Why Chemical Biology!! School of Chemistry, Seoul National University Seung Bum Park, Ph.D. What is Chemical Biology? CHEMISTRY: a science that deals with the composition, structure, and properties of substances and with the transformations that they undergo BIOLOGY: a branch of knowledge that deals with living organisms and vital processes BIOCHEMISTRY: chemistry that deals with the chemical compounds and processes occurring in organisms Reference: 1 What is Chemical Biology? CHEMICAL BIOLOGY: The use of chemical approaches to study or emulate biological systems. CHEMICAL APPROACH: The creation or perturbation of structure to generate or alter function. Roadmap in Chemical Biology Generate a new functional molecule DESIGN Engineer Natural Solution • De novo • Protein Protein Design Engineering • Rational Drug (GFP, inteins) Design • Cell Surface Engineering EvolutionBased Approaches Study a Natural Molecule Chemical Combinatorial Approach on Approaches Biopolymers • Protein • Site-directed Evolution Mutagenesis • Catalytic Igs • Unnatural • Combinatorial Amino acid Small molecule Mutagenesis Approaches • Molecular Evolution • Genomics • Proteomics Perturb Using Small Molecules • Substrate Analogs • Chemical Genetics 2 Roadmap in Chemical Biology Generate a new functional molecule DESIGN • De novo Protein Design • Rational Drug Design Engineer Natural Solution EvolutionBased Approaches • Protein Engineering (GFP, inteins) • Cell Surface Engineering Study a Natural Molecule Chemical Combinatorial Approach on Approaches Biopolymers • Site-directed • Protein Mutagenesis Evolution • Catalytic Igs • Unnatural • Combinatorial Amino acid Small molecule Mutagenesis Approaches • Molecular Evolution • Genomics • Proteomics Perturb Using Small Molecules • Substrate Analogs • Chemical Genetics How Viagra Works ViagraTM (sildenafil citrate, Pfizer); 2002 sales: >$1,000,000,000 Discovered via rational drug design aimed at inhibiting “type 5” cGMP Phosphodiesterase (PDE) for hypertension treatment Fortuitously discovered that type 5 PDE’s also hydrolyze cGMP in the human corpus cavernosum (penile smooth muscle) 3 Type 5 cGMP Phosphodiesterase H N H2N O O P O O OH N OH N H N H2N O N O HO P O OH HO O N phosphodiesterase O N N OH Type 5 PDE’s discovered in 1976 A dimer with two Zn2+-binding motifs essential for activity cGMP binds at two allosteric sites and one active site per monomer cGMP binding allows phosphorylation of Ser 92 by PKG or PKA, which enhances phosphodiesterase activity J. Corbin & S. Francis J. Biol. Chem. 274, 13729 (1999) Chemical Lineage of Viagra: Great Grandparents and Grand parents Before Viagra, Zaprinast (anti-allergy drug) was the major known type 5 cGMP PDE inhibitor Zaprinast shows little selectivity, modest affinity for type 5 PDE’s First Attempt: explore a range of 2-alkoxyphenyl heterocycles Conclude: pyrazolopyrimidinone structure is promising, yielding higher potency and selectivity than Zaprinast N. Terrett & P. Ellis et al. Bioorg. Med. Chem. Lett. 6, 1819 (1996) 4 Viagra’s Parents Next, answer two questions: 1) Can we fill an enzyme binding pocket which normally binds ribose (in cGMP) with a C-3 substituent? 2) What is the effect of removing the 1-Me group? Conclude: 3-Me to 3-Pr increases affinity >10-fold, but removal of the 1-Me group reduces PDE inhibition N. Terrett & P. Ellis et al. Bioorg. Med. Chem. Lett. 6, 1819 (1996) Viagra’s Less Able Uncles and Aunts Now explore the 2’ (ortho-phenyl) substituent: Conclude: ethoxy group is strongly preferred over many other groups, including H-, HO-, nitro-, sulfonamide-, etc. Thus the H-bond between the pyrimidinone NH and the 2’ oxygen lone pair may be important, along with a small 2’ alkyl group 5 Viagra at Last Final efforts to improve the properties of these PDE inhibitors: 1) Fill the space in the PDE normally occupied by the phosphate of cGMP using substituents at the 5’ position of the phenyl ring 2) Improve the solubility of the drug by reducing its lipophilicity Conclude: a variety of Structure are tolerated at this position Sildenafil (ViagraTM) shows a strong mixture of potency, selectivity, and in vivo characteristics Why Viagra is Exciting A Successful example of rational drug design (but treats an unforeseen condition) Arteries supplying the corpus cavernosum with blood are lined with smooth muscle cells; relaxation dilates these arteries Expansion of the resulting spaces in the penis restrict the outflow of blood, increasing blood pressure and inducing an erection 6 Novel Natural Proteins (Green Fluorescent Protein, GFP) Chemistry of protein backbone rearrangements Green Fluorescent Protein - GFP chromophore formation - GFP structure - Generating new GFP variants Green Fluorescent Protein GFP: a 31kDa, 238 amino acid monomeric fluorescent protein isolated from the jellyfish Aequoria victoria Normally, the chemiluminescent protein aequorin binds to GFP; its blue emission spectra is converted by GFP to green light GFP requires no cofactors; the fluorophore is protein-based Upon denaturation of GFP, fluorescent is lost Scientific history of GFP 1964-present: Shimamura, Prasher, Tsien, Aurora, Vertex 7 Application of GFP Protein Tagging: express a protein of interest linked covalently with GFP via gene fusion - Localize protein in time and space within a living cell - The 27kDa minimal GFP may perturb protein function Gene Expression: can monitor gene expression in single living cells, but the signal cannot be amplified and GFP in vivo folds into fluorescent form slowly In vivo screening: rapidly find cells expressing desired genes - FACS sort transgenic stem cells Plant Golgi: ERD2-GFP Structure of Chromophore GFP denatured with heat and digested with papain (thiol protease) Resulting chromogenic peptide purified by reverse phase HPLC The absorption spectrum of the protease fragment is identified to that of denatured GFP and is pH dependent Boil in 6 N HCl for 24h: find 1 mol Gln, 1 mol Val, 1 mol Phe, 2 mol Gly, 1 fructuating mol of Ser Digest the peptide with carboxypeptidase (C terminal peptidase): lose Gln and Val Digest with pronase (endopeptidase): lose Gln and Phe Therefore the N-terminus is Phe and the Cterminus is Val-Gln 8 Structure of the Chromophore Core Hydrolysis librates a compounds which is identical in chromatographic properties to p-hydroxybenzaldehyde 2D-NMR and mass spectral analysis suggest the following complete structure of the chromatogenic peptide: O NH2 HO O O H N N O N H O N O O OH O N N O H2N N N O O N H NH OH O N H OH OH GFP made anaerobically is not fluorescent, suggesting O2 oxidation Mechanism of Hydrolysis of GFP chromophore in 6N HCl HO O O N H2N H+ O H2O HN N H O H2N HO OH HO O HO N OH H O HN OH H2N O H2N OH H2O HN H2N O OH O HN H OH OH HO OH OH H+ HN O O OH - glycine O OH H2N O N H H2N OH OH H+ H+ HO OH HO O H+ OH HO H+ HO H2N H+ N O N HO O OH N O N HO OH HO HO OH N HO H2O O HO OH N H2O HO HO H+ H2N O H+ HN OH OH OH OH H2N Glycine and Serine OH 9 Mechanism of Chromophore Formation Fluorescence of anaerobically preformed GFP developed with a single exponential time course after air is readmitted Rate of Fluorescence development is independent of [GFP], [cofactor] Imidazolinones spontaneously autooxidize in air Asn-Gly sequence cyclize to imides by a similar pathway Mass spectra indicates loss if 1±4 Da upon exposure to air Crystal Structure of GFP (Ormö and Tsien, 1996; Yang and Phillips, 1996) GFP structure solved by X-ray diffraction to 1.9Å by two groups GFP is a “β-can” of 11 β-strands Chromophore is completely buried from solvent 135 Å3 cavity lies next to the chromophore (O2-binding pocket?) 10 Structural Basis of GFP Properties High Quantum Yield: protection from O2 and H2O Two absorbance peak (395nm and 475nm): correspond to two GFP-stabilized forms of the chromophore: neutral and anionic Resistance of the chromophore to changes in external pH: solvent inaccessibility Mutation of Thr203 to aromatic polar residues (H, Y, W) redshifts the emission maximum: lowering of the energy of the excited state of the chromophore Engineering New Colors of GFP (Heim and Tsien, 1996) Why engineer GFP to have altered emission and excitation spectra? 1) Provide distinguishable marker to follow multiple process 2) Fluorescence resonance energy transfer (FRET) requires two fluorophores with overlapping excitation/emission spectra 3) Illuminate the structure-function relationship in GFP Error-prone PCR and in vivo screening on plates (7,000 colonies per round) resulted in the isolation of interesting GFP mutants 11 Genetic Basis for Altered Spectra 1) 2) 3) 4) 5) 6) Six Classes of GFPs: Wild-type like Excit. only at 488nm - mutation at Ser 65 Excit. only at 399nm - mutation at Thr 203 Red-shifted emission - replace Thr 203 with aromatic residues Blue-shifted emission - Tyr 66 Trp mutation Very blue-shifted em. - Tyr 66 His mutation Chemical Basis for Altered Spectra 12 GFP FRET-based Protease Assay (Heim and Tsien, 1996) FRET: a through-space effect that can occur when two fluorophores are located within in ~100Å of each other The emission spectrum of one fluorophore (“donor”) must overlap the excitation spectrum of the second fluorophore (“acceptor”) The mutual orientation of the fluorophores’ transition dipoles also affect FRET efficiency, which = 1/(1+[R/R°]6) - R, the distance at which FRET is 50% efficient, depends on the quantum yield of the donor, the extinction coefficient of the acceptor, and the overlap between their spectra (can calc.) Use FRET between a class 6 (blue) GFP and a class 2 (green) GFP to monitor proteolysis Chemical Genetics Approach wt protein gene mutation treated Genetics -CTG-CAC-CAT-GCA-AAG-Leu-His-His-Ala-Lys-CTG-CAC-GCT-GCA-AAG-Leu-His-Ala-Ala-Lys- Chemical Genetics phenotype small molecule Genetic Approach: 1. Permanent alteration/deletion of whole protein functions 2. No temporal control of protein functions 3. Gene Knockout: discrete, no observable, obscure phenotype or Embryonic lethal 13 Chemical Genetics Approach wt protein gene mutation treated Chemical Genetics Genetics -CTG-CAC-CAT-GCA-AAG-Leu-His-His-Ala-Lys-CTG-CAC-GCT-GCA-AAG-Leu-His-Ala-Ala-Lys- phenotype small molecule Genetic Approach: 1. Permanent alteration/deletion of whole protein functions 2. No temporal control of protein functions 3. Gene Knockout: discrete, no observable, obscure phenotype or Embryonic lethal Chemical Genetic Apprach: 1. Using small molecule to alter protein functions 2. Temporal and reversible control 3. Specific alteration of single function among multiple roles in a protein 4. Lack of generality (case-by-case basis) Chemical Genetics Approach (interdisciplinary approach) Biological Warfare for survival over billion years small molecule Technology High Throughput Screening 1. Forward Chemical Genetics (Cell based Phenotypic Screening) 2. Reverse Chemical Genetics (Proteomic Screening using SMP) Natural Selection Collections of natural product (created by random mutation) Biological Probes Drugs Selection Process Chemistry Large collection of complex and diverse March 3 2003 small molecules Volume 81, issue 9 (Diversity-Oriented Synthesis) rd Biology Protein Modulators (Biological Probes) Protein Localizers Medicine Chemistry: Diversity-Oriented Synthesis Technology: High Throughput Screening Biology 14 Drug Discovery Process Target Discovery Assay Development In vitro Assay Target Identification Target Validation Lead Discovery Preclinical Study Phase I Phase II Phase III Pharmacology Safety Efficacy Safety Efficacy Safety Long-term use ADME-Tox 20-1000 Healthy Volunteers Chemistry Cell Based Assay HTS Efficacy In vivo Assay ? 100-5000 Patient Volunteers 1.5 Yrs 6.5 Yrs 2 Yrs Drug 1000~50000 Patient Volunteers 3.5 Yrs Hit to Lead Process: Essence of Drug Discovery Hit Generation Target & Hit Identification Hits Leads Generation Leads Hit Refinement Leads Optimization Clinical Candidates New Drug Lead Refinement Chemical Genetics and DiversityOriented Synthesis Random mutation – Natural selection Diversity-Oriented Synthesis – High throughput screening phenotypic screening proteomic screening small molecule and protein microarrays Arrested in mitosis Presence of extra spindle Detached chromosomes Schreiber, S. L. Bioorg. Med. Chem. 1998, 6, 1127; Schreiber, S. L. Science, 2000, 287, 1964. 15 Diversity-Oriented Synthesis (vs. Target Oriented Synthesis or Combinatorial Chemistry) Combinatorial Chemistry Target Oriented Synthesis Diversity-Oriented Synthesis Criteria of Diversity-Oriented Synthesis Complexity Diversity Efficiency - Diversity-Oriented Synthesis (subjects for biological evaluation) R2 R3 R1 R2 O n R3 N n N O O N N O N R4 O R3 N R3' O H O O O H N O H N N R2 H R3 H H O R2 O N N R2 R4 O R1 R1 O i-Pr i-Pr Si R1 X O O * R1 R4 O 500~600 micron Polystyrene resin H or O O R1 O R1 R3 O N H O O R3 N O N R2 R2 Br R1 R2 R1 O H H R1 O O O O R1 N N N R 4 H R2 H N R3 R3' O R3 O N R4 16 In-Silico Analysis of Library Molecular descriptor Molecular descriptor Biological assay data Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Structure Analysis (Afferent) Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor Molecular descriptor 222 Molecular Descriptors (QSARIS) Principle Component Analysis (PCA) (Spotfire) Target values in chemical space (Spotfire) What Questions will a Virtual Library Answer? How different is the chemical space occupied by each core structure in the library? How do the target values of my library compare with known molecules or other libraries? Which building blocks will provide the most diverse library for a selected size (approx. 4000 molecules)? How will biological assay data correlate with the target values in chemical space? 17 Virtual Library Representations in Spotfire view 11342576 view view view view view view 8 8 4 2 cLog(P) 6 0 –2 300 500 700 900 molecular weight (Da) 1100 Principle Component Analysis (PCA) Bicyclic diene products Tetracyclic monoene products Maximize the diversity of library in the stage of building block selection 18 Small Molecule Printing (Proteomic Screening) print stock solutions onto derivatized glass microscope slide cleave small molecules from solid support probe slides with proteins prepare stock solutions in DMF (~1 mM) beads from a split-pool library 384-well stock plate scan slides for fluorescence protein 7,000 printed compounds tag small molecule protein-ligand interaction on a printed microscope slide fluorescent spots reveal putative binding interactions Pathway-Dependent Distribution of small molecules in Biology Space (Chemical Genomics) Diversity-Oriented Synthesis (DOS) in biological space 6 different chemical genetics modifier screens Six collections of small molecules resulting from six diversity oriented synthesis pathway assays DOS-small molecules Collaboration with Paul Clemons at Harvard ICG 19 Small Molecule Microarraying Process 1. OmniGrid Microarrayer 2. 1. OmniGrid Microarrayer: Up to 100 chemically-derivatized slides are placed on the slide platform. Stock plates are placed on the corner of the platform. The robot is enclosed for printing under an argon atmosphere. 3. 2. Print-head containing a single pin: Using a 48-pin print-head, 6,912 solutions may be printed onto 137 slides in under 3 hours. 3. Close-up of a single split pin: Each split pin picks up ~200 nL of stock solution and delivers 1 nL of solution to each slide on the platform. Pins are washed in acetone and acetonitrile before each dip. Each pin tap results in a 250-300 μm diameter spot. Chemical Genetics: Emulating the Logics of Classical Genetics using Small Molecules 1) Chemical perturbations resulting in phenotypic differences in biological systems 2) Chemical genetic modifier screens:suppressor / enhancers 3) Distance metric: map the relative position of small molecules vs. biological systems in space 4) Chemical genetic profiling Chemical Genomics Network 20 Small Molecule Microarray Technology (High Throughput Proteomic Screening) 21