Conus geographus) 435.2 O1 III ALRSTKVSKSPPCLVAGSSCRG​TTR​
VCCGFCSHYGYKCRDRPTS
17 BtIIIA conotoxin (Conus betulinus) 352.21 M III CCKQSCTTCMPCCW​
18 Con-BKA conantokin (Conus striolatus) 385.16 unknown NO cys GD(Gla)(Gla)YS(Gla)FI(Gla)RER(Gla)
LVSSKIPR
19 SxIIIA conotoxin (Conus striolatus) 565.77 M III RCCTGKKGSCSGRACKNLKCCA(nh2)
20 Eb3-R02 conotoxin (Conus ebraeus) 423.22 M III EFILPAVGRTCCDLTWCDGNCRCCTP
21 GlB conotoxin (Conus geographus) 637.79 A I ECCNPACGRHYSCKG
22 TxMMSK-01 conotoxin (Conus textile) 380.21 M III CCRLLCLSCNPCC
23 FVIA conotoxin (Conus fulmen) 452.23 omega VI/VII CKGTGKSCSRIAYNCCTGSCRSGKC
(nh2)
24 Lv3-YH04 conotoxin (Conus lividus) 803.02 M III MKLMLSALRQQECCKPSTCDGGCY-
HCC
25 Im5.5 conotoxin (Conus imperialis) 466.24 T V CCIKFHPCCHN
26 TxMMSK-01conotoxin (Conus textile) 381.68 M III CCRLLCLSCNPCC
27 Gla-MrIII conotoxin (Conus marmoreus) 528.74 T V FCCRTQ(Gla)VCC(Gla)AIKN (nh2)
28 S8.1 conotoxin (Conus striatus) 448.68 S VIII GCSGTCHRREDGKCRGTCDCSGYSY-
CRCGDAHHFYRG​CTC​S
CQ
29 Co3-S01 conotoxin (Conus coronatus) 415.73 M III TCCPPGICSSDKCSCC
30 ArXIA conotoxin (Conus arenatus) 466.2 I1 XI RTCSRRGHRCIRDSQCCGGMCCQGN-
RCFVAIRRCFHlPF
31 Mr15.2 conotoxin (Conus marmoreus) 451.19 N XV CRSGKTCORVGODVCC(Gla)RSDCF-
CKLVOARPFWRYRCICL
32 Ca15a conotoxin (Conus caracteristicus) 401.72 O2 XV CRVENKCOHTVCCDRSRCSCKLIR-
TRPLMYHVCVC
33 Bu25 conotoxin (Conus bullatus) 1018.77 A IV YWTECCGRIGPHCSRCICPGVVCPK
R(nh2)

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390 International Journal of Peptide Research and Therapeutics (2021) 27:385–395

Table 1  (continued)
S. no Description m/z value Super family Cysteine frame work Peptide sequence

34 Vc5.22Conotoxin (Conus victoriae) 610.27 T V GICCSKHPPCCSQFQ

35 Mr6.11 conotoxin (Conus marmoreus) 525.25 UNKNOWN VI/VII SCAQSGLSCDTRPCCDDKPCVPN-
GRQSMCG
36 Mr14.1 conotoxin (Conus marmoreus) 635.31 J XIV VPAEPILEIICPEMCDEGVGEEPF-
CHCTEKRDAVSSRI
37 Mi025 conotoxin (Conus miles) 587.29 O1 VI/VII CTDSGQFCDPTDHDCCSGN-
CIDEGGNGVCAFVREDVPKLY
38 Pu11.1 conotoxin (Conus pulicarius) 549.27 I1 XI ASICRG​TGG​RCTKDKHCCGWLCCG-
GPSVGCATSFAPCN
39 MiEr95 conotoxin (Conus miles) 407.73 O1 VI/VII ECREKGQGCTNTALCCP-
GLECEGQSQGGLCVDN
40 Vx3 conantokin (Conus vexillum) 515.7 B2 NO CYS FPQRRDGAPAENLKSFDPAG-
MQAMAGGMPNMQGMQPMGN-
MAPGQMLPFNPTWLWDSRGPWRT​
41 Conantokin-Pr2 conantokin (Conus 366.62 NA NO CYS DEP(Gla)YA(Gla)AIR(Gla)YQLKYGKI
parius)
42 GVIIIA conotoxin (Conus geographus) 436.23 S VIII GCT​RTC​GGOKCTG​TCT​
CTNSSKCGCRYNVHPSG(BTr)GCG​
CAC​S(nh2)
43 G33 conotoxin (Conus geographus) 847.29 O1 VI/VII CTQDFDPCMPVCHECCTRSHFV-
VCRRPICL
44 LiC121 conotoxin (Conus lividus) 948.42 T V ALCCY​GYR​FCCPNFR
45 Tx3f conotoxin (Conus textile) 501.75 M III RCCKFPCPDSCRYLCC(nh2)
46 Tx5.14 conotoxin (Conus textile) 571.31 T V GIFALAKSVCCTAAKLSFCC
47 Di6.9 conotoxin (Conus distans) 762.38 O1 VI/VII ATPECSRAGCKNVPCCSGLKCTG-
PQNGPVCQPE
48 Mn1.1 conotoxin (Conus monachus) 409.6 A I NGHCCHPACGGKYVKC
49 G33 conotoxin (Conus geographus) 601.29 O1 VI/VII CTQDFDPCMPVCHECCTRSHFV-
VCRRPICL
50 Co6.5 conotoxin (Conus coronatus) 601.79 O1 VI/VII CTPAGKACDATA​TCC​VLFCNLVTNKC-
QVPRFP

2015; Wang et al. 2015). The LC–MS data revealed proteins
with disulfide linkages of different molecular mass distrib-
uted in the range of 352–1263 m/z. belonging mostly to O1,
O2, T, M, J, S, I1, A and N superfamilies with cysteine
framework pattern I, III, VI, VII, VIII, XIV, V, and IV. The
identified superfamilies and cysteine frameworks of the
venom proteins from C. loroisii were also observed in C.
miles, C. lividus, C. textile, C. vexillium, C. geographus. The
cysteine frameworks are determined by the organization of
C–C–CCC–CC in the conopeptides primary sequences(Kaas
et al. 2008).
The identification of chemical characteristics of func-
tional groups and chemical bonds present in the venom sam-
ples were studied using FT-IR spectroscopy (Tarlai et al.
2017). The results of FT-IR spectra are reported in Fig. 4.
The observed peak at 3359.30 cm−1 was assigned to N–H
stretching of aliphatic primary amine. The peak observed
at 2095.41 cm−1 attributed to N=C=S stretch belonged to
isothiocyanate. The band observed at 1634.94 cm−1 was
assigned to C=C stretching of alkene and the small peak
at 1543.60  cm−1 was assigned to N–O stretching of the
nitro compound. The peak at 1402.84 cm−1 belongs to S=O
sulfonyl chloride. The peak at 1079.36 belongs to C-O for
primary alcohol, and 561.61 belongs C-I halo compound.
Earlier FT-IR study on C. loroisii venom (Janeena and Lyla
2015) showed amide, alkenes, sulfonic acid, hydrohalides,
azides, and alkyl mercaptans. The spectral determination of
specimens of the identical species permits to evaluate diver-
sity in their chemical contours. Consequently, FT-IR spec-
troscopy can be used as a rapid, easy and confined method
in the determination of chemical compounds between spe-
cies (Naseer et al. 2019). The Brine Shrimp lethality test
examination of the venom peptide on Brine shrimp showed
(Fig. 5) that half inhibitory concentration was at 30 µg/ml.

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International Journal of Peptide Research and Therapeutics (2021) 27:385–395 391

Fig. 4  Infrared absorption spectrum showing chemical profile of isolated venom protein

This is almost similar with earlier reported results on C.

amadis (Ramesh et al. 2014) the I­ C50 was seen at 25 µg/ml.
This test was considered as the preliminary evaluation of
toxicity (Primahana et al. 2015) which probably paved the
path for the anticancer potential of the biological extracts.
The chicken embryo chorioallantoic membrane assay with
venom peptide showed an increase in the thickness of the
blood vessels after the incubation period was over. The
chicken chorioallantoic membrane (CAM) system has been
wildly used to study human tumour growth and tissue regen-
eration research. However, in the present work, we have used
to study the angiogenic activity of the venom peptide from
C. loroisii toxin. The angiogenic activity of venom peptide
indicates that these peptides can be very beneficial in the
growth of new blood vessels, organ growth and repair. A
similar study was reported on marine mollusc invertebrates
extracts (Gupta et al. 2014), the CAM assay is applied to
study in vivo angiogenesis assay (Subauste et al. 2009). It
helps to study tumour angiogenic and anti-angiogenic activ-
ity of molecules (Fig. 6).
The conopeptide, derived from C. loroisii was ana-
lyzed for cytotoxic assay against MCF-7 breast cancer
Fig. 5  Brine-shrimp lethality assay cell lines: Based on the results, it is confirmed that the C.

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392 International Journal of Peptide Research and Therapeutics (2021) 27:385–395

Fig. 6  Chorio Allantoic Mem-

brane assay on chick embryo

Fig. 7  Cytotoxicity effect of
Conus loroisii venom peptides
against MCF-7 breast cancer
cell lines: a Control cells treated
with Acridine Orange/Etbr
Staining (AO/EB) appearing
green; b venom peptide (­ IC50)
treated cells inferring apoptotic
bodies inferring red due to
chromatin condensation; c con-
trol cells treated with Hoechst
33,344 staining with no signifi-
cant apoptotic morphology; d
venom peptide (­ IC50) treated
cells inferring pyknotic nucleus
inferring bright blue

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International Journal of Peptide Research and Therapeutics (2021) 27:385–395 393
Table 2  pyDockWEB results of protein–protein docking between (Am2766 conotoxin-E2 HPV type 2 protein) showing electrostatics and desol-
vation score values
loroisii showed a half-maximum inhibitory concentration
of 32 µg mL−1 upon incubation for 24 h. Further, the ­IC50
treated conotoxin from C. loroisii treated breast cancer cells
showed morphological evidence of apoptosis and nuclear
beading. The dual staining by AO/EtBr revealed the for-
mation of apoptotic bodies inferring red due to chromatin
condensation. Meanwhile, the control cells display no sig-
nificant red emission inferring healthy cells in green. In the
same, Hoechst 33,344 staining was performed to evaluate
the nuclear morphology upon treatment with conotoxin from
C. loroisii Fig. 7 shows control cells with no blue emission
with healthy nuclear morphology, whereas, the conotoxin
treated cells show pyknotic nucleus with blue emission
inferring the progression of apoptosis. Keeping this in mind,
we predict that conotoxin from C. loroisii inducing apoptosis
by damaging the nucleus as a result cell occurs.
Earlier reported results revealed that α-conotoxin RgIA
prevents neuropathic pain from antagonist to α9α10 nico-
tinic receptors (Pacini et al. 2016). To the best of our knowl-
edge anticancer potential of the peptide from C. loroisii on
MCF-7 cancer cell lines were reported for the first time
(Fig. 7). Hence, this study concludes these conopeptides
could be used as a new drug for anticancer applications.
The toxicity test, angiogenic activity and anticancer poten-
tial from the conotoxin extracts of C. loroisii can provide
a better option in pharmaceutical research. The Insilico
studies were carried out to study the anticancer potential of
conotoxin. The binding of conotoxin delta-Am2766 having
a sequence length of 27 amino acid chain residues with E2
regulatory protein from HPV type 31 was analyzed load-
ing receptor protein and ligand–protein in the GRAMM-
X docking tool and AutoDock method was applied to see
the H-bond interactions. The results were depicted in and
Table 2 and Fig. 8a–c analyzed by using Discovery studio
tool. Based on the interaction between the receptor protein
and the ligand–protein interaction, The in silico analysis
concluded that the 1YZ2 is having low bond length as 2.529
with 1A7G. Based on this interaction we have concluded
that the protein 1A7G is considered as a protein to minimize
the inflammation caused by cervical cancer cells.
Conclusion
It was concluded that the conopeptide extracted from
cone snail C. loroisii species showed an excellent antican-
cer activity and peptide showed low toxicity with normal
cells remained biologically antagonist for the cancer cell.
The proteome characterization by LC–MS/MS could be
described and followed as the easiest way for proteomics
research. Hence, these reports argue that the pharmaceutical
diversity of the conopeptides available in this conus species
have been used as a source for development of drug cancer
cells. Therefore, this novel conopeptide and their biomedical
impact would lead to the discovery of valuable drugs against
life-challenging diseases of cancer.
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394
Fig. 8  a Secondary structure of 1YZ2, b secondary structure of
1A7G, c protein–protein interactions: the low bond length as 2.529
Acknowledgements  This work is partially supported by the PG &
Research Department of Biotechnology, Mahendra Arts and Science
College (Autonomous), and the Department of Science and Tech-
nology, Government of India (DST-FIST sponsored—Ref. No. SR/
FST/College-232/2014). The authors extend their appreciation to The
Researchers supporting project number (RSP-2019/56), King Saud
University, Riyadh, Saudi Arabia.
Compliance with Ethical Standards  Laemmli UK (1951) SDS-PAGE, Bio-Protocol, 1970. Protein measure-
Conflict of interest  On behalf of all authors, the Corresponding author
states that there is no conflict of interest.
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International Journal of Peptide Research and Therapeutics (2021) 27:385–395
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