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1 Development and Screening of Transition Metal Complexes as CXCR4 Antagonists Dr. Tim Hubin Department of Chemistry and Physics.

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Presentation on theme: "1 Development and Screening of Transition Metal Complexes as CXCR4 Antagonists Dr. Tim Hubin Department of Chemistry and Physics."— Presentation transcript:

1 1 Development and Screening of Transition Metal Complexes as CXCR4 Antagonists Dr. Tim Hubin Department of Chemistry and Physics

2 2 CXCR4 chemokine receptor Important role in embryonic development: – Organogenesis (liver, heart) – Stem cell movement – Cerebellar neuron migration (formation of brain) Seven transmembrane G- protein-coupled receptor 27% of amino acids are Asp, His or Tyr. Expressed in : » Leukocytes » T-lymphocytes » Endothelial cells » Neuronal cells Khan, A.; Greenman, J.; Archibald, S. J. Curr. Med. Chem. 2007, 14, 2257.

3 3 CXCL12 67 residue highly basic protein Only known natural ligand (chemokine) for CXCR4 Secreted by stromal, lung and liver cells, and lymph nodes Attracts leukocytes to sites of inflammation and lymphoid organs

4 4 Disease states Role in disease – Human Immunodeficiency Virus (along with CCR5) – Tumour growth and metastasis – Stem cell mobilization – Autoimmune disorders (rheumatoid arthritis)

5 5 Inhibitors against 5 steps of HIV replication cycle Still need for new targets Prototype bicyclams AMD3100 CXCR4-antagonists and HIV

6 6 Gerlach et al., 2001

7 7 Blocking receptor functions Cell Drug CXCL12/HIV

8 8 Plerixafor/ AMD3100 AMD3100  The first bicyclams were discovered as impurities in a sample of cyclam. Amongst the most active anti-HIV agents in vitro.  Likely a prodrug; complexation of Zn 2+ will occur in plasma  Anti-HIV clinical testing discontinued.  Stem cell mobilization For example: Mol. Pharm., 1999, 55, 67. J. Med. Chem., 1995, 38, 366. Biochemistry, 2003, 42, 715.

9 9 Molecular shape Bosnich, B.; Poon, C. K.; Tobe, M. L. Inorg. Chem.,1965, 4,1102

10 10 Restrict to one configuration Only cis V Only trans-II

11 11 Side-Bridged Synthesis Reagents: (a) acetonitrile, RT, 24 h (89%); (b) NaBH 4, EtOH reflux, 1 h (65%).

12 12 Cross-Bridged Synthesis Reagents: (a) acetonitrile, RT, 7 d (80%); (b) NaBH 4, 95%EtOH, RT, 7 d (70%). (a) (b)

13 13 AMD3100 Lewis, E. A.; Hubin, T. J.; Archibald, S. J. European Patent A2.

14 14 Cu-O12.28(1) Å Side bridged (SB) Cross bridged (CB) Cu-O11.95(1) Å AxialEquatorial

15 15

16 [Cu 2 (p-CB-cyclams)] = SJA5 16

17 17 CXCR4 Binding Assays Use anti-CXCR4 antibodies to screen cell lines Two identified: Jurkat and Molt-4 Four anti-CXCR4 antibodies used (variation in binding epitopes)

18 18 Binding by flow cytometry CXCR4 Drug molecule Receptor specific antibody Fluorescent antibody KeyNameParameter - control.001FL1-H + Control FL1-H L FL1-H L FL1-H

19 mAB Inhibition 19 Summary of mAb 12G5 binding to CXCR4 in the presence of bound antagonists.

20 Competitive Binding Studies 20 IC 50 and EC 50 concentrations for CXCR4 antagonists in competition with mAb in Jurkat cells.

21 21 Residence time G. McRobbie, A. Khan, G. Nicholson, L. Madden, J. Greenman C. Pannecouque, E. De Clercq, T. J. Hubin and S. J. Archibald, J. Am. Chem. Soc, 2009, 3416.

22 22 CompoundConcentration (16 nM) ( τ ) (hr)t 1/2 diss (hr) AMD Cu 2 -AMD Cu 2 -Cross Bridged Relative ( τ ) and t 1/2 dissociation constants for compounds binding to CXCR4 receptor in Jurkat cells.

23 HIV Infection Assays 23

24 Ca 2+ Ion Signaling Assays ng/ml 8 ng/ml Time (sec) Fluorescence Change (counts) Ca-signaling data for AMD3100 CXCR4 experiment by collaborator Schols.

25 25

26 CXCR4 and Cancer Cell Metastasis oCXCL12 is normally responsible for trafficking of lymphocytes oCXCL12 is secreted by stromal, lung and liver cells, and lymph nodes oThe interaction at the cell membrane is through CXCR4, which is over- expressed in some cancers oPotential mechanism of metathesis Normal cellCancer cell

27 27 Invasion assays Cell invasion assays in response to a chemokine gradient. Initially used SJSA cells. Experiments run in presence and absence of antagonist. ANTI-CANCER ACTIVITY

28 28 Control Drug/ no CXCL12 CXCL12 Drug + CXCL12

29 Cancer Cell Invasion Assay 29 Invasion of SJSA cells in matrigel with CXCL12 (12.5 nM) and CXCR4 antagonists ( nM). Cells were counted in five different fields (x40 obj) in duplicates. Mean of the values plotted. Asterisk represents significance (p < 0.01) from B. A = no CXCL12 and no antagonist; B = CXCL12 only; C = 20 nM Cu-Cross Bridged antagonist; D = 200 nM Cu-Cross Bridged antagonist; E = 20 nM AMD3100; F = 200 nM AMD3100.

30 An acute administration of AMD3100 is known to rapidly mobilize bone marrow stem cells and progenitors. This mobilization is due to inactivation of the CXCR4-CXCL12 axis which holds progenitors in the bone marrow. AMD3100 induces neutrophilia and leukocytosis, which reach their maximum 2 hours post-injection. AMD3100 has not been noted to have an impact on monocytes. C57BL/6 strain = a common strain of lab mouse, probably the most widely used "genetic background“ for use as models of human disease. They are the most widely used lab mouse strain, due to the availability of congenic strains, easy breeding, and robustness. 30 Stem Cell Mobilization—OMRF (Barlic)

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33 Time (h): Total leukocyte count (x10 3 /  l) Total neutrophil count (x10 3 /  l) Total monocyte count (x10 3 /  l) saline AMD3100SAJ5 baseline

34 34 Acknowledgements Funding – OK-INBRE (NIH) – Research Corporation – SWOSU Dr. Steve Archibald (Hull) Dr. Abid Khan (Hull) Prof. Erik De Clercq (Leuven) Dr. Christophe Pannecouque(Leuven) Dr. Dominique Schols (Leuven) Prof. Tony Ng (KCL) Dr. Jana Barlic (OMRF) Current research group: Courtney Garcia (Chemistry/Medicine) Paul Won(Chemistry/Pharmacy) Justin Le (Chemistry/Pharmacy) Past members: Robert Ullom—University of Kansas (Medicine) Joe Blas—Creighton (Medicine) Danny Maples—OSU (Chemistry) Randall Maples—OSU (Chemistry) Dallas Matz—Arizona State University (Chemistry) Mike McClain—OU (Chemistry) Amy Cain—U. British Columbia (Chemistry) Orry Birdsong—UT Galveston (Medicine) Kimberly Roewe—OSU (Chemistry) Kiet Ngyuen—SWOSU (Pharmacy) Josh Priddle—OSU (Medicine) Desiray Cannon (Chemistry) Katherine Coats (Chemistry) Natalie Simpson (Chemistry) Kevin Wilson (Chemistry)

35 35 United States Weatherford—synthesis/characterization (Hubin) Oklahoma City—stem cell mobilization, atheroregression, obesity (Barlic) United Kingdom Hull—synthesis/characterization, CXCR4 binding, imaging, cancer metastases (Archibald) London—cancer cell imaging (Ng), PET Imaging, pharmacology (Blower) Belgium Leuven—anti-HIV properties (DeClercq, Pannecouque), Ca-Signaling (Schols)

36 Collaborations Steve Archibald, University of Hull – Central connection between all other collaborators Expertise – Synthetic Organic and Inorganic Chemistry – Bioinorganic and Medicinal Chemistry – X-Ray Crystallography Contributions – Synthesis and Characterization of New Compounds – Antibody binding studies on CXCR4 expressing cell lines – Cancer Cell Invasion Assays – X-Ray Crystal Structures 36

37 Collaborations Tim Hubin, Southwestern Oklahoma State University Expertise – Synthetic Organic and Inorganic Chemistry – Bioinorganic and Medicinal Chemistry Contributions – Synthesis and Characterization of New Compounds 37

38 Collaborations Dominique Schols, Christophe Pannecouque, University of Leuven – Eric De Clercq (retired, but still active) Expertise – Virology – Chemokine Receptors Contributions – HIV Infection Assays—Pannecouque – Ca 2+ Ion Signaling Assays—Schols – CXCR4 and CCR5 binding studies—Schols – Viral mutation studies—Pannecouque 38

39 Collaborations Tony Ng, King’s College London – Gilbert Fruhwirth Expertise – Cancer – Fluorescence Imaging of CXCR4 in vitro and in vivo – Immunology Contributions – Initial mouse toxicity studies – Imaging of CXCR4 expression – Confirmation of antagonism using fluorescence imaging 39


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