E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide targets abrin and ricin toxicity: Hitting two toxins with one stone

Biomedicine & Pharmacotherapy 143 (2021) 112134

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Biomedicine & Pharmacotherapy

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E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide targets abrin and ricin toxicity:

Hitting two toxins with one stone

,

*

Pooja Phatak^a, Vinita Chauhan^b, Ram Kumar Dhaked^b, Uma Pathak^c, Nandita Saxena^a

^aDivision of Pharmacology & Toxicology, Defence Research Development & Establishment, Defence Research Development Organization, Gwalior 474002, India

^bBiotechnology Division, Defence Research Development & Establishment, Defence Research Development Organization, Gwalior 474002, India

^cSynthetic Chemistry Division, Defence Research Development & Establishment, Defence Research Development Organization, Gwalior 474002, India

A R T I C L E I N F O

A B S T R A C T

Keywords:

The efﬁcacy of small molecule inhibitors (SMIs) against the enzymatic activity of Shiga toxin prompted the

evaluation of their efﬁcacy on related toxins viz. ricin and abrin. Ricin, like Shiga toxin, is listed as a category B

bioweapon and belongs to the type II family of ribosome inactivating proteins (RIPs). Abrin though structurally

and functionally similar to ricin, is considerably more toxic. In the present study, 35 compounds were evaluated

in A549 cells in in vitro assays, of which 5 offered protection against abrin and 2 against ricin, with IC₅₀values

Abrin

Ricin

Shiga toxin

Small molecule inhibitor

Ribosome inactivating proteins (RIPs)

ranging between 30.5-1379 μM and 300-341 μM, respectively. These ﬁndings are substantiated by ﬂuorescence

based thermal shift assay. Moreover, the binding of the promising compounds to the toxin components has been

validated by Surface Plasmon Resonance assay and in vitro protein synthesis assay. In vivo studies reveal com-

plete protection of mice with compound 4 E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide against orally adminis-

tered lethal doses of, both, abrin and ricin. The present study thus proposes the emergence of E-N-(2-acetyl-

phenyl)-3-phenyl-acrylamide as a lead compound against RIPs.

1. Introduction

administration of antitoxins, following exposure, could disrupt the

intoxication process and improve the prognostic outcome [1,4,5].

Abrin and ricin, the most lethal plant toxins known, are derived from

rosary peas (Abrus precatorius L.) and castor beans (Ricinus communis L.),

respectively. Their potency, stability, relative ease of production, and

the worldwide availability of their source plants, qualify them as po-

tential biological weapons. Ricin is monitored as a schedule 1 agent

under the Chemical Weapons Convention (CWC) and is also classiﬁed as

a Category B agent by the US Centre for Disease Control and Prevention

(CDC). Despite structural and functional similarities, abrin is consider-

ably more lethal than ricin [1]. These two, type 2 ribosomal inactivating

proteins (RIPs) are dipeptides, the catalytic A chain and the lectin B

chain being held by a disulﬁde bond [2,3]. Due to the unavailability of

speciﬁc treatment protocols, the current treatment options for abrin and

ricin poisoning are largely symptomatic and supportive. Medical coun-

termeasures against these toxins have yet to be granted regulatory

approval. Published animal data suggest that vaccination and/or prompt

The focus of recent research on the development of vaccines, devoid

of cytotoxicity or other deleterious effects, led to the development of

abrin and ricin mutant A-chain vaccines. The former has been shown to

offer 100% protection against 10XLD₅₀abrin dose in mice [6]. Though

ricin mutant A chain vaccine (RiVax) was immunoprotective in mice

challenged by both oral gavage and aerosol inhalation, its low thermo

stability and tendency to undergo partial unfolding render it unsuitable

as a potential vaccine candidate. RVEc, also an engineered RTA vaccine

with increased protein stability, has been shown to offer full protection

to animals against supralethal aerosol challenge [7,8]. Although,

vaccination is a promising approach, but the issues of mass immuniza-

tion, cost effectiveness, intravenous administration, unpredictable

therapeutic efﬁciency limit their clinical utility.

The discovery and development of inexpensive and stable small

molecule inhibitors (SMIs) amenable to oral administration, has

Abbreviations: CC₅₀, 50% Cytotoxic Concentration; CDC, Centre for Disease Control and Prevention; FTSA, Fluorescence-based Thermal Shift Assay; IC₅₀, 50%

Inhibitory Concentration; i.p., intraperitoneal; IVTS, In Vitro Translation System; K_a, Fast on rate; K_D, Dissociation Constant; LDH, Lactate Deydrogenase; MTD,

Maximum Tolerated Dose; MTT, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; NR, Neutral Red; RIPs, Ribosome Inactivating Proteins; SI,

Selectivity Index; SMI, Small Molecule Inhibitor; SPR, Surface Plasmon Resonance; SRL,

* Corresponding author.

α-sarcin/ricin loop; Stx, Shiga toxin.

E-mail address: nanditasaxena@drde.drdo.in (N. Saxena).

https://doi.org/10.1016/j.biopha.2021.112134

Received 3 June 2021; Received in revised form 25 August 2021; Accepted 25 August 2021

Available online 31 August 2021

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

P. Phatak et al.

Biomedicine & Pharmacotherapy 143 (2021) 112134

revolutionized drug development over the last decade. Retro-1 and

Retro-2 SMIs have been found to inhibit the intracellular retrograde

transport of ricin from early endosomes to the Golgi apparatus, thus

inhibiting cytotoxic activity in A549 cells. Further, Retro-2 has also been

seen to exhibit prophylactic effects in mice against lethal intranasal ricin

challenge [9]. Since targeting trafﬁcking pathways may be detrimental

for the transport of normal proteins, speciﬁc targeting of enzyme activity

of toxins would be harmless and remedial.

nitrobenzoylchloride (2.5 mmol). The reaction was monitored by GC-MS

and TLC. After completion of the reaction, the reaction mixture was

washed with NaHCO3 solution and then with water. The DCM layer was

collected and dried over anhydrous sodium sulfate. On evaporation of

the organic layer an oily residue was obtained which solidiﬁed on

cooling. The product was further puriﬁed by recrystallization from

ethanol. White solid Dec. > 200 ^◦C. ¹H NMR (400 MHz, CDCl3): δ 8.28

(d,2H, J = 8.8 Hz), 8.072 (d,2H, J = 8.8 Hz), 7.98 (d,2H, J = 8.8 Hz), 7.5

(bs, 1H), 7.34 (d,2H, J = 8.4 Hz), 7.17–7.11 (m, 5H), 3.87–3.81 (m,4H),

3.51 (t, 2H, J = 8 Hz), 3.00(t, 2H, J = 8 Hz). ¹³C NMR (CDCl₃, 100.6

MHz) 169.61, 169.52, 165.53, 149.47, 147.86, 147.68, 143.08, 140.50,

139.75, 136.13, 134.66, 129.86, 128.85, 124.10, 47.38, 39.48, 34.35,

29.71. EIMS: m/z 494 (M + H) ⁺Anal. Calcd for C₂₄H₂₂N₄O₆S: C, 58.29;

H, 4.48; N, 11.33; S, 6.48 Found C, 57.92; H, 4.41; N, 11.86; S, 5.92.

The mechanism of abrin and ricin toxicity involves B chain facilitated

toxin internalization, [10] followed by the A chain mediated excision of

A4324 of the α-sarcin/ricin loop (SRL) of the 28S rRNA, abolishing the

EF-2 binding site, resulting in the cessation of protein synthesis and

hence cell death [11,12] Thus, inhibition of A chain activity by

small-molecule inhibitors may be a more speciﬁc therapy. Although a

few speciﬁc catalytic blockers, which effectively protect cells against the

cytotoxic effects of ricin and Shiga toxin (Stx), have been reported, their

efﬁcacy on animal models is yet to be proven [13]. In an earlier study in

our laboratory 4 out of 20 SMI tested, offered protection against Stx in

mice [14]. Similarities of abrin and ricin with Stx prompted the evalu-

ation of these molecules against abrin and ricin as well, with the quest to

discover a common inhibitor effective against these RIPs. The present

study thus involves the evaluation of the SMIs studied against Stx, and

their novel structural analogs, for their ability to neutralize abrin and

ricin toxins. It is hypothesized that an effective and essentially irre-

versible A chain inhibitor could be a common antidote against all three

toxins used in bio-warfare.

2.2.2. Synthesis of N-(2-acetyl-phenyl)-4-methoxy-benzamide (compound

21)

Two-aminoacetophenone (1 mmol) was taken in a round bottom

ﬂask and to which 10 ml of dry DCM was added followed by slow

addition of triethylamine (2 mmol). The contents were stirred till a clear

solution was formed. To this 4-Methoxybenzoyl chloride (1.2 mmol) in

10 ml of DCM was added slowly (in 2 h) at 20–25 ^◦C. The reaction was

monitored by GC-MS and TLC using hexane: ethylacetate (1:1) as the

mobile phase. After completion of the reaction, the reaction mixture was

washed with NaHCO₃solution followed by water. DCM layer was

collected and dried over anhydrous sodium sulfate. On evaporation of

the organic layer a solid residue was obtained which was further puriﬁed

by recrystallization from ethanol. White solid, m.p 96–99 ^◦C. ¹H NMR

(400 MHz, CDCl₃): δ 9.00 (d, 1H, J = 4 Hz), 8.14–7.01 (m, 8H), 3.91 (s,

3H), 2.75 (s, 3H); ¹³C NMR (CDCl₃, 100.6 MHz) 203.3, 165.7, 162.6,

141.7, 135.4, 132.8, 131.8, 129.4 127.2, 122.2, 121.85, 120.7, 114.1,

114.0, 55.4, 28.6. EIMS: m/z 77.0, 135.1, 226.1, 269. Anal. Calcd for

2. Material and methods

2.1. Reagents

SYPRO Orange dye, RPMI-1640, Dimethyl sulphoxide (DMSO), Fetal

Bovine Serum (FBS), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-

tetrazolium bromide (MTT), Neutral Red (NR), Crystal Violet Dye

Exclusion (CVDE), in vitro Lactate Dehydrogenase (LDH) release assay

kit and polyethylene glycol (PEG)-300 were purchased from Sigma

Chemicals (Lake Placid, NY). Chemicals required for toxin puriﬁcation

were also procured from Sigma Chemical (Lake Placid, NY). In Vitro

Translational Assay kit (IVT) was obtained from Promega.

C₁₆H₁₅NO_3:C, 71.36; H, 5.61; N, 5.20. Found C, 71.96; H, 5.11; N, 5.12.

2.2.3. Synthesis of N-(2-acetyl-phenyl)-4-chloro-benzamide (compound

22)

The procedure for the synthesis of this compound was the same as for

compound 21except that 2-aminoacetophenone and 4-chlorobenzoyl

chloride were the starting materials. white solid m.p 102–104 ^◦C. ¹H

NMR (400 MHz, DMSO): δ 8.60 (d, 1H, J = 4 Hz), 8.11 (d, 1H, J = 4 Hz),

7.98–7.94 (m, 2H), 7.68 (d, 3H, J = 4 Hz), 7.56 (d, 2H, J = 8 Hz),

7.30–7.27(m,1H), 2.70 (s,3H); ¹³C NMR (CDCl₃, 100.6 MHz) 204.0,

164.5, 139.9, 137.6, 135.1, 133.6, 132.6, 131.6, 129.6, 129.5, 129.2,

124.2, 123.8, 120.9, 29.2. EIMS: m/z 75.0, 111.0, 139.0 230.0, 273

Anal. Calcd for C15H12ClNO2: C, 65.82; H, 4.42; N, 5.12. Found C,

64.97; H, 4.13; N, 4.92.

2.2. Procedure for the synthesis of compounds

Reagents were obtained from commercial suppliers and were of

analytical grade (> 98%), and were used without further puriﬁcation.

Column chromatographic puriﬁcation of products was performed on

silica gel (60–120 mesh). ¹H, 31P and ¹³C NMR spectra were recorded on

a Bruker AVANCE II 400 MHz and Bruker AV III 600 MHz NMR.

Chemical shifts were expressed in parts per million (δ) downﬁeld from

the internal standard tetramethylsilane and were reported as s (singlet),

d (doublet), t (triplet), q (quartet) and m (multiplet). Melting points

were measured by scientiﬁc-MP-DS melting point apparatus. Mass

spectra were obtained from Agilent 5975C GC-MS and elemental anal-

ysis was performed on Elementar vario MICRO cube CHNS analyser.

Purity was checked by elemental analysis and was > 98% for all the

compounds synthesized. Total 35 compounds were included in the

study. These molecules were either amides, chalcones, esters or quino-

lones. Among these compounds, the synthesis procedure for compound

1–15 and compounds 17–20 is the same as in Chauhan et al. [1]. The

Synthesis procedures for compounds 16 and 21–35 are given below.

2.2.4. Synthesis of 3-phenyl-N-(3-phenylsulfanyl-propyl)-acrylamide

(compound 23)

Phenyl-S-propylamine hydrochloride (1 mmol) and cinnamoyl

chloride (1.2 mmol) were dissolved in 10 ml of DCM. Triethylamine (2

mmol) diluted in 10 ml of DCM was added slowly over a period of 15

min at 20–25 ^◦C. The reaction was monitored by GC-MS and TLC with

hexane: ethylacetate (1:1) as the mobile phase. After completion of the

reaction, the reaction mixture was washed with NaHCO₃solution fol-

lowed by water. The DCM layer was then collected and dried over

anhydrous sodium sulfate. On evaporation of the organic layer a solid

residue was obtained. The product was further puriﬁed by recrystalli-

zation from ethylacetate- hexane. white solid m.p 81–82 ^◦C. ¹H NMR

(400 MHz, CDCl₃): δ 8.20 (t, 1H, J = 4 Hz), 7.56 (d, 2H,J = 4 Hz),

7.44–7.31 (m, 8H), 7.21–7.18(m, 1H), 6.60(d,1H,J = 12 Hz), 3.30(t,2H,

J = 0.4 Hz), 3.01 (t, 2H, J = 4 Hz), 1.79–1.75 (m,2H); ¹³C NMR (CDCl₃,

100.6 MHz) 165.4, 139.0, 136.6, 135.4, 129.8, 129.5, 129.4, 128.5,

127.9, 126.1122.6, 38.2, 30.0, 29.1. EIMS: m/z 51.0, 77.0, 103.0, 160.0,

297. Anal. Calcd for C₁₈H₁₉NOS: C, 72.69; H, 6.44; N, 4.71; S, 10.78

Found C, 72.01; H, 5.94; N, 5.01; S, 10.98.

2.2.1. Synthesis of 4-nitro-N-(2-(4-nitrobenzamido) ethyl)-N-(2-

(phenylthio) ethyl) benzamide (compound 16)

N1-(2-Phenylsulfanyl-ethyl)-ethane-1, 2-diamine, (1 mmol) was

taken in a round bottom ﬂask and to this 10 ml dry DCM was added. To

this mixture triethylamine (7 mmol) was added very slowly and stirred

till a clear solution was formed. This was followed by the addition of 4-

2

P. Phatak et al.

Biomedicine & Pharmacotherapy 143 (2021) 112134

2.2.5. Synthesis of 4-nitro-N-(3-phenylsulfanyl-propyl)-benzamide

(compound 24)

C₁₅H₁₂FNO₂C, 70.03; H, 4.70; N, 5.44; Found C, 70.98; H, 4.56; N,

5.481.

The procedure for the synthesis of compound 23 was followed, the

starting materials being Phenyl-S-propylamine hydrochloride and 4-

Nitrobenzoylchloride.white solid m.p 135–137 ^◦C. ¹H NMR (400 MHz,

DMSO): δ 8.86 (t, 1H,J = 4 Hz),8.31 (d,2H,J = 8 Hz), 8.07 (d, 2H,J = 4

Hz), 7.35–7.31 (m, 4H), 7.20–7.18 (t, 1H, J = 4 Hz), 3.43–3.40 (m,2H),

3.04(t, 2H, J = 4 Hz), 1.88–1.83 (m, 2H); ¹³C NMR (CDCl₃, 100.6 MHz)

165.1, 149.3, 140.6, 136.6, 129.5, 129.2, 128.5, 126.1, 123.9, 38.9,

30.05, 28.9. EIMS: m/z 76.0, 104.0, 120.0, 149.9, 316. Anal. Calcd for

2.2.9. Synthesis of N-(2-acetyl-phenyl)-2,5-diﬂuoro-benzamide

(compound 28)

The procedure for the synthesis of compound 28 is similar to that of

compound 21, except that the starting chemicals are 2- amino-

acetophenone and 2, 5 diﬂuorobenzoyl chloride. White solid 89–90 ^◦C.

¹H NMR (600 MHz, CDCl3): δ 12.5 (bs, 1H), 8.90 (d, 1H, J = 6 Hz)7.95

(d, 1H, J = 6 Hz), 7.94 (s, 1H), 7.63–7.62 (m,1H), 7.21–7.19 (m,3H),

2.69 (s,3H); ¹³C NMR (CDCl₃, 100.6 MHz) 202.47, 161.43, 140.17,

135.02, 131.65, 123.25, 122.96, 121.74, 120.33, 120.26, 120.10,

118.00, 117.87,117.82,28.53. EIMS: m/z 63.0, 113.0, 141.0, 232.0,

260.0, 275.0. Anal. Calcd for C₁₅H₁₁F₂NO₂C, 65.45; H, 4.03; N, 5.09.

Found 64.89; H, 4.76; N, 5.22.

C

₁₆H₁₆N₂O₃S: C, 60.74; H, 5.10; N, 8.85; S, 10.14 Found C, 59.52; H,

5.22; N, 8.05; S, 9.97.

2.2.6. Synthesis of N-(2-acetyl-phenyl)-2, 3-diﬂuoro-benzamide

(compound 25)

Synthesized as compound 21 starting from 2-aminoacetophenone

and 2, 3 diﬂuorobenzoyl chloride. white solid m.p 109–111 ^◦C. ¹H

NMR (600 MHz, CDCl3): δ 12.5 (bs, 1H), 8.95 (m,1H), 7.98(m,1H), 7.78

(m, 1H), 7.66 (m, 1H), 7.38–7.37 (m, 1H), 7.25–7.24 (m, 2H), 2.73 (s,

3H); ¹³C NMR (CDCl₃, 100.6 MHz) 202.6, 161.8, 140.3, 135.1, 131.7,

125.8, 125.2, 124.5, 124.4, 123.3, 122.7, 121.5, 120.5, 120.4,28.5.

EIMS: m/z 63.0, 113.0, 141.0, 232.0, 260.0, 275. Anal. Calcd for

2.2.10. Synthesis of N-(2-acetyl-phenyl)-3-(2-chloro-phenyl)-acrylamide

(compound 29)

Same as described for compound 21, 2-aminoacetophenone and 2-

chlorocinnamoyl chloride being the starting chemicals. White solid

118–120 ^◦C. ¹H NMR (400 MHz, CDCl3): δ 8.95 (d, 1H, J = 8.4 Hz),

8.20–7.16 (m,9H), 6.62 (d, 1H, J = 16 Hz), 2.73 (s, 3H); ¹³C NMR

(CDCl₃, 100.6 MHz) 203.08, 164.42, 141.27, 138.14, 135.32, 135.02,

133.04, 131.76, 130.75, 130.19, 127.67, 127.04, 124.86, 122.60,

121.83, 120.99,28.65. EIMS: m/z 51. 0, 75.0, 101.0, 120.0, 137.0,

164.9, 264.1, 299.0Anal. Calcd for C17H14ClNO2 C 68.12; H, 4.71; N,

4.67. Found C, 67.87; H, 4.22; N, 4.02.

C

₁₅H₁₁F₂NO₂: C, 65.45; H, 4.03; N, 5.09. Found C, 64.91; H, 4.21; N,

45.92.

2.2.7. Synthesis of 2-methyl-N-[2-(2-phenylsulfanyl-ethylamino)-ethyl]-

benzamide (compound 26)

DRDE-07 (1 mmol) was taken in a round bottom ﬂask and to which

10 ml dry DCM was added. To this mixture triethylamine (2 mmol) was

added very slowly. The contents were stirred till the clear solution was

formed. Two methoxybenzoyl chloride (1.1 mmol) in 10 ml of DCM was

then added slowly (in 2 h) at 20–25 ^◦C. The reaction was monitored by

GC-MS and TLC with MeOH: CHCl3: NH₃(10:10: 1) as the mobile phase.

After completion of the reaction, the reaction mixture was washed with

NaHCO₃solution and then with water. The DCM layer was collected and

dried over anhydrous sodium sulfate. On evaporation of organic layer an

oily residue was obtained which was dissolved in acetone and dry HCl

gas was passed. Hydrochloride salts of amide derivatives appeared as a

white precipitate. The product was further puriﬁed by recrystallization

from ethanol-acetone. Physical state- white solid m.p decomp. > 178 ^◦C.

¹H NMR (400 MHz, CD3OD): δ 7.49–7.23 (m, 9H), 3.31–3.27(m, 8H),

2.42 (s,3H) 13C NMR (CDCl₃, 100.6 MHz) 172.88, 136.02, 135.08,

133.41, 130.66, 130.31, 130.09, 129.14, 127.14, 126.95, 125.45, 46.51,

36.28, 29.42, 18.57. ESIMS (m/z): 315 (M+H) ⁺Anal. Calcd for

2.2.11. Synthesis of N-(2-acetyl-phenyl)-2,6-dichloro-benzamide

(compound 30)

Similar procedure was performed as described for compound 21

Starting from 2-aminoacetophenone and 2,6 dichlorobenzoyl chloride.

White solid 155–157 ^◦C. ¹H NMR (400 MHz, CDCl3): δ 12.03 (s, 1H),

8.94 (d,1H, J = 8 Hz), 7.96 (d,1H, J = 8 Hz), 7.68–7.65 (m,1H),

7.39–7.23(m, 3H), 2.68 (s,3H); ¹³C NMR (CDCl₃, 100.6 MHz) 202.84,

163.40, 140.24, 136.32, 135.35, 132.19, 131.77, 130.88, 128.26,

123.40, 122.22, 121.24, 23.56. EIMS: m/z 63.0, 75.0, 92.0, 109.0,

144.9, 264.0, 291.0, 307.0, 308. Anal. Calcd for C₁₅H₁₁C_l2NO₂C, 58.46;

H, 3.60; N, 4.55. Found C, 59.12; H, 3.85; N, 4.34.

2.2.12. Synthesis of 3-methyl-thiophene-2-carboxylic acid (4-acetyl-

phenyl)-amide (compound 31)

As described for compound 21, the synthesis of compound 31 is

begun with 4-aminoacetophenone and 3-Methyl-thiophene-2-carbonyl

chloride.White solid 144–145 ^◦C. ¹H NMR (400 MHz, CDCl3): δ

7.48–7.45 (m, 5H), 4.93–4.87(m, 1H), 4.10–4.02(m, 4H), 3.50–3.95 (m,

4H), 3.18–3.12(m, 4H), 1.31–1.27 (t,6H, J = 6.8 Hz). EIMS: m/z 53.1,

77.0, 97.0, 259.1Anal. Calcd for C₁₄H₁₃NO₂S C, 64.84; H, 5.05; N, 5.40;

S, 12.37. Found C, 65.23; H, 4.96; N, 5.87; S, 11.87.

C

₁₈H₂₃C_lN₂OS C, 61.61; H, 6.61; N, 7.98; S, 9.14 Found C, 62.11; H,

6.14; N, 7.22; S, 9.58.

2.2.8. Synthesis of N-(3-acetyl-phenyl)-2-ﬂuoro-benzamide (compound

27)

Three-aminoacetophenone (1 mmol) was taken in a round bottom

ﬂask and to which 10 ml of dry DCM was added. To this triethylamine (2

mmol) was added slowly. The contents were stirred till a clear solution

was formed. The desired 2-ﬂuorobenzoyl chloride (1.1 mmol) in 10 ml

of DCM was then added slowly (in 2 h) at 20–25 ^◦C. The reaction was

monitored by GC-MS and TLC with ethylacetate: hexane (1:1) as mobile

phase. After completion of the reaction, the reaction mixture was

washed with NaHCO₃solution and then with water. The DCM layer was

then collected and dried over anhydrous sodium sulfate. On evaporation

of the organic layer a solid residue was obtained. The product was

further puriﬁed by recrystallization from ethylacetate-hexane. White

solid 108–110 ^◦C. ¹H NMR (600 MHz, CDCl3): δ 8.56 (bs, 1H), 8.21–8.18

(m, 2H), 8.00 (d, 1H, J = 6 Hz), 7.75 (d, 1H, J = 6 Hz), 7.55–7.48 (m,

2H), 7.36–7.33 (m, 1H), 7.21–7.20 (m,1H), 2.64(s, 3H); ¹³C NMR

(CDCl₃, 100.6 MHz) 197.75, 159.66, 138.20, 137.95, 134.14, 132.33,

129.45, 125.27, 125.11, 124.62, 120.99, 120.08, 116.34, 116.17, 26.75.

EIMS: m/z 51.0, 63.0, 75.0, 95.0, 123.0, 257. Anal. Calcd for

2.2.13. Synthesis of [2-(2-phenylsulfanyl-ethylamino)-ethyl]-

thiophosphoramidic acid O,O^′-diethyl ester (compound 32)

A two-neck ﬂask was charged with dry dichloromethane (DCM) and

DRDE-07. The mixture was stirred and then triethylamine was added

very slowly to the reaction mass. The reaction mixture was stirred

further for another 5–10 min. This was followed by drop wise addition of

diethyl chlorothiophosphate dissolved in dichloromethane at 20–25 ^◦C.

On complete addition of diethyl chlorothiophosphate, the reaction

mixture was stirred for an additional 1–2 h. The reaction was monitored

by GC-MS. After completion of the reaction, the organic layer of DCM

was washed thrice with NaHCO₃, and the DCM layer was dried over

anhydrous Na₂SO₄. The solvent (DCM) was evaporated and an oily

residue was obtained. The residue was dissolved in acetone and acidiﬁed

by dry HCl gas. The acidic acetone solution was evaporated to a half

volume by purging nitrogen. Then diethyl ether was added. A white

precipitate was appeared which was ﬁltered and washed with diethyl

3

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Biomedicine & Pharmacotherapy 143 (2021) 112134

ether & dried under vacuum. Compound was recrystallized by acetone-

ether solvent system. White solid Dec. > 200 ^◦C. ¹H NMR (600 MHz,

CD3OD): δ 7.97–7.91(m,3H)7.72 (d, 2H, J = 12 Hz), 7.36 (d, 1H, J = 12

Hz), 6.96 (d,1H, J = 6 Hz), 2.60–2.59 (m,6H); ¹³C NMR (CDCl₃, 100.6

MHz) 197.00, 161.27, 143.15, 142.30, 132.99, 132.45, 130.24, 129.76,

127.46, 119.31, 26.45, 15.91. ESI MS (m/z): 349 (M + H)⁺. Anal. Calcd

for C₁₄H₂₆C_lN₂O₂PS₂C, 43.68; H, 6.81; N, 7.28; S, 16.66 Found C, 42.87;

H, 6.52; N, 7.82; S, 15.87.

resulting docking conformations compared to determine similarities,

and then they are clustered accordingly and ranked on the basis of

energy.

2.5. Screening of SMI using ﬂuorescence based thermal shift assay

(FTSA)

FTSA is a cost-effective high throughput method for the identiﬁca-

tion of inhibitors of target protein. FTSA is based on monitoring the shift

of melting temperature (ΔT_m) of the protein in presence of the ligand

using an environmentally sensitive dye [16]. The ﬂuorescent SYPRO

orange dye has been used to monitored protein unfolding. The FTSA was

conducted in the real time PCR detection system (Applied Biosystems,

Life Technologies, USA) as described previously [14]. ΔT_m2.5 ^◦C was

selected as a cut off to exclude non-speciﬁc interactions.

2.2.14. Synthesis of [2-(2-propylsulfanyl-ethylamino)-ethyl]-

thiophosphoramidic acid O,O^′-diethyl ester (compound 33)

Same as described for compound 32, starting from diethyl chlor-

othiophosphate and DRDE-30. White solid 172–178 ^◦C. ¹H NMR (400

MHz, CDCl3): δ 4.84–4.02 (m,4H), 3.34–3.13 (m,6H), 2.86–2.83(m,2H),

2.61–2.57 (m,2H), 1.67–1.61(m,2H),1.34–1.29(m,6H), 1.02(t,3H, J =

7.2 Hz). ¹³C NMR (CDCl₃, 100.6 MHz). ³¹P NMR (CDCl₃, 162 MHz) δ

72.19.¹³C NMR (CDCl₃, 100.6 MHz) δ 77.36, 77.04, 76.73, 63.55, 63.50,

47.53, 45.89, 38.13, 34.10, 27.34, 22.81, 16.03. ESI MS (m/z): 315 (M +

H) ⁺. Anal. Calcd for C₁₁H₂₈C_lN₂O₂PS₂C, 37.65; H, 8.04; N, 7.98; S,

18.28 Found C, 38.12; H, 8.87; N, 7.12; S, 17.89.

2.6. In vitro evaluation of compounds

All 35 compounds were evaluated in vitro in A549 cell line using

MTT, NR and LDH release assay. The human lung endothelial cell line,

A549 (passage no.12) was obtained from National Centre for Cell Sci-

ence, Pune and maintained in RPMI-1640 medium supplemented with

10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin,

2.2.15. Synthesis of N-(4-acetyl-phenyl)-2-ﬂuoro-benzamide (compound

34)

100 μ

g/ml streptomycin and 2 mM L-glutamine at 37 ^◦C in a humidiﬁed

Same as described for compound 21, starting from 4-aminoacetophe-

none and 2-Fluorobenzoyl chloride. White solid 159–160 ^◦C. ¹H NMR

(600 MHz, CDCl3): δ 8.65–8.62 (bs, 1H), 8.20–8.17 (m, 1H), 8.01–7.99

(m, 2H), 7.80–7.78 (m, 2H), 7.58–7.55 (m, 1H), 7.36–7.33 (m, 1H),

7.23–7.21(m, 1H), 2.60 (s, 3H). ¹³C NMR (CDCl₃, 100.6 MHz) 196.90,

161.42, 142.03, 134.31, 134.24, 132.39, 129.81, 125.31, 120.86,

119.70, 116.36, 116.19, 26.49. EIMS: m/z 75.0, 95.0, 123.0, 257.0.

Anal. Calcd for C₁₅H₁₂FNO₂C, 70.03; H, 4.70; N, 5.44. Found C, 69.86;

H, 4.21; N, 5.13.

atmosphere containing 5% CO₂and 95% air in an incubator.

2.6.1. Determination of 50% toxic concentration (CC₅₀) of abrin and ricin

The concentration of abrin and ricin toxic to 50% (CC₅₀) of the cells

was determined from the plots of viability of cells by MTT and NR assay.

A549 cells were dispensed in 96-well ﬂat-bottomed microtiter plates

(NUNC, Roskilde, Denmark) at a density of 1 × 10⁴cells/well in 200

μl.

Cells were incubated with various concentrations of abrin (0.5–4 ng/ml)

and ricin (1–20 ng/ml) for 24 h following which 0.5 mg/ml MTT in

RPMI-1640 medium was added to the cells and incubated for 4 h. The

resulting formazan crystals were dissolved in the DMSO. The absorbance

was measured using an ELISA reader (Chameleon Multi-detection Plat-

form, Finland) at a wavelength of 570 nm. For NR assay after 24 h of

2.2.16. Synthesis of 3-methyl-thiophene-2-carboxylic acid (2-acetyl-

phenyl)-amide (compound 35)

The procedure for the synthesis ofcompound35 is similar to that of

compound 21, 2-aminoacetophenone and 3-Methyl-thiophene-2-

carbonyl chloride being the starting material. White solid 128–129 ^◦C.

¹H NMR (600 MHz, CDCl₃): δ 12.27 (s,1H), 8.83(d,1H, J = 12 Hz), 7.93

(t, 1H, J = 6 Hz), 7.58 (d,1H, J = 6 Hz), 7.36 (d,1H, J = 6 Hz)7.15–7.13

(m,1H),6.95–6.94 (m,1H), 2.69 (s, 3H), 2.64(s, 3H). ¹³C NMR (CDCl₃,

100.6 MHz) 202.92, 162.12, 143.32, 141.25, 135.14, 132.32, 131.72,

131.50, 127.62, 122.45, 122.10, 121.07, 28.53, 15.98. EIMS: m/z 53.0,

70.0, 97.0, 216.0, 259. Anal. Calcd for C₁₄H₁₃NO₂SC, 64.84; H, 5.05; N,

5.40; S, 12.37 Found C, 65.12; H, 4.93; N, 5.78; S, 11.92.

toxin treatment, 0.33% neutral red solution (100 μl) was added to the

culture and incubated for another 4 h. The incorporated dye was solu-

bilized with 1% acetic acid in 50% methanol and absorbance was

measured at 540 nm. Data were normalized to the measurement from

control cultures which were considered as 100% survival. On the basis of

control values, CC₅₀concentration was determined. For all subsequent

time course studies, CC₅₀of abrin and ricin was used.

2.6.2. LDH release assay

2.3. Isolation of abrin and ricin

LDH activity in the culture media of abrin and ricin treated cells was

measured spectrophotometrically at 490 nm according to manufac-

turer’s protocol as an index of plasma membrane damage and loss of

membrane integrity. Enzyme activity was expressed as the percentage of

extra cellular LDH activity of the total LDH activity of the cells.

Abrin and ricin were isolated from the Abrus precatorius and Ricinius

communis seeds respectively. Puriﬁcation of both toxins was done by

using sepharose 6B afﬁnity and gel ﬁltration chromatography [15]. The

purity and molecular weight of abrin and ricin were conﬁrmed by

coomassie blue staining and MALDI-TOF (data not shown). The stock

protein solution was diluted with PBS (pH 7.2) to a concentration of 2

mg/ml.

2.6.3. Determination of CC₅₀and maximum tolerated doses (MTD) of

compounds

SMI listed in Table 1 were dissolved in DMSO and 200 mM stock

solution was prepared. These compounds were 10-fold serially diluted

2.4. In silico analysis of SMI against abrin and ricin toxin

(ranging from 2500 to 0.0025 μM) in media for their toxicity evaluation.

CC₅₀of compounds were determined by dose response curve analysis.

We have also determined highest concentration of compounds that is

non-toxic to cells and known as MTD.

In silico docking of compounds was done using AutoDock 4.2. Small

molecule structures in ligand.pdb format was prepared using Argus.lab

4.0.1 (http://www.arguslab.com) The 1DM0.pdb ﬁle of abrin and ricin

was obtained from RCBS (http://www.ncbi.nlm.nih.gov/pub

med/xxxxx). The Grid Box was cantered on the active site amino acid

residues Tyr74, Tyr113, Glu164, Arg167, Trp198 of abrin and Asn78,

Tyr80, Val81, Phe93, Gly121, Asn122, Tyr123, Arg134, Ile172, Glu177,

Arg180, Glu208, Asn209, Trp211 of ricin. AutoDock was launched and

2.6.4. Determination of efﬁcacy of compounds against abrin and ricin

All 35 compounds were tested for their protective effect against abrin

and ricin toxicity. For determining the same, cells were treated with

varying concentrations (ranging from 2500 μM to 0.0025 μM) of com-

pounds for 1 h prior to abrin or ricin treatment. The cell viability assay

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Table 1

Chemical structure and name of the synthesized compounds evaluated as N-glycosidase activity inhibitor of abrin and ricin. Binding energy (BE) and inhibitory

constant (ki) of each compound was obtained using AutoDock 4.2.

Compound

number

Compound structure

Compound name

Binding energy

(BE) (kcal/

mole)

Inhibitory constant Ki

(µM)

Abrin

-5.80

Ricin

Abrin

53.80

Ricin

1

2

N-(2-(phenylthio) ethyl)-2-(pyrrolidin-1-

-10.11

0.038

3.33

yl) ethanamine dihydrochloride

O, O-diethyl (2-{[2-(butylsulphanyl)

ethyl] amino}ethyl)phosphoramido-

thioate hydrochloride salt

-4.60

-5.26

-7.47

-6.67

600.0

3

4

5

N-[2-(2-(phenylthio)ethylamino)ethyl]-

140.28

12.98

10.84

14.31

4-nitrobenzamide hydrochloride

E-N-(2-acetyl-phenyl)-3-phenyl-

acrylamide

-7.63

-5.26

-6.77

-6.66

2.57

E-N-(2-hydroxy-phenyl)-3-phenyl-

acrylamide

139.9

6

7

E-N-(2-hydroxy-ethyl)-3-phenyl-

acrylamide

-5.53

-9.40

-6.47

-7.01

88.51

0.129

18.19

7.25

3-phenyl-acrylic acid 4-oxo-2-phenyl-4H-

chromen-3-yl ester

8

9

(E)-1-(2-hydroxyphenyl)-3-(4-

-6.49

-6.96

-6.95

-5.87

17.61

7.90

8.05

methoxyphenyl) prop-2-en-1-one

(E)-3-(4-chlorophenyl)-1-(2-

50

hydroxyphenyl) prop-2-en-1-one

(continued on next page)

5

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Table 1 (continued)

Compound

number

Compound structure

Compound name

Binding energy

(BE) (kcal/

mole)

Inhibitory constant Ki

(µM)

Abrin

-7.54

Ricin

-7.56

Abrin

2.98

Ricin

10

(E)-3-(2-ﬂuorophenyl)-1-(2-

2.89

hydroxyphenyl) prop-2-en-1-one

11

12

(E)-1-(2-hydroxyphenyl)-3-(thiophen-2-

yl) prop-2-en-1-one

-6.88

-7.52

-8.13

-3.80

9.01

3.09

1.10

1.64

4-chloro-N-[2-(2-propylsulfanyl-

ethylamino)-ethyl]-benzamide

13

14

2-styryl-1H-quinolin-4-one

-8.90

-5.50

-7.03

-7.24

0.28

7.08

4.97

2-(2-ﬂuro-phenyl)-1H-quinolin-4-one

92.89

15

16

N-(2-acetyl-phenyl)-2-ﬂuoro-benzamide

-7.12

-7.87

-5.82

-4.92

6.02

1.70

54

4-nitro-N-(2-(4-nitrobenzamido) ethyl)-

248

N-(2-(phenylthio) ethyl) benzamide

17

2-(3-methyl-thiophen-2-yl)-1H-quinolin-

-6.75

-6.23

11.34

27.15

4-one

18

19

Cyclohexanecarboxylic acid[2-(2-

-5.25

-7.45

-4.8

141.40

3.45

304

phenylsulfanyl-ethylamino)-ethyl]-amide

N-(2-acetyl-phenyl)-4-nitro-benzamide

-7.42

3.63

(continued on next page)

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Table 1 (continued)

Compound

number

Compound structure

Compound name

Binding energy

(BE) (kcal/

mole)

Inhibitory constant Ki

(µM)

Abrin

Ricin

Abrin

Ricin

20

21

22

23

N-(2-acetyl-phenyl)-4-ﬂuoro-benzamide

N-(2-acetylphenyl)-4-methoxybenzamide

N-(2-acetylphenyl)-4-chlorobenzamide

-7.40

-6.94

-7.34

-5.19

-7.52

-4.53

-5.91

-5.83

3.77

3.06

8.20

4.14

476.72

46.91

3-Phenyl-N-(3-phenylsulfanyl-propyl)-

acrylamide

156.15

53.41

24

25

4-Nitro-N-(3-phenylsulfanyl-propyl)-

benzamide

-6.62

-7.14

-4.41

-4.05

14.09

5.88

588.96

N-(2-acetylphenyl)-2,3-

1000

diﬂuorobenzamide

26

27

2-methyl-N-(2-(2-(phenylsulphanyl)

-7.06

-7.23

-4.38

-4.66

6.68

612.79

ethylamino) ethyl) benzamide

N-(3-acetylphenyl)-2-ﬂuorobenzamide

4.98

384.79

(continued on next page)

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Table 1 (continued)

Compound

number

Compound structure

Compound name

Binding energy

(BE) (kcal/

mole)

Inhibitory constant Ki

(µM)

Abrin

Ricin

Abrin

Ricin

28

29

N-(2-acetylphenyl)-2,5-

-7.20

-6.93

5.24

8.34

diﬂuorobenzamide

(E)-N-(2-acetylphenyl)-3-(2-

-7.77

-6.42

2.03

19.73

chlorophenyl) acrylamide

30

31

N-(2-acetylphenyl)-2,6-

-8.20

-4.35

-6.92

-2.88

0.98

8.52

dichlorobenzamide

3-Methyl-thiophene-2-carboxylic acid (4-

acetyl-phenyl)-amide

649.56

76,900

32

33

[2-(2-Phenylsulfanyl ethylamino)-ethyl]-

thiophosphoramidic acid O, O^′-diethyl

ester

-6.94

-5.87

-6.3

8.13

24.24

1.41

[2-(2-Propylsulfanyl-ethylamino)-ethyl]-

thiophosphoramidic acid O, O^′-diethyl

ester

-3.89

49.45

34

35

N-(4-acetylphenyl)-2-ﬂuorobenzamide

-6.78

-7.38

-6.74

-7.57

10.70

3.90

11.56

2.81

3-Methyl-thiophene-2-carboxylic acid (2-

acetyl-phenyl)-amide

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Biomedicine & Pharmacotherapy 143 (2021) 112134

was performed 24 h post-treatment employing MTT and NR assay. The

half the members of a tested population (LD₅₀), was determined by

intraperitoneal (i.p.) and oral route for 14 days using up and down

method of TG-425 of OECD-5 guidelines.

dose at which the compound inhibits 50% toxicity of abrin or ricin (IC₅₀

)

value was also estimated using dose response curve analysis. Further,

Selectivity Index (SI) as an estimate of therapeutic window of com-

pounds was calculated from CC₅₀and IC₅₀values of compounds (SI =

CC₅₀/IC₅₀). Highest DMSO amount used in a study was tested as a

vehicle control and showed no toxicity to cells.

2.9.3. Treatment regimen

Compounds that offered protection in in vitro study were further

evaluated in mice for their efﬁcacy against abrin and ricin. Compounds

were dissolved in PEG 300 and they were soluble up to 200 mM. Hun-

dred microliters of 200 mM of compounds was ﬁxed for administration

in mice, keeping in mind that volume above to this may cause discomfort

2.6.5. Efﬁcacy evaluation of compound using LDH release assay

Compounds that offered protection against abrin and ricin by MTT

and NR assay were further evaluated by LDH release assay to validate

their efﬁcacy.

to mice. All compounds were tested (100 μl of 200 mM) for their toxicity

and were found safe at their highest soluble dose. Even repetitive dosing

of compounds was also found to be safe (Data not shown). For efﬁcacy

evaluation of SMIs, three approaches were followed i.e. premixed

(toxins and SMIs were pre-incubated for 1 h and then administered in

mice); pre-treatment (SMIs were treated 1 h prior to abrin or ricin); and

post-treatment (SMIs were treated after 1 h of toxins treatment). For all

the three approaches, both toxins and SMIs were administered through i.

2.7. Surface plasmon resonance (SPR) analysis of shortlisted compounds

The interaction between SMIs and abrin or ricin was further

conﬁrmed using a ProtOn XPR36 instrument (Bio-Rad, Hercules, CA) as

per the previously described protocol [14]. In Brief, amine coupling

p. route. SMIs were tested at a ﬁxed dose of 100 μl of 200 mM against a

chemistry was used to immobilize abrin and ricin (100 and 200 μg/ml)

single dose of 5XLD₅₀of abrin or ricin. Control animals received the

same volume of PEG 300 as the experimental group. Toxin alone treated

group was administered a single dose of 5XLD₅₀of either abrin or ricin.

Compounds that offered protection in the post-treatment approach for

1 h were further evaluated by repetitive treatment after 4 and 8 h. The

repetitive post-treatment, oral vs i.p., and i.p.vs oral for compound and

toxin respectively has also been evaluated. For all treatments four or six

animals in each group were used. Experiments were repeated at least

three times. The animals were observed for toxicity related symptoms,

mean survival time and mortality till the 14th day of exposure following

administration of abrin or ricin and compounds. Protection Index

(PI=LD₅₀of compound with toxin/LD₅₀of toxin alone) was also calcu-

lated for the compounds that offered complete protection.

on the EDAC/Sulfo-NHS activated GLH sensor chip at a density of ~

6000 resonance unit (RU) as per the manufacturer’s instructions. Solu-

tions of SMIs were prepared in phosphate buffered saline-Tween 20 and

injected at 100

μ

l min^{ꢀ 1}for 200 s at concentrations of 250–12.5

μM.

The dissociation was monitored for 600 s. Chip surfaces were regener-

ated by injecting 50 mM NaOH for 30 s for reuse. All the SPR experi-

ments were performed at 25 ^◦C. The sensogram data obtained were

analyzed using ProtOn Manager 3.1 software and ﬁtted to the simplest

Langmuir 1:1 interaction model.

2.8. Functional characterization of SMIs via in vitro translation system

(IVTS)

To detect the activity of toxin i.e. protein synthesis inhibition and

effect of SMI on reversing the activity of toxin, microtitre-based cell free

translational assay was done as described elsewhere [17]. Initially

concentration of toxin, inhibiting 50% translation or protein synthesis

was determined and at this dose SMIs were evaluated to observe their

efﬁciency to reverse translation inhibition. In brief, translation lysate

was prepared by mixing nuclease-treated, rabbit reticulocyte lysate,

amino acid mixture, RNasin ribonuclease inhibitor nuclease treated

deionised water and luciferase mRNA in 35:1:1:35: ratio respectively.

This translation reaction was performed at 4 ^◦C. In a microtitre plate,

2.9.4. Statistical analysis

The complete data was expressed as a mean ± SEM of three or more

independent experiments with triplicate samples. Statistical compari-

sons between groups were performed using one-way ANOVA followed

by Dunnett’s test or Student’s t-test or Student–Newman–Keuls multiple

comparison test. Differences with p-value less than 0.05 were taken as

signiﬁcant.

3. Result

25 μl/well translation lysate was dispensed and 5 μl of abrin or ricin

3.1. In silico screening of SMI

alone or along with different concentrations of the compounds were

added. Complete reaction mixture was incubated at 37 ^◦C in a moist

Thirty-ﬁve compounds were evaluated for efﬁcacy against abrin and

ricin. The minimum binding energy (kcal/mol) and inhibitory constant

chamber for 90 min. Post incubation, 10 μl of mixture was transferred to

a black microtitre plate and bioluminescence assay buffer containing the

luciferin substrate was added to the wells and luminescence was

measured. Data were normalized to the measurement from control well

(translation lysate and PBS) which was considered as 100% translation.

(μM) of these compounds were calculated by docking on the active site

of abrin and ricin (catalytic site of A chain) using AutoDock 4.2. The

binding energy of compounds against abrin and ricin varied from ꢀ 4.35

to ꢀ 9.40 and ꢀ 2.88 to ꢀ 10.11 kcal/mole, respectively. For abrin,

inhibitory constant values were ranging from 0.129 to 649.56 μM and

2.9. In vivo evaluation of compounds

for ricin 0.038–76,900 μM (Table 1). These molecules were found to

interact with the active site residues of abrin and ricin, suggesting the

probability of efﬁcient binding and thus inhibition of abrin and ricin

toxicity. The docked models of top scoring compounds were visualized

using UCSF Chimera to observe the interactions between the compounds

and toxins [18]. To analyse the possibility of nonspeciﬁc or multi-targets

binding of these molecules, pan assay interference compounds (PAINS)

ﬁlter was applied. None of the compound was recognized as potential

PAINS.

2.9.1. Animals

Balb/c male mice randomly bred in animal facility of the institute,

weighing between 22 and 25 g were used in this study. The animals were

housed under standard conditions of temperature and humidity and

were fed with the standard pellet diet (Ashirwad Brand, Chandigarh,

India). Food and water were given ad libitum. The animals were handled

according to the guidelines of the Committee for the Purpose of Control

and Supervision on Experiments on Animals (CPCSEA, India). All the

doses used and protocols were approved, by the Institutional Animal

Ethics Committee (IAEC) and the protocol number was TOX-73/53/NS.

3.2. Synthesis of SMIs

2.9.2. Determination of median lethal dose (LD₅₀) of abrin and ricin

All compounds were synthesized as described in methodology and

are largely amides, chalcones, quinolinones and their derivatives.

For treatments, median lethal dose of abrin and ricin, required to kill

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Compound 21, 22, 25, 27, 28, 30, 34 are the derivatives of compound 15

while compound 23 and 29 are derived from compound 4. Compound

24, 26 and 16 are the derivatives of compound 3 and compound 31, 32,

33 and 35 are derived from compound 2.

concentrations of abrin ranging between 0 and 4 ng/ml for 24 h and

viability was determined by MTT and NR assay. Abrin caused signiﬁcant

dose dependent toxicity at 24 h. CC₅₀was found to be 1 ng/ml by both

MTT and NR assays. Similarly, cells were treated with different con-

centration of ricin (0–20 ng/ml) and 10 ng/ml doses were estimated as

CC₅₀by both assays. Abrin and ricin toxicity was further conﬁrmed by

LDH release assay. Both toxins cause dose dependent increase in LDH

release indicating damage to the cell membrane. At CC₅₀concentration

of the toxin, LDH release was more than 2 folds compared to control

(Fig. 2).

3.3. Initial screening of compound using ﬂuorescence-based thermal shift

assay (FTSA)

FTSA was done to screen the compounds on the basis of their inter-

action with abrin and ricin. FTSA utilizes the characteristic of protein

that it unfolds at deﬁnite temperature and interaction with ligand,

change the thermal stability of the protein which causes shift in

unfolding temperature. Here SYPRO orange dye has been used, which

exhibits ﬂuorescence on binding to hydrophobic surfaces resulting from

the unfolding of protein. All 35 compounds were evaluated against abrin

and ricin for their interaction and ΔTm (change in temperature) of abrin

and ricin protein was recorded for each compound. ΔT_m> 2.5 ^◦C was

considered to be signiﬁcant. Among the 35 compounds, 15 compounds

showed signiﬁcant interaction with abrin. Compound 4 binds with

highest afﬁnity with abrin (ΔT_m5.60 ^◦C) followed by 15 (ΔT_m4.25 ^◦C),

5(ΔT_m4.01 ^◦C), 25 (ΔT_m3.95 ^◦C), 3, 30 (ΔT_m3.50 ^◦C), 10, 19, 32, 33,

34 (ΔT_m3.25 ^◦C), and 6, 20, 24, 28(ΔT_m2.75 ^◦C) (Fig. 1A). Similarly,

interactions of all compounds with ricin were evaluated and of which 4

compounds exhibited signiﬁcant shift in unfolding temperature. These

are compound 4(ΔT_m4.80 ^◦C), 5(ΔT_m3.80 ^◦C), 15(ΔT_m3.26 ^◦C), and 25

(ΔT_m2.70 ^◦C) (Fig. 1B). For both toxins, compound 4 showed highest

afﬁnities compared to the other compounds.

3.5. Determination of MTD of compounds

For determination of MTD, A549 cells were treated with varying

concentrations (0.0025–2500 μM/well) of compounds for 24 h and then

cell viability was measured by MTT assay. MTD values of compounds

varied from 1500 μM (102 ± 0.9% viability) to 2.5 μM (104 ± 1.7% cell

viability). All compounds were tested within MTD range for their efﬁ-

cacy as an inhibitor (Table 2).

3.6. In vitro efﬁcacy evaluation of compounds as an abrin and ricin

inhibitor

For analysing the efﬁcacy of compounds as abrin and ricin inhibitors,

cells were pre-treated with compounds for 1 h followed by abrin and

ricin exposure, at its 50% toxic dose. Among the 35 compounds evalu-

ated, ﬁve compounds (compounds 4, 5, 15, 25 and 28) and two com-

pounds (4 and 15) showed signiﬁcant reversal of abrin and ricin induced

toxicity, respectively using MTT assay (Fig. 3A). Efﬁcacy of these

promising compounds was further conﬁrmed by NR assay. The com-

pounds those found promising by MTT assay were also found to protect

cells from abrin and ricin induced toxicity in NR assay (Fig. 3B). In

addition to this, promising compounds also showed signiﬁcant

3.4. Abrin and ricin induced cytotoxicity in a dose and time dependent

manner in A549 cells

To determine the CC₅₀(concentration that causes 50% toxicity to

cells) of abrin in A549 cells, cells were treated with varying

Fig. 1. Fluorescence based thermal shift assay showing interaction with small molecule inhibitors and abrin or ricin. The ΔTm represents the difference between

melting Tm of abrin or ricin incubated with compounds and Tm of abrin or ricin only. Values are mean of three replicates. ΔTm values above 2.5 were considered to

be signiﬁcant. Fifteen compounds against abrin and 4 compounds against ricin showed signiﬁcant shift in melting temperature.

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Fig. 2. Abrin and ricin induced toxicity in A549 cells. Cytotoxic effect of toxins in A549 cells was determined by MTT and NR assay. For determination of CC₅₀, the

cells were dispensed on to a 96 well plate and treated with varying concentration of toxins for 24 h. (A) Abrin; The 50% cytotoxic concentration (CC₅₀) was calculated

as 1 ng/ml. Viability of untreated cells assumed as 100%. (B) Ricin; the 50% cytotoxic concentration (CC₅₀) was calculated as 10 ng/ml. Viability of untreated cells

considered as 100%. (C) Abrin and (D) Ricin caused signiﬁcant dose dependent increase in LDH release. More than 2 folds release of LDH at CC₅₀concentrations of

both toxins was observed. Data are the mean of three independent experiments each in triplicates with SEM. *p < 0.05 compared to un-treated cells.

inhibition of abrin and ricin induced LDH release and maintains the cell

membrane integrity (Fig. 3C). The doses of compounds inhibiting 50%

toxicity of abrin or ricin (IC₅₀) were also calculated by analysing the

dose response curves obtained by MTT assay. The IC₅₀values of com-

pounds 4, 5, 15, 25 and 28 were estimated to be 160.6 ± 7.1,

binds to abrin, with strong afﬁnity, in a dose dependent manner with

strong afﬁnity and yielding an equilibrium dissociation constant (K_D) of

1.15 × 10^{ꢀ 7}M with a fast on rate (k_a) = 2.35 × 10⁶1/Ms and fast off

rate k_d= 2.71 × 10^{ꢀ 1}1/s. Compound 4 also interact with ricin with high

afﬁnity with K_D= 4.59 × 10^{ꢀ 7}M; k_a= 2.35 × 10⁶1/Ms; k_d

= 2.07 × 10^{ꢀ 1}values (Fig. 6F). Compound 5 (K_D= 4.64 × 10^{ꢀ 6}M; k_a=

5.92 × 10⁶1/Ms; k_d= 2.75 × 10¹), compound 15(K_D= 9.62 × 10^{ꢀ 7}M;

191 ± 13.0, 290 ± 5.4, 30.5 ± 5.1 and 305 ± 12.6 μM against abrin

toxicity, respectively. The IC₅₀values of compounds 4 and 15 against

ricin toxicity were found to be 300 ± 8.2 M and 341 M ± 6.1 M,

μ

k_a= 3.12 × 10⁷1/Ms; k_d= 3.00 × 10¹), compound 25 (K_D

=

respectively. These compounds (4, 5, 15, 25 and 28 against abrin and 4,

5 against ricin) were also tested for their 50% cytotoxic concentration

(CC₅₀) using MTT assay. On the basis of CC₅₀and IC₅₀values, the

selectivity index (SI = CC₅₀/IC₅₀) of each compound was determined,

which reﬂects the quantity of compound, that is active against the toxin

but is non-toxic towards the cells. The SI values of compound 4, 5, 15, 25

and 28 were 15.5, 13.0, 8.6, 16.0 and 4.5, respectively against abrin and

of compounds 4 and 15 were 8.3 and 7.3 against ricin (Table 2).

7.95 × 10^{ꢀ 7}M; k_a= 9.47 × 10⁷1/Ms; k_d= 7.53 × 10¹), and compound

28 (K_D= 3.10 × 10^{ꢀ 7}M; k_a= 9.65 × 10⁷1/Ms; k_d= 3.06 × 10¹) were

interacted with abrin with high afﬁnity (Fig. 5B–F). Compound 15 was

interacting with ricin (K_D= 1.42 × 10⁰M; k_a= 9.15 × 10^{ꢀ 2}1/Ms; k_d=

1.30 × 10^{ꢀ 1}) with high afﬁnity thus conﬁrm its interaction with ricin

(Fig. 5G).

3.8. Effect of compounds on the inhibition of protein synthesis

3.6.1. Dose dependent potential of promising compounds

Initially, the effect of abrin and ricin on protein synthesis was

observed using IVTS. Different doses of toxins (1, 10, 100, 1000 ng/ml)

were used to determine the concentration required for 50% inhibition of

protein synthesis or translation. Abrin at 1 ng/ml and ricin at 10 ng/ml

showed 50% inhibition of protein synthesis and these doses of abrin and

ricin were further used in the efﬁcacy study of the compounds (Fig. 6).

A dose dependent effect of promising compounds (4, 5, 15, 25 and 28

against abrin and 4, 15 ricin toxicity) was also analysed and compounds

were found to inhibit abrin or ricin induced toxicity in a dose dependent

manner. Among all compounds either 250 or 25 μM dose was found to

be optimum for maximum inhibition of abrin or ricin cytotoxicity

(Fig. 4). After efﬁcacy evaluation, compounds that offered protection in

cells were further validated for their interactions and biological activity

against toxins using SPR and IVTS, respectively.

Three doses of each compound (2.5, 25 and 250 μM) were used to

observe the efﬁcacy to revert the toxin induced translation inhibition.

The compounds short listed through cell viability assays i.e. compounds

4, 5, 15, 25 and 28 were analysed against abrin and compounds 4 and 15

were studied against ricin toxicity. All compounds were seen to signif-

icantly restore the protein synthesis (ranging from 65 ± 2.3% to

3.7. Binding studies of SMIs with abrin and ricin

To quantitatively assess the binding afﬁnity of promising compounds

with abrin and ricin, SPR was performed using immobilized abrin or

ricin on GLH sensor chip at ~ 6000RU. As shown in Fig. 5A compound 4

70 ± 4%) at 250 μM dose against abrin induced toxicity. Compound 4

(91 ± 6.3%) and 15 (79 ± 2.4%) were seen to signiﬁcantly reverse the

inhibition of protein synthesis and restored the translation inhibition

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Table 2

Safety and efﬁcacy parameters of compounds. Plated cells were treated with varying concentrations of compounds for the estimation of maximum tolerated dose (MTD;

highest concentration of compounds that does not affect cell, viability). For efﬁcacy analysis, cells were pre-treated with compounds followed by toxin exposure. Only

those compounds which offered protection against toxins were also calculated for 50% Cytotoxic concentration (CC₅₀) and 50% inhibitory constant (IC₅₀; measure of

inhibitory potential of compound against toxins). Selectivity Index (SI; capability of compounds to produce differential effect against toxin) of compounds were

calculated as a ratio of CC₅₀to IC₅₀. Data are the mean±SEM of three independent experiments each in triplicate.

Compound number

MTD (µM) (% Viability ± SD)

CC₅₀(µM)

IC₅₀(µM)

SI

Abrin

Ricin

Abrin

Ricin

1

500 (99.6 ± 13.7)

50 (98.8 ± 4.1)

25 (94.9 ± 7.0)

500 (106.8 ± 5.6)

250(92.2 ± 1.4)

250 (99.6 ± 7.0)

25 (95.0 ± 1.08)

250 (95.9 ± 3.5)

2.5 (97.5 ± 5.3)

500 (99.4 ± 8.1)

1000 (96.8 ± 4.0)

25 (95.9 ± 7.3)

2.5 (104 ± 1.7)

1000 (96.3 ± 2.9)

500 (95.2 ± 0.7)

25 (91.5 ± 1.2)

100 (103.7 ± 1.7)

250 (98.2 ± 0.1)

500 (97.7 ± 3.8)

1000 (94.3 ± 3.8)

1500 (102.1 ± 0.9)

500 (97.0 ± 6.5)

250 (95.4 ± 3.1)

2.5 (103.1 ± 0.2)

50 (96.3 ± 1.07)

500 (93.6 ± 0.9)

250 (94.9 ± 3.2)

500 (98 ± 4.1)

2

3

4

2500

160.6 ± 7.1

300 ± 8.24

15.5

13.0

8.3

5

191.6 ± 13.0

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

2500

290 ± 5.4

341.3 ± 6.1

8.6

7.3

489.5 ± 11.6

1379.3 ± 12.0

30.5 ± 5.1

305 ± 12.6

16.0

4.5

100 (98.0 ± 0.8)

100 (93.2 ± 0.06)

2.5 (100.5 ± 0.1)

50 (100.5 ± 2.4)

2.5 (106.8 ± 1.7)

250 (95.1 ± 2)

250 (93.7 ± 1.4)

induced by ricin (51 ± 6.2%) (Table 3).

method section. All three approaches i.e. pre-mixed, pre-treatment and

post-treatment resulted in the extension of survival time for all ﬁve

compounds tested against abrin toxicity. In pre-mixed strategy, exten-

sion of survival time (in days) was highest with compound 4 (8.6 ± 5.7;

50% survival) followed by 5 (6.8 ± 5.7; 33% survival), 15 (5.9 ± 5.8;

31% survival), 25 (3.6 ± 3.5; 9% survival) and 28 (2.6 ± 1.6% survival)

compared to abrin treatment where all animals were died within a day.

Compound 4 was most effective with an estimated protection index (PI)

value of 5. In the pre-treatment group all mice died within 2–3 days

compared to abrin exposed mice. In post-treatment strategy, mice sur-

vived for 6.2 ± 5.7 (33% survival), 5.0 ± 5.7 (25% survival), 3.3 ± 2.6

(0% survival), 2.7 ± 1.3(0% survival) and 2.4 ± 1.7 (0% survival)days

with compounds 4, 15, 25, 5 and 28 treated group after abrin intoxi-

cation (Table 4A). In a similar pattern, compounds 4 and 15 were

evaluated against ricin toxicity. In the pre-mixed group, compounds 4

(5.9 ± 4.7 days; 22% survival) and 15(5.3 ± 3.6 days; 13% survival)

signiﬁcantly enhanced the survival time compared to ricin exposed

(1.0 ± 0 days) mice. In the pre-treatment group all mice were survived

up to 2–3 days. Post-treatment with the compound, extended the sur-

vival time (compound 4: 6.7 ± 5.1 days; 30% survival and compound

15: 4.3 ± 4.8 days; 17% survival) compared to ricin treated mice

(Table 4B). Among all the compounds tested, compound 4 offered

maximum protection against both toxins. Efﬁcacy of compound 4 was

further validated using repetitive dosing at 1, 4 and 8 h via post-

treatment strategy. For post-treatment three combinations of route of

administration were evaluated which i.e. (i) toxin and compound both

via i.p. route; (ii) toxin via oral and compound via i.p. route; (iii) toxin

3.9. Determination of median lethal dose (LD₅₀) of abrin and ricin

The LD₅₀dose for abrin and ricin was estimated to be 1.0

μ

g/kg

(0.5–1.5 μg/kg, 95% conﬁdence limit) and 10 μg/kg (8.5–11.2 μ

g/kg,

95% conﬁdence limit) of body weight respectively through intraperi-

toneal (i.p.) route. LD₅₀dose of abrin 36.7 mg/kg (27.5–42.6 mg/kg,

95% conﬁdence limit) and ricin 13.4 mg/kg (10.5–17 mg/kg, 95%

conﬁdence limit) were estimated via oral route. For all subsequent

studied 5XLD₅₀doses were used for both toxins. For oral administration

of abrin, LD₅₀of abrin was estimated to be 36.7 mg/kg which is very

high value and it is not feasible to administer the dose beyond 2XLD₅₀in

mice. Thus, the 2XLD₅₀dose of abrin has been used for oral adminis-

tration in this study.

3.10. Assessment of in vivo efﬁcacy of compounds

To examine whether efﬁcacy of SMIs restricted to in vitro studies,

compounds 4, 5, 15, 25 and 28 against abrin toxicity and compound 4,

15 against ricin toxicity were further evaluated in mice against 5XLD₅₀

of toxins. All animals, after abrin or ricin exposure showed shivering,

cachexia bloody diarrhea. Their time of onset of symptoms varied be-

tween 6 and 8 h. 5XLD₅₀dose of abrin and ricin consistently produced

100% lethality and the mean time to death varied from 1 to 2 days via

both i.p. and oral route. Initial screening was done via i.p. route for

compounds and both toxins, using three approaches as described in the

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Fig. 3. Efﬁcacy evaluation of small molecule inhibitors in A549 cells. Cells were pre-treated with various doses of compounds for 1 h followed by treatment with the

CC₅₀value of abrin or ricin for 24 h. Cytotoxicity was measured by MTT, NR and LDH release assay. (A) Compounds 3, 4, 15, 25, 28 and compounds 4, 5 signiﬁcantly

reduced the abrin and ricin induced cytotoxicity by MTT assay; respectively; (B) similar observation was found via NR assay; (C) pre-treatment of compounds 3, 4, 15,

25, 28 against abrin and compounds 4, 5 against ricin exposure, signiﬁcantly restored the cell membrane integrity as assessed by lactate dehydrogenase (LDH) release

assay. Data are the mean ± SEM of three independent experiments each in triplicate. *p < 0.05 compared to untreated cells. •p < 0.05 compared to abrin or ricin

treated cells. Control cells are untreated cells. Each compound offering maximum protection is shown in the ﬁgure.

via i.p. and compound via oral route. Intraperitoneal administration of

compound 4 at 4 and 8 h post abrin exposure (i.p.) offered additional

protection (6.9 ± 6.1 days; 38% survival) compared to single dosing at

1 h (Fig. 7A). Intraperitoneal administration of compound 4 at 1 h

extended the survival time (6 ± 5.7 days; 33% survival) compared to

abrin induced lethality after oral administration. Further, repetitive

dosing at 4 and 8 h signiﬁcantly extended the survival time to 7.1 ± 6.2

days with 42% survival. Surprisingly, cumulative dosing of 1, 4 and 8 h

increased the survival percentage up to 92% with a mean survival time

of 13 days (Fig. 7B). However, oral administration of compound 4, fol-

lowed by abrin (i.p.) exposure did not offer any protection. Similar to

abrin, ip administration of ricin and oral administration of compound

did not offer any protection in spite of repetitive treatment (Table 5).

Repetitive dosing of compound 4 (i.p.) enhances the survival time

(7.3 ± 6.0 days) against ricin toxicity (i.p.) in mice with signiﬁcant in-

crease in the percentage survival (38%) (Fig. 7C). Intraperitoneal

treatment of compound 4 protected the mice exposed to ricin via oral

route with protection index 5 even at a single dose suggesting the efﬁ-

cacy of compound 4(Fig. 7D). Further repetitive dosing did not add any

beneﬁcial effect. Considering ﬁndings against both toxins, compound 4

i.e. E-N-(2-acetyl-phenyl)-3-phenyl-acrylamidewas found to be the

promising specially against oral toxicity and can be taken as the lead as

common inhibitor against RIPs.

abrin poisoning. The likelihood abrin and ricin attack thus mandates the

identiﬁcation of countermeasures to combat the ensuing toxicity.

The use of Structure based small molecule inhibitors is a promising

approach for the treatment of abrin and ricin intoxication. The identi-

ﬁcation of SMIs countering the toxicity of Stx, in an earlier study [14]

prompted the evaluation of the antidotal effect of these SMIs as their

structural analogs against abrin and ricin. In the present study, 35

compounds were screened against the two toxins, targeting their cata-

lytic activity. Structural analogs were synthesized by introducing

different functional groups or changing their position on the parent

molecule. Initial screening of these compounds was done using FTSA

and in in vitro assay using A549 cells. Compound those offered protec-

tion were further validated employing SPR and IVTS assay for their

binding and biological efﬁcacy against abrin and ricin toxins. Shortlisted

compounds were ﬁnally evaluated in Balb/c for their in vivo protection.

FTSA is cost effective, less time-consuming method for screening of

small molecule inhibitors that based on shift in thermal stability of the

protein after binding to the ligand [19]. Among 35 compounds studied,

total 15 and 4 compounds were found to increase the stability of abrin

and ricin, respectively; conﬁrming the interaction with toxins. Com-

pounds 4,5,15 and 25 were effective against both abrin and ricin by

FTSA. All the compounds which signiﬁcantly raised the melting point of

toxins are having amide functional group except compound 10 which

falls in the chalcones category.

4. Discussions and conclusion

All 35 compounds were further evaluated using MTT and NR assay.

Compound 4 (E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide), 5(E-N-(2-

hydroxy-phenyl)-3-phenyl-acrylamide) 15(N-(2-acetyl-phenyl)-2-ﬂu-

oro-benzamide), 25(N-(2-acetylphenyl)-2, 3-diﬂuorobenzamide) and 28

(N-(2-acetylphenyl)-2, 5-diﬂuorobenzamide) not only offered protec-

tion against abrin toxicity but also averted the abrin induced cell deaths.

Abrin and ricin are potential bio warfare agents that inhibit protein

synthesis thus causing toxicity. Given its global availability, high

toxicity, and ease of isolation, both toxins are viewed as a probable

terrorist threat. Presently, there is no approved therapy for ricin and

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Fig. 4. Dose dependent potential of compounds 4 against abrin and ricin toxin in A549 cells. Pre-treatment (1 h) of compounds at the indicated concentration

followed by abrin or ricin (CC₅₀) exposure for 24 h. Cells were analyzed by MTT assay. Evaluation against abrin toxicity, (A) Compound 4; (B) Compound 5; (C)

compound 15; (D) compound 25; (E) compound 28, likewise (F) compound 4 and (G) compound 15 evaluated against ricin toxicity. Data are the mean ± SEM of

three independent experiments each in triplicate. •p < 0.05 compared with toxin treated cells.

LDH release assay revealed the restoration of abrin induced cell mem-

brane integrity by these 5 compounds. Compounds 4 and 5 offered

protection against ricin cytotoxicity and also showed high afﬁnity

interaction with ricin by FTSA. The efﬁcacy of compounds was further

substantiated by NR and LDH release assays.

compounds against abrin and for 2 compounds against ricin, when

compared with their IC₅₀values against Stx in an earlier study [14]. This

difference in IC₅₀values of same compounds to different toxins may be

attributed to sequence variability of enzymatic site of toxins resulting in

the variation in electrostatic and molecular interactions with com-

pounds, exactly similar to variation in toxin interaction with ribosomal

proteins [20]. Another in vitro study also reported a highly effective

Since IC₅₀denotes the potential of a compound to inhibit a speciﬁc

biological or biochemical function the lower the value, the more potent

the compound. The present study reveals a higher IC₅₀for all 5

small molecule with IC₅₀value of 30 μM on the basis of IVTS assay [14].

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Fig. 5. Surface plasmon resonance sensogram represents the binding curves and ﬁtted results of compounds to immobilize abrin or ricin on GLH sensor chip. The

concentrations of compounds were ranging from 12.5 to 250 μM. PBS with Tween-20 was used as a control. The data were ﬁtted to the simplest Langmuir 1:1

interaction model. (A) Compound 4; (B) Compound 5; (C) Compound 15; (D) Compound 25; (E) Compound 28 against abrin toxin and (F) Compound 4; (G)

compound 15 against ricin toxin K_a: association rate; k_d: dissociation rate; K_Dequilibrium dissociation constant.

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Biomedicine & Pharmacotherapy 143 (2021) 112134

The present study reports the protective efﬁcacy of compounds both in in

vitro and in vivo studies, the latter being mandatory for successful drug

development.

The SI values, which are an index of the compounds ability to induce

a protective effect without undesirable side effects, were calculated.

Among all the 5 compounds, 4 and 15 showed maximum SI against abrin

toxicity while in ricin both compounds showed equal efﬁcacy. Contrary

to this, compound 3 was reported as the most effective compound with

at par SI value i.e. 22.23 [14]. Taking in to consideration the efﬁcacy of

compounds against all three toxins, it is certain that compounds effec-

tive against all three RIPs must possess an amide functional group.

However, only 5 out of the 27 amide group containing compounds were

seen to exhibit a protective effect suggesting that the position of the

functional group as well as the presence of other moieties may also have

a role in determining the protective efﬁciency of compounds. This was,

thus the basis for the synthesis of structural analogs of compounds with

promise. Since, compounds 3, 4 and 15 appeared promising against Stx,

their structural analogs were screened for their action against abrin and

ricin. Compound 25 and 28 are the variants of compound 15 with

ﬂuorine at positions 3 and 5 respectively and were found to be beneﬁcial

to some extent against abrin toxicity. Compounds 21, 22, 27, 30, 34 and

35 are also structural analogous of compound 15. Compound 21, pre-

pared by the removal of an electron donating ﬂuoro group from position

2 and the introduction of an electron withdrawing methoxy group at

position 4 of compound 15, failed to offer protection. Compound 22,

with chlorine introduced at 4th position and ﬂouro group removed from

2nd position in compound 15, to assess the efﬁciency of electron

donating group at position 4, also proved a non-beneﬁcial change. This

could be attributed to the larger size of the molecule hindering the

binding of the compound with the toxin. Similarly, the addition of

chloro group at 2nd and 6th positions (compound 30) on compound 15

was also found to be ineffective. Acetyl phenyl group which was initially

present at 2nd position in compound 15 was shifted to 3rd (compound

27) or 4th position (Compound 34) in order to determine the positional

effect of this group in toxin binding, but no protection was offered which

suggest that the position of functional group plays important role. These

modiﬁcations also indicate that acetyl group is effective only at ortho

position. Compounds 31 and 35 are structurally similar to compound 34

and having 4-aminoacetophenone or 2-aminoacetophenon as the com-

mon moiety but both compounds did not offer protection. Compound 29

is the derivative of compound 4 and possesses additional chlorophenyl

group to enhance the electronegativity of the molecule but this modi-

ﬁcation also failed to offer protection. Similarly, the inefﬁcacy of com-

pound 23, formed by fusion of compounds 3 and 4, implies that the

backbone of the parent molecule is essential for efﬁcacy of compounds.

Since compound 3 showed promise against of Stx [14], compounds 16,

24 and 26 which are structural derivatives of compound 3, were tested

against abrin and ricin toxicity in the present study. Acylation of both

amines of compound 3 at 4-nitro benzoxyl group (compound 16) did not

give any promising effect. Similarly, acylation of primary amine of

compound 3 on 2-methyl benzoxyl group (compound 26) was not found

effective. Compound 24 another structural analog of compound 3,

lacking the diamine phenyl sulﬁde backbone was also ineffective,

emphasizing the functional importance of the backbone of the molecule.

Compound 2 was not found to be promising against all three toxins but

has been randomly selected for synthesis of its structural analogs i.e.

compounds 32 and 33 with different di amino sulﬁde backbone. How-

ever, these compounds are also failed to offer beneﬁcial effect against

abrin and ricin toxicity. These ﬁndings somewhat ensured that the

likelihood of procuring effective derivatives of other compounds is a

remote possibility. Compounds 4 and 5 differ only by an acetyl phenyl

group in compound 4 and a hydroxyl phenyl group in compound 5.

While the former was effective against all three toxins, the latter was

effective against abrin and ricin through FTSA and the efﬁcacy being

further restricted to abrin when analysed by cytotoxicity assay. Using

FTSA around 50% compounds against abrin and 11% against ricin were

Fig. 6. Effect of abrin and ricin on the inhibition of protein synthesis. Different

concentrations of abrin and ricin were mixed with the translation mix and

incubated for 90 min. Aliquot of the reactions was added to luciferin reaction

buffer and luminescence was detected. Values are calculated and compared

with control. Abrin at 1.0 ng/ml and ricin at 10 ng/ml showed 50% inhibition

in protein synthesis and these 50% inhibitory doses of abrin and ricin were

further used to study the efﬁcacy of compounds. Data are the mean ± SEM of

three independent experiments each in triplicate. *p < 0.05 compared with

toxin treated cells.

Table 3

Compounds restore the abrin or ricin induced inhibition of protein synthesis.

Abrin and ricin were incubated with compounds and then mixed with the

translation mixture and incubated for 90 min. Aliquots of the reaction were

added to lucifer in reaction buffer and luminescence was detected. Data are the

mean ± SEM of three independent experiments each in triplicate. *p < 0.05

compared with toxin treated cells and •p < 0.05 compared with toxin treated

cells.

Abrin

Compound

number

%

Ricin

Compound

number

%

Translation

–

100 ± 0

–

100 ± 0

+

–

52 ± 2*

+

–

51 ± 6.2*

4

70 ± 4^•*

65 ± 2.3^•*

67 ± 5.4^•*

67 ± 5.9^•*

67 ± 6.7^•*

4

–

91 ± 6.3^•

5

–

15

25

28

+

15

–

79 ± 2.4^•*

–

Table 4

In vivo efﬁcacy evaluation of shortlisted compounds against abrin and ricin

toxicity. Compounds (20 mM) and toxins (5XLD₅₀) both were administered

intraperitoneally. Primary screening was done using three approaches i.e. pre-

mixed: toxin and compound were preincubated for 1 h then administered in to

mice; Pre-treatment: compound was given 1 h before toxin administration; Post-

treatment: compound was given 1 h after toxin treatment. Survival of mice was

recorded and compared with toxin treated group. (A) Survival against abrin

toxicity and (B) survival against ricin toxicity.

Abrin (ip; 5XLD₅₀) and compounds (ip; 20 mM)

Compound No.

Treatment (1 h) (mean survival in days & percent survival after 14

days)

Pre-mixed

Pre-treatment

Post-treatment

(A)

Abrin

4

1.0 ± 0.0 (0%)

8.6 ± 5.7 (50%)^•

6.8 ± 5.7 (33%)

5.9 ± 5.8 (31%)

3.6 ± 3.5(09%)

2.6 ± 1.6 (0%)

1.0 ± 0.0 (0%)

2.3 ± 0.5 (0%)

2.5 ± 0.5 (0%)

2.0 ± 0 (0%)

1 ± 0.0 (0%)

6.2 ± 5.7 (33%)

2.7 ± 1.3 (0%)

5.0 ± 5.7 (25%)

3.3 ± 2.6 (0%)

2.4 ± 1.7 (0%)

5

15

25

28

2.0 ± 0.8 (0%)

1.4 ± 0.5 (0%)

Ricin (ip; 5XLD₅₀) and compounds (ip; 20 mM)

Compound No.

Treatment (mean survival in days & percent survival after 14 days)

Pre-mixed

Pre-treatment

Post-treatment

(B)

Ricin

4

1.0 ± 0.0 (0%)

5.9 ± 4.7 (22%)

5.3 ± 3.6 (13%)

1.0 ± 0.0 (0%)

2.5 ± 0.5 (0%)

2.0 ± 1.1 (0%)

1 ± 0.0 (0%)

6.7 ± 5.1 (30%)

4.3 ± 4.8 (17%)

15

^•PI-Protection Index-5.

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Biomedicine & Pharmacotherapy 143 (2021) 112134

Fig. 7. Compound 4 protects against abrin and ricin toxicity. Comparison of survival curves of mice that were treated with compound 4 and then exposed to abrin or

ricin. (A) Abrin treated mice received a single dose of abrin 5XLD₅₀intraperitoneally 1 h prior to compound 4 (20 mM; intraperitoneally) administration. Repeated

dose of the compound was administered at 1 h, 4 h and 8 h. Control animals received the vehicle only and observed for survival; (B) mice were treated with abrin

2XLD₅₀orally followed by repetitive dosing of compound 4 (20 mM; intraperitoneally) at 1 h, 4 h and 8 h and survival was noted; (C) ricin treated mice received a

single dose of abrin 5XLD₅₀intraperitoneally 1 h prior to compound 4 (20 mM; intraperitoneally) administration. Repeated dose of compound was administered at 1,

4 and 8 h; (D) mice were treated with ricin 5XLD₅₀orally followed by repetitive dosing of compound 4 (20 mM; intraperitoneally) at 1, 4 and 8 h and survival

was recorded.

preincubation reduced the ricin quantity available for binding to cell

Table 5

surface receptors, leading to signiﬁcant decreases in the inhibition of

Abrin or ricin (5XLD₅₀; i.p.) treated mice received a single or repeated dose of

protein synthesis by ricin [21]. SPR is highly reliable label free sensitive

compound 4 at 1, 4 and 8 h (20 mM) via oral route. Oral administration of

technique to quantify the binding interaction between compounds and

compound 4 did not offer protection against even repetitive dosing of compound

toxins. The equilibrium constant (K_D) for compounds against abrin was

estimated to be in a range of 1.15 × 10^{ꢀ 7}to 4.64 × 10^{ꢀ 6}M and that

against ricin was found to be between 4.5 × 10^{ꢀ 7}to 1.4 × 10⁰M.

Among all effective compounds, compound 4 showed least K_Dvalues

against both abrin (1.15 × 10^{ꢀ 7}M) and ricin (4.59 × 10^{ꢀ 7}M) sugges-

tive of efﬁcient binding with these toxins. Both, SPR and IVTS assay

advocate the strong binding of compound 4 with the toxins and its

ability to avert the toxin mediated inhibition of protein synthesis.

Further, docking studies also showed the formation of hydrogen bond

between the amide group in compounds 4 and 15 with Tyr113 of abrin

4 did not add beneﬁcial effects compared to toxin alone group.

Ricin (ip; 5XLD₅₀) and compounds (ip; 20 mM)

Compound No.

Treatment (mean survival in days & percent survival after 14 days)

Pre-mixed

Pre-treatment

Post-treatment

Ricin

4

1.0 ± 0.0 (0%)

5.9 ± 4.7 (22%)

5.3 ± 3.6 (13%)

1.0 ± 0.0 (0%)

2.5 ± 0.5 (0%)

2.0 ± 1.1 (0%)

1 ± 0.0 (0%)

6.7 ± 5.1 (30%)

4.3 ± 4.8 (17%)

15

shortlisted. In in vitro assay, compounds were further narrowed down to

14% against abrin and 6% against ricin. Although number of shortlisted

compounds were more in FTS assay but there were 4 compounds (4, 5,

15, 25) were found to be common against both abrin and ricin. To verify,

whether our ﬁndings are restricted to only FTSA and cell culture-based

assays, the compounds offering protection were further subjected to

IVTS assay and SPR analysis. IVTS is a cell-free translation system and

can reproduce physiological cytoplasmic environment for protein syn-

thesis. Considering the protein synthesis inhibition mechanism of abrin

and ricin and their reversal by SMIs, IVTS assay is an attractive tool for

functional screening of compounds. All the shortlisted compounds

effective against abrin and ricin signiﬁcantly reversed the protein syn-

thesis inhibition caused by toxin. Compound 4 was most efﬁcient and

was seen to signiﬁcantly restore the protein synthesis suggestive of

blockage of enzymatic activity of toxins due to high binding afﬁnity of

compounds. Thus, data obtained by IVTS strongly support the fact that

the compounds are able to restore protein synthesis. Further, our IVTS

approach is in agreement with previous study where lactose and ricin

and π-π bond with Tyr74 of abrin. Though a π-π bond was seen with

Trp211 of ricin with compound 4 but not with compound 15, which

appeared to dock at a pocket near the active site in its closed confor-

mation. Compound 5 against abrin showed no hydrogen or π-π inter-

action thus may possess weak interaction as also observed in SPR

analysis. Compound 5 against ricin formed one hydrogen bond but was

not found to be effective in our screening procedure. Similarly, com-

pound 25 against abrin showed only hydrogen bond with Val75 and no

π-π bond. While in case of ricin, no such bonds were observed which may

be one of the reasons for weak or no interaction between compound and

ricin and thus offered no protection in cytotoxicity assays. Compound 28

showed hydrogen bond with Tyr113 and a π-π bond with Tyr74 which

suggests comparatively strong interaction and thus provides protection

against abrin in cellular model. In spite of similar in silico interactions of

compound 4 and 28 to abrin, compound 4 was more effective, sug-

gesting conformational differences and other electrostatic interactions

are equally important in efﬁcacy determination. This is further backed

17

P. Phatak et al.

Biomedicine & Pharmacotherapy 143 (2021) 112134

by the inability of compound 28 to reverse the ricin induced toxicity

repetitive dosing of compound compensated the loss occurred due to

metabolic destruction of compound and thus enhance the protection

(Fig. 9).

despite hydrogen bond formation and π-π interaction (Fig. 8).

Based on various screening and validation approaches, the number of

compounds was reduced from 35 to 5 (compounds 4, 5, 15, 25 and 28)

for abrin and 2(compounds 4 and 15) for ricin for in vivo studies. Thus,

reducing the number of animals in compliance with the concept of 3R

(Replacement, Reduction and Reﬁnement). The possible routes of

exposure for both toxins include respiratory (inhaled aerosol), gastro-

intestinal (ingested), and parenteral (injected). In the present study, the

oral and parenteral routes were used for both toxins and all shortlisted

compounds, using three schemes of treatment viz. pre-treatment, pre-

mixed and post-treatment in mice. The dose of both toxins for animal

exposure was ﬁxed at 5XLD₅₀. LD₅₀dose of abrin and ricin estimated in

present study is either in line or lower than previously reported else-

where [22,23]. In present study, abrin was found 10 folds more toxic

than ricin via i.p. route but, 3 folds less toxic than ricin via oral route

with an estimated value of 36.7 mg/kg. Interestingly, pre-treatment

with short listed compounds did not offer protection against both

toxins, which may attribute to the biological degradation of compounds

before encountering the toxin. Increased survival rate in the premixed

group conﬁrms the efﬁcient binding of compounds with the toxin and its

consequent neutralization. Compound 4 exhibited maximum neutral-

izing activity with a PI value of 5. Limitations in the solubility of the

compounds barred the calculation of higher PI values. Solubility of the

compounds was one of the biggest limitations of the study. It is proposed

that increasing the solubility of compounds without altering its prop-

erties might enhance their potency. Post-treatment strategy mimicking

warfare scenario is the most relevant approach for developing a thera-

peutic drug. Signiﬁcant extension of the survival time of mice following

exposure to the toxins by compound 4 suggested an increase in the dose

with the quest to further increase the longevity of the animals. Solubility

restrictions could be overcome by repetitive dosing. Since compound 4

exhibited a signiﬁcant edge over the other compounds in extending the

life span of exposed animals, subsequent post-treatment studies focused

on compound 4 as the SMI.

To the best of our knowledge, thus far no treatment regime has been

reported to protect mice at lethal doses of RIPs. The analysis of animal

data, in this study, reveals protection against lethal doses of toxins by

compound 4. A similar study involves the in silico digging of a peptide

fragment to block the binding of A chain catalytic site to the ribosomal P

stalk protein of host cells. These peptides, mimicking the c-termini of

ribosomal P protein, have been shown to hinder ricin A chain and ri-

bosomal binding. The ability of a peptide to inhibit ricin activity holds

hope for the development of antidotes against RIPs [27]. Retro-1 and

Retro-2 compounds have also been tested for their anti-RIP activity.

Both compounds were designed to target retrograde pathway of toxin

trafﬁcking rather toxin itself. Though these compounds held promise

due to their protective effect on mice challenged with lethal doses of

ricin via the intranasal route, the test was performed as a prophylactic

measure [9]. In an in vivo study involving the therapeutic potential of

Retro 2 derivatives, though one of the derivatives increased the

longevity of mice and none of them restored the normal life span of the

animals [28]. Furthermore, DA2MT an achiral analog of retro 2 de-

rivatives also showed cell protection against ricin toxicity [29]. The

strong electrostatic interactions at the RIPs-ribosomal subunit interface

offering resistance to the r RNA-candidate inhibitor interaction makes

small-molecule RIP therapeutics challenging [30]. The present study

supports the concept that targeting the enzymatic site of the toxin with

small molecules is the right approach to reverse the RIP toxicity.

Moreover, the identiﬁcation of a common inhibitor against three RIPs

brightens the prospects for the development of a single drug to target

three toxins.

Though pharmacokinetic and toxicokinetic studies will give us a

clearer picture of these compounds, the speciﬁc inhibition of toxin

enzyme activity predicts a targeted effect of these compounds. Further,

one notable issue with these compounds was their solubility. The com-

plete safety of these compounds even in repeated dose regimes gives

room for dose enhancement. Moreover, a computational based approach

may help in the identiﬁcation of solubility, and hence, efﬁcacy

enhancing functional groups. The use of antioxidants, anti-inﬂammatory

agents and cytoprotectants, neutralizing antibodies [31] along with

compounds 4 and 5 as a combinatorial treatment regime to counter the

known oxidative stress, inﬂammation, apoptosis inducing effects [32,

33] of these toxins is suggested. This may further enhance the protection

against abrin, ricin and Stx toxicities via either synergistic or additive

effects.

Just a modest improvement in the survival rate by repetitive dosing

of compounds advocates the need for a single high dose. This would

prevent the possibly faster manifestation of the toxic effects in the liver

and other organs via oxidative stress and related signaling pathways

(Fig. 9) reported to manifest in barely thirty minutes [24]. Decreasing

the time interval of repetitive dosing along with the inclusion of anti-

oxidants in treatment regiments could offer complete neutralization of

the toxin. In present study, the failure of oral dosing to provide pro-

tection against toxicity administered via i.p.route could be due to the

complete degradation of the compound via its digestive enzymes and

making them unavailable for toxin neutralization. Considering the

possibility of spiking of food sample and beverages with toxins, we have

also included oral route of toxin administration. Oral exposure of

supralethal doses of ricin in mice was completely reversed by compound

4 with PI Value 5. The same compound also protected all mice against

abrin toxicity at 2XLD₅₀when orally administered orally. The efﬁcacy of

the compound beyond 2XLD₅₀dose of abrin could not be determined

due to non feasibility of administration of a high dose in mice, the oral

LD₅₀being 36.7 mg/kg. Literature search also revealed the paucity of

data of oral toxicity studies of these toxins. Available reports estimated

2.3 mg/kg abrin toxicity via oral route with 1000–10, 000 less toxicity

than the injectable routes [25]. Wide variation in these two ﬁndings may

owe to variability in mode of forced feeding which may invoke a

different toxicity outcome as compared to an actual scenario of human

poisoning involving consumption of toxin-contaminated food samples

[26]. Oral administration of toxins followed by i.p. administration of

compound offered the best protection. Probable reason of protection

conferred by this combination is that toxin and compound both followed

the different path with different rates, encountered successfully some-

where in circulatory system. Well timed interaction between compound

and toxin was able to neutralize the most of the toxicity. Further,

The modiﬁcation of the drug delivery method and the inclusion of a

combination of compounds targeting different pathways of ricin toxi-

cosis in the treatment regime, could act as an efﬁcacy booster. The de-

livery of NAC, to target cells poisoned by ricin A-chain, by Liposome-

encapsulated N-acetylcysteine (Lipo-NAC) has recently been reported

[34]. The delivery of liposome encapsulated SMI along with antioxi-

dants is likely to enhance their delivery to toxin affected target cells.

Further studies aimed at reversing the abrin, ricin, stx induced lethality

involving optimization of the chemistry of SMIs, targeted drug delivery

approach along with antioxidants and cytoprotectants are

recommended.

CRediT authorship contribution statement

Nandita Saxena: Conceptualization, experiment performed, data

analysis, Writing – original draft. Ram Kumar Dhaked: Conceptuali-

zation, experiment performed, data analysis, Writing – review & editing.

Uma Pathak: synthesis and characterization the compounds. Pooja

Phatak: Experiment performed. Vinita Chauhan: Experiment per-

formed. All authors read and approved the ﬁnal version of manuscript.

18

P. Phatak et al.

Biomedicine & Pharmacotherapy 143 (2021) 112134

Fig. 8. Representative images are showing docked conformations of shortlisted compounds in the active site of toxins. (A) Active site of abrin (pdb ID: 1ABR); (B)

active site of ricin (pdb ID: 2AAI).

19

P. Phatak et al.

Biomedicine & Pharmacotherapy 143 (2021) 112134

Fig. 9. Possible mechanism of compound 4 against abrin and ricin toxicity in Balb/c mice. Compound and toxins both administered intraperitoneally; (A) pre-mixed

treatment: pre-incubation of toxins and compound offered signiﬁcant protection due to neutralization of most of the toxin molecules outside the body resultant of

efﬁcient binding with compound; (B) pre-treatment: pre-treatment of compound could not protect mice from toxin induced lethality as compound may metabolized

in circulatory system before it counteract with toxin; (C) post-treatment-repetitive dosing of compound, post toxin exposure signiﬁcantly extended the survival time,

compared to toxin treated mice. Toxin after reaching to the circulatory system, liver and other organ, initiate toxicity. In post-treatment also, most of the compounds

may got degraded but, some of the compound could able to reach the toxin to neutralize the toxicity. Further, repetitive dosing also increases the availability of

compounds, thus toxin neutralization. (D) Compound (i.p.) vs toxin (oral); post-treatment of compound protects from oral toxicity as toxin and compound both

followed the different path with different rate but encounter at the same site and neutralization occur before toxin could establish its toxicity. Further, repetitive

dosing of compound compensates the loss occurred due to metabolic destruction of compound and thus enhances the protection.

[6] Y.H. Han, S. Gao, W.W. Xin, L. Kang, J.L. Wang, A recombinant mutant abrin A

Conﬂict of interest statement

chain expressed in Escherichia coli can be used as an effective vaccine candidate,

Hum. Vaccines 7 (2011) 838–844, https://doi.org/10.4161/hv.7.8.16258. Epub

None.

2011 Aug 1. PMID: 21817853.

[7] C.A. McHugh, R.F. Tammariello, C.B. Millard, J.H. Carra, Improved stability of a

protein vaccine through elimination of a partially unfolded state, Protein Sci. 13

(2004) 2736–2743, https://doi.org/10.1110/ps.04897904. Epub 2004 Aug 31.

PMID: 15340172; PMCID: PMC2286567.

Acknowledgments

[8] D.E. McLain, T.L. Horn, C.J. Detrisac, C.Y. Lindsey, L.A. Smith, Progress in

biological threat agent vaccine development: a repeat-dose toxicity study of a

recombinant ricin toxin A-chain (rRTA) 1-33/44-198 vaccine (RVEc) in male and

female New Zealand white rabbits, Int. J. Toxicol. 30 (2011) 143–152, https://doi.

org/10.1177/1091581810396730. Epub 2011 Mar 4.

We thank Dr. DK Dubey, Director, DRDE, for providing facilities and

support required for the study. Dr. Vinita Chauhan is a RA working in

CSIR research scheme. Dr Pooja Phatak is thankful to Defence Research

and Development Organisation for the award of Senior Research

Fellowship. DRDE accession no. of manuscript is DRDE/P&T/51/2020.

[9] B. Stechmann, S.K. Bai, E. Gobbo, R. Lopez, G. Merer, S. Pinchard, L. Panigai,

D. Tenza, G. Raposo, B. Beaumelle, D. Sauvaire, D. Gillet, L. Johannes, J. Barbier,

Inhibition of retrograde transport protects mice from lethal ricin challenge, Cell

141 (2010) 231–242, https://doi.org/10.1016/j.cell.2010.01.043.

[10] J.M. Lord, L.M. Roberts, J.D. Robertus, Ricin: structure, mode of action, and some

current applications, FASEB J. 8 (1994) 201 –208 .

References

[1] H.Y. Chen, L.Y. Foo, W.K. Loke, Abrin and ricin: understanding their toxicity,

diagnosis, and treatment, in: P. Gopalakrishnakone, M. Balali-Mood, L. Llewellyn,

B. Singh (Eds.), Biological Toxins and Bioterrorism. Toxinology, Springer,

Dordrecht, 2015, pp. 1–20, https://doi.org/10.1007/978-94-007-6645-7_1-1.

[2] J. Schrot, A. Weng, M.F. Melzig, Ribosome-inactivating and related proteins,

Toxins 7 (2015) 1556–1615, https://doi.org/10.3390/toxins7051556 (Basel).

[11] D.R. Franz, N.K. Jaax, Chapter 32 ricin toxin, in: F. Sidell, T. Takafuji, D. Franz

(Eds.), Medical Aspects of Chemical and Biological Warfare, Ofﬁce of The Surgeon

General, TMM Publications, Washington, DC, 1997 .

[12] J. Audi, M. Belson, M. Patel, J. Schier, J. Osterloh, Ricin poisoning: a

comprehensive review, JAMA 294 (2005) 2342–2351, https://doi.org/10.1001/

jama.294.18.2342.

´

[3] P. Jimenez, J. Tejero, D. Cordoba-Diaz, E.J. Quinto, M. Garrosa, M.J. Gayoso,

[13] P.G. Wahome, Y. Bai, L.M. Neal, J.D. Robertus, N.J. Mantis, Identiﬁcation of small-

molecule inhibitors of ricin and shiga toxin using a cell-based high-throughput

screen, Toxicon 56 (2010) 313–323, https://doi.org/10.1016/j.

´

T. Girbes, Ebulin from dwarf elder (Sambucus ebulus L.): a mini-review, Toxins 7

(2015) 648–658, https://doi.org/10.3390/toxins7030648 (Basel).

[4] J.E. Smallshaw, J.A. Richardson, E.S. Vitetta, RiVax, a recombinant ricin subunit

vaccine, protects mice against ricin delivered by gavage or aerosol, Vaccine 25

(2007) 7459–7469, https://doi.org/10.1016/j.vaccine.2007.08.018. PMID:

17875350; PMCID: PMC2049008.

toxicon.2010.03.016. Epub 2010 Mar 27. PMID: 20350563; PMCID: PMC2902711.

[14] V. Chauhan, D. Chaudhary, U. Pathak, N. Saxena, R.K. Dhaked, In silico discovery

and validation of amide based small molecule targeting the enzymatic site of shiga

toxin, J. Med. Chem. 59 (2016) 10763–10773, https://doi.org/10.1021/acs.

jmedchem.6b01517. Epub 2016 Nov 29.

[5] P.V. Lemley, B.S. Thalley, D.C. Stafford, Prophylactic and therapeutic efﬁcacy of an

avian antitoxin in ricin intoxication, Ther. Immunol. 2 (1995) 59 –66 .

20

P. Phatak et al.

Biomedicine & Pharmacotherapy 143 (2021) 112134

[15] O. Kumar, A. Kannoji, R. Jayaraj, R. Vijayaraghavan, Puriﬁcation and

characterization of abrin toxin from white Abrus precatorius seeds, J. Cell Tissue

Res. 8 (2008) 1243 –1248 .

[26] C.J. Roy, K. Song, S.K. Sivasubramani, D.J. Gardner, S.H. Pincus, Animal models of

ricin toxicosis, Curr. Top. Microbiol. Immunol. 357 (2012) 243–257, https://doi.

org/10.1007/82_2011_173. PMID: 21956160; PMCID: PMC4302748.

[27] X.P. Li, R.K. Harijan, J.N. Kahn, V.L. Schramm, N.E. Tumer, Small molecule

inhibitors targeting the interaction of ricin toxin A subunit with ribosomes, ACS

Infect. Dis. 6 (2020) 1894–1905, https://doi.org/10.1021/acsinfecdis.0c00127.

Epub 2020 Jun 8. PMID: 32428396; PMCID: PMC7678679.

[16] M.C. Lo, A. Aulabaugh, G. Jin, R. Cowling, J. Bard, M. Malamas, G. Ellestad,

Evaluation of ﬂuorescence-based thermal shift assays for hit identiﬁcation in drug

discovery, Anal. Biochem. 332 (2004) 153–159, https://doi.org/10.1016/j.

ab.2004.04.031.

[17] M.L. Hale, Microtiter-based assay for evaluating the biological activity of

ribosome-inactivating proteins, Pharmacol. Toxicol. 88 (2001) 255 –260 .

[18] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,

T.E. Ferrin, UCSF Chimera–a visualization system for exploratory research and

analysis, J. Comput. Chem. 25 (2004) 1605–1612, https://doi.org/10.1002/

jcc.20084.

[28] X. Zhao, H. Li, J. Li, K. Liu, B. Wang, Y. Wang, X. Li, W. Zhong, Novel small

molecule retrograde transport blocker confers post-exposure protection against

ricin intoxication, Acta Pharm. Sin. B 10 (2020) 498–511, https://doi.org/

10.1016/j.apsb.2019.08.005. Epub 2019 Aug 22. PMID: 32140395; PMCID:

PMC7049615.

[29] S. Yu, J.G. Park, J.N. Kahn, N.E. Tumer, Y.P. Pang, Common pharmacophore of

structurally distinct small-molecule inhibitors of intracellular retrograde

trafﬁcking of ribosome inactivating proteins, Sci. Rep. 3 (2013) 3397, https://doi.

org/10.1038/srep03397. PMID: 24292269; PMCID: PMC3844963.

[19] M.W. Pantoliano, E.C. Petrella, J.D. Kwasnoski, V.S. Lobanov, J. Myslik, E. Graf,

T. Carver, E. Asel, B.A. Springer, P. Lane, F.R. Salemme, High-density miniaturized

thermal shift assays as a general strategy for drug discovery, J. Biomol. Screen. 6

(2001) 429–440, https://doi.org/10.1177/108705710100600609.

[20] N.E. Tumer, X.P. Li, Interaction of ricin and shiga toxins with ribosomes, Curr. Top.

Microbiol. Immunol. 357 (2012) 1–18, https://doi.org/10.1007/82_2011_174.

PMID: 21910078; PMCID: PMC4103753.

[30] Y.P. Pang, J.G. Park, S. Wang, A. Vummenthala, R.K. Mishra, J.E. McLaughlin,

R. Di, J.N. Kahn, N.E. Tumer, L. Janosi, J. Davis, C.B. Millard, Small-molecule

inhibitor leads of ribosome-inactivating proteins developed using the doorstop

approach, PLOS One 6 (2011), 17883, https://doi.org/10.1371/journal.

pone.0017883. PMID: 21455295; PMCID: PMC3063779.

[21] S.E. Lumor, B.D. Deen, I. Ronningen, K. Smith, N.R. Fredrickson, F. Diez-Gonzalez,

T.P. Labuza, Assessment of the inhibition of ricin toxicity by lactose in milk,

J. Food Prot. 76 (2013) 2037–2039, https://doi.org/10.4315/0362-028X.JFP-13-

091.

[31] M.L. Orsini Delgado, A. Avril, J. Prigent, J. Dano, A. Rouaix, S. Worbs, B.G. Dorner,

C. Rougeaux, F. Becher, F. Fenaille, S. Livet, H. Volland, J.N. Tournier, S. Simon,

Ricin antibodies’ neutralizing capacity against different ricin isoforms and

cultivars, Toxins (2021) 100, https://doi.org/10.3390/toxins13020100 (Basel).

[32] P. Phatak, D.P. Nagar, N. Saxena, Dose dependent acute toxicity of abrin in Balb/c

mice after intraperitoneal administration, Toxicon 167 (2019) 49–59, https://doi.

org/10.1016/j.toxicon.2019.06.008. Epub 2019 Jun 8.

[22] J.Y. Lin, C.C. Chen, L.T. Lin, T.C. Tung, Studies on the toxic action of abrin, Taiwan

Yi Xue Hui Za Zhi 68 (1969) 322 –324 .

[23] K. Chaturvedi, S.E. Jadhav, Y.D. Bhutia, O. Kumar, R.K. Kaul, N. Shrivastava,

Puriﬁcation and dose-dependent toxicity study of abrin in swiss albino male mice,

Cell. Mol. Biol. 61 (2015) 36 –44 (Noisy-le-grand).

[33] N. Saxena, P. Yadav, O. Kumar, The Fas/Fas ligand apoptotic pathway is involved

in abrin-induced apoptosis, Toxicol. Sci. 135 (2013) 103–118, https://doi.org/

10.1093/toxsci/kft139. Epub 2013 Jun 20.

[24] N. Saxena, P.V. Rao, A.S. Bhaskar, Y.D. Bhutia, Protective effects of certain

pharmaceutical compounds against abrin induced cell death in Jurkat cell line, Int.

Immunopharmacol. 21 (2014) 412–425, https://doi.org/10.1016/j.

intimp.2014.05.028. Epub 2014 Jun 2.

[34] C. Buonocore, M. Alipour, A. Omri, K. Pucaj, M.G. Smith, Z.E. Suntres, Treatment

of ricin A-chain-induced hepatotoxicity with liposome-encapsulated N-

acetylcysteine, J. Drug Target. 19 (2011) 821–829, https://doi.org/10.3109/

1061186X.2011.582645. Epub 2011 May 26.

[25] E.A. Garber, Toxicity and detection of ricin and abrin in beverages, J. Food Prot. 71

(2008) 1875–1883, https://doi.org/10.4315/0362-028x-71.9.1875.

21

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E-N-(2-acetyl-phenyl)-3-phenyl-acrylamide targets abrin and ricin toxicity: Hitting two toxins with one stone

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