convenor: dr. fawaz aldabbaghglycam.org/legacy_courses/2013/molecularmodeling2013/... · 2013. 8....
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Some Heterocyclic DrugsSome Heterocyclic Drugs
Convenor:Dr. Fawaz AldabbaghDr. Fawaz Aldabbagh
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Learning Outcomes• The student should be able to identify and write the structure of all nucleotides and nucleosides derived
f DNA d RNA Al k th t t f ATP d NAD+from DNA and RNA. Also know the structure of ATP and NAD+.
• Student should know the numbering and stereochemistry about a ribose or deoxyribose ring.
• The student should be familiar with primary and secondary structure of DNA, biosynthesis and p y y , y
replication.
• The student should be familiar with the Watson‐Crick Model of DNA (B‐DNA)
• The student should have an understanding of the mode of action of AZT used on HIV patients.
• The student should be able to propose a synthesis of AZT from thymidine
Th t d t h ld b bl t bi th i f S d l thi i d d ib it• The student should be able to propose a biosynthesis for S‐adenosyl methionine, and describe its
methylation to form caffeine in terms of a reaction mechanism.
• The student should describe the biosynthesis of cAMP from ATP with a reaction mechanism.y
• The student should be able to derive the structure of NADPH from NADH, and write mechanisms for
asymmetric reductions.
• The student should be able to write the “ping‐pong” mechanism for NQO1 reduction of quinones if
provided with the isoalloxazine ring of FADH2.
• The student should be able to write the mechanism for mitomycin C bioreductive activation (one and• The student should be able to write the mechanism for mitomycin C bioreductive activation (one and
two‐electron), and explain the formation of cross‐linked adducts with DNA.2
Two Nucleic Acids (Polymers ‐ polynucleotides) –deoxyribonucleic acid (DNA) anddeoxyribonucleic acid (DNA) and ribonucleic acid (RNA)
Mild degradation yields monomeric units Nucleotides
Complete degradation yields1 A Heterocyclic Base
Pyrine or 1. A Heterocyclic Base
2. A five Membered MonosaccharidePyrimidine
3. A Phosphate ion
OD‐Ribose or 2’‐deoxy‐D‐ribosePO
O
O
O3
The Phosphate esterThe Phosphate ester can be at C-5’ or C-3’
ONO
5'
Heterocyclic Base
N- Glycosidic linkage
Hydolysis of Phosphate
OCOP
O
O 1'
2'3'
4'
N- Glycosidic linkage
HHNucleosideOH OH
Heterocyclic Base
ON
COP
O
O
O 1'4'
5' N- Glycosidic linkage
H H
OH H
O 2'3'H
4
H
O
N
NH2 PurinesN
N N
N
NH
HN
N N
N
N N NH2
H Guanine (G)
NH Adenine
(A)
H
O
N
NH2Pyrimidines
H3C H
O
N
N
ON
N
O N
N
O
Thymine (DNA only) (T)
Cytosine(C)
H HUracil (RNA only)
(U)
H
(T) (C) (U)
5
N
NH2
NHN
O
N
NN
N
HO
NH
N
N
NH2N
HO
OHH
HH
HOO
HHHH
HO
Nucleosides that can be obtained
HOHHH HOH
NH2
2'-Deoxyadenosine 2'-DeoxyguanosineOobtained
from DNA N HN
O
O
HONO
O
HONO
HOH
HH
HH
O
HOH
HH
HH
2'-Deoxycytidine6
ThymidineDraw Uridine.
DNA and RNA Biosynthesis
Nucleotide Triphosphate
New nucleotides are added at the 3’‐end5’‐3’ direction growth
PhosphodiesterDNA is synthesized by enzymes called DNA‐polymerase
RNA is synthesized by enzymes called RNA‐polymerase
pbond
y y y p y
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NN
NH2Adenine
NN
OHH
OHN
O5'
i SHOHH
PO
O O
NO
O OThymine
5'
3'
Base Sequence is
Primary Structure
O O
HO
HHHH
PO
NH
N
N
NH2N
O
3'
Base Sequence is the Genetic Code
PO
O O
HO
HHHH
O Guanine5'
3' HO
PO
O
OA T G5' 3'
Nucleotides are held together by phosphate ester linkages.Phosphate esters link 3’‐ OH of one ribose (or deoxyribose) with the 5’‐OH of another. This makes the nucleic acid a long unbranched h i i h b kb f d h h i i h h lichain with a backbone of sugar and phosphate units with heterocyclic bases protruding from the chain at regular intervals.
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Secondary Structure The width of a double‐stranded
OH3C NNH
H
The width of a double stranded molecule is constant:Purine must pair up with a pyrimidineIf larger purines are paired the strand
N H
3
N N
N If larger purines are paired, the strand would bulgeIf smaller pyrimidine hydrogen‐bond the
d ldNO
N
Thymine
Adeninestrand would contract
Chargaff’s rule:[adenine] = [thymine]y
N NO
H
H
[guanine] = [cytosine]
Explained by pairing them up on
NN
NN NH
Complementary Strands
If you know the sequence of bases on N
ON
Cytosine
GuanineNH
H
one strand, you can figure the sequence of bases on another strand
H
Two Complementary Chains Result 9
Double Helix is the Secondary Structure of DNATwo nucleic acid chains are held together by weak H-bonds between bases of opposite strands
Wound into a helix with a common axisThe strands are anti-parallel running in opposite directions
The base pairs are on the inside of the helix and the sugar-phosphate backbone is on the outsidethe sugar-phosphate backbone is on the outside34A repeating unit contains 10 successive nucleotide pairs
Phosphate Sugar backbone is regular basePhosphate-Sugar backbone is regular, base pairs can assume many different permutations
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Watson-Crick Model of DNA
(1953)
3’‐end 5’‐end 11
Features of the DNA‐double helix
•The DNA strands are not linear, but are twisted into a helix around a common axis.
•The base pairs are planar and parallel to each other on the inside of the helix.
•There are favourable Van der Waals interactions between mutually induced dipoles of adjacent
base pairs. These weak attractive forces are known as stacking interactions, when added
together contribute significantly to the stability of the double helix.
•The sugar‐phosphate backbone is wrapped around the bases.
•The phosphate OH group has a pKa of about 2, so it is in its basic form (negatively charged) at
physiological pH. This repels nucleophiles, so preventing phosphate linkages from cleavage.
•The DNA must remain intact for the lifetime of the cell.
Q tiQuestion:5’‐G‐G‐A‐C‐A‐A‐T‐C‐T‐G‐C‐3’
a What is the sequence of bases in the complementary stranda. What is the sequence of bases in the complementary strand.b. What base is the closest to the 5’‐end of the complementary strand.
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Biosynthesis of DNA: Replication
Prior to cell division, the DNA material in the original cell must be duplicated so that after cell division, each new cell contains the full amount of DNA material. The process f d l ll ll d l h l dof DNA duplication is usually called replication. The replication is termed semi‐
conservative since each new cell contains one strand of original DNA and one newly synthesized strand of DNA. The original polynucleotide strand of DNA serves as a t l t t id th th i f th l t l l tid f DNAtemplate to guide the synthesis of the new complementary polynucleotide of DNA.
Fragments are joined by DNA‐ligase
h f f h ll d f hThe genetic information of a human cell is contained in 23 pairs of chromosomes.Each chromosome is composed of several thousand genes (segments of DNA).The total DNA of a human cell – the human genome‐contains 3.1 billion base pairs. 13
Alkaline hydrolysis of RNA
‐ RNA is easily cleaved; synthesized when needed, and degraded once it has served its purpose.
Important: 2’ and 3’‐OH are cis in ribose
Question:
2 3‐Cyclic phosphodiester formed upon RNA hydrolysis can react further with water to
Important: 2 and 3 ‐OH are cis in ribose
2,3‐Cyclic phosphodiester formed upon RNA hydrolysis can react further with water to
give a mixture of 2’‐ and 3’‐phosphates.
Propose a mechanism for the reaction.Propose a mechanism for the reaction.
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Summary of Nucleic Acids Function
DNA encodes an organisms hereditary information and controls cell’s growth and division
DNA genetic information is transcribed (transcription) to RNA
RNA translates (translation) the information for the synthesis of all proteins
Proteins are required for cellular structure and function
Each three‐base sequence‐a codon‐specifies the particular amino acid to be incorporated
into a protein
The codons and the amino acids they specify are known as the genetic code
E.g.
‐ACA‐ACT‐ ‐UGU‐UGA‐ add a cysteine and then stop
DNA triplet RNA triplet end of a proteinDNA triplet RNA triplet end of a protein
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Modified Nucleosides: Targeting Retrovirsuses
AIDS (acquired immunodeficiency syndrome) & herpes are caused by retroviruses
Acyclovir(Zovirax)
Anti‐herpes drugAZTBrand Name: Retrovir (Retrovis)
HN
O
CH3
Manufacturer: GSK
HONO
OHH
HH
HOAZT
3‐TCLamivudine (anti‐AIDS drug)
HN3HH
3'-azido-2'-deoxythymidineActive against AZT‐resistant viruses 16
AZT interferes with retrovirus DNA synthesis (reverse transcription).
3’‐Azido‐2’‐deoxythymidine (AZT)
AZT interferes with retrovirus DNA synthesis (reverse transcription).AZT are taken up by T‐lymphocytes, cells which are particularly susceptible to human immunodeficiency virus (HIV), the retrovirus that causes AIDS. AZT is converted to AZT‐triphosphate.p p
NH
O
ON
OOP
O
OPOPO
O O
O
HN
HH
HH
N
O O O
N
Reverse transcriptase of the virus binds more strongly
dTTP
Reverse transcriptase of the virus binds more stronglyTo AZT‐triphosphate than to dTTP
AZT rather than T is added to the DNA chain, because there is no 3’‐OH in AZT, no additional nucleotides can be added to the DNA chain.
The host cell’s DNA‐polymerase should prefer dTTP for its DNA synthesis17
Stereochemistry of Nucleosides
AZTSN2thymidine
Cyclic nucleoside
O O
HN
NOI
N
NOthymidine
AgOAc, MeCN
O
I
O
Oy
HO HO Cyclic nucleoside18
NH2
ATP Chemistry
NN
NN
O
OP
HOO
Adenosine‐5’‐monophosphate
OHOH
OH
Adenosine Monophospahe (AMP)
NN
NH2Adenosine Monophospahe (AMP)
NN
OP
OO
PO
O
PHO
OY
X
O
OHOH
P
OH
P
OH
P
OH
Y i h d l hilOHOH
Adenosine Triphosphate (ATP)
Y‐ is a hard nucleophileX‐ is a soft nucleophile
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S‐Adenosyl methionine (SAM): Nature’s Methylating AgentNH2
NN
2
SMe
HO2C
NH2H
NN
O
OP
OO
PO
O
PHO
O
2
O
OHOH
OHOHOH
Adenosine Triphosphate (ATP)
NH2SN2SAM is formed by reaction of methionine with ATP
NN
NH2HMe
NN
O
SHO2C
Me
OHOHSAM
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S‐Adenosyl methionine (SAM): Nature’s Methylating Agent
Purine Stimulants
Theobromine(chocolate) Caffeine
(coffee & tea)(coffee & tea)
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cAMP (cyclic adenosine monophosphate)
cAMP helps control of blood clotting and acid secretion in the stomach
ATP is used to make cAMPATP is used to make cAMP
Cyclic AMP (cAMP)Adenylate cyclase
N.B. Fully account for ring‐stereochemistry in cAMP
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Biology RevisionMany enzymes require a component, other than the protein for their catalytic activity:‐co‐factor (e.g. FAD in NQO1)The remaining protein (apoenzyme) has no catalytic activity.
A co‐factor that is firmly bound to the apoenzyme is termed a prosthetic group, and most contain a metal centre
A co‐factor that is bound loosely to the apoenzyme, and can be readily separated from it, is called a co‐enzyme (e.g. NAD(P)H)
Vit i ti l f lif H t t k it i i th i di t b th tVitamins are essential for life. Humans must take vitamins in their diet, because they are not made by the body. There are 14 vitamins.
Nicotinamide(Vit i B )
Pyridoxine(Vit i B )
Vitamin B (complex):Series of water soluble vitamins important
(Vitamin B3) (Vitamin B6)p
in metabolism, amongst many other things& prevent pancreatic cancer 23
Nicotinamide Adenine Dinucleotide (NAD+) O
In metabolism compounds
N
NH2
PO
OO
O Nicotinamide (from vitamin B3)
In metabolism, compounds accept and donate electrons in redox reactions.
NH2O
O
OHOH= NAD The reaction is reversible. The
co‐enzyme can continuously cycle between NAD+ and NADH
N
NN
N
NH2
O
cycle between NAD and NADH forms without being consumedadenine
NN
OOPO
O NH2
Oreduction
H H
NH2
O
OHOH N
Ribo
oxidation N
RiboNADP has a phosphate Ribo
ADPADP
NAD 2 H + 2e NADH 24
Enzyme Active Site allows NADH Stereoselective Reduction
Racemic lactic acidRacemic lactic acidPyruvic acid
S(+)lactic acid
The enzyme binds both the substrate and NADH in a specific way so that the hydride is delivered to one enantiotopic face of the ketone
Write mechanisms for both reactions25
Flavin adenine dinucleotide : FADH2
Ribitol made from the reduction of Ribose
Isoalloxazine ringg
Riboflavin (vitamin B2)FADH2FAD
Ribitol + islloxazine
ADP
FADH2 not really a dinucleotide26
deficiency results in skin inflammation
NAD(P)H: Quinone Oxidoreductase 1 (NQO1)
Functions of NQO1
•Detoxification by removal of quinones,nitro, azo, iminoquinone compounds
•Bioreductive activation of anti‐tumorquinone prodrugs
• 4 homomonomers interlocked as dimers
• Two noncovalently bound FAD, held by residues from both monomers
• The FAD is held such that its isoalloxazine ring forms the floor of the active site cavity
• Ping‐pong mechanism has two distinct steps: 1. hydride transfer from NAD(P)H to the
FAD cofactor. 2. Release of NAD(P)+ and hydride transfer to the substrate from the
reduced cofactor.
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Catalytic Cycle of NQO1: Ping‐Pong Mechanism O
N
O
OH H
Gly149N
O
OH H
Gly149
N NR
O N NR
OH
NH HH
H
NN
OH
NH
NN
OH
HO OH OH
FADH2FAD
N
H H
O
NH2OH OH
H+
substrateN
RiboseADP
O
NH2
O O OHNAD(P)Hsubstrate
N
Ribose
O
NAD(P)+
RiboseADP
R. Li et al, PNAS 1995, 92, 884628
NQO1 is over‐expressed in solid tumours
6
5.5
6
-logGI50
4.5
5logGI50
4
Cancer type
Although NQO1 is expressed in normal tissues, elevated levels occur in many human tumour g Q p , ycell lines – certain prostate cancers, melanoma & CNS cancerCorrelation of NQO1 activity with cell lines at the NCI (shown)
S. A. Fitzsimmons et al, J. Nat. Cancer Instit. 1996, 88, 259V. Fagan et al, Bioorg. Med. Chem. 2012, 20, 3223.
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Bioreductive Quinones
O
R
X
+ 1e
O
R
X- X
O
R
Nu
Semiquinone
O O OO2O2
Semiquinoneradical anion
Quinone methide
O
R
Nu
O
R
alkylated moietyO
The ,‐unsaturated enone is strongly activated towards attack by nucleophilic DNA
The biored cti e dr g design in ol es ha ing a ben lic lea ing gro p (X)The bioreductive drug design, involves having a benzylic leaving group (X)
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Hypoxic Cells associated with Solid TumoursOne‐Electron (SET) reduction is reversible and happens under hypoxic conditionse.g. NADPH‐cytochrome c reductase
~100μm
Solid T mo rs
O2 Blood Vessel
Solid Tumours
Viable Cells Hypoxic Cells
Anoxic Cells
In phagocytes (our immune system) superoxide (O ‐●) is produced in large quantities byIn phagocytes (our immune system) superoxide (O2 ) is produced in large quantities by the enzyme, NADPH oxidase for use in oxygen‐dependent killing mechanisms of invading pathogens.
Because superoxide is toxic, nearly all organisms living in the presence of oxygen contain the superoxide scavenging enzyme, superoxide dismutase, or SOD.
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Mitomycin C (MMC)
Isolated in the 1950s from Streptomyces caespitous by Japanese scientistsUsed to treat various solid tumours, also has antibiotic activity.Bioreductive: reduction leads to electrophilic sites at C‐1 and C‐10 for alkylation of DNAp yThis cross‐linking of complementary DNA strands prevents replication, thus leads to a cytotoxic response 32
Isolated MMC‐DNA Alkylation AdductsReductively activated MMC reacts with N2 position of guanine in the minor groove of DNA .
A & B are mononalkylated adducts
C is so‐called cross‐linked DNA adduct; alkylated by dG on opposite strands of DNA in 5’‐d(CG)‐3’alkylated by dG on opposite strands of DNA in 5 ‐d(CG)‐3
M. Tomasz, R. Lipman, C. Chowdary, J. Pawlak, G. L. Verdine, K. Nakanishi, Science 1987, 235, 1204 33