Download 5FdU10 For Treatment of Cancer: Pre

Improved Fluoropyrimidine
Chemotherapy
William H. Gmeiner, Ph.D.
Professor and Chairman
Department of Biochemistry, WFUSM
Colon Cancer: 2nd Leading Cause of
Cancer Deaths in U.S.
 56,000 deaths in U.S. each year
 Highly treatable if detected early
 Poor prognosis if diagnosed at late stages
 Treatment: Surgery, Radiation and/or
Chemotherapy
 Most frequently used chemotherapy:
5-Fluorouracil + Leucovorin (5FU/LV)
Web Sites For Reliable Information
About Cancer
 National Cancer Institute
(www.cancer.gov)
 American Cancer Society
(www.cancer.org)
 American Institute of Cancer Research
(www.aicr.org)
 Wake Forest University Cancer Center
(www.wfubmc.edu/cancer)
Cancer Drug Development
 Identify Molecular Target: Must Differ Between Malignant




and non-Malignant Cells (e.g. involved in cell division).
Determine which types of tumor are most sensitive
Cell culture studies to investigate mechanism of action
Animal studies to demonstrate efficacy in vivo and to
determine PK and PD profile.
Clinical studies: Phase I – Safety; Phase II – Optimal
Dose; Phase III – Efficacy.
5FUra
- A Clinically Useful Anticancer Drug
For More Than 40 Years
O
F
HN
O
N
H
Widespread Clinical Experience
Active Against Many Solid Tumors
Among the Most Effective Drugs
Dose-Limiting Toxicities
Rarely Curative
New Strategies Required
Malignant and Non-Malignant Cells
Often Differ in Cell Division
Malignant Cells
Non-Malignant Cells
The Cell Cycle: Identifying Drug
Targets
Fluoropyrimidines
Ara-C, Hydroxyurea
DNA Synthesis
S
Pre-Mitotic Interval
G2
Mitosis
G1
M
Preparation
for Cell Division
Vincrisitine, Vinblastine
G0
Resting Phase
TS & RR Are Targets For S-Phase
Blocking Anticancer Drugs
G1
S-Phase
O
F
HN
O
O
OH
P
-O
O
F
H H O
HO
H
HH
N
H
N
O
NH
O
FdUMP
TS
DNA
G2
dUMP
dTMP
ADP
ADP
GDP
dUDP
GDP
RR
CDP
CDP
UDP
O
HOHN
Hydroxyurea
NH2
Toxicities Associated With 5-FU
GI-Tract Toxicity
O
F
HN
O
FUTP
N
H
5-FU
Cardio- & Neurotoxicity
FBAL
RNA
FdUMP[N] Serves As A ProDrug of FdUMP
Recursion Applied to Factorials
recursion n. 1 the act of returning.
2 (Math) the repeated application
of a mathematical procedure to a
10! = 10(9!)
However, 9 factorial is 9 times 8 factorial:
9! = 9(8!)
preceding result to generate a
sequence of values.
Using the recursive concept:
8! = 8(7!)
7! = 7(6!)
6! = 6(5!)
5! = 5(4!)
4! = 4(3!)
3! = 3(2!)
2! = 2(1)
(((((
)))))
10! = 10 9 8 7 6 5(4(3(2(1))))
Important Initial Questions
About FdUMP[10]
 Does it work? (i.e. is FdUMP[10] either
cytotoxic or cytostatic?).
 Is there selectivity for certain
malignancies?
 Is it safe? (i.e. can FdUMP[10] be used in
vivo?)
 Is it efficacious? (i.e. can FdUMP[10]
inhibit growth of a human tumor?)
Does FdUMP[10] Work? GI50 Values and
Relative Potency in the NCI 60 Cell Line Screen




Drug
IC50
Relative
5FU
2.4 x 10-5 1
FdU
1.8 x 10-6 13.3
FdUMP[10] 7.1 x 10-8 338.0
Is FdUMP[10] Selective For
Certain Malignancies?
 Ovarian, NSCLC
Cell Line
FdUMP[10]
5FU
1.00 x 10-8
3.98 x 10-6
5.01 x 10-7
6.31 x 10-6
COLO205
1.25 x 10-7
6.31 x 10-6
SW620
5.01 x 10-5
2.51 x 10-5
especially sensitive to
FdUMP[10]
HCT116
 Colorectal less
sensitive to
HT29
FdUMP[10]
FdUMP[10] Efficiently Inhibits TS in
HT29 Colon Cancer Cells
24 h
48 h
72 h
5FU (4 x10-6)
5.3%
11.5%
23.4%
FdUMP[10] (4 x 10-8)
0.01%
2.2%
14.2%
Inhibition of TS by FdUMP[10]
Results in DNA Damage
-O
3'-OH
H
HO
H
H
H
H
O
N
O
N
H
O
-O
P
O
H
H
H
F
O
O
-O
H
N
H
H
F
O
O
H
O
-O
N
H
O
H
O
N
O
H
F
N
H
O
-O
H
H
OH
H
O
H
F
N
O
H
O
N
O
O
P
OH
H
H
H
O
H
F
O
P
O H
H
N
O
O
P
O H
H
H
O
N
O
O
P
O H
N
H
O
O
N
H
FdUMP[6]
O
OH
P
-O
DSBs
O
H H O
HO
H
HH
N
O
NH
O
FdUMP
TS
replication
DNA
F
dUMP
dTMP
5'-OH
OH
F
O
COMET Assay of HT29 Cells
FdUMP[10] Causes Substantially Greater
DNA Damage Than 5FU
A
FdUMP[10]
1 x 10-7 M
B
5FU
1 x 10-6 M
FdUMP[10] Causes S-Phase Arrest
of HT29 Cells
Fluoropyrimidines
Ara-C, Hydroxyurea
DNA Synthesis
S
Pre-Mitotic Interval
G2
Mitosis
G1
M
Preparation
for Cell Division
Vincrisitine, Vinblastine
G0
Resting Phase
FdUMP[10] Inhibits Proliferation
and Induces Apoptosis
Apoptotic and Proliferative Activity From 40 HT29 Cells
Experiment
Control
5FU 4 x 10-6 M
FdUMP[10] 4 x 10-8 M
Apoptotic activity
0 apoptotic events
3 apoptotic events
20 apoptotic events
Proliferation
111 cell divisions
20 cell divisions
2 cell divisions
Is FdUMP[10] Safe?
 MTD for FdUMP[10] > 200 mg/kg/dose
 MTD for 5-FU ~ 40 mg/kg/dose
FdUMP[10] Inhibits Growth of
Human Tumor Xenografts
Tumor Size (mm3)
1600
Control
5-FU
FdUMP10
FdUMP10 + 5-FU
1200
800
400
0
0
7
14
21
28
35
42
Days After Tumor Inoculation
49
56
FdUMP[10] Causes Less Damage
to GI-Tract Than 5FU
Control
FdUMP[10]
5FU
FdUMP[10] + 5FU
Pre-Clinical Studies With FdUMP[10]:
Current Research Objectives
 Maximize cellular uptake and target
specifically to tumor cells.
 Validate TS-Inhibition and DNA Damage
as Mechanisms Responsible For Activity.
 Determine the Pathway Responsible For
Apoptotic Cell Death
 Demonstrate Efficacy Towards TSOverexpressing, 5FU-Resistant Tumors.
Establishing the Link Between TS
Inhibition and DNA Damage
 TS Inhibition Results in Elevated
dUTP/dTTP Ratios
 dUTP Misincorporation Initiates Futile
Cycles of DNA Repair (UDG; BER)
 Direct FdUTP Misincorporation May Also
Be Important
 Involvement of Topoisomerase I?
Top I Regulates Topological State of
DNA For Transcription & Replication
 Circular DNA is supercoiled for
compaction
 Supercoils relieved for access,
regenerated for compaction
 Top1 Breaks One Strand, Passes Intact
Strand Through Break
 Alters Linking Number by +/- 1
Topoisomerase I: Target for
Nucleoside Analogs?
 Top1 is the cellular target for
camptothecins (CPTs).
 CPTs Stabilize Top1 Cleavage Complexes
 DNA Damage Occurs When Stabilized
Cleavage Complexes Interact With
Replication or Transcription Apparatus
 Do Nucleoside Analogs Affect the Stability
of Top1 Cleavage Complexes?
AraC and dFdC: 2’-Deoxycytidine
Analogs with Distinct Mechanisms and
Spectrums of Activity
NH2
N
O5'
H2'
H3'
N
N
N
OH
O
N
O5'
O
O5'
N
H3'
H3'
O
O
O
H2''
H2''
O3'
NH2
NH2
O3'
F
O3'
F
O
Nucleoside Analogs Inhibit Key Enzymes
And Are Incorporated Into DNA
FdUMP
dFdCDP
UDP
CDP
GDP
ADP
RR
TS
dUDP
dCDP
dGDP
dADP
dUMP
dTMP
dTTP
dCTP
dGTP
dATP
S-Phase
AraCTP
dFdCTP
FdUTP
Fluoropyrimidines
Ara-C, Hydroxyurea
DNA Synthesis
DNA
S
Pre-Mitotic Interval
Polymerase Pausing
G2
Mitosis
G1
M
Preparation
for Cell Division
Vincrisitine, Vinblastine
G0
Resting Phase
Top1 Inhibition
Relevance of Top1 Inhibition For
Efficacy of dFdC, AraC
 Top1 Cleavage Complexes Detected in
Human Leukemia CEM Cells Treated With
dFdC, AraC
 P388/CPT45 Top1-Deficient Cells 5-fold
resistant to dFdC, 7-10 fold resistant to
AraC
Induction of Top1 Cleavage
Complexes In Vitro
37mer
14mer
23mer
5' - GATCTAAAAGACTTGGAAAAATTTTTAAAAAAGATCA*
3' - CTAGATTTTCTGAACCTTTTTAAAAATTTTTTCTAGT
+1 +2
A – DNA alone
B - top1
C - top1, CPT
Site-Specific Effects of AraC and
dFdC on Top1 Cleavage
 4-6 Fold Enhancement of Top1 Cleavage




Complexes When AraC in +1 Position
AraC Enhancement Primarily Due to Inhibition of
Top1-Mediated DNA Religation
5-7 Fold Enhancement of Top1 Cleavage
Complexes When dFdC in +1 Position
dFdC Enhances Formation of Top1 Cleavage
Complexes
dFdC Induced One New Top1 Cleavage Site
Observed In Presence of CPT
Structure of Top1 Cleavage Complex
With AraC in +1 Position of the NonScissile Strand
RO
OP
O
O
-1
OH
O
Top1
N
N
O
NH2
+1
N
OR
P O
-O
N
HN
OH
OP
O
O
NH2
O
O
O
H3'
O
NH2
H2''
N
O
N
N
H3'
O
H2''
O3'
-O P O
OR
-1
O
H2'
O5'
N
HN
N
O3'
-O P
OP
O
O
N
O5'
O
+1
OR
Structural Basis For Effects of AraC
and dFdC On Top1 Cleavage
 NMR Structures of AraC- and dFdCSubstituted Model Okazaki Fragments
 NMR Studies of 31mer DNA Hairpin
Containing a Single Top1 Cleavage Site
 Preparation of 31mer DNA Hairpin With
dFdC and AraC Substitution
AraC-, dFdC-Substituted Model
Okazaki Fragments
NMR Structures of dFdC-, and AraCSubstituted Model Okazaki Fragments
AraC-, dFdC-Substitution
Destabilizes Duplex Structure
 [OKA] Tm 46.8 oC
 [AraC] Tm 42.4 oC
 [GEM] Tm 41.2 oC
AraC Sugar Pucker Differs From
dC in Model Okazaki Fragment
P ~ 110o
P ~ 160o
AraC Sugar Pucker Disrupts Base
Stacking in Adjacent Strand
Structural Effects of AraC:
Implications For Top1 Cleavage
 AraC Adopts C2’-endo Sugar Pucker
 Stereoelectronic Effects Cause Arabinosyl
Sugar to be More Rigid Than 2’Deoxyribose
 Rigidity of Arabinosyl Sugar May Inhibit
Religation of Top1 Cleavage Complexes
dFdC Adopts C3’-endo Sugar Pucker
With Altered Electrostatic Surface
Relative to dC
P ~ 30o
P ~ 110o
Electrostatic Surface of dFdC and
dC in [GEM] and [OKA]
Structural Effects of dFdC:
Implications For Top1 Cleavage
 dFdC Adopts C3’-endo sugar pucker with
altered electrostatic surface relative to dC
 Electronegativity of Fluorines in dFdC may
contribute to Enhanced Formation of Top1
Cleavage Complexes
 Relative Flexibility of dFdC Allows Facile
Re-Ligation to Occur
Top1: Target for FdUMP[10]?
Conclusions
 FdUMP[10] Is a Prototype of a New
Fluoropyrimidine That Is Safer and More
Effective Than 5FU
 FdUMP[10] Blocks HT29 Cells in S-Phase
and Causes DNA Damage
 Top1 May Be Involved in Mediating
Damage Resulting From FdUMP[10]
Exposure
Acknowledgements
 Current Group: Cui Wei, Xi-an Mao,




Debbie Boles.
Former Group: Jinqian Liu, Changnian Liu,
Parag Sahasrabudhe.
Top1: Yves Pommier (NCI); Phillipe
Pourquier (Bordeaux).
Structural Biology: Tom James (UCSF);
Dave Konerding UCSF.
Funding: WFUSM; CCCWFU