4. RESULTS 4.1 Cloning of stxA gene 4.1.1 Genomic DNA isolation from Shigella dysenteriae Total genomic DNA was isolated from the overnight grown S. dysenteriae type 1 culture obtained from NICED, Kolkata. Genomic DNA was used as a template for amplification of full length StxA subunit gene (Fig. 1). PCR yielded an amplification of expected size of 955 bp. 4.1.2 Recombinant plasmid construction and sequencing of StxA Primers were designed, PCR was performed and amplicon of expected size (955 bp for full length stxA gene) was obtained (Fig. 2). The product was cloned into pQE30UA vector, where in three attempts clones obtained were found to be in opposite orientation. Second set of primers were designed to amplify stxA gene with Nde/BamH1 restriction endonuclease sites and PCR was performed to amplify stxA gene. After digestion the purified product was cloned into pUC18 cloning vector at Nde/BamH1 restriction endonuclease sites. DNA sequencing confirmed the presence of full length gene in both pQE30UA expression and pUC18 cloning vectors. The pUC18 construct was digested and released purified insert was subsequently cloned in pET28a vector at Nde/BamH1 restriction endonuclease sites. The recombinant pET28a construct was transformed into E. coli BL21(DE3) and positive clones were selected on the bases of releases of insert and specific restriction digestion. Upon induction with 1mM IPTG at 37°C for different time period there was no expression of recombinant protein was seen on 10% SDS-PAGE (data not shown). Finally, new set of primers were designed with the insertion BamH1//EcoR1 restriction sites. Position of the StxA forward primer is located in the gene at -18 to 3 with BamH1 RE site. The position of the StxA reverse primer is located in the gene at 952-935 with EcoR1 RE site. The purified PCR product was ligated into the pGEX5X-2 vector at BamH1/EcoR1 restriction sites (Fig. 2). The recombinant vector pGEX5X-2-StxA was introduced into the competent E. coli BL21(DE3) cells using electroporation method. This construct was predicted to express recombinant protein
1
with a molecular weight of ~58 kDa.
2
3
1
2
3
4
rStxA 955 bp
Fig. 3 : Screening of rStxA clones by colony PCR Lane 1: 100 bp DNA ladder Lanes 2, 3, 4: PCR product of size 955 bp
1
2
Fig. 4 : Plasmid of rStxA Lane 1: 100 bp DNA ladder Lane 2: Plasmid of rStxA
4.1.3 Nucleotide sequencing of rStxA The nucleotide sequences of the cloned PCR products were determined (data not provided) and compared with other known sequences from the databases using BLASTn software for verification. DNA sequencing results confirmed the successful cloning of rStxA. The nucleotide sequence thus obtained has been submitted to GenBank, the public sequence database of National Centre for Biological Information (NCBI), National Institute of Health, USA and was assigned the accession number: HM017965 (Fig. 5).
4
Shigella dysenteriae Shiga toxin subunit A (stxA) gene, partial cds GenBank: HM017965.1 Feature Sequence LOCUS HM017965 955 bp DNA linear BCT 15-MAY-2010 DEFINITION Shigella dysenteriae Shiga toxin subunit A (stxA) gene, partial cds. ACCESSION HM017965 VERSION HM017965.1 GI:295882026 KEYWORDS . SOURCE Shigella dysenteriae ORGANISM Shigella dysenteriae Bacteria; Proteobacteria; Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae; Shigella. REFERENCE 1 (bases 1 to 955) AUTHORS Singh,P., Singh,M.K. and Dhaked,R.K. TITLE In vitro reconstruction of active Shiga toxin from recombinant A and B subunits JOURNAL Unpublished REFERENCE 2 (bases 1 to 955) AUTHORS Dhaked,R.K., Singh,P. and Singh,M.K. TITLE Direct Submission JOURNAL Submitted (18-MAR-2010) Biotechnology, DRDE, Jhansi Road, Gwalior, MP 474002, India FEATURES Location/Qualifiers source 1..955 /organism="Shigella dysenteriae" /mol_type="genomic DNA" /db_xref="taxon:622" gene 1..>955 /gene="stxA" CDS 1..>955 /gene="stxA" /codon_start=1 /transl_table=11 /product="Shiga toxin subunit A" /protein_id="ADG56725.1" /db_xref="GI:295882027" /translation="MKIIIFRVLTFFFVIFSVNVVAKEFTLDFSTAKTYVDSLNVIRS AIGTPLQTISSGGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTG FVNRTNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLTTS LDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSGRSYVMTAED VDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALILNCHHHASRVARMAS DEFPSMCPADGRVRGITHNKILWDSSTLGAILMRRTISSACV" ORIGIN 1 atgaaaataa ttatttttag agtgctaact tttttctttg ttatcttttc agttaatgtg 61 gttgcgaagg aatttacctt agacttctcg actgcaaaga cgtatgtaga ttcgctgaat 121 gtcattcgct ctgcaatagg tactccatta cagactattt catcaggagg tacgtcttta 181 ctgatgattg atagtggcac aggggataat ttgtttgcag ttgatgtcag agggatagat 241 ccagaggaag ggcggtttaa taatctacgg cttattgttg aacgaaataa tttatatgtg 301 acaggatttg ttaacaggac aaataatgtt ttttatcgct ttgctgattt ttcacatgtt 361 acctttccag gtacaacagc ggttacattg tctggtgaca gtagctatac cacgttacag 421 cgtgttgcag ggatcagtcg tacggggatg cagataaatc gccattcgtt gactacttct 481 tatctggatt taatgtcgca tagtggaacc tcactgacgc agtctgtggc aagagcgatg 541 ttacggtttg ttactgtgac agctgaagct ttacgttttc ggcaaataca gaggggattt 601 cgtacaacac tggatgatct cagtgggcgt tcttatgtaa tgactgctga agatgttgat 661 cttacattga actggggaag gttgagtagt gtcctgcctg actatcatgg acaagactct 721 gttcgtgtag gaagaatttc ttttggaagc attaatgcaa ttctgggaag cgtggcatta 781 atactgaatt gtcatcatca tgcatcgcga gttgccagaa tggcatctga tgagtttcct 841 tctatgtgtc cggcagatgg aagagtccgt gggattacgc acaataaaat attgtgggat 901 tcatccactc tgggggcaat tctgatgcgc agaactatta gcagtgcatg cgtat
Fig. 5: DNA sequence of rStxA (955 bp) submitted to NCBI (Accn. No. HM017965)
4.2 Expression 4.2.1 Expression and localization of rStxA To obtain the protein products of rstxA gene for further characterization, expression of cloned gene was attempted. Small scale cultures of the positive clones (selected on the basis of PCR screening) were subjected to IPTG induction to identify clones capable of expressing the predicted ~58 kDa recombinant protein. The protein expression was checked at different concentration of IPTG (0.5 mM, 0.75 mM, 1.0 mM, 1.5 mM and 2.0 mM) and 1 mM was found to show the best expression (Fig. 7). After induction with different concentration, the induced culture was allowed to grow for different time periods i.e. 1h, 2h, 3h, 4h to12 h to obtain protein expression at 37°C (Fig. 6 and Fig. 8). The expression conditions were also optimized with respect to different media i.e LB, TB, SB and NB. Amongst these media, the best expression of the recombinant 5
protein was shown in LB media and thus, was used for all the further experiments (Fig.9). The expression of recombinant protein was found to be maximum at 37°C after 3 h of induction with 1 mM IPTG using BL21(DE3) expression host. The size of the recombinant proteins was found to be 58 kDa. No over-expression was seen in the uninduced cells. To devise an appropriate purification strategy, the relative distribution of the expressed recombinant protein in the soluble and insoluble fractions was examined. For this purpose, presence of protein in supernatant as well as pellet after sonication and centrifugation was checked (Fig. 10). The expression of the fusion protein was found to be in the form of inclusion bodies. Western blot developed with anti-GST antibody of pellet and supernatants obtained after sonication further confirms the localization of rStxA protein in the form of inclusion bodies (Fig. 11).
kDa 97.0
1
2
3
4 5
6
7 8
9 10 11 12 13 14
66.0
45.0 30.0 25.0
A
1
2
3
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5
6 7
8
9 10 11 12 13
kDa 97.0 66.0
45.0 30.0 25.0 B Fig. 6 :Optimization of time dependent expression profile of rStxA protein in BL21DE3 at 37 ˚C (A) Lane 1: Protein molecular weight marker (kDa) Lanes 2, 3, 5, 7, 9, 11, 13: Uninduced cell pellet harvested at 0, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h respectively Lanes 4, 6, 8, 10, 12, 14: Induced cell pellet harvested at 1 h, 2 h, 3 h, 4 h, 5 h, 6 h respectively (B) Lane 1: Protein molecular weight marker (kDa) Lanes 2, 4, 6, 8, 10, 12 : Uninduced cell pellet harvested at 7 h, 8 h, 9 h, 10 h, 11 h, 12 h respectively Lanes 3, 5, 7, 9, 11, 13: Induced pellet harvested at 7 h, 8 h, 9 h, 10 h 11 h, 12 h respectively
6
1
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9
kDa 97.0 66.0 45.0 30.0 A 25.0
kDa
1
2
3
4
5
6
7
8
9
97.0 66.0
45.0
30.0
25.0
B
Fig. 8 : Optimization of temperature dependent expression profile of rStxA protein in BL21DE3 at 20˚C, 25˚C, 30˚C, 37˚C (A) Lane 1 : Protein molecular weight marker Lanes 2, 3, 4, 5: Induced cell pellet after 2 h at 20˚C, 25˚C, 30˚C, 37˚C respectively Lanes 6,7, 8, 9: Induced cell pellet after 3 h at 20˚C, 25˚C, 30˚C, 37˚C respectively (B) Lane 1 : Protein molecular weight marker Lanes 2, 3, 4, 5: Induced cell pellet after 4 h at 20˚C, 25˚C, 30˚C, 37˚C respectively Lanes 6, 7, 8, 9: Induced cell pellet after 5 h at 20˚C, 25˚C, 30˚C, 37˚C respectively
7
1 2
3 4
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6
7 8
9 10 11 12 13 14 15
kDa 97.0 66.0
45.0 30.0 25.0 A
14.4 kDa 97.0
1
2
3
4
5
6
7
8
9
10 11
66.0 45.0
30.0 25.0 14.4
B
Fig. 9 : Optimization of media for expression of rStxA (A) Lane 1: Molecular weight marker Lanes 2, 3, 4, 5, 6, 7: Induced pellet and supernatant after 2 h, 3 h, 4 h, in LB media respectively Lanes 8, 9, 10, 11, 12, 13: Induced pellet and supernatant after 2 h, 3 h, 4 h, in 2YT media respectively Lanes 14, 15: Induced pellet and supernatant after 2 h in NB media (B) Lane 1: Molecular weight marker Lanes 2, 3, 4, 5: Induced pellet and supernatant after 3 h, 4 h in NB media respectively Lanes 6, 7, 8, 9, 10, 11: Induced pellet and supernatant after 2 h, 3 h, 4 h, in SB media respectively
4.3 Purification of rStxA Purification of the rGST-StxA protein was attempted from the isolated inclusion bodies. The urea and GuCl were used for solubilization of inclusion bodies, however no binding with glutathione resin was observed due to the denatured GST enzyme (data not shown). Finally, sarkosyl was used to solubilize inclusion bodies for purification by affinity gel column chromatography. Incubation of inclusion bodies with 10% sarkosyl containing buffer effectively solubilized 90% of protein. The sarkosyl concentration was lowered to 2% before binding with the resin (precipitation was observed at 1% sarkosyl concentration). Recombinant GST-StxA was purified using elution buffer without sarkosyl and eluents revealed the presence of a 58 kDa protein as a major band in all the eluted fractions on SDS-PAGE analysis of the as shown in (Fig. 12). Elutes were pooled together and dialyzed in PBS, after dialysis the yield of fusion protein from 1 litre bacterial culture was found to be ~5 mg.
8
1
2
3
4
5
kDa 97.0 66.0 45.0 30.0 25.0 14.4 Fig. 10: Localization of expressed rStxA protein at 37˚C Lane 1: Molecular weight marker Lane 2: Uninduced pellet Lane 3: Induced pellet Lane 4: Pellet after sonication Lane 5: Supernatant after sonication kDa 117 130
1
2
3
4
5
95 72 56 43
34 26 Fig. 11: Western blot analysis of rStxA protein with anti-GST Lane 1: Prestained protein molecular weight marker Lane 2: Uninduced cell pellet Lane 3 : Induced cell Lane 4: Pellet after sonication Lane 5: Supernatant after sonication
9
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9
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11 12 13
14
kDa 116.0
66.0
45.0
35.0
Fig. 12: Purification of the rStxA by GST-affinity column Lane 1: Molecular weight marker
Lane 2: Pellet before sonication Lane 3: Lysate for purification Lane 4: Flow through Lane 5-8: Washes Lane 9-14: Elutes with 50 mM reduced Glutathione
4.3.1 Factor Xa cleavage for removal of GST-tag from GST-StxA For purification rStxA (removal of GST tag) rGST-StxA lysate was allow to bind with glutathione resin (2 hrs) and after washing it was treated with factor Xa for 16 hours at RT as mentioned in material and methods. Protein was recovered after centrifuged at 1000 xg for 2 min in the supernatant removed carefully without disturbing the resin and factor Xa was removed from the elutes by passing through factor Xa-removal resin. The size of purified recombinant StxA protein was found to be ~32 kDa after removal of GST-tag (Fig. 13). After dialysis in PBS, the yield of rStxA from 1 litre bacterial culture was found to be ~2 mg. This protein was stable, did not precipitated during dialysis and was used for the immunization of mice and rabbit.
10
1
2
3
4
5
kDa 97.0 66.0 45.0 32 KDa
30.0
25.0 14.4 Fig. 13: Factor Xa mediated endoproteolytic cleavage of GST from GST-StxA. Lane 1: Protein molecular weight marker Lane 2: Lysate of rStxA Lane 3: Wash Lane 4: GST-bound rStxA Lane 5: Factor Xa cleaved rStxA (32 kDa) kDa 130 100 70
1
2
3
4
5
55 35 25 15
10 Fig. 14: Confirmation of Factor Xa mediated endoproteolytic cleavage of GST-tag from GST-StxA by Western Blot Lane 1: Prestained protein molecular weight marker Lane 2: UI pellet of rStxA Lane 3: Induced pellet of rStxA Lane 4: GST-bound rStxA Lane 5: Factor Xa cleaved rStxA (32 kDa)
4.4 Protein identification 4.4.1 Identification by Western blot with commercially available antibody. Western blot was developed by using commercially available anti-GST antibody at a dilution of 1:2000 revealed a single and specific band of a size of ~58 kDa on the nitrocellulose membrane (Fig. 16).
11
kDa 250 130
1
2
3
4
5
6
7
8
9
10
100 70 55
35 25
Fig. 16: Western blot analysis of rStxA protein using anti-GST antibody (1:2,000 dilution) Lane 1: Molecular weight markers Lane 2: Uninduced pellet Lane 3: Induced pellet Lane 4-10: Purified recombinant rStxA protein
4.4.2 Identification by peptide mass fingerprinting For the analysis of protein by mass spectrometry, the recombinant protein was digested with trypsin and the peptides thus generated were identified by MALDITOF/MS analysis. The spectra were subjected to MASCOT data base search at http://www.matrixscience.com. MASCOT search parameters and protein identities were as per the guidelines of Human Proteome Organization (HUPO) and protein was considered identified when the PMF search results provided either >30% sequences coverage or MS/MS spectra of two or more peptides of the given protein had significant hit to data of same protein available in the database. In the MASCOT analysis 13 peptides were matched with 50% coverage of the sequence of rStxA available in database (Fig. 15).
12
Fig. 15 : MASCOT search result of rStxA protein: Match to :gi|6730408 Score: 161 Expect: 5.2e-10 Chain A, Shiga Toxin matched peptides shown in Bold Red
4.5 Immunization 4.5.1 Production of Antisera Mice and rabbit were given intra-muscular injections. Initially, the priming of the animals was done with the lower dose which was increased in the subsequent booster doses. Animals remained healthy during the immunization period. The test bleed and subsequently, final bleed was carried out to obtain antisera from both mice as well as the rabbit. Western blot analysis using corresponding antisera at a dilution of 1:4000 revealed a single and specific band of a size of ~32 kDa (Fig. 17). It was also observed that antiserum was specifically reacting with Shiga toxin A subunit present in lysate of Shigella dysenteriae and STEC and there was no cross reactivity found with bacterial cell lysates S. aureus, E. coli and V. cholerae. No reaction was observed with the serum collected from the animals before immunization (data not shown).
13
kDa 1 55
35
2
3
4
5
Stx
25
15 10 Fig. 17: Western blot of purified rStxA and S. dysenteriae lysates using mice antisera (1:4,000). Lane 1: Prestained marker Lane 2 : Factor Xa cleaved rSxA Lane 3-5: Lysates of S. dysenteriae
kDa 250 130 100 70 55 35 25 15 10 Fig. 18: Western blot showing the titer of mice anti-rStxA sera in two-fold serial dilution. Lane 1: Prestained marker Lane 2-10: Mice antisera titer– 1:128,000
4.5.2 Antibody titration by Western blot and ELISA To estimate the titer of antisera obtained from the final bleed during immunization of mice and rabbit, both Western blot as well as indirect ELISA were carried out. In the case of mice antisera, the titer estimated by ELISA was 1:256000 and that of the rabbit antisera was 1:128000 (Fig. 19). In the Western blot the amount of rStxA antigen used was 1μg/well. Four successive immunizations of female BALB/c mice with 10 μg and fifth with 40 μg recombinant proteins induced significantly elevated levels of antibodies, the titer of mice antisera was 1:128000 (Fig. 18).
14
OD (450 nm)
4 3.5 3 2.5 2 1.5 1 0.5 0
Mice Antisera
A
Dilutions of Antisera Cut off = 0.14 Titer= 1:256000
Rabbit Antisera
B
3
OD (450 nm)
2.5 2 1.5 1 0.5 0
Dilutions of antisera Cut off = 0.12 Titer= 1:128000 Fig. 19 : Indirect ELISA showing antisera titer after final bleed A. Mice antisera titer (1:256,000) B. Rabbit antisera titer (1:128,000)
4.5.3 Isotyping and protection assay In order to determine the nature of the specific immune response generated against rStxA in mice, isotyping of IgG in the hyper immune serum was carried out. IgG1 and IgG3 antibody subtypes were found to be dominant and 4-5 fold increased amount was detected compare to control (Fig. 20). The protection was monitored by challenging animals with rStxA (5 LD/mouse) through intraperitoneal route. It was observed that mice immunized with the highest dose i.e. 40 μg of rStxA showed protection after 3rd dose, hence eliciting protective immune response.
15
2.5
OD at 450 nm
2
1.5 controls IgG sub types
1
0.5
0
IgG1
IgG2a
IgG2b
IgG3
Fig. 20: Profile of IgG subclass in sera from mice immunized with rStxA formulated with FCA/FIA. Sera samples collected on 21 st day from group I and analyzed for the presence of rStxA specific IgG1, IgG2a, IgG2b, IgG3, subclass antibodies by ELISA. Group II used as control.
4.6 Construction of recombinant protein Recombinant StxA from S. dysenteriae was produced using pGEX5X-2 vector and recombinant StxB from S. dysenteriae was produced using pQE30UA-StxB constructs under IPTG induction in E. coli (Fig. 21). Interaction between rStxA and rStxB subunits is studied by GST-pull-down assay and surface plasmon resonance.
16
kDa 97.0 66.0
kDa 45.0 GST-StxA
45.0
31.0
30.0 21.5 25.0 14.4 18.0 His6x-StxB
14.0
A
B
Figure 21: Purification of rStxA and rStxB for reconstitution of rStx A. Purified GST-StxA B. Purified His6x-StxB
4.6.1 Pull down assay Interaction between rStxA and rStxB was shown by pull down assay. Supernatant of rStxA containing GST-StxA was loaded on Gluthione column after incubation and washing the sample was incubated with recombinant His-StxB as mentioned in material and methods. After elution analyzed on SDS-PAGE (15%) revealed two bands one 58 kDa rStxA and 7.7 kDa rStxB, later confirmed by Western blotting (Fig. 22 and Fig. 23).
17
1 7
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8
rStxA
rStxB
Fig. 22: GST-pull down assay to confirm reconstitution of rStx (15% SDS PAGE) Lane 1: Lysate rStxA Lane 2: Purified rStxB Lanes 3-6: Washes Lanes 7-8: Elutes of rStx with 50 mM Glutathione-reduced 1
2
3
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6
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8
rStxA
rStxB A
B
Fig 23: Western blot to confirm reconstitution of rStx by GST pull down assay A. Developed with anti GST (1:5000) B. Developed with anti His (1:5000) Lanes 1-3: Elutes of rStx Lane 5: Prestained protein molecular weight marker Lanes 6-8: Elutes of rStx
4.6.2 SPR binding analysis of rStxA and rStxB Surface plasmon resonance binding analysis was used to study reconstitution between rStxB and rStxA proteins. Immobilization of His-tag rStxB protein on HTG chip comprising several steps. Ligand (rStxB) was allowed to bind with different dilutions (1:100 and 1:200). An increase in resonance units was detected with the increased rStxB protein concentration. Binding of different concentration of rStxB and rStxA in different dilution (1:2 in PBS, 1:5 in PBS, 1:10 in PBS; 1:10 in sarkosyl buffer) was used as an analyte in order to know the reconstitution of recombinant Shiga toxin. The sensorgrams are shown in Fig. 24A and Fig. 24B represents the binding of rStxA and rStxB proteins. BSA used
18
as negative control showed no binding with immobilized rStxB in comparison of rStxA protein.
A Fig. 24: Reconstitution of recombinant Shiga toxin was confirmed by surface plasmon resonance (SPR) A. rStxB was used as a ligand for interaction Dilutions L1-L3: 1:100 Dilutions L4-L6: 1:200
19
B Fig. 24 B: Reconstitution of recombinant Shiga toxin was confirmed by surface plasmon resonance (SPR) B. rStxA was used as analyte for interaction A1-A2: Control (BSA) A3: rStxA 1:2 in PBS A4: rStxA 1:5 in PBS A5: rStxA 1:10 in PBS A6: rStxA 1:10 in sarkosyl buffer
4.6.3 Biological activity of rStx Cytotoxicity to Vero cells of sonic lysate of S. dysenteriae, purified rGST-StxA, reconstituted rStx and pentameric rStxB were examined. The cytotoxicity was found in reconstituted rStx holotoxin (2 μg/ml) and in sonic lysate of S. dysenteriae, but not in purified rGST-StxA (0.85 mg/ml) or pentameric rStxB (0.3 mg/ml) (Fig. 25). The in vitro reconstituted recombinant Shiga toxin (AB5) showed lethality in mice after 24h as compare to their respective control.
20
A
B
C
D
Fig. 25 : Biological activity of rStx was confirmed by Vero cell cytotoxicity assay A. Healthy Vero cells B. rStx A (0.85 mg/ml) C. rStx B (0.3 mg/ml) D. rStx (2.0 µg/ml)
4.7 Cell culture studies 4.7.1 Vero cell cytotoxicity The cytotoxicity to Vero cells was observed microscopically after 24h incubation with Stx for change in the morphology and cell death. Cell free S. dysenteriae type 1 lysate used for cytotoxicity assay containing 2.5 mg/ml protein, produced cytotoxicity. The morphological data was also compared with the colorimetric assay (neutral red dye uptake assay). 1:20 dilution of the cell lysate was found cytotoxic to the cells where >50% cell death was recorded (Fig. 26A). Hence, the same dilution of cell free lysate was used for cytoprotection assay. 4.7.2 Cytoprotection assays The antiserum produced against recombinant protein rStxA efficiently inhibited the cytotoxicity of Stx towards Vero cells by prior incubation for 1 h at 37°C (Fig. 26 B).
21
Toxin neutralization was determined from the dose response curve of Stx on 96-well plate and titer was expressed as the highest serum dilutions. The most effective inhibition was brought about by antiserum at a dilution of 1:100 led to ~80% reduction of the cytotoxic effect. The production of antibodies by rStxA immunized mice was considered as in vitro indicator of a protective immune response. The Vero cells were observed microscopically after 24h incubation and neutralization also estimated by colorimetric assay using neutral red.
Fig. 26: A. Cytotoxicity assay showing cell cytotoxicity of Stx towards Vero cells B. Cytoprotection assay showing neutralization titers of murine antibodies to Stx produced by S. dysenteriae type 1 in Vero cells
4.8 Molecular Docking Studies 4.8.1 In silico screening to identify small molecule inhibitors In an effort to identify potential inhibitors, initially various molecules were considered to in silico studies and docked on the active A chain of Shiga toxin using ADT. Scoring was done on the basis of minimum binding energy (kcal/mol) of proteinligand complex formation. The results of the docking study of the 20 compounds are 22
summarized in table 1. The binding energy varied from -7.06 to -5.54 and Ki values were from 3.21 µM to 439.63 µM. These molecules were found to interact with the active site residues of the structure of Shiga toxin A subunit which indicated their probability of efficient binding thus inhibition. Twelve small molecules reported as Shiga/ricin toxin inhibitors were included in order to perform the comparative analysis and their BE varied from -6.68 to -3.18 (Table 1.). The compound 3 docked in the pocket of the StxA active site is shown in Fig. 28, where nitro group of compound is forming single hydrogen bond with Ser112 and is interacting with all the molecules of active site i.e. Tyr77, Val78, Tyr114, Glu167, Ala168, Trp203 and Arg170. Additionally, in 4 the nitro group of amide forms a hydrogen bond with Arg170 (Fig. 28). The molecule 15 has been found to interact with Arg170 and Tyr114 of the active site residues (Fig. 28). As shown in figure 28, the 16 in docked conformation is interacting with Ser112 and Val78, the residues lies in active site of Shiga toxin.
23
Fig.27 : In silico screening of small molecule inhibitors on the active side residue of Shiga toxin(Active amino acid residue: Tyr77, Val78, Ser112, Tyr114, Glu167, Ala168, and Arg170 )
Table: 1 in silico screening of small molecules inhibitors
Previously reported compound S. no. Name of compound
Binding energy (kcal mol-1)
Inhibitory Constant (Ki)
1
3-[(4-Isopropylphenoxy)methyl]-4methoxybenzaldehyde
-12.52
666.07 pM
2
3-Ethoxy-4-[2-oxo-2-(pyrrolidin-1-yl) ethoxy] benzaldehyde
-11.92
1.83 nM
3
3-Methoxy-4-(2-oxo-2-pyrrolidin-1yl)ethoxy benzaldehyde
-11.66
2.85 nM
4
3-{2-[4-(Dimethylamino)benzylidene] hydrazino}benzoic acid
-12.14
1.26 nM
5
2-(2-Furyl)quinoline-4-carboxylate
-12.14
1.27 nM
6
N-Cyclohexyl-N-(4-fluorobenzyl)-2-(1H1,2,4-triazol-5-ylsulfanyl)acetamide
-11.25
1.56 nM
24
7
2-(4,4,5,5-Tetramethyl-1,3,2dioxaborolan-2-yl)benzaldehyde
-12.34
1.35 nM
8
3-Benzyl-4-hydroxy-5methoxybenzaldehyde
-11.9
1.9 nM
9
(2E,6S,10E,13S,14aS)-13,14a-Dihydroxy6-methyl-1,6,7,8,9,11a,12,13,14,14adecahydro-4H-cyclopenta[f] oxacyclotridecin-4-one
-12.85
380.66 pM
10
6-[(1R,2S)-1,2-Dihydroxypropyl]-1methyl-2,4(1H,3H)-pteridinedione
-12.19
1.15 nM
11
4-[2-(4-Methylpiperidin-1-yl)-2oxoethoxy]benzaldehyde
-12.85
380.66 pM
12
Fenbendazole
-12.2
1.15 nM
13
Pteridine
-10.36
25.46 nM
14
Pterin
-10.46
21.46 nM
S. no.
Name of compound
Binding energy (kcal mol-1)
Inhibitory constant (Ki)
15
4-Aminopyrazolo(3,4-d)pyrimidine
-10.88
10.63 nM
16
3-Methoxy-4-(2-morpholin-4-yl-2-oxoethoxy)-benzaldehyde
-12.03
1.51 nM
17
3-(5,6-Dichloro-1,3-dioxo-1,3-dihydro2H-isoindol-2-yl)propanoic acid
-11.23
2.45 nM
18
4-Hydroxy-3-iodo-5methoxybenzaldehyde
-10.54
1.35 nM
19
3-(2,6-Dimethyl-phenoxymethyl)-4methoxy-benzaldehyde
-11.96
1.7 nM
20
Purine and pyrimidine derivatives 6-Methylpurine
-4.2
840.50 μM
21
6-Mercaptopurine
-4.4
598.78 μM
22
2-Aminopurine
-4.42
580.18 μM
25
23
2,6-Diaminopurine
- 4.22
805.50 μM
24
Xanthine
-4.57
450 .59 μM
25
Hypoxanthine
-4.32
560.32 μM
26
Guanine
-4.37
622 μM.
27
Allopurinol
-4.4
596.72 μM
28
4-Aminopyridine
- 3.18
3.18 mM
29
8-Azaadenine
-4.44
555.58 μM
30
Adenosine
-6.68
12.61 μM
31
Adenosine monophosphate
-6.41
20.06 μM
32
5,6-Diaminopyrimidine-2,4-diol
-17.01
342.25 fM
Binding energy (kcal mol-1) -18.54
Inhibitory constant (Ki) 25.89 fM
S. no.
Name of compound
33
Methylguanosine
34
8-Methyl-9-oxoguanine
-5.05
198.56 fM
35
7,8-Dihydro-8-oxoguanine
-17.56
133 μM
36
2-Amino-3-hydroxy-7-methyl-3,7dihydro6H-purin-6-one
-17.48
154.48 fM
37
2-Amino-7-methyl 1-7,9-dihydro-3Hpurine-6,8-dione
-17.11
287.81 fM
38
2-Amino-1,5,7,8-tetra hydro-4,6pteridinedione
-16.72
558.18 fM
39
8-Amino-6-methyl[1,2,4]triazole[4,36]pyrimidine-5,7(1H,6H)-dione
-15.56
445.62 fM
40
5-Amino-2-methyl[1,3]oxazolo[5,4d]pyrimidin-7-ol
-4.3
706.63 nM
41
Amide derivatives N-(2-Aminocyclohexyl)benzamide
-8.56
533.18 nM
42
N-(3-Aminopropyl)benzamide
-7.13
5.92 µM
26
43
N-(4-Aminobutyl)benzamide
-7.29
4.55 µM
44
N-(6-Aminohexyl)benzamide
-7.51
3.12 µM
45
N-((1R,2R)-2Aminocyclohexyl)benzamide
-8.3
830.46 nM
46
N-((1r,4r)-4-Aminocyclohexyl)benzamide
-8.28
852.56 nM
47
3-Methyl-thiophene-2-carboxylic acid (2acetyl-phenyl)-amide
-6.66
13.33µM
48
Cyclohexanecarboxylic acid [2-(2phenylsulfanyl-ethylamino)-ethyl]-amide
-6.09
34.5µM
49
N-(2-Acetyl-phenyl)-4-nitro-benzamide
-7.23
5.0 µM Inhibitory constant (Ki) 3.36 µM
S. no.
Name of compound
50
N-(2-Acetyl-phenyl)-4-fluoro-benzamide
Binding energy (kcal mol-1) -6.16
51
N-(2-Acetyl-phenyl)-2-fluoro-benzamide
-4.59
439.63 µM
52
3-Phenyl-N-[2-(2-propylsulfanylethylamino)-ethyl]-acrylamide
-5.08
189.36 µM
53
N-(2-Hydroxyphenyl)cinnamamide
-6.45
18.68 µM
54
N-(2-Hydroxethyl)cinnamamide
-6.05
37.05 µM
55
N-(2-Acetylphenyl)cinnamamide
-7.06
6. 69 µM
56
Amine derivatives N-Benzoyl ethylene diamine
-7.17
5.55 µM
57
N-Benzoyl-N,N'-diethylethylene diamine
-7.89
1.65 µM
58
N-Benzoyl-N,N'-dipropylethylene diamine
-7.19
5.33 µM
59
N-Benzoyl-N,N'-dimethylethylene diamine
-7.89
1.65 µM
60
N-Benzoyl-N,N'-dipropylethylene diamine
-8.1
1.15 µM
61
N-Benzoyl-N'-methylethylene diamine
-7.19
5.33 µM
62
Chalcone derivatives 1-(2-Hydroxyphenyl)-3-(4-
-6.83
9.93 µM 27
methoxyphenyl) prop-2-en-1-one
63
3-(4-Chlorophenyl)-1-(2-hydroxyphenyl) prop-2-en-1-one
-6.77
10.88 µM
64
3-(2-Fluorolorophenyl)-1-(2hydroxyphenyl) prop-2-en-1-one
-7.35
4.1 µM
65
1-(2-Hydroxyphenyl)-3-(thiophen-2-yl) prop-2-en-1-one
-7.16
5.96µM
S. no. 66
Benzothiazol derivatives Name of compound Binding energy (kcal mol-1) (1S,4S)-N1-(Benzothiazol-2-8.4 yl)cyclohexane-1,4-diamine
Inhibitory constant (Ki) 699.85 nM
67
(1S,2R)-N1-(Benzothiazol-2yl)cyclohexane-1,2-diamine
-7.87
1.71 µM
68
N-Benzylbenzothiazol-2-amine
-8.4
698.33 nM
69
2-(4-(6-Chlorobenzothiazol-2-yl)piperazin1-yl)ethanol
-8.1
1.16 µM
70
2-(4-(6-Nitrobenzothiazol-2-yl)piperazin1-yl)ethanol
-8.46
632.79 nM
71
N-Benzothiazolepyrollidine
-7.77
2.03 µM
72
N1-(Benzothiazol-2-yl)butane-1,4-diamine
-7.33
4.22 µM
73
N1-(Benzothiazol-2-yl)butane-1,2-diamine
-7.07
6.55 µM
74
N1-(Benzothiazol-2-yl)propane-1,3diamine N1-(Benzothiazol-2-yl)-N1,N2dimethylethane-1,2-diamine
-7.32
4.32 µM
-8.24
905.19 nM
N1-(Benzothiazol-2-yl)-N1,N2diethylethane-1,2-diamine
-7.91
1.59 µM
-7.62
2.61 µM
75 76
Piperazine Derivatives 77
1-Tosylpiperazine
28
78
1-Tosyl-1,4-diazepane
-7.97
1.45 µM
79
1-(2-Chlorophenylsulfonyl)piperazine
-8.09
1.18 µM
80
1-(2-Fluorophenylsulfonyl)piperazine
-7.52
3.06 µM
81
1-(3-Fluorophenylsulfonyl)piperazine
-7.73
2.17 µM
82
1-(4-Cynophenylsulfonyl)piperazine
-8.41
680.26 nM Inhibitory constant (Ki) 2.65 µM
S. no. 83
1-(3-Tolylsulfonyl)piperazine
Binding energy (kcal mol-1) -7.61
84
1-(2-Tolylsulfonyl)piperazine
-7.9
1.63 µM
85
3-(4-(4-Chlorophenylsulfonyl)piperazin-1yl)-N-phenylpropanamide
-9.83
62.66 nM
86
3-(4-(2-Fluorophenylsulfonyl)piperazin-1yl)-N-phenylpropanamide
-9.68
80.53 nM
87
3-(4-(3-Fluorophenylsulfonyl)piperazin-1yl)-N-phenylpropanamide
-9.63
87.8 nM
88
3-(4-(4-Cyanophenylsulfonyl)piperazin-1yl)-N-phenylpropanamide
-9.72
75.24 nM
89
3-(4-(4-Fluorophenylsulfonyl)piperazin-1yl)-N-phenylpropanamide
-10.18
34.7 nM
90
N-(4-Cyanophenyl)-3-(4-(2-fluoro phenylsulfonyl)piperazin-1yl)propanamide
-10.21
32.66 nM
91
N-(4-Cyanophenyl)-3-(4-(3-fluoro phenylsulfonyl)piperazin-1yl)propanamide N-(4-Cyanophenyl)-3-(4-(4fluorophenylsulfonyl)piperazin-1yl)propanamide
-10.56
18.14 nM
-10.13
37.65 nM
93
N-Phenyl-3-(4-(o-tolylsulfonyl)piperazin1-yl)propanamide
-8.23
924.11 nM
94
N-Phenyl-3-(4-tosylpiperazin-1-yl) propanamide
-10.24
31.16 nM
92
Name of compound
29
95
S. no.
3-(4-(2-Chlorophenylsulfonyl)piperazin-1yl)-N-phenylpropanamide
Others Name of compound
57.38 nM -9.88
Binding energy (kcal mol-1) -5.04
Inhibitory constant (Ki) 53.78 µM
96
O,O-Diethyl (2-{[2-(butylsulfanyl) ethyl]amino}ethyl)phosphoramido-thioate hydrochloride salt
97
4-Oxo-2-phenyl-4H-chromen-3-yl cinnamate-2-(2-Fluoro-phenyl)-1Hquinolin-4-one
-6.64
8.78 µM
98
2-Styryl-1H-quinolin-4-one
-10.06
41.9 9µM
99
3-[(5-Chloro-2-oxo-2H-indol-3-yl)amino] benzoic acid
-7.49
32.34 µM
30
Table 2: List of molecules used for in vitro studies (1-4) S. No.
Name and Score of Ligands
1
N- (2-(Phenylthio)ethyl)-2(pyrrolidin-1-yl)ethanamine dihydrochloride
Structure of Ligands
Molecular Weight: 323.32 B.E. : -5.64 Ki : 73.78 µM
2
O,O-Diethyl (2-{[2(Butylsulfanyl)ethyl]amino }ethyl) phosphoramidothioate hydrochloride salt Molecular Weight: 364.94 B.E. : -5.04 Ki : 53.78 µM
3
N-(2-(2-(Phenylthio)ethyl)4-nitrobenzamide hydrochloride Molecular Weight: 364.94 B.E. : -5.94 Ki : 42.46 µM
4
N-(2Acetylphenyl)cinnamamide Molecular Weight: 265.31 B.E. : -7.06 Ki : 6. 69 µM
31
Table 2: List of molecules used for in vitro studies (5-8) S. No.
Name and Score of Ligands
5
N-(2Hydroxyphenyl)cinnamami de
Structure of Ligands
Molecular Weight: 239.27 B.E. : -6.45 Ki : 18.68µM 6
N-(2Hydroxethyl)cinnamamide
Molecular Weight:191.23 B.E. : -6.05 Ki : 37.05µM
7
4-Oxo-2-phenyl-4Hchromen-3-yl cinnamate Molecular Weight:368.38 B.E. : -6.64 Ki : 8.78µM
8
1-(2-Hydroxyphenyl)-3-(4methoxyphenyl) prop-2-en1-one Molecular Weight: 254.28 B.E. : -6.83 Ki : 9.93µM
32
Table 2: List of molecules used for in vitro studies (9-12) S. No.
Name and Score of Ligands
9
3-(4-Chlorophenyl)-1-(2hydroxyphenyl) prop-2-en1-one
Structure of Ligands
Molecular Weight: 258.7 B.E. : -6.77 Ki : 10.88µM 10
3-(2-Flulorophenyl)-1-(2hydroxyphenyl) prop-2-en1-one Molecular Weight: 242.25 B.E. : -7.35 Ki : 4.1µM
11
1-(2-Hydroxyphenyl)-3(thiophen-2-yl) prop-2-en1-one Molecular Weight: 230.28 B.E. : -7.16 Ki : 5.96µM
12
4-Chioro-N-[2-2propylsulfanyl-ethylamio)ethyl]-benzamide Molecular Weight:337.31 B.E. : -5.49 Ki : 93.86µM
33
Table 2: List of molecules used for in vitro Studies (13-16) S. No.
Name and Score of Ligands
13
2-Styryl-1H-quinolin-4-one
Structure of Ligands
Molecular Weight: 247.29 B.E. : -10.06 Ki : 41.99µM
14
2-(2-Fluoro-phenyl)-1Hquinolin-4-one Molecular Weight: 239.24 B.E. : -7.49 Ki : 3.21µM
15
N-(2-Acetyl-phenyl)-2fluoro-benzamide Molecular Weight: 257.26 B.E. : -4.59 Ki : 439.63µM
16
3-Phenyl-N-[2-(2propylsulfanyl-ethylamino)ethyl]-acrylamide
Molecular Weight: 328.90 B.E. : -5.08 Ki : 189.36µM
34
Table 2: List of molecules used for in vitro studies (17-20) S. No.
Name and Score of Ligands
17
3-Methyl-thiophene-2carboxylic acid (2-acetylphenyl)-amide
Structure of Ligands
Molecular Weight: 259.32 B.E. : -6.66 Ki : 13.33µM
18
Cyclohexanecarboxylic acid [2-(2-phenylsulfanylethylamino)-ethyl]-amide
Molecular Weight:342.93 B.E. : -6.09 Ki : 34.5µM
19
N-(2-Acetyl-phenyl)-4nitro-benzamide Molecular Weight: 284.27 B.E. :-7.23 Ki : 5.0µM
20
N-(2-Acetyl-phenyl)-4fluoro-benzamide Molecular Weight: 257.26 B.E. : -6.16 Ki : 3.36µM
35
1
2
8 Fig 28 : A close view to show the interaction and active site of Shiga toxin (PDB structure 1DM0) with the docked ligands using UCSF chimera ( Compounds 1-8)
36
9
10
11
12
13
14
15
16
Fig 28 : A close view to show the interaction and active site of Shiga toxin (PDB structure 1DM0) with the docked ligands using UCSF chimera (Compounds 9-16)
37
17
19
R7
18
20
R8
Fig. 28 : A close view to show the interaction and active site of Shiga toxin (PDB id: 1DM0) with the docked ligands using UCSF chimera (Compounds 17-20, R7 and R8)
4.8.2 Synthesis of small molecules Chalcones 8, 9, 10 and 11 were prepared by the reported literature method. Amides 4, 15, 19 and 20 were prepared by direct acyaltion of appropriate amine with acyl haide. Compound 1 was prepared by treating Phenyl-S-ethylamine hydrochloride with 1-(2Chloroethyl)-pyrrolidine in the presence of triethylamine as a base. Compound 2 was prepared by selective thiophosphorylation of N1 -(2-Butylsulfanyl-ethyl)-ethane-1,2diamine
with
Diethylchlorothiophosphate.
Selective
amidation
of
N1-(2-
Phenylsulfanyl-ethyl)-ethane-1,2-diamine with corresponding acyl halide resulted in formation of compounds 3 and 18. Similarly, selective amidation of N1-(2Propylsulfanyl-ethyl)-ethane-1,2-diamine with 4-Chlorobenzoyl chloride, Cinnamoyl chloride yielded compounds 12 and 16. Acylation of 2-Aminophenol and 2Aminoethanol with Cinnamoyl chloride in water utilizing Sodium carbonate as base resulted in the formation of 4 and 5 respectively. Compound 7 was prepared by esterifying 3-Hydroxyflavone with cinnamoyl chloride. Quinolones 13, 14 and 17 38
were accessed by cyclisation of the amides obtained from 2-Aminoacetophenone and corresponding Acyl halides.
4.8.3 Stx induced cytotoxicity in a dose and time dependent manner in Vero Cells To examine the cytotoxic effect and determine the CC50 of Stx in Vero cells, the cells were treated with various concentration of Stx ranging from 0 to 120 ng/ml for 24h and the viability was determined by NR assay. Stx caused a dose dependent decrease in viability with increase in Stx concentration with 50% cytotoxicity at 100ng/ml at 24h (Fig. 29). The CC50 was calculated to 100 ng/ml and was used in further experiments.
4.8.4 Effect of compound on cells and Stx induced toxicity Effect of small molecules on cells was calculated by two parameters maximum tolerated dose (MTD) and CC50 of the compounds. The cells were treated with varying concentration of compounds for 24h and than cell viability was measured for determination of MTD by NR assay. MTD values of our synthesized compounds varied from 5.0±1.23 to 123±2.45µM and for reported compounds 9.3±1.53 to 250±1.75µM. (Table 3 and 4). Compounds were also tested for their 50% cytotoxic concentration (CC50) and results are mentioned in table 2. Further, to test the efficacy of the small molecules as an inhibitor of Stx, cells were pre-treated with compounds for 1h followed by Stx exposure. Among 32 compounds, four new compound (3, 4, 15, 16) and two reported compounds (R7, R8) showed 75 - 90% inhibition of Stx induced toxicity. The compounds offered protection in dose dependent manner. Other compounds showed no significant reversal of Stx induced toxicity in Vero cells (Fig. 30 & 31). The IC50 for the compounds (3, 4, 15, 16, R7 and R8) were also determined in cells treated with different concentrations of compounds (started from MTD) and CC50 of the Shiga toxin and the results obtained are provided in table 2A and 2B. Based on CC50 and IC50 values of compounds, SI values (CC50/IC50) were calculated and it varied from 11.29 to 22.23. Although, compounds 3, 4, 15 and 16 significantly inhibited the Stx toxin induced cell death, but the compounds 3 and 15 were found to be more potent with SI value of 22.23 and 15.5, respectively (Table 2A). From the
39
previously reported compounds included in the study, Guanine (R7) showed 88% inhibition with IC50 value of 11.5µM, however, 76% inhibition was observed with Allopurinol (R8) with an IC50 value of 23.9 µM. The reported compounds showed selectivity indices 11.8 and 17.9 for R7 and R8, respectively (Table 4).
100
% Cell Viability
80
60
40
20 0
0
20
40
60
80
100
120
Stx (ng/ml) Fig. 29: Cytotoxic effect of stx in Vero cells was determined by neutral red assay. For determination of CC50, the cells were seeded on to a 96 well plate and treated with varying concentration of stx for 24h. The 50% cytotoxic concentration (CC50) was calculated as 100ng/ml. Data are the mean of three independent experiments each in triplicate with SEs.p<0.05 compared with untreated cells.
40
Fig. 30: Evaluation of small molecule inhibitors for in vitro protection in Vero cells; compound 3,4,15 and 16 were found to be promising; ;Data are the mean of three independent experiments each in triplicates with standard error. p<0.05 compare to Stx treated cells.
41
Compound no.
MTD (μM)
CC50 (μM)
IC50 ( μM)
SI
1
62.5
-
-
-
2
31.0
-
-
-
3
15.5
177
07.96
22.23
4
07.2
29.71 5.4
02.63
11.29
5
31.0
-
-
-
6
31.0
-
-
-
7
62.5
-
-
-
8
07.2
-
-
-
9
03.6
-
-
-
10
03.6
-
-
-
11
15.5
-
-
-
12
125
-
-
-
13
9.3
-
-
-
14
5.0
-
-
-
15
37.5
120
7.7
15.5
16
37.5
123
8.2
15
17
5.0
-
-
-
18
37.5
-
-
-
19
18.5
-
-
-
20
9.3
-
-
-
Table 3 : The minimum tolerated doses of small molecules, CC50 , IC50 and selectivity index of the small molecules.
42
Compound ID
MTD (μM)
CC50 (μM)
IC50 (μM)
SI
R1
9.3 2.3
-
-
-
R2
125 3.24
-
-
-
R3
18.7 5.6
-
-
-
R4
125 1.5
-
-
R5
9.3 2.8
-
-
-
9.3 1.53
-
-
-
R7
125 1.87
136.5 2.5
11.5
11.8
R8
250 1.75
428 5.6
23.9
17.9
R9
250 3.6
-
-
-
R10
18.7 4.2
-
-
-
R11
125 3.2
-
-
-
R12
75 3.45
-
-
-
R6
Table 4: The Maximum tolerated dose (MTD), CC50, IC50 and selectivity index of the reported 12 small molecules.
43
% Cell Viability
(A)
120
100
80
60 40 20 0 0
% LDH Release
(B)
Stx
Stx+ 3 Stx+4 Stx+15 Stx+16
R-7
R-8
Compound (µM) 120 100 80 60 40
R-7
R-8
20 0 0
Stx
Stx+3 Stx+4 Stx+15 Stx+16
Compound (µM) Fig. 32. Compounds 3, 4, 15, 16, R7 and R8 significantly reduced stx induced toxicity (A) cells were treated with stx CC50 in presence or in absence of compounds (3, 4, 15, 16, R7 and R8) for 24h and analyzed by MTT assay. (B) All six compounds significantly block stx induced LDH release and decreased back to normal level. Data are the mean of three independent experiments each in triplicates with standard error. p<0.05 compare to stx treated cells.
4.9 In vivo evaluation compound 3, 4, 15, 16 and R7, R8 in mouse model To assess whether or not our finding were restricted to long term cultured cell lines, we analyzed the efficacy of compound in mice against supralethal dose of Stx (5LD50).In Stx treated group all mice were died between 12 to 24hours with symptoms of shigellosis. In both groups where mice were exposed to Stx followed by either pre or post treatment of compounds were also died within 24 to 48 hr. In the group where compounds were mixed with 5LD50 of Stx and then administered, no death was observed in this group of animals and no animal showed any symptomatic effect of toxin (Fig. 33). Treatment with compound alone did no show any toxicity (data not shown).
44
45