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Chemical biology – the biologist’s perspective

Chemical biology – the biologist’s perspective. Drug development 2007 Per Sunnerhagen. What are the goals of chemical biology?. To develop chemical tools and applying them to answer questions in cell biology. The object is not to develop drugs, although results

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Chemical biology – the biologist’s perspective

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  1. Chemical biology – the biologist’s perspective Drug development 2007 Per Sunnerhagen

  2. What are the goals of chemical biology? • To develop chemical tools and applying them • to answer questions in cell biology • The object is not to develop drugs, although results • obtained with chemical biology may well become • useful in drug development

  3. The roles of chemical biology in drug development

  4. Workflow of chemical biology at the Broad Institute, MIT

  5. What arguments can be used for chemical, rather than genetic, intervention as a tool for investigations in cell biology? 1) Permanent shut-off (or overexpression) of a gene will cause secondary adaptations in the mutant cell, leading to changes in e.g. expression levels of other genes. By contrast, short-term inhibition using a chemical will only produce primary effects • 2) Certain cellular pathways and function (e.g. cytoskeleton, translation) • have a large proportion of essential genes, making genetic analysis difficult. • Chemical inhibitors open two possibilities for analysis of such pathways: • Partial continuous inhibition • Complete but transient inhibition, • in both cases preserving viability 3) Conditional mutants (e.g. temperature-sensitive alleles) require conditions (e.g. temperature shift) that cause major changes in cell state, making appropriate controls difficult to evaluate

  6. cont’d 4) Continuous variability - by applying different amounts of inhibitor, it is possible to get a graded response from an enzyme or a pathway. This is essential for a systems biology analysis of a pathway, with the intention of identifying the components, the activity of which are critical for the overall output of the pathway. Such components are potential drug targets. 5) Speed – it is possible to shut off the activity of a target (protein) within seconds – a few minutes using small molecules as inhibitors. Cf: transcriptional shut-off 30 – 60 minutes; RNAi hours – days. Necessary to follow cellular events with fast kinetics – signal transduction, cell cycle etc. 6) Selective inhibition of the interaction between a specific protein pair, targeting one interaction domain, leaving other interactions intact. This allows shutdown of only one branch of a pathway 7) Chemicals acting as fluorescent tags and e.g. ”caged substrates” can be developed, adding further versatility

  7. What about drawbacks? • side effects (small molecules are never entirely specific; but • gene disruptions are) • limited bioavailability: penetrating different cell types from different organisms • will vary; a small molecule developed will to some degree be species- • specific

  8. Protein kinases constitute a large protein family The selectivity of kinase inhibitors can be evaluated using panels of immobilised recombinant kinase enzyme

  9. ”Orthogonal chemical genetics” compared with ordinary genetics

  10. Selective inhibition of a genetically engineered protein kinase allele using a designed ”orthogonal” inhibitor that can be accommodated only by the modified kinase. All other ”wild-type” kinases are unaffected

  11. Analogue-sensitive kinase alleles can be used for fingerprinting the response (e.g. transcript profile) to inhibition of a kinase. This can be utilised in the search for new wild-type kinase inhibitor lead compounds Analog sensitive kinase allele (ASKA)-based gene expression ‘blueprints’ for kinase inhibitor lead profiling. Using the ASKA system, changes in gene expression resulting from potent, specific kinase inhibition can be measured by DNA microarrays. The open circles represent cells with either ASKA or wildtype kinases; the compounds are either the ASKA-specific orthogonal inhibitor or a lead compound of a wildtype kinase (lead). The checkerboards represent gene expression patterns. The far-left treatment shows the pattern that results from inhibition of the ASKA. The middle two treatments show the small changes that result from either the ASKA or the analog inhibitor that can be ‘subtracted’ from the ASKA + inhibitor pattern. The far-right treatment shows the pattern obtained by profiling a lead compound. By comparing gene expression patterns, such ASKA-based blueprints can be used to determine the possible off-target effects of kinase inhibitor lead compounds. In the example shown here, the lead compound gives the same profile as the ASKA blueprint; thus, the lead has a similar potency and specificity to the ASKA.

  12. Use of analogue-sensitive kinase alleles in mice Analogue-sensitive kinase allele (ASKA) mice can be used either directly (in various chemically induced disease models) or by mating to appropriate disease model strains. Specific kinase inhibition can be achieved by injection of an orthogonal inhibitor that inhibits only the ASKA and not any wildtype kinases.

  13. Identification of protein kinase substrates using orthogonal mutations and ATP analogues Only the genetically modified protein kinase – in this case Src – will be able to accept the bulky ATP analogues, which are radiolabeled As a result, only the direct substrates of Src will be radiolabeled, allowing their identification

  14. Second generation ATP analogues and orthogonal mutations would be specific (excluding normal ATP from reaction)

  15. Chemical Biology at Göteborg University

  16. Current focus of the Chemical Biology Platform Can we create several independent orthogonal pairs of sensitive kinase alleles and inhibitors? This would allow simultaneous and independent manipulations in vivo of two or more protein kinases in a signalling pathway – approaching true systems biology analysis

  17. A chemical genetics approach to direct proteolysis to selected targets in vivo (”chemical knockout”) A heterofunctional synthetic molecule (PROTAC) includes ligand functionality for the target protein (white triangle), a linker moiety (grey hourglass shape) and a ligand for E3 ligase (black rectangle)

  18. Expansion of the genetic code through incorporation of unnatural amino acids through chemical genetics Schematic representation of orthogonal chemical genetic strategy for site specifically incorporating unnatural amino acids. An orthogonal synthetase is evolved that specifically acylates a cognate orthogonal suppressor tRNA with a desired unnatural amino acid. To introduce an unnatural amino acid site specifically in a target protein, an appropriate codon is mutated to an amber, opal or four base codon and transfected in the host cells. The orthogonal tRNA/synthetase pair in the host cells responds to the mutated codon by incorporating the desired unnatural amino acid at the site of interest in vivo.

  19. Biological uses of unnatural amino acids incorporated in specific proteins • a photo-crosslinking amino acid can be used to probe dynamic • protein-protein interactions at different stages of a signalling cascade; • transient enzyme-substrate complexes can be covalently bonded and • isolated • fluorescent amino acids can be used to probe the microenvironment • created during the signalling processes (pH, redox etc.) • amino acids bearing a photo-cleavable side chain provide a • temporal switch to regulate activity in vivo using laser • ”killer” functionalities (e.g. azido groups) can be used to inactivate • a target enzyme at a specific time and place in the cell • heavy atom-containing amino acids for protein structure studies

  20. Incorporation of unnatural UV-crosslinkable amino acids into protein can be used to reveal protein-protein interactions, with high resolution in space and time

  21. The bioorthogonal chemical strategy extended A chemical reporter linked to a substrate is introduced into a target biomolecule through cellular metabolism. In a second step, the reporter is covalently tagged with an exogenously delivered probe. Both the chemical reporter and exogenous probe must avoid side reactions with nontarget biomolecules.

  22. Specific sequences of natural amino acids can work as a specific • base for chemical reporters • Cys-Cys-X-X-Cys-Cys reacts selectively with biasenicals • This amino acid sequence can be introduced genetically into • any protein. This works in instances where GFP tagging disrupts • function of the protein. HeLa cells expressing tetracysteine-fused connexin were treated with FlAsH (green), incubated for 4 hours, then treated with ReAsH (red). This two-color pulse-chase labeling experiment shows that newly synthesized connexin is incorporated at the outer edges of existing gap junctions

  23. Fluorescent probes: monitoring enzyme-substrate intermediates by FRET (fluorescence resonance energy transfer) (Top) A photolabile chemical protection group ("cage") on the phosphate moiety of the phosphotyrosine-containing synthetic peptide prevents binding to the active site of the PTP. (Middle) UV-induced photolysis of the cage induces substrate binding at the PTP active site and FRET, monitored by FLIM and/or emission intensity changes. (Bottom) After catalysis, the reaction product dissociates from the PTP, resulting in loss of FRET. Yudushkin et al., Science 315:115 (2007)

  24. “A map of Km/S for the enzyme (PTP1B) reveals that the peripheral pool, near the plasma membrane, of PTP1B operates in a near-saturation regime (i.e., low Km/S)” Yudushkin et al., Science 315:115 (2007)

  25. Combining genetics and chemical screening A compound that causes cell death on its own can be used in a genetic suppressor screen that identifies cDNAs (or siRNAs) that prevent death caused by the compound. Such suppressor reagents are likely to encode proteins involved in the response to the compound, including the direct protein target of the compound

  26. Cell wall (staurosporin inhibits PKC;caspofungin inhibits synthesis of -glucan) Crude extract clusters together with pure chemicals Synthesis of ergosterol (membrane lipid) DNA-damaging compounds Classification of crude extracts from nature containing bioactive compounds Clustering of mutants and chemicals according to sensitivity profiles groups compounds with similar mechanisms of action Mutants Chemicals (Data are cleaned through eliminating mutants in membrane pumps and membrane lipid metabolism, which give multidrug sensitivity) Parsons et al., Cell 126:611 (2006)

  27. Actinomycin, a DNA-damaging compound, which also inhibits RNA polymerase, is grouped here together with other DNA-damaging compounds Verrucarin and neomycin both inhibit translation. They are grouped together in a factoring analysis but not with hierarcical clustering. Mutants in this group affect the 40S sub- unit of ribosomes Factoring analysis finds correlations between compounds based on responses in only a small cluster of genes

  28. High correlation between sensitivity profiles for extracts 00-192 and 00-132 Spontaneously mutated strains co-resistant to both chemicals Active compound of extract 00-192 Active compound of extract 00-132

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