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Mass spectrometry &
drug discovery
5조
Shin Jihye
Kim Boram
Lim Jongsun
Lee Sangwon
Basic principle in MS
The uses of mass spectrometry
Doping-test
Pesticide residue-test
Disease marker discovery
Drug discovery
Bibliographical Analysis
1st nanoMate paper
Development automated
HDX methods
Novel prize
for ESI
Commercially MS availabel
Applications of MS
in the Drug Discovery Process
Paper #1
Characterization of Platinum Anticancer Drug ProteinBinding Sites Using a Top-Down Mass Spectrometric
Approach
Christian G. Hartinger,*,†,‡ Yury O. Tsybin,† Jens Fuchser,§ and Paul J.
Dyson*,†
• Fourier transform ion cyclotron resonance mass(FTICR-MS)
spectrometric approach
• collision-induced dissociation (CID)
• Infrared multi-photon dissociation(IRMPD)
Introduction
•
Characterizing directly the
binding sites of platinum
complexes to Ub using a topdown approach employing
Electrospray ionization Furier
transform ion cyclotron
resonance( FT-ICR-MS/MS).
The high resolving power
and mass accuracy of FTICRMS have been used to
study metallodrug-protein
interactions, although no
attempts were made to locate
metallodrug binding sites.
Result
•
•
Broad-band ESI-FT-ICR-MS
spectra of Ub incubated with
cisplatin, transplatin, and
oxaliplatin demonstrate
distinct differences in the
extent and composition of the
observed species.
A series of mono-, bis-, and
trisadducts were observed.
• Oxaliplatin was found
attached to the Nterminal 1Met-Gln2
sequence in which the
chxn ligand remained
bound to the platinum
moiety.
• Pt-[1Met-Gln-Ile-Phe4]
- The smallest platinumcontaining fragment
-the platinum moiety is
probably bound to the
methionine residue,
confirming the Nterminal attachment of
the platinum center
- fragmentation of Ub +
[Pt(NH3)] resulted in the
cleavage of the ammine
ligands from the
platinum center.
•
19Pro-Ser-Asp-Thr-Ile-Glu24
peptide
(a few platinum-modified Cterminal peptides
-accessible at the protein
surface
-containing a number of
potential donor atoms.
-potentially coordinate to
platinum so the precise
binding site can be
speculated.
conclusion
• The standard method for determining metal
binding sites to proteins, especially in the domain
of metal-based drugs.
• It should ultimately provide data that facilitate drug
design and discovery.
Paper #2
Target
Telomere :
Protects the end of the chromosome.
Necessarily occurs during chromosome
replication.
Telomerase & Cancer
Telomerase, the enzyme complex
responsible for elongating telomeres, is
activated in approximately 90% of
tumors.
The Formation of quadruplexes in
telomeres has been shown to decrease
the activity of the enzyme telomerase,
which is responsible for maintaining
length of telomeres and is involved in
around 85% of all cancers. This is an
active target of drug discovery.
Fig.1 Human telomere caps
http://en.wikipedia.org/wiki/File:Telomere_caps.gif
Telomeric DNA sequence & BRACO-19
Molecular modeling of drug–DNA
interactions: Virtual screening to
structure-based design
Dik-Lung Maa, , , Daniel Shiu-Hin Chana,
Paul Leea, Maria Hiu-Tung Kwana, ChungHang Leung Biochimie 2011
Fig. 2 (Left) Representation of a 45-mer telomeric DNA sequence, with two Gquadruplexes and a trisubstituted ligand (compound 15) in a low-energy
docked position at the interface between the two quadruplexes. The ligand is
shown as a solvent-accessible surface
colored by charge. (Right) The BRACO-19 molecule.
DNA : 22-mer human telomeric G-quadruplex sequence d[AGGG(TTAGGG)3] (tel22)
Synthesis of ligands
Scheme 1 Synthesis of ligands 5–15: (i) 4-chlorobutyryl chloride (4 eq.), 4℃ to rt, overnight; (ii) pyrrolidine (5 eq.), 4℃ to rt,
overnight; (iii) chloroacetyl chloride/3-chloropropionyl chloride, TEA (2 eq.), THF, 4℃ to rt, 2 h; (iv) piperidine/pyrrolidine/
diethylamine (3 eq.), THF, 4℃ to rt, overnight; (v) anhydrous THF, H2, Pd/C (10% m/m), rt, overnight; (vi) tBuONO (2.5 eq.),
HCl (5.5 eq.), THF, 0℃, 1.5 h; (vii) NaN3 (3 eq.), H2O, 0℃ to rt, overnight; (viii) 1,3,5-triethynylbenzene, compound (4a–k) (4
eq.), CuSO4 (0.05 eq.), sodium ascorbate (0.2 eq.), bathophenanthrolinedisulfonic acid disodium salthydrate (0.1 eq.), H2O–
tBuOH, 110 ℃, microwave, 15 min.
List of Ligands
General procedure for the synthesis of tri-click compounds
MS Experiment & Affinity calculate
MS Experiment schme
Determination of the equilibrium
dissociation constants (Kd)
Electrospray mass spectrometry to study drug-nucleic acids interactions
F. Rosu, E. De Pauw and V. Gabelica, Biochimie, 2008, 90, 1074
MS peak
Figure S1 Representative ESI-MS spectra of mixtures between 5 μM telomeric Gquadruplex
dAGGG(TTAGGG)3 (tel22) and 5 μM ligand 8 (a), 12 (b), 15 (c), or 10 μM of the same ligand (d-f).
The free tel22 and the complexes were detected at charge states 6- to 4-The most intense charge
state (5-) was used for determining the Kd’s.
Dissociation constants
Figure S2 The excellent correlation between the Kd for
ligand binding to tel22 and determination and the
fraction free tel22 found in the competition experiment
shows that the competition experiment is a reliable way
to assess relative ligand binding affinities for several
DNA structures at the same time.
Paper #3
Detection, Characterization, and
Screening of Heme-Binding
Molecules by Mass Spectrometry
for Malaria Drug Discovery
Sangwon Lee
Introduction
• Malaria
– A recurring challenge for health policies, medicinal research,
and the pharmaceutical industry
– Vector-born
• The fact that most of the drugs
currently used to cure or prevent
contributes to the
critical situation in terms of resistance
• The emerging resistance to artemisinine analogs is of
primary concern
malaria have been known and used for decades also
Target-based approaches
• Target in malaria is the strictly parasite-specific, heme
detoxification pathway.
• The massive release of free, toxic ferriprotoporphyrin
IX is handled by the parasite via its biomineralization
into hemozoin, a supramolecular assembly of heme
dimers
Target-based approaches
• Consequently, the inhibition of synthetic hemozoin
formation is the basis for a variety of tests aimed at
the discovery of antimalarial compounds
Term
• Fragmentor voltage
– The transfer voltage applied between the end of the transfer
capillary and the first skimmer placed in front of the focusing
octopole and the quadrupole analyzer
– The higher the voltage, the faster ions are accelerated and the higher
the energy of subsequent intermolecular collisions
Simple/triple quadrupole
• Single quadrupole instrument
– decrease of heme abundance signal until a reproducible plateau of 41%
– Heme molecules are resistant to fragmentation
•
Triple quadrupole instrument
– The aceeleration is performed by the second quadrupole
– Collisions with the inert gas are much more energetic
– Complete disapperance of the heme signal
Relative stabilities of heme-drug
adducts
Conclusion
• This method
– based on mass spectrometry to detect and
characterize compounds able to bind heme.
– based on the interaction of small molecules with
heme which is the main mechanism of action of
most efficient known antimalarials. (not correlate to
an activity in vivo)
– can detect a heme-binding compound within a
complex mixture
– gives structural information on the compound,
opening the path for biological dereplication of
natural extracts