II. Use Of Photoaffinity Label For Off Target Id P39


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Identification & Validation of Drug Target by Photo-affinity label and transfection studies

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  • ‘ Double Reverse Pharmacology – DRP’ Research to bedside and back again!!!! Instead of asking questions on what causes the observed low reticulocytes cell count in patients, in contrast, we address that by posing the question to the drug itself. Let the drug tell us what the target is or are in the host system. How do you do it? Via photo-probe, I’d say. In the next half hour, I will show you how we do that and what conclusion can one draw from it. First I must get a point across. Slide 1 What is a photoprobe? 1. ‘Reporter’ – Radiolabel. 2. ‘Crosslinker’- Azide 3. Ligand- Drug (Bioactivity, specificity,stability) Antibiotic (drug) --- In the bacterium, Mode of action is protein synthesis inhibition (Normal application of target identification) Toxicity (drug) ----In the human body (mammalian), Mode of action is UNKNOWN (Hopefully in the host system, this would point us to the direction of explaining the cause of side effects observed.)
  • Nucleophilic attack to generate the covalent adduct.
  • Transfected MOA-A are localized to the mitochondrial membrane as expected.
  • In vitro versus in vivo approach. Separates into two components, cell membrane and cytosol. Each is photo lablled in the presence or absence of cold drug.
  • Note the preference of photoprobe of labeling MAO-A over MAO-B.
  • Again MAO-A avidity to PHA 276454 is well demonstrated.
  • How does the chemical structure of rotenone compares with the photo probe and the potent MAO.A inhibitor Harmine. Flat heterocyclic ring structures?
  • We embarked on a proteomic approach to identify the fingered target. The rotenone competed protein target is much more clearly demonstrated by a 2D gel system. Point out where MAO.A and Serum albumin are on the two D gels.
  • What does a stain gel like that looks like? A typical gel at its loading capacity.
  • Impossible to identified the spot because of other protein merging into the spots. Noise greater than signal. The turning point is that we have find a way to enrich the target, p39. All gel studied and shown so far was with the membrane pellet. We found that if cell were disrupted by dounce homogenization in sucrose. P39 is partitioned exclusively into the cytosolic fraction (data not shown.)
  • A procedure is derived to enrich this target . This will not be discuss. From these spots, three different peptides were identified by mass spect. of its tryptic digest and the full sequence of a human p39 un-annotated protein were retrieved from the data base.
  • We prove that p39 is indeed the identified target by in vivo labeling the p39 transfected histagged product. Again, it should be emphasized that labeling p39 has to be performed with intact cells. Partially enriched p39 is not labeled by the photoprobe in vitro. CINDY WOLFE Attempt to express the human p39 in COS7 by transfection were unsuccessful. Expression in HEK, a human embryonic kidney cell line was successful albeit at low level. Here, in this composite, the stain gel image on the upper left represent column fraction of an enriched p39. The extract was generarted from HEK transfected with p39(histagged) and after in vivo photolabel treatment.
  • All cross reacted proteins are slightly smaller in size (37kD), an exception is that seen in pancreas whiach has a molecular weight of ~40kD.
  • II. Use Of Photoaffinity Label For Off Target Id P39

    1. 1. Target Identification and Validation: A native approach using a photo-affinity ligand <ul><li>Method Validation – Monoamine Oxidase A </li></ul><ul><li>Definition of drug target </li></ul><ul><li>Target identification – Proteomics </li></ul><ul><li>Validation of target – Transfection study </li></ul><ul><li>Characterization of target – Data mining </li></ul>
    2. 2. Statement of problem <ul><li>Drug A – target organism, bacterium. Bioactivity and mechanism defined. </li></ul><ul><li>What would Drug A do in mammalian system? </li></ul><ul><li>-- Classical toxicology </li></ul><ul><li>-- Cell biology </li></ul><ul><li>-- Protein Chemistry???? </li></ul><ul><li>Photo-probe, Medicinal Chemistry support </li></ul>
    3. 3. I-125 N 3 I-125 N 3 1 . Photo probe must traverse cell membrane and binds to its target(s). I-125 2. UV exposure for covalent adduct(s) formation. UV 254nm What is a photo-probe? Drug A Drug A Target Covalent adduct
    4. 4. Ref: Y. Hatanaka & Y. Sadakane (2002). Photoaffinity Labeling in Drug Discovery and Developments: Chemical Gateway for Entering Proteomic Frontier. Current Topics in Medicinal Chemistry 2: 271-288. (1) AZIDE (2) DIAZIRINE (3) BENZOPHENONE Typical Photoreactive Groups: Photolabeling success depends on binding affiniity of the ligand for a receptor, combined with the positioning of the photoreactive group in the receptor. Photolysis & nucleophilic attack by a target to form a X-link is generally low efficiency. Therefore, detection requires that the ligand also carry a radioactive probe [ 3 H or 125 I]
    5. 5. Human MAO-A Human MAO-B Inhibition of recombinant human MAO-A and MAO-B by Azido-Drug A and Drug A. Concentrations are nanomolar Azido-Drug A Azido-Drug A 276 Drug A Drug A Monoamine Oxidases A, B (MAO-A, B)
    6. 6. Mock transfected MAO-A transfected Nuclear membrane
    7. 7. Intact cell wall – A prerequisite for a meaningful outcome in cross-link experiment.
    8. 8. Mock transfected MAO-A MAO-B - + - + - + COS cells transfected with: Mock rhMAO-A rhMAO-B 50 nM 2 uM 50 nM 2 uM 50 nM 2 uM [Conc of Azido- Drug A] In vivo whole cell labelling Note that unlike the in vitro case, only a single band is noted with MAO-A using intact cell labelling: therefore, integrity of the cell is critical for specificity A B 125 I Azido-Drug A = 50 nM nMAO (no tag)
    9. 9. Titration study (COS7 – Non transfected cells) <ul><li>Exquisite affinity for MAO.A </li></ul><ul><li>Establish drug label concentration </li></ul><ul><li>Possible targets emerging </li></ul>
    10. 10. Identification of MAO.A in COS7 cells <ul><li>Chlorgyline – a MAO.A specific irreversible inhibitor compete well with Drug A. </li></ul><ul><li>Other labeled proteins are NOT competed against with chlorgyline </li></ul><ul><li>What are these proteins???? </li></ul>
    11. 11. Effect of electron transport inhibitors in drug competition <ul><li>Both Harmine and Cimoxatone are potent inhibitors against monoamine oxidase A. </li></ul><ul><li>Rotenone interferes photo probe but not other electron transporter inhibitors. </li></ul><ul><li>MAO.A is located on the outer membrane of the mitochondria and rotenone disrupts the mitochondrial membrane and its electron pathway (Complex I) located within. </li></ul>
    12. 12. Rotenone Harmine Greenamyre (2000) observed Parkinsonism in rats(??) dosed (fed) with large amount of rotenone. Bertarbet R, et al. Nature Neuroscience 3 , 1301 - 1306 (01 Dec 2000)
    13. 13. 2 Dimension gel analyses Control Rotenone (50uM)
    14. 14. BSA Narrow range pI 7-4 ????
    15. 17. Phosphor image Anti-6 his probed Stained image
    16. 18. A survey of P39 tissue distribution in mouse (BalbC – female) using UP492, an N- terminal peptide antibody. 1.lung 2. Heart 3. Thymus 4. Muscle 5. Spleen 6. Liver 7. L. intest. 8. S Intest 9. Spinal column 10. Kidney 11. Pancreas 12. Stomach 12. uterus
    17. 19. What is known about P39? <ul><li>Not annotated- No references, mostly by sequence homology (Function unknown). </li></ul><ul><li>Orthologs- bacteria to mammals but not in fruit fly (it is in mosquito however). </li></ul><ul><li>Transcript #(Kamath et. al. ) – C. elegan low and constant expression, ~10ppm. </li></ul><ul><li>RNAi (Kamath et. al .)– Developmentally non-lethal. </li></ul><ul><li>Two patents claims filed. </li></ul>Kamath RS, et al Systematic functional analysis of the Caenorhabditis elegans genome using RNAi Nature 2003 Jan 16; 421:231-7
    18. 20. Acknowledgements <ul><li>Cindy Wolfe -- Cloning, Transfection and all Cell Cultures Works. </li></ul><ul><li>Larry Alward– Proteomics. </li></ul><ul><li>Lisa Thomasco – Chemistry </li></ul><ul><li>Martin R. Deibel Jr. -- Mentoring </li></ul>