FUNCTIONAL PEPTIDOMICS OF AMPHIBIAN VENOMS The dermal granular - - PowerPoint PPT Presentation

FUNCTIONAL PEPTIDOMICS OF AMPHIBIAN VENOMS

• The dermal granular (venom) gland

The dermal granular (venom) gland

The venom producing cells of the granular gland produce secretions, which are stored in the cytoplasmic continutiy of the syncytium Stimulation/injury causes opening of the granular gland and emptying of the syncitium (containing cellular constituents) onto the dermal surface of the frog; this includes poly A+ mRNA

Focused interests Focused interests

• antimicrobials

antimicrobials

• anticancer

anticancer

• novel

novel neurohormones neurohormones

• vasoactives

vasoactives

• cell growth regulators

cell growth regulators

• anthelmintics

anthelmintics

• insecticides

insecticides

• molecular targets in pathology

molecular targets in pathology

Pharmaceutical Biotechnology Pharmaceutical Biotechnology Research Group, UUC Research Group, UUC

• 1 x LCQ

1 x LCQTM

TM “Classic” Ion Trap

“Classic” Ion Trap

• 1 x Voyager DE MALDI

1 x Voyager DE MALDI -

• TOF

TOF

• 1 x QToF

1 x QToF Ultima Ultima

LCQ Ion Trap LCQ Ion Trap

Fractionation of venom Fractionation of venom

RT: 0.00 - 70.02 SM: 7B 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Time (min) 20 40 60 80 100 20 40 60 80 100 R e l a t i v e A b u n d a n c e 56.81 44.46 45.55 59.47 46.29 63.14 48.49 55.50 66.08 40.63 34.95 26.53 54.48 27.15 36.48 33.82 17.42 6.50 19.41 15.65 5.54 8.08 56.39 50.10 46.07 42.79 26.46 33.87 59.25 17.36 27.07 34.90 63.07 36.40 14.22 27.85 19.36 54.58 65.93 5.77 6.59 31.36 19.96 11.23 4.04 NL: 5.43E8 TIC MS Feb25_Pac hymed_dac nic_5mg- 500ul NL: 1.00 Analog 1 Analog Feb25_Pac hymed_dac nic_5mg- 500ul

LC LC-

• UV

UV-

• MS Analysis of

MS Analysis of Litoria aurea Litoria aurea skin secretion skin secretion

RT: 0.01 - 240.00 20 40 60 80 100 120 140 160 180 200 220 Time (min) 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Relative Abundance 124.99 112.42 129.37 97.41 37.15 132.54 73.77 88.21 3.44 2 24.77 223.50 151.80 176.14 208.29 63.87 198.30 43.24 24.55 3.47 28.86 124.86 37.04 122.40 126.17 40.69 85.58 4.25 98.08 13.42 43.13 73.68 132.46 220.73 187.03 2 210.99 174.80 165.45 58.78

LC-MS chromatogram LC-UV chromatogram

(214nm) 200ul injection. 0-80% ACN in 0.05% TFA water in 240 minutes.

LC LC-

• UV

UV-

• MS Analysis of

MS Analysis of Litoria aurea Litoria aurea skin secretion skin secretion LC-MS chromatogram LC-UV chromatogram

(214nm)

RT: 10.73 - 44.08 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Time (min) 10 20 30 40 50 60 70 80 90 100 10 20 30 40 50 60 70 80 90 100 Relative Abundance 37.15 37.28 34.89 37.45 40.78 32.41 24.77 33.14 36.23 25.16 38.39 31.19 25.47 27.16 41.04 21.96 38.59 30.40 17.56 19.79 22.69 16.45 13.49 11.05 24.55 28.86 37.04 27.59 34.81 40.69 32.85 36.17 10.77 38.27 13.42 4 31.36 19.65 16.72 15.12 21.75 24.18

Expanded area

200ul injection. 0-80% ACN in 0.05% TFA water in 240 minutes.

Serotonin Serotonin-

• based Compounds

based Compounds

Serotonin Serotonin R = NH2 N’ N’ Methyl Methyl serotonin R=NHCH3 Bufotenine Bufotenine R = N(CH3)2

N’N’N’ N’N’N’ Trimethyl serotonin Trimethyl serotonin

R = N+(CH3)3

N R H O H

MS/MS of isobaric compounds MS/MS of isobaric compounds

Trimethyl serotonin Trimethyl serotonin m/z m/z 219.1 219.1 m/z m/z 160.1 CID product 160.1 CID product ion ion

Energy Energy Energy Energy

m/z m/z 59.1 CID fragment 59.1 CID fragment

• N+(CH3)3

5 5-

• MeO

MeO DMT DMT m/z m/z 219.1 219.1 m/z m/z 174.1 CID product 174.1 CID product ion ion m/z m/z 45 CID fragment 45 CID fragment

• N(CH

N(CH3

3)

)2

2

MS analysis of N’N’N’ trimethyl serotonin and 5-methoxy dimethyl tryptamine

S#: 1-36 RT: 0.02-0.70 AV: 36 NL: 1.20E7 F: + c Full ms [ 50.00 - 500.00] 50 100 150 200 250 300 350 400 450 500 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 219.2 160.2 220.2 391.1 413.3 161.2 421.3 284.4 369.3 352.2 453.3 495.3 221.2 481.3 249.2 149.2 313.2 190.2 132.2 74.0 115.1 215.2 95.2 S#: 4-15 RT: 0.06-0.25 AV: 12 NL: 7.34E7 F: + c Full ms [ 50.00 - 500.00] 100 150 200 250 300 350 400 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 219.1 174.3 220.1 175.3 390.2 217.3 354.8 221.1 130.3 378.1 159.3 277.0 421.2 176.3 249.1 326.5 292.9 304.0 118.2 104.1 85.0 71.1

N N H O H CH3 CH3 CH3 +

N N O H CH3 CH3 C H3 H+

160 219 174 219 N’N’N’ trimethyl serotonin 5-methoxy dimethyl tryptamine DISTINGUISHED! DISTINGUISHED!

N N H O H CH3 CH3 CH3 N H O H NH3 MS N' methyl 5-HT 191 N NH H O H CH3 N N H O H CH3 CH3 N H H+ MS3 132 N H O H MS 5-HTQ 219 + MS 5-HT 177 H+ H+ MS bufotenine 205

• 17
• 31
• 45
• 59
• 28
• 17

H+ MS2 160 +

• NH3
• CH3NH2
• (CH3)2NH
• (CH3)3N
• CO
• NH3

MS4 115 ?

MS analysis of peptide mixtures

Mar02_eleanor_bo_fr97b#50-79 RT: 1.30-1.59 AV: 19 NL: 2.25E6 F: + c Full ms [ 150.00-1100.00] 550 600 650 700 750 800 850 900 950 1000 1050 m/z 10 20 30 40 50 60 70 80 90 100 R e l a t i v e A b u n d a n c e 531.0 538.7 1074.3 549.8 556.6 630.1 562.5 973.5 904.3 864.7 675.5 918.4 1005.5 697.4 837.6 811.3 752.5

Bradykinin and threonine-6 bradykinin from Bombina orientalis

+1 charge states +2 charge states

MS/MS Sequencing of Bradykinin

Mar02_eleanor_bo_fr98#69-92 RT: 1.31-1.82 AV: 14 NL: 5.42E3 F: + Z ms [ 526.00-536.00] 526 527 528 529 530 531 532 533 534 535 536 m/z 10 20 30 40 50 60 70 80 90 100 R e l a t i v e A b u n d a n c e 530.8 531.2 531.7 534.7 532.3 535.2 527.3 532.7 530.6 526.8 529.4 533.2 528.4 533.6 527.8 535.7 526.1

ZoomScan high resolution mass analysis

This isolates the ion of interest, even in a complex mixture. Fragmentation can then take place.

MS/MS Sequencing of Bradykinin

Mar02_eleanor_bo_fr97b#70-122 RT: 1.63-2.95 AV: 48 NL: 9.83E4 F: + c Full ms2 531.00@40.00 [ 145.00-1100.00] 200 300 400 500 600 700 800 900 1000 1100 m/z 10 20 30 40 50 60 70 80 90 100 R e l a t i v e A b u n d a n c e 904.3 452.8 886.3 642.2 807.3 858.3 710.2 624.2 402.1 643.2 1001.3 175.1 906.3 555.2 501.3 370.3 333.2 254.2 711.2 607.2 1002.4 653.3 288.2 748.2 217.2 907.3 1004.3

MS/MS fragmentation profile can be used to call the peptide sequence

SEQUEST SEQUESTTM

TM

Predicted MS/MS Spectra

SEQ 1 SEQ 2 SEQ 3 SEQ 4

STEP 2.

Mass Search of Indexed Protein and DNA Databases based on the peptide parent molecular weight MS and MS/MS Spectrum

STEP 1.

Compares and correlates predicted and experimental MS/MS spectra

STEP 3. STEP 4.

Rank hits, sequence peptides, create summary and Protein I.D.

MS/MS peptide sequencing software

(a)

RPPGFSPFR

S#: 75-108 RT: 1.63-2.56 AV: 34 NL: 9.80E4 F: + c Full ms2 531.00 [ 145.00 - 1100.00] 200 300 400 500 600 700 800 900 1000 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 904.3 452.8 642.2 886.3 905.3 807.3 858.3 710.2 624.2 402.1 643.2 404.3 808.3 1001.4 175.1 555.2 453.7 906.3 370.3 333.2 254.2 711.2 841.3 1002.4 419.2 614.3 237.2 506.4 653.3 492.4 288.2 748.2 217.2 1003.4 907.3 983.4

b 1 y 3 y 7 y 6 y 8 y 5 y 4 b 2 b 3 b 5 b 6 b 4 b 7 b 8 y 1 y 2

(b)

RPPGFTPFR

S#: 25-33 RT: 0.41-0.65 AV: 9 NL: 6.24E4 F: + c Full ms2 538.00 [ 150.00 - 1100.00] 200 300 400 500 600 700 800 900 1000 m/z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 918.3 656.2 459.9 900.3 919.3 821.2 402.1 515.9 724.2 657.2 612.3 872.3 508.3 411.4 822.3 638.2 1015.4 507.5 157.1 377.4 725.2 316.2 419.2 855.3 555.2 254.1 920.3 333.2 237.2 667.3 274.2 777.5 998.2 971.3 1017.4

b 3 b 1 y 3 y 7 y 6 y 8 y 5 y 4 b 2 b 5 b 6 b 4 b 7 b 8 y 1 y 2 b 9

Transcriptomic Transcriptomic strategy strategy

Extraction of Extraction of peptide mixtures peptide mixtures from from frog frog sample sample followed by solvent followed by solvent extraction, centrifugation, extraction, centrifugation, lyophilization lyophilization, etc , etc Fractionation of Fractionation of the mixtures the mixtures by HPLC by HPLC and v and verification erification of peptide mass

• f peptide mass

by MALDI by MALDI-

• TOF mass spectrometry

TOF mass spectrometry V Verification erification of existing sequence by MS/MS (LCQ

• f existing sequence by MS/MS (LCQ) and d

) and determination etermination of

• f

novel peptide sequence by novel peptide sequence by Edman Edman degradation degradation Screening of collected fraction Screening of collected fraction Extraction of Extraction of polyA polyA RNA/total RNA RNA/total RNA for biological activity for biological activity for for cDNA cDNA library library

1 CCAATTAAGA TTTCCAGGTG GTATCCATTC TCAGAACCAG GTGAACCACC GGTTAATTCT AAAGGTCCAC CATAGGTAAG AGTCTTGGTC CACTTGGTGG 1 M F T L K K S L L L L F F 51 AGAGCCCAAA GATGTTCACC TTGAAGAAAT CCCTGTTACT CCTTTTCTTC TCTCGGGTTT CTACAAGTGG AACTTCTTTA GGGACAATGA GGAAAAGAAG 14 L G T I N L S L C Q D E T N A E E · 101 CTTGGGACCA TCAACTTATC TCTTTGTCAG GATGAGACAA ATGCCGAAGA GAACCCTGGT AGTTGAATAG AGAAACAGTC CTACTCTGTT TACGGCTTCT 31 . E R R D E E V A K M E E I K R G I · 151 AGAAAGAAGA GATGAAGAAG TTGCTAAAAT GGAAGAGATA AAACGCGGTA TCTTTCTTCT CTACTTCTTC AACGATTTTA CCTTCTCTAT TTTGCGCCAT 48 .. L S G I L G A G K S L V C G L S 201 TATTAAGTGG CATCCTCGGT GCGGGGAAGA GCTTAGTATG TGGACTTAGC ATAATTCACC GTAGGAGCCA CGCCCCTTCT CGAATCATAC ACCTGAATCG 64 G L C * 251 GGGCTGTGCT AAAGCTTGCA ATACCGAAAT CATCTGATGT GGAATATCAT CCCGACACGA TTTCGAACGT TATGGCTTTA GTAGACTACA CCTTATAGTA 301 TTAGCTAAAT GCTAAATGTC TTATAAATAA TAAAAATGTC GCATACACTG AATCGATTTA CGATTTACAG AATATTTATT ATTTTTACAG CGTATGTGAC 351 AAAAAAAAAA AAAAAAAAAA AAAAAA TTTTTTTTTT TTTTTTTTTT TTTTTT

1 50 Bm8a (1) MKCFAQIVVLLLVIAFSHGAVITGVCDRDAQCGSGTCCAASAFSRNIRFC Bm8b (1) MKCFAQIVVLLLVIAFSHGAVITGVRDRDAQCGSGTCCAASAFSRNIRFC Bm8c (1) MKCFAQIVVLLLVIAFSHGAVITGVCDRDAQCGSGTCCAASAFSRNVRFC Bm8d (1) MKCFAQIVVLLLVIAFSHGAVITGVCDRDAQCGSGTCCAASAFSRNIRFC Bm8e (1) MKCFAQIVVLLLVIAFSHGAVITGVCDRDAQCGSGTCCAASAFSRNIRFC Bm8f (1) MKCFAQIVVLLLVIAFSHGAVITGVCDRDAQCGSGTCCAASAFSRNIRFC 51 96 Bm8a (51) VPLGNNGEECHPASHKVPYNGKRLSSLCPCNTGLTCSKSGEKFQCS Bm8b (51) VPLGNNGEECHPASHKVPYNGKRLSSLCPCNTGLTCSKSGEKYQCS Bm8c (51) VPLGNNGEECHPASHKVPYNGKRLSSLCPCNTGLTCSKSGEKFQCS Bm8d (51) VPLGNNGEECHPASHKVPYNGKRLSSLCPCNTGLTCSKSGEKSQCS Bm8e (51) VPLGNNGEECHPASHKVPYNGKRLSSLCPCNTGLTCPKSGEKFQCS Bm8f (51) VPLGNNGEECHPASHKVPSDGKRLSSLCPCNTGLTCSKSGEKYQCS

RT: 0.00 - 260.01 SM: 7B 20 40 60 80 100 120 140 160 180 200 220 240 260 Time (min) 20 40 60 80 100 20 40 60 80 100 Relative Abundance 138.68 137.19 204.35 139.87 136.61 194.11 135.21 179.51 130.46 144.67 173.54 83.18 150.57 223.36 95.88 127.40 227.21 80.77 107.29 239.60 71.76 50.16 25.18 42.57 15.69 89.50 82.22 30.34 91.16 130.75 137.44 25.35 95.17 153.56 13.07 205.01 178.31 170.92 188.99 97.18 73.86 107.24 220.27 234.62 244.44 118.85 68.88 63.00 52.06 NL: 2.75E8 Base Peak F: + c Full ms [ 50.00 - 2000.00] NL: 1.00E6 Analog UV 1

6 7 8 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 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 5354i56 57 58 59 60 61 62 63

• 100
• 75
• 50
• 25

25 50 75 100

Peptide Number % Relaxation

Vasoactivity screen on rat tail artery

BMAX030101_01#2360-2636 RT: 78.29-85.34 AV: 277 Avg MW: 2179.0 +- 0.4 NL: 1.73E7 T: + c Full ms [ 50.00-2000.00] 500 1000 1500 2000 m/z 10 20 30 40 50 60 70 80 90 100 Relative Abundance +2 1090.3 +3 727.3 +4 545.9

RT: 0.00 - 260.01 SM: 7B 20 40 60 80 100 120 140 160 180 200 220 240 260 Time (min) 20 40 60 80 100 20 40 60 80 100 Relative Abundance 82.81 83.77 158.75 143.48 137.10 87.15 191.36 204.43 167.06 224.19 108.90 245.29 78.87 67.06 25.15 53.83 12.64 89.50 82.22 30.34 91.16 130.75 137.44 25.35 95.17 153.56 13.07 205.01 178.31 170.92 188.99 97.18 73.86 107.24 220.27 234.62 246.72 118.85 68.88 63.00 52.06 NL: 5.40E7 m/z= 1089.5-1090.5 F: + c Full ms [ 50.00 - 2000.00] NL: 1.00E6 Analog UV 1

Mass filter 1090 m/z

S #: 100-115 R T: 3.61-4.24 A V : 16 N L: 1.67E 4 F: + c Fu ll m s2 1090.30 [ 305.00 - 2000.00] 400 600 800 1000 1200 1400 1600 1800 2000 m /z 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 1011.9 1032.4 954.4 1081.4 945.6 904.2 811.3 699.5 1233.5 1691.8 486.1 548.2 837.2 797.6 1186.5 1325.4 1988.0 416.1 1851.4 1576.6 648.8 1439.4

b4 b5 b6 b7 b8 b9 b10 b11 b12 b13 b14 b15 b16 b17 y2 y3 y4 y5 y7 y8 y6 y9 y10 y11 y12 y13 y14 y15 y17

C ycle num ber

1 2 3 4 5 6 7 8 9 10 P TH

• am

ino acid D L P K I N R K G P Y ield (pm

• l)

1194.3 787.9 560.0 416.9 585.9 514.6 542.0 299.1 374.5 279.7

C ycle num ber

11 12 13 14 15 16 17 18 19 P T H

• am

ino acid R P P G F S P F R

Y ield (pm

• l)

328.7 295.4 361.4 164.0 137.1 48.7 98.0 76.3 14.1

Artery

5 10 15 20 25 30 35 40 45 50 Bradykinin Maximakinin Relative Potency

Small Intestine

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Bradykinin Maximakinin Relative Potency

M R L W F C L · 1 TAGTTCTCAG TGTCACTTCC AGCTCTGATC ATGAGACTGT GGTTCTGTCT . S F F I V L C L E H F P G T L A D · 51 AAGTTTTTTC ATCGTCCTGT GCCTGGAGCA TTTTCCAGGA ACCCTGGCAG .. E R N N R D Y T I R T R L H G H 101 ATGAAAGGAA TAATCGTGAC TACACCATCA GAACCCGCTT ACATGGCCAT H K P S R N N R Y A I K T S I H G · 151 CATAAACCAA GCAGGAATAA CCGTTACGCC ATCAAAACCA GCATACATGG . H H I P R N V P E S E E K T E Q L · 201 CCATCATATA CCAAGGAATG TTCCAGAGAG TGAAGAAAAA ACTGAGCAGC .. L R D L P K I N R K G P R P P G 251 TCCTGAGGGA TTTGCCTAAG ATCAACCGCA AAGGACCACG TCCACCGGGG F S P F R G K F H S Q S L R Q I P · 301 TTCTCCCCTT TTCGAGGAAA ATTCCATAGC CAGTCCCTAC GACAAATTCC . G L G P L R G * 351 TGGTTTAGGC CCTCTGCGTG GATAACGAAG CTCAGGGATA AGAATCTGCC 401 CTATGTGTAT GCCATGTTCA CCATAGGCTA AAAAGTAGCG TCCCCTGCTA 451 TAAATAAGCA TTGTTATGTC ACCTCTGTAA TACCAGCTCT GACTGACATG 501 GTTTATTAAA CAGCAGATTT GTGCTCTCTA AAAAAAAAAA AAAAAAAA

Kinestatin pGlu-Ile-Pro-Gly-Leu-Gly-Pro-Leu-Arg.NH2 Bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg[4,9] (Thr-6)-bradykinin Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg[5,9] (Leu-8)-bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu-Arg[17] (Val-1,Thr-6)-bradykinin Val-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg[5] (Ala-3, Thr-6)-bradykinin Arg-Pro-Ala-Gly-Phe-Thr-Pro-Phe-Arg[10] (Val-1,Thr-3,Thr-6)-bradykinin Val-Pro-Thr-Gly-Phe-Thr-Pro-Phe-Arg[10]

References References

The characterisation and determination of indole alkaloids in frog skin secretions by electrospray ionisation ion trap mass spectrometry

• S. McClean, R.C. Robinson, C. Shaw and W.F. Smyth.

Rapid Communications in Mass Spectrometry 16 (2002) 346-354

Bradykinins and their precursor cDNAs from the skin of the Fire-Bellied Toad (Bombina

• rientalis)
• T. Chen, D.F. Orr, A.J. Bjourson, S.McClean, M. O’Rourke, D.G. Hurst, P. Rao

and C.Shaw

Peptides 23 (2002) 1547-1555

Novel bradykinins and their precursor cDNAs from the European Yellow-Bellied Toad (Bombina variegata) skin.

• T. Chen, D.F. Orr, A.J. Bjourson, S. McClean, M. O'Rourke, D.G. Hirst, P. Rao and C.

Shaw.

European Journal of Biochemistry 269 (2002) 4693-4700

Heuristic charge assignment for deconvolution of electrospray ionization mass spectra.

• H. Zheng, P.C. Ojha, S.McClean, N.D. Black, J.G. Hughes and C. Shaw

Rapid Communications in Mass Spectrometry 17 (2003) 429-436

Granular gland transcriptomes in stimulated amphibian skin secretions. T.Chen, S.Farragher, AJ.Bjourson, DF, Orr, P.Rao and C.Shaw

Biochemical Journal 371 (2003) 125-130

Cloning of maximakinin precursor cDNAs from Chinese toad (Bombina maxima) venom. T.Chen, AJ.Bjourson, S.McClean, P.Rao and C.Shaw

Peptides 24 (2003) 853-861

AmphiBiotics Ltd

GENOMICS (Genes; Mutations) PROTEOMICS (Proteins: Identity; Expression; Activity) MEDICINAL CHEMISTRY (Orally Active Drugs: Natural Products; Synthetic; Combinatorial) DRUG ACTIVITY/SPECIFICITY (Protein/Drug Assays: Receptor Binding; Enzyme Inhibition) BIOAVAILABILITY/METABOLISM (In Vitro/In Vivo Preclinical) CLINICAL PHARMACOKINETICS/METABOLISM (Human Pharmacokinetics/Metabolism) METHODS DEVELOPMENT FOR PRODUCTION (Formulation, Stability) PRODUCTION QC (Purity)

Acknowledgements Acknowledgements

• Prof Chris Shaw

Prof Chris Shaw

• Prof Franklin Smyth

Prof Franklin Smyth

• Prof David

Prof David Hirst Hirst

• Dr Tony

Dr Tony Bjourson Bjourson

• Dr David Orr

Dr David Orr

• Dr

Dr Piyush Ojha Piyush Ojha

• Dr

Dr Tianbao Tianbao Chen

• Dr VN

Dr VN Ramachandran Ramachandran

• Dr Martin O’Rourke

Dr Martin O’Rourke

• Dr Jane

Dr Jane Zheng Zheng

• Mr

Mr Eddie Eddie O’Kane O’Kane

• Mr

Mr Danny Coulter Danny Coulter

• Mrs

Mrs Velma Hayes Velma Hayes Chen Pharmaceutical Biotechnology Research Group www.FrogPharm.com

‘ The toad, ugly and venomous, wears yet

a precious jewel in his crown’

Trollius & Cressida Shakespeare