catalytic antibodies and their use as therapeutic agents
TRANSCRIPT
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Catalytic Antibodies and Their Use in Therapeutics
Samantha M. FrawleyOrganic Seminar
February 9th, 2005
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Seminar OutlineBackground information
What are antibodies?What are catalytic antibodies?
Catalytic antibodies used in therapeuticsTransition-state analogue approach
Antibody catalyzed cocaine degradation
Hapten-substrate approachOxidative degradation of nicotine using catalytic antibodies
Reactive immunizationUsing catalytic antibodies to activate prodrug
Conclusion
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Important TermsAntibody: An immunoglobulin protein produced by the immune system in response to the invasion of a foreign substanceHapten: A small molecule that reacts with a specific antibody but cannot induce the formation of antibodies unless it is bound to a carrier protein Antigen: Any substance that elicits an immune response when introduced into an animalIgG: The most common immunoglobulin, which is distributed between the blood and extravascular fluid. The IgG is used in the formation of catalytic antibodies
Voet, D.; Voet, J.; Pratt, C. Fundamentals of Biochemistry, Rev. Ed., John Wiley & Sons, New York, 1999.
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Structure of Antibodies
Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K.; Watson, J. Molecular Biology of the Cell, 3rd Ed., Garland Publishing Co., 1994.
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Antibodies vs. EnzymesHigh affinity and specific bindingStabilization of transition state vs. ground state
Tramontano, A.; Janda, K.D.; Lerner, R.A. Science 1986, 234, 1566.
http://bio.winona.edu/berg/308s04/Lec-note/chap6.htm
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Catalytic AntibodiesCatalytic antibodies utilize
Antibody specificityEnzyme’s catalytic power
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Methods of Forming Catalytic Antibodies
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Seminar OutlineBackground information
What are antibodies?What are catalytic antibodies?
Catalytic antibodies used in therapeuticsTransition-state analogue approach
Antibody catalyzed cocaine degradation
Hapten-substrate approachOxidative degradation of nicotine using catalytic antibodies
Reactive immunizationUsing catalytic antibodies to activate prodrug
Conclusion
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What is Cocaine?An alkaloid found in the leaves of Erythroxylon cocaBlocks removal of dopamine from a synapse in the reward pathway of the central nervous system
N O
OCH3
O O
(-)-Cocaine
Baird, T.; Deng, S.; Landry, D.; Winger, G.; Woods, J. J. Pharmacol. Exp. Ther. 2000, 295, 1127.
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Previous Method to Treat Cocaine Addiction
This method depletes and inactivates available antibodies
Bunce, C.; Loudon, R.; Akers, C.; Dobson, J.; Wood, D. Curr. Opin. Mol. Ther. 2003, 5, 58.
Sherer, E.; Yang, G.; Turner, G.; Shields, G.; Landry, D. J. Phys. Chem. A 1997, 101, 8526.
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Degradation of CocaineThe hydrolysis of cocaine’s benzoyl ester yields two biologically inactive productsThis transformation is an attractive approach to destroy cocaine prior to its absorption into the brain
N O
OCH3
O O
(-)-Cocaine
N
OH
O
OCH3
CO2H
EcgonineMethyl Ester
BenzoicAcid
+
Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.
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Transition-State Analogue Approach to the Formation of Catalytic Antibodies
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Transition-State Analogue ApproachPrevious work indicated transition-state analogue using the phosphonate monoester, yielded artificial esterases with the greatest activity
N O
OCH3
O OOH
N O
OCH3
O ON
OH
O
OCH3
CO2H
EcgonineMethyl Ester
BenzoicAcid(-)-Cocaine Transition State
+
NH3C O
OCH2R
OP
O
O
Phosphonate Mono-EsterTransition State Analog
R = tether carrier
Landry, D.; Zhao, K.; Yang, G.; Glickman, M.; Georgiadis, T. Science 1993, 259, 1899.
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Importance of Using a LinkerAllows for exposure of the epitope to antibodyStimulate immune responseLinker variations
Tether siteLength
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Formation of Transition State Analogue
N O
OH
(-)-Ecgonine
I(CH2)4N3
1) tetrazole PhP(O)Cl2
2) MeOH
1) P(CH3)3
2) 1,4-14C-succinic anhydride
3) DCC/DMAP Benzyl alcohol
4) H2, Pd/C
1) DCC N-OH phthalimide
2) TMSBr
N O
O(CH2)4N3
N
O
O
O(CH2)4N3
POCH3
O(CH3)4NOH
78% 89%
N
O
O
O
POCH3
O
HN
OOH
O
**
70%
N
O
O
O
PO
O
HN
OO
O
** N
O
O
~70%
OH OH
Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.
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Coupling Transition-State Analogue to a Carrier Protein
Carrier protein BSA or ovalbumin
N
O
O
O
POCH3
O
HN
ONH
O
TSA1
* *N
O
O
O
POCH3
O
HN
OO
O
** N
O
O
Coupling ratio: 6:1 BSA15:1 ovalbumin
N OH
O
O
Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.
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Other Synthesized Transition-State Analogues
NH3C O
O
OP
HN
ONH
O
Transition State Analogue 1
Transition State Analogue 3
Transition State Analogue 2
NH3C O
OCH3
OP
O
OH
NH
O
O
NH
N O
OCH3
OP
O
OH
HN
OHN
O
O
OH
Yang, G.; Chun, J.; Arakawa-Uramoto, H.; Wang, X.; Gawinowicz, M.; Zhao, K.; Landry, D. J. Am. Chem. Soc. 1996, 118, 5881.
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How Catalytic Antibodies are Synthesized
http://www.scripps.edu/~yunfeng/personal/researchweb.html
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Formation of Monoclonal AntibodiesThe half-life of regular IgGantibodies could not withstand testing-immortalize and culture
http://biology.kenyon.edu/courses/biol114/Chap08/Chapter_08b.html
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Formation of Active Cocaine Degrading Antibodies
Deng, S.; Prada, P.; Landry, D. J. Immunol. Meth. 2002, 269, 299.
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Results of Cocaine Degradation Using 15A10
LD90 (16 mg/kg) of cocaine was administered to rats previously injected with catalytic antibody 15A10
Deng, S.; Prada, P.; Landry, D. J. Immunol. Meth. 2002, 269, 299.
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Results of Cocaine Degradation Using 15A10
0
20
40
60
80
100
120
0.000 0.015 0.030 0.060 0.125 0.250 0.500
Cocaine (mg/kg/injection)
Self-
Adm
inis
tere
d In
fusi
ons Saline
10 mg/kg Mab15A1030 mg/kg Mab15A10100 mg/kg Mab15A10
Deng, S.; Prada, P.; Landry, D. J. Immunol. Meth. 2002, 269, 299.
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Conclusions for Cocaine Degrading Antibody 15A10
Efficacy Km = 220 µMKcat = 2.3 min-1
ImprovementsLinker lengthButyrylcholinesterase studiesSite-directed mutagenesis
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Seminar OutlineBackground information
What are antibodies?What are catalytic antibodies?
Catalytic antibodies used in therapeuticsTransition-state analogue approach
Antibody catalyzed cocaine degradation
Hapten-substrate approachOxidative degradation of nicotine using catalytic antibodies
Reactive immunizationUsing catalytic antibodies to activate prodrug
Conclusion
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Facts about NicotineNicotine is the most widely addictive drug in the world
Using nicotine gum and nicotine patches to quit smoking afforded less than 20% success rates
Nicotine binds to nicotinic cholinergic receptors in the mesolimbic dopamine system
N
N
Nicotine
H
Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.
Carrera, M.; Ashley, J.; Hoffman, T.; Isomura, S.; Wirsching, P.; Koob, G.; Janda, K. Bioorg. Med. Chem. 2004, 12, 563.
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Previous Work on Nicotine AddictionNicotine gum/patchesNicotine vaccine
Bunce, C.; Loudon, R.; Akers, C.; Dobson, J.; Wood, D. Curr. Opin. Mol. Ther. 2003, 5, 58.
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Oxidative Degradation of Nicotine
N
N
N
N
N
N
N
N
N
NH
N
N
N
N
OHO
OHHO CO2
-
O
O-
Nicotine
H
Six main metabolites of nicotine, many are formed from P-450 oxidation
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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Hapten-Substrate Approach
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Hapten-Substrate Approach for Nicotine Degradation
Dickerson, T.; Yamamoto, N.; Janda, D. Bioorg. Med. Chem. 2004, 12, 4981.
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Requirements for Nicotine Hapten(S)-configuration stereochemistry Similar structural properties Sufficient alkyl linker length, ~12 Å, to expose all structural features
NH
HN
O
OH
O
NIC hapten 3
N
NH
Nicotine
Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.
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Synthesis of Hapten 3
N
Br CO2H
N
Br CO2Et
N
BrN
N
BrNH
H
EtOHH2SO4, reflux
1) 1-vinyl-2-pyrrolidinone, NaH, THF, reflux2) HCl(aq), reflux3) NaOH
NaBH4
1) MTPA salts
2) H2, Pd/C, Et3N, EtOH
DIEA, acetonitrileN
NH
H IO
HN
O
OBn
N
NH
O
HN
O
OBnN
NH
O
HN
O
OHH2, Pd/C,
MeOH
Hapten 3
94%72%
81%
54%48%
66%
Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.
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Coupling Hapten 3 to a Carrier Protein
N
NH
O
HN
O
OH
Hapten 3
N
NH
O
HN
O
NHCarrier proteinKLH or BSA
Coupling ratio:19:1 BSA
Isomura, S.; Wirsching, P.; Janda, K. J. Org. Chem. 2001, 66, 4115.
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Screening of Catalytic AntibodiesThe elicited monoclonal antibodies were screened against nicotine and irradiated with UV light in the presence of riboflavin
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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Preliminary ResultsNo catalysis was observedConclusion
Antibodies might be binding too strongly to substrateDesign hapten that binds weaker
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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Synthesis and Reasoning Behind Hapten 4
Substrate binding by antibodies to hapten 4 would not be as tight as hapten 3
N
N
O OH
O
Hapten 4
N
NH
O OO
DIEA72%
H H
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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Antibodies vs. OzonolysisThe two most proficient monoclonal antibodies, TD1-36H10 and TD1-10E8 were tested vs. ozonolysis
N
N
O3
N
N
N
N
N
NH
OO
N
N
N
N
NOH N
H
N
NH
OH
O O
O
1 2
or
TD1-36H10/TD1-10E8 andlight, riboflavin
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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Results of Nicotine DegradationAfter 6 hr of white light irradiation in the presence of either antibody, no detectable nicotine remained
0
10
20
30
40
50
60
0 45 88 180 270 375
Time (min)
Prod
uct 1
Con
cent
ratio
n ( µ
M)
TD1-36H10TD1-10E8w/o antibody
N
NOH
1
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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Further Results of Nicotine DegradationThere was a 10-fold rate enhancement vs. uncatalyzedreaction
0
10
20
30
40
50
60
70
0 45 88 180 270 375
Time (min)
Prod
uct 2
Con
cent
ratio
n ( µ
M)
TD1-36H10TD1-10E8w/o antibody
NH
N
OH
O O
O
2
Dickerson, T.; Yamamoto, N.; Janda, K. Bioorg. Med. Chem. 2004, 12, 4981.
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Conclusions on Nicotine-Degradation Using Catalytic Antibodies
Antibodies with modest affinity may be more efficient Efficacy
Requires 20 µM antibody for 200 µM of nicotine
Practical applicationsNicotine mimic metabolitesBiologically active productsIn vivo systems
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Seminar OutlineBackground information
What are antibodies?What are catalytic antibodies?
Catalytic antibodies used in therapeuticsTransition-state analogue approach
Antibody catalyzed cocaine degradation
Hapten-substrate approachOxidative degradation of nicotine using catalytic antibodies
Reactive immunizationUsing catalytic antibodies to activate prodrug
Conclusion
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Prodrug ActivationAdministration of a drug in its non-toxic form (prodrug), which is enzymatically converted into its active form
X
OHO
XHEnzymatic cleavage
Inactive drug Active drug
drugdrug
Guo, X.; Lerner-Tung, M.; Chen, H.; Chang, C.N.; Zhu, J.; Chang, C.P.; Pizzorno, G.; Lin, T.; Cheng, Y. Biochem. Pharmacol. 1995, 49, 1111.
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Catalytic Antibodies and Their Use in Prodrug Activation
Catalytic antibodies can access reactions that are not catalyzed by endogenous enzymes
Campbell, D.; Gong, B.; Kochersperger, L.; Yonkovich, S.; Gallop, M.; Schultz, P. J. Am. Chem. Soc. 1994, 116, 2165.
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Reactive Immunization Approach
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Selecting a Hapten for Aldolase AntibodyAppropriate reactivity Requires a mechanism-based “trap” in the active site of the antibody Ability to form the enamineProvide appropriate binding sites for intermolecular reaction
HN
OO
O
OH
O
Hapten 1
Wagner, J.; Lerner, R.; Barbas III, C. Science 1995, 270, 1797.
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Formation of Hapten-Antibody Complex-Trapping Lysine
R
O O
Hapten 1
R
O
H2N Lys
BH
Ab
NH Lys Ab
HO
R
O N
H H
H Lys Ab
B R
O N
H
LysH Ab
R
O N
H
Lys AbH
Barbas III, C.; Heine, A.; Zhong, G.; Hoffmann, T.; Gramatikova, S.; Bjornestedt, R.; List, B.; Anderson, J.; Stura, E.; Wilson, I.; Lerner, R. Science 1997, 278, 2085.
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Formation of Catalytic AldolaseAntibody
Antibodies are screened for the formation of the enaminoneintermediate
R
O N
H
LysH Ab
λ = 316 nm
Wagner, J.; Lerner, R.; Barbas III, C. Science 1995, 270, 1797
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Mechanism Using Catalytic AldolaseAntibody
O HOHN N
H
N
R
O
H R
OH N
R
OOHH2N Lys Ab+
H2N Lys Ab -H2O
H2O
H Lys Ab H Lys Ab
H Lys Ab
Lys Ab
B
+N
H Lys Ab
Hoffmann, T.; Zhong, G.; List, B.; Shabat, D.; Anderson, J.; Gramatikova, S.; Lerner, R.; Barbas III, C. J. Am. Chem. Soc. 1998, 120, 2768.
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Diversity of the 38C2 Catalytic AntibodyMore than 100 different aldoladditions or condensations have been formed by a single catalyst
Barbas III, C.; Heine, A.; Zhong, G.; Hoffmann, T.; Gramatikova, S.; Bjornestedt, R.; List, B.; Anderson, J.; Stura, E.; Wilson, I.; Lerner, R. Science 1997, 278, 2085.
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Prodrug Activation by 38C2 via a Tandem Retro-Aldol-Retro-Michael Reaction
O
OHO
XH
Inactive drug
Active drug
drug
drug
O
O
38C2N
ON
O
X
H H
B
Odrug
N
ON
O
X
H H
O
drug XN
N
O
BH
CO2
NN
O
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
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EtoposideEtoposide is an antitumor drug used in the treatment of the early stages of pediatric neuroblastomaCurrent survival rates of stage 4 neuroblastoma are quite poor
OO O
OHO OH
OO
OO
OCH3H3COOH
Etoposide
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
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Activation of Prodrug 6 to Etoposide
OO O
OHO OH
OO
OO
OCH3H3CO
O
O
NN O
O OHO
O ONN
O
CO2
Ab38C2O
O OO
HO OH
OO
OO
OCH3H3COOH
Prodrug 6 Etoposide
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
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Prodrug 6 Activation ResultsComparison in growth inhibition activity of prodrug 6 in the absence and presence of 38C2
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
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Prodrug 6 Activation Results The effect of 38C2-mediated prodrug 6 activation
Shabat, D.; Lode, H.; Pertl, U.; Reisfeld, R.; Rader, C.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 2001, 98, 7528.
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Activation of Doxorubicin and Camptothecin Prodrugs by 38C2
NN
O
O
OO
ON O
O OHO
O O
CO2
Camptothecin
Camptothecin-prodrug
38C2 NN
O
O
OOH
N
O
O
O
OH
OH
OH
OCH3O
OOH
O
HOHN O
O OHOO
38C2
O
O
O
OH
OH
OH
OCH3O
OOH
O
HO NH2
Doxorubicin
CO2
Doxorubicin-prodrug
Shabat, D.; Rader, C.; List, B.; Lerner, R.; Barbas III, C. Proc. Natl. Acad. Sci. 1999, 96, 6925.
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A New Type of Prodrug Activation-Enediynes
Biologically active enediynes cleave DNA Benzenoid diradical abstracts hydrogen atoms from the DNA backbone
HN
OH
OOH
OH O
O
OMe
CO2H
Dynemicin A
OMe
Me
OH
O
O
O
O
O
O
OHO
OH
NH
O
Neocarzinostatin chromophore
Nicolaou, K.; Dai, W.; Tsay, S.; Estevez, V.; Wrasidlo, W. Science, 1992, 256, 1172.
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Activation of Endiyne Prodrugs Under Basic Conditions
HN
X
HO
Nu
HN
X
HO
Nu
DNAcleaved DNA
O2
Bergmancyclization
N
O
O
X
OS
Ph
O O
HN
X
ON
X
PhS
O O
CO2
HN
X
HO
Nu
Nu-H
Base
HO
Sinha, S.; Li, L.; Miller, G.; Dutta, S.; Rader, C.; Lerner, R. Proc. Natl. Acad. Sci. 2004, 101, 3095.
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Pathway of Activation of Prodrugs of Dynemicin Analogs
N
O
OO
HO
X
ON
O
O
X
O
N
O
O
X
OS
Ph
O O
HN
X
ON
X
PhS
O O
CO2
HN
X
HO
Nu
O
mAb 38C2
O
O
CO2
Nu-
HO HN
X
HO
Nu
HN
X
HO
Nu
DNAcleaved DNA
O2
Bergmancyclization
Sinha, S.; Li, L.; Miller, G.; Dutta, S.; Rader, C.; Lerner, R. Proc. Natl. Acad. Sci. 2004, 101, 3095.
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Results Using 38C2 to Activate Dynemicin
N
O
OO
HO
H
O
N
O
O
H
O
3a
4a
Sinha, S.; Li, L.; Miller, G.; Dutta, S.; Rader, C.; Lerner, R. Proc. Natl. Acad. Sci. 2004, 101, 3095.
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Other Approaches for Prodrug DeliveryADEPT-Antibody-directed enzyme prodrugtherapy
Johnston, N. Today’s Chemist at Work 2001, 10, 30.
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Other Approaches to Activate ProdrugsADAPT: Antibody-directed abzymeprodrug therapy
Use of catalytic antibody instead of enzyme in drug activation
Transformations which cannot be performed by enzymesActivity of catalytic antibodies is lower
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Conclusions on Aldolase ProdrugActivation
Catalyze transformations not available to enzymesEfficacy
Km = 54 µMKcat = 4.4 x 10-3 min-1
ConsequencesSubstrate selectivityLocalization
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Final ThoughtsDifferent approaches can be employed to synthesize catalytic antibodies
Basic transformations-Transition-state analogue approachExternal energy source required-Hapten-substrate approachDiversity in substrates-Reactive immunization
Improvements need to be made before catalytic antibodies can be used in a clinical setting
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Acknowledgements Dr. BorhanDr. TepeTepe group members
VasudhaTeriManasiAdamChrisJasonGwenJamesSamantha 2