1. Mass spectrometry & drug discovery 5 조 Shin Jihye Kim Boram Lim Jongsun Lee Sangwon

2. Basic principle in MS

3. The uses of mass spectrometry Doping-test Pesticide residue-test Disease marker discovery Drug discovery

4. Bibliographical Analysis Novel prize for ESI 1 st nanoMate paper Development automated HDX methods Commercially MS availabel

5. Applications of MS in the Drug Discovery Process

6. Paper #1 Characterization of Platinum Anticancer Drug Protein- Binding 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)

7. Characterizing directly the binding sites of platinum complexes to Ub using a top- down approach employing Electrospray ionization Furier transform ion cyclotron resonance( FT-ICR-MS/MS).  The high resolving power and mass accuracy of FT-ICRMS have been used to study metallodrug-protein interactions, although no attempts were made to locate metallodrug binding sites. Introduction

8. 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. Result

9. Oxaliplatin was found attached to the N-terminal 1Met-Gln2 sequence in which the chxn ligand remained bound to the platinum moiety.

10. Pt-[1Met-Gln-Ile-Phe4] - The smallest platinum- containing fragment -the platinum moiety is probably bound to the methionine residue, confirming the N-terminal attachment of the platinum center - fragmentation of Ub + [Pt(NH3)] resulted in the cleavage of the ammine ligands from the platinum center.

11. 19Pro-Ser-Asp-Thr-Ile-Glu24 peptide (a few platinum-modified C- terminal 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.

12. 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.

13. Paper #2

14. 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

15. Telomeric DNA sequence & BRACO-19 Fig. 2 (Left) Representation of a 45-mer telomeric DNA sequence, with two G- quadruplexes 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) 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, Chung- Hang Leung Biochimie 2011

16. 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, H 2, Pd/C (10% m/m), rt, overnight; (vi) t BuONO (2.5 eq.), HCl (5.5 eq.), THF, 0 ℃, 1.5 h; (vii) NaN 3 (3 eq.), H 2 O, 0 ℃ to rt, overnight; (viii) 1,3,5-triethynylbenzene, compound (4a–k) (4 eq.), CuSO 4 (0.05 eq.), sodium ascorbate (0.2 eq.), bathophenanthrolinedisulfonic acid disodium salthydrate (0.1 eq.), H 2 O– t BuOH, 110 ℃, microwave, 15 min.

17. List of Ligands General procedure for the synthesis of tri-click compounds

18. MS Experiment & Affinity calculate Determination of the equilibrium dissociation constants (K d ) MS Experiment schme Electrospray mass spectrometry to study drug-nucleic acids interactions F. Rosu, E. De Pauw and V. Gabelica, Biochimie, 2008, 90, 1074

19. 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.

20. 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.

21. Detection, Characterization, and Screening of Heme-Binding Molecules by Mass Spectrometry for Malaria Drug Discovery Sangwon Lee Paper #3

23. 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 malaria have been known and used for decades also contributes to the critical situation in terms of resistance The emerging resistance to artemisinine analogs is of primary concern

24. 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

25. 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

26. 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

27. 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

28. Relative stabilities of heme-drug adducts

29. 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