1. Synthesis of a Bi-substrate Inhibitor of Thymidylate Synthase
Jessica Hossa, Svetlana Kholodar and Amnon Kohen
Department of Chemistry, University of Iowa, Iowa City IA, 52242
Thymidylate Synthase Enzyme Intermediate B Proposed Synthesis of an Analog
of Int.-B• Thymidylate Synthase catalyzes the conversion of 2ʹ-
deoxyuridine 5ʹ -monophosphate (dUMP) to 2ʹ-deoxythymidine 5 -ʹ
monophosphate (dTMP), an essential bioprecursor of DNA
replication
Two Alternative Mechanistic
Pathways of Thymidylate Synthase
Catalyzed Reaction
• Thymidylate synthase (TS) is targeted for potential cancer therapy
treatment as well as antibacterial chemotherapy.
• A known ‘anti-cancer’ drug that targets Thymidylate Synthase is 5-
fluorouracil, however, some colon cancers have gained resistance
against 5-fluorouracil and the drug lacks specificity to Thymidylate
synthase.
• Many studies have been performed and are currently in progress
involving development of new inhibitors that are more specific to the
enzyme and have potential to help patients who have acquired a
resistant strain of cancer.
Figure (2) Metabolic Pathway to DNA biosynthesis
• In Figure (3) the Thymidylate Synthase catalytic mechanism is shown.
The reaction is initiated by nucleophilic activation of the substrate upon
formation of a covalent bond to the active site cysteine (Step 1) followed
by an attack by the methylene of the co-substrate with C5 of dUMP
(Step 2).
• The following mechanism involves a proton abstraction (steps 3A or 3B)
with water serving as the direct acceptor and a hydride transfer to form
the exocyclic methylene intermediate. Step 5 proceeds with a concerted
hydride transfer and C-S cleavage to form dTMP (reported from previous
QM/MM calculations)(1)
.
• Two alternative mechanisms have been considered for the proton
abstraction step 3. Major difference between these two mechanisms is
the nature of generated intermediates.
• Previously reported QM/MM calculations (1)
suggested that deprotonation
of C5 leads to the cleavage of the C-S bond which shows a new non-
covalently bound reaction intermediate (Intermediate B).
• Further studies(2)
demonstrated that chemically synthesized Int.-B is
behaving like a reaction intermediate. Specifically, Int.-B was found to
partition between substrates (dUMP and CH2H4Folate) and products
(dTMP and CH2Folate) of Thymidylate Synthase catalyzed reaction.
Additionally, Int.-B was found to react at a rate faster than the substrate
of the reaction (CH2H4Folate).
• Int.-B provides new drug design opportunities since it is non-covalently
bound do the enzyme.
• Previously, few analogs of Int.-B have been synthesized and found to
inhibit Thymidylate Synthase:
• The proposed synthesis is for diethyl (4-(((e,4-diamino-5,6,7,8-
tetrahydropyrido[3,2-d]pyrimidin-6yl)methyl)amino)benzoyl)
glutamate
• Similar analog of Int.-B has already been synthesized as a mixture of
diastereomers and reported as being a potent competitive inhibitor of
Thymidylate Synthase.
Figure (4) Structure of Intermediate A
Figure (5) Structure of Intermediate B
References
Figure(7) Synthesis for Analog of Intermediate B
1. Wang Z. et al, Biochemistry, 2013, 52, 2348.
2. Kholodar S. and Kohen A., J.Am.Chem.Soc., (Communication), 2016,
138, 8056.
3. (a) Gupta V.S. and Heunnekens F.M., Biochemistry, 1967, 6, 2168;
(b) Park J.S. et al., J. Med. Chem. , 1979, 22, 1134; (c) Srinivasan A.
et al., J. Med. Chem., 1984, 27, 1710.
• Although compound (4) is a potent nanomolar inhibitor it has only been
tested as a mixture of diastereomers at the position C6.
• Molecular basis of inhibition by analogs (1)-(4) still remains unclear (i.e.
covalent or non-covalent inhibition).
Ki - 37 μM 0.75 μM 58 nM
(human TSase) (mixture of diastereomers)
Figure (6) Structures of Previously Synthesized Analogs of
Int-B and their corresponding inhibition constants3
(1) (2) (3) (4)
1. Synthesize analog of Int.-B as a single diastereomer
2. Test this analog as inhibitor of Thymidylate Synthase
3. Obtain a crystal structure of Thymidylate Synthase complex
with this analog
•According to the traditional mechanism (A) proton abstraction step 3A is
followed by formation of covalently-bound enolate intermediate Int.-A.
Intermediate A
Figure (1) Crystal Structure of Thymidylate Synthase
Ternary Complex with 5-fluoro dUMP (green) and
methylenetetrahydrofolate (yellow)
Current Study Objectives
O
OH
O
N
NH
O
O
O
OH
O
N
NH
O
O Thymidylate Synthase
CH2H4folate H2 folate
H4folate
HN
N N
H
H
N
HN
N N
H
H
N
HN
N N
H
N
O
O
N
H
N
H2N
O
R'
H
N
R'
NADPH
NADP+
Dihydrofolate reductase
Serine
Glycine
Serine hydroxymethyl
transferase
R'
P
O
O
HO
P
O
HO
O
O
OH
O
N
NH
O
O
P
O
O
O
3
DNA
dTTP
dTMP
dUMP
R'=
O
N
H
O OH
OH
O
H2N
H2N
Figure (3) Thymidylate Synthase Mechanism