Development and Screening of Transition Metal Complexes as CXCR4 Antagonists

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

2. 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 CXCR4 chemokine receptor Khan, A.; Greenman, J.; Archibald, S. J. Curr. Med. Chem. 2007, 14, 2257.

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

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

6. Gerlach et al., 2001

7. Blocking receptor functions CXCL12/HIV Drug Cell

8. Plerixafor/ 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 Zn2+ 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. AMD3100

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

10. Restrict to one configuration Onlytrans-II Onlycis V

11. Side-Bridged Synthesis Reagents: (a) acetonitrile, RT, 24 h (89%); (b) NaBH4, EtOH reflux, 1 h (65%).

12. Cross-BridgedSynthesis (a) (b) Reagents: (a) acetonitrile, RT, 7 d (80%); (b) NaBH4, 95%EtOH, RT, 7 d (70%).

13. AMD3100 Lewis, E. A.; Hubin, T. J.; Archibald, S. J. European Patent 1765826A2 .

14. Side bridged (SB) Cross bridged (CB) Axial Equatorial Cu-O1 2.28(1) Å Cu-O1 1.95(1) Å

16. [Cu2(p-CB-cyclams)] = SJA5

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. Key Name Parameter - control.001 FL1-H + Control 717.019 FL1-H L2 717.010 FL1-H L1 717.009 FL1-H Binding by flow cytometry Fluorescent antibody Receptor specific antibody Drug molecule CXCR4

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

20. Competitive Binding Studies IC50 and EC50 concentrations for CXCR4 antagonists in competition with mAb 44717 in Jurkat cells.

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.

23. HIV Infection Assays

24. 8000 Control 7000 1000 ng/ml 200 ng/ml 6000 5000 4000 3000 2000 1000 0 -1000 0 50 100 150 Ca2+ Ion Signaling Assays 40 ng/ml 8ng/ml Fluorescence Change (counts) Time (sec) Ca-signaling data for AMD3100 CXCR4 experiment by collaborator Schols.

26. CXCR4 and Cancer Cell Metastasis • CXCL12 is normally responsible for trafficking of lymphocytes • CXCL12 is secreted by stromal, lung and liver cells, and lymph nodes • The interaction at the cell membrane is through CXCR4, which is over-expressed in some cancers • Potential mechanism of metathesis Normal cell Cancer cell

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

28. CXCL12 Control Drug/ no CXCL12 Drug + CXCL12

29. Cancer Cell Invasion Assay Invasion of SJSA cells in matrigel with CXCL12 (12.5 nM) and CXCR4 antagonists (20-200nM). 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. Stem Cell Mobilization—OMRF (Barlic) • 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.

33. saline baseline AMD3100 SAJ5 Total neutrophil count (x103/ml) Total leukocyte count (x103/ml) Time (h): - 2 4 8 Time (h): - 2 4 8 Total monocyte count (x103/ml) Time (h): - 2 4 8

34. 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) OrryBirdsong—UT Galveston (Medicine) Kimberly Roewe—OSU (Chemistry) KietNgyuen—SWOSU (Pharmacy) Josh Priddle—OSU (Medicine) Desiray Cannon (Chemistry) Katherine Coats (Chemistry) Natalie Simpson (Chemistry) Kevin Wilson (Chemistry) 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)

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

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

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 • Ca2+ Ion Signaling Assays—Schols • CXCR4 and CCR5 binding studies—Schols • Viral mutation studies—Pannecouque

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