Medicinal Chemistry. February - June, Prof. Elena Goun

Transcription

1 Medicinal Chemistry February - June, 2018 Prof. Elena Goun

2 Laboratory of Bioorganic Chemistry and Molecular Imaging (LCBIM) Imaging beyond the Proteome Elena Dubikovskaya 12/15/10

3 Short Biography Copyright:

4 Please introduce yourself What is your name? Where are you from? What is your field of study? What are your career plans?

5 Course Objective Objective: To provide students with relevant background in the field of medicinal chemistry, drug development, and diagnostics.

6 The Main Book* * The course will also include additional material not included in the book Copyright: wiley.com

7 The second book* * The course will also include additional material not included in the book Copyright: amazon.com

8 Grading system Exam: Written exam 70% of the grade Homework: Presentation in the class 30% of the grade min, 3 people per group any medicinal chemistry topic of your choice Will take place 8-9 week of the semester Need to select the title before week 7

9 Novel Chemical Tools and Their Application in Medicine Imaging beyond the Proteome Laboratory of Bioorganic Chemistry and Elena Molecular Dubikovskaya Imaging 12/15/10

10 Molecular Imaging enables the interrogation of molecules in living systems Copyright: web.rollins.edu/~jsiry/memb; blogs.nature.com

11 Molecular Imaging enables the interrogation of molecules in living systems 2008 Nobel Prize in Chemistry Copyright:

12 Molecular Imaging enables the interrogation of molecules in living systems Copyright: cas.uoregon.edu/photos/sciencephoto, bsc.ua.edu/site/images

13 Development of Probes Fatty Acid Transport Cell Surface Glycosylation Protein functions H 2 O 2 signaling Copyright: millville.sps.edu

14 Development of probes Image guided surgery Mitochondria membrane potential Activity of Glucose Transporters Small ligand-receptor interactions Copyright: micro.magnet.fsu.edu; wikimedia.org/wikipedia; nature medicine

15 Near-infrared probes for Imaging of fatty acid uptake Near-Infrared imaging uses light in the range of nm: - minimum absorption by tissues - low interference from tissue autofluorescence The NIR window in biological tissues It allows increasing tissue depth penetration and minimizing background signal Frangioni J.V., Curr. Opin. Chem. Biol., 2003, 7, Weissleder R. Nature Biotech., 2001, 19,

16 Developing of Probes for Image-Guided- Surgery and Diagnostic in Oncology

17 - Antibody - Peptide - Small molecule What is Probe?

18 Conjugates of Interest Optical probes Optoacoustic probes PET probes Dual modality probes (PET/Optical) Drug conjugates

19 Development of tumor-specific fluorescent probes Cancertargeting molecule Small molecules, peptides, antibodies... clickable NIR fluorescent dye Conjugation via click chemistry PROBE for intra-operative imaging Cancertargeting molecule

20 Tumor Detection in Ovarian Cancer Folate receptor-α (FA-α) is overexpressed in % of epithelial ovarian cancers FA-α binds folate with high affinity which is then endocytosed and shuttled into the cytoplasm Folate + Fluoresceine = folate-fitc van Dam GM et al. Nature Medicine 2011

21 Tumor Detection in Ovarian Cancer van Dam GM et al. Nature Medicine 2011

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23 Tumor Detection in Ovarian Cancer van Dam GM et al. Nature Medicine 2011

24 Tumor Detection in Ovarian Cancer van Dam GM et al. Nature Medicine 2011

25 - Antibody - Peptide - Small molecule What is Probe?

26 Integrin Receptors Transmembrane receptors to recognize the components of extracellular matrix Critical for cell adhesion, migration, proliferation and angiogenesis Upon activation triggers a number of signaling cascades (FAK, Src, ERK and AKT pathways) leading to new blood vessel formation Highly expressed in aggressive solid tumors and their function is important for the invasion and metasthesis of cancer cells

27 RGD peptide: introduction RGD is a tripeptide composed of L-arginine, glycine, and L- aspartic acid α v β 3 integrins are counter parts of RGD-Peptides E. Ruoslahti, M.D. Pierschbacher, Cell 1986, 44 (4),

28 RGD (Arg-Gly-Asp) Extracellular segment of integrin α V β 3 in complex with an Arg-Gly-Asp ligand

29 Possible other markers for image-guided surgery of GBM: RGD peptides Integrins α v β 3 and α v β 5 were expressed in many glioma vessels; the intensity of vascular expression of >va3 increased with grade of malignancy

30 Possible other markers for image-guided surgery of GBM: Cilengitide Cilengitide was in clinical trials the treatment of GBM Small molecule and potent binder (high intratumoral concentrations were detected in humans) Conjugates of Cilengitide with ICG might comprise a valuable tool for image-guided surgery of GBM Biological evaluation in animal models of GBM

31 Diagnostic RGD-Ga68 imaging reagent had been previously developed and validated in clinic by Dr. John Prior (CHUV) Copyright:

32 Multimeric crgd Cy7 1-cRGD-Cy7 2-cRGD-Cy7 4-cRGD-Cy7

33 Copyright: themyersonthemove.blogspot.com

34 Copyright:

35 Multimeric crgd Cy7 1-cRGD-Cy7 2-cRGD-Cy7 4-cRGD-Cy7

36 Advantages of Multimeric crgd Copyright: themyersonthemove.blogspot.com In collaboration with Dr. Anita Wolfer

37 RGD peptide: a multimeric approach RGD-ICG 2RGD-IR820

38 Fatty acid uptake plays a key role in Extensive uptake is linked: biology of many diseases Insulin resistance in skeletal muscle leads to type 2 diabetes Cardiomyopathy (heart muscle disease leads to heart failure) Fatty liver disease (large vacuoles of triglyceride fat accumulate in liver cells liver failure) Lipotoxicity in heart, liver, and pancreatic beta cells (major cause of heart failure, obesity, and diabetes) Other pathologies: cardiac hypoxia intestinal melabsorption and many others

39 Using ICG-FA probe for imaging of GBM? 39

40 Accumulation of ICG-FA in tumor vs healthy brains ICG-FA High grade astrocytoma tumor models (pathology is GBM-like) Brains with Tumor y665 Healthy brain y667 ICG-FA ICG-FA injected IV (200 ul, 100 um) Mice were sacrificed 2 h postinjection Mouse model: GFAPHaRas;GFAP-CRE;GFAP- LUC;p53WT/FLOX line that crosse-bred and then bred 10N on FVBNJ background - In collaboration with Doug Hanahan

41 Accumulation of ICG-FA in U87 tumor vs healthy brains BLI U87 brain tumor WT nude mice U87 brain tumor WT nude mice BLI ICG In collaboration with PerkinElmer

42 Targeting of brain tumors with ICG-FA probe In collaboration with PerkinElmer

43 ICG-FA DLIT Reconstruction

44 Development of Probes Fatty Acid Transport Cell Surface Glycosylation Protein functions H 2 O 2 signaling Copyright: millville.sps.edu

45 Problem: - No methods that would allow studies of fatty acid fluxes in living organisms exist Urgent need to develop new assays for drug discovery

46 A novel approach for imaging metabolic fluxes in living animals with Prof. Andreas Stahl Group Nutritional Science and Toxicology Department, UC Berkeley

47 Obesity is a real problem worldwide

48 Health effects of obesity Obesity is associated with about 30 diseases heart disease high blood pressure arthritis sleep apnea stroke type 2 diabetes It is the second leading cause of death in the US Economic costs are estimated of 123 billion per year

49 Body Mass Index Categories: Underweight = <18.5 Normal weight = Overweight = Obesity = BMI of 30 or greater Copyright: freebmicalculator.net

50 Trends of obesity among different countries Copyright:

51 Trend of obesity among US adults 45% 33% 67% Copyright:

52 What are the molecular signatures of obesity related disorders that can be targeted to create new therapeutics?

53 Fatty acids are a very important source of energy = ENERGY FAT Copyright:

54 Uptake of fatty acids into cells O OH Passive diffusion Fatty Acid Transporter Proteins (FATP) Cytosol Pflugers Arch. (2004) 447, 722

55 Blocking FATP in the intestine reduces caloric uptake O OH X = Decreased Fatty Acid uptake Cytosol Pflugers Arch. (2004) 447, 722

56 Mice with no muscle/fat FATP are protected from diet induced obesity These mice are also protected High Fat Diet from insulin desensitization 6 weeks Knock out FATP High Fat Diet 6 weeks J. Lipid Res. (2005), 46, 597

57 Development of assays that can be used for drug discovery Problem: - No methods that would allow studies of fatty acid fluxes in vivo exist

58 Bioluminescent imaging (BLI) mol/l 100,000 times more sensitive than fluorescence HO N S N S CO 2 H Firefly luciferase + HO N S N S O Firefly Luciferin ATP, O 2, AMP, PP i, Mg 2+ CO 2 Oxyluciferin

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60 Bioluminescence in Nature Find Food Attract Mates Defend against predators Camouflage Chemical & Engineering News, 2006, 84, 36-38; Science, 2005, 309, 263

61 Molecular Imaging enables the interrogation of molecules in living systems Luciferin

62 Molecular Imaging enables the interrogation of molecules in living systems Luciferin

63 Applications of BLI to probe molecular signatures of target tissues CAGE X N S X = O, NH N S CO 2 H UNCAGING CAGE HX N S N S Free Luciferin CO 2 H Firefly luciferase ATP, O 2, AMP, PP i, Mg 2+ CO 2 + HO N S N S Oxyluciferin O (1) Nat Methods, 2006, 3, 295. (2) Mol Ther, 2005, 11, 926. (3) Proc. Natl. Acad. Sci. 2010, (4) Angew. Chem. Int. Ed. 2007, 46, 7031.

64 SAR of FATP in respect to their substrates O n > 10 HO R O HO O O NR F B N F not a substrate for FATP BODIPY - Lauric acid J. Lipid Res. (2005), 46, 597

65 FFA (LIPID) Releasable Linker Luciferin (IMAGING PROBE) CCD Camera HO O S S O O O N S N S CO 2 H FATP FATP Cytosol HO O S S O O O N S N S CO 2 H Glutathione (GSH) HO O SH HS O O O N S N S CO 2 H S O O HO N S N S CO 2 H Luciferese Mg 2+, ATP + oxyluciferin

66 FFA (LIPID) Linker Luciferin (IMAGING PROBE) HO O S O O O N S N S CO 2 H FATP FATP CCD Camera Cytosol HO O S O O O N S N S CO 2 H HO Hydrolysis S OH O + CO 2 HO N S N S CO 2 H Luciferese Mg 2+, ATP oxyluciferin +

67 Imaging Intestinal absorption Probes behave just like a natural fatty acid Colon Small Intestine Copyright:

68 Intestinal fatty acid uptake in real time in live animals

69 Where does the light come from? The duodenum is the first section of the small intestine Stomach Duodenum Colon

70 MicroCT and BLI overlay of uptake BaSO 4 radiocontrast agent

71 Comparison to the control compound HO O Polyethylene Glycol S O O O N S N S CO 2 H 60 min 60 min Copyright:

72 Brown adipose tissue generates body heat White adipocyte Brown adipocyte The only known mammals lacking BAT (Boars) Copyright:

73 Recent discovery of BAT in adult humans Copyright: N Engl J Med, (2009) 360, 1509

74 If activated, brown adipose tissue Drug ~ 25% of daily calories next promising target for the treatment of obesity N Engl J Med, (2009) 360, 1509 Copyright: sce.uhcl.edu/puzdrowski/vitruvian

75 Current technologies to image BAT Positron Emission (PET) and Single Photon emission Computed Tomography (SPEC) Both require radioisotope labeled tracers ( 18 F and 123 I) Very expensive and not suitable for high throughput screening of drugs

76 Imaging of Brown adipose tissue in living mice control FFA-luc FFA-luc + CL HO H - first example N of O imaging CO2 H BAT O CO without using radioactivity 2 H Cl CL first detection of FFA flux Increases BAT thermogenesis and metabolic in live mice rate Copyright:

77 Near-infrared probes for Imaging of fatty acid uptake Near-Infrared imaging uses light in the range of nm: - minimum absorption by tissues - low interference from tissue autofluorescence The NIR window in biological tissues It allows increasing tissue depth penetration and minimizing background signal Frangioni J.V., Curr. Opin. Chem. Biol., 2003, 7, Weissleder R. Nature Biotech., 2001, 19,

78 Optoacoustic imaging Based on laser energy absorption by tissues and ultrasound waves ICG containing molecules represent an ideal class of compounds for optoacoustic imaging Conjugation of fatty acid with ICG allows creation of novel NIR probe for imaging of fatty acid uptake Indocyanine Green (ICG) (FDA approved in 1959) Current status: 40 microns and 2 cm penetration depth Copyright: 79

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80 Imaging BAT Activation with ICG-FA Probe With and without β-3 adrenergic stimulation (CL316243) Interscapular BAT No Stimulation Interscapular BAT CL Stimulation CL31624 was injected i.p. After 20 minutes mice were injected with ICG-FA and 1 hour later biodistribution was determined In collaboration with Vasilis Ntziachristos (TUM) 81

81 Imaging BAT Activation with ICG-FA probe With and without β-3 adrenergic stimulation (CL316243) Interscapular BAT No Stimulation Interscapular BAT CL Stimulation Conclusion: the probe mimics natural fatty acids uptake In collaboration with Vasilis Ntziachristos (TUM) 82

82 Developing of Probes for Image-Guided- Surgery and Diagnostic in Oncology

83 Conclusions Fatty Acid Transport Cell Surface Glycosylation Protein functions H 2 O 2 signaling Copyright: millville.sps.edu