Design of Macrocyclic Chelators for Biomedical Applications

1. Sept. 16, 2010 Oklahoma State University Design of Macrocyclic Chelators for Biomedical Applications Dr. Tim Hubin Department of Chemistry and Physics

2. Metal containing drugs • Therapeutic and diagnostic • Cisplatin • Magnevist/ Dotarem • Zevalin (Indium, Yttrium) DOTA

3. Generally used chelators

4. CHEMOKINE RECEPTORS

5. 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 on : Leukocytes T-lymphocytes Endothelial cells Neuronal cells CXCR4 chemokine receptor Khan, A.; Greenman, J.; Archibald, S. J. Curr. Med. Chem. 2007, 14, 2257.

6. 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

7. Disease states • Role in disease • Tumor growth and metastasis • Human Immunodeficiency Virus • Stem cell mobilization • Autoimmune disorders (rheumatoid arthritis)

8. Blocking receptor functions CXCL12/HIV Drug Cell

9. Over expression of CXCR4 receptors Cancer cell Normal cell

10. CXCR4 antagonists • Peptide based • Side chains protonated at physiological pH

11. 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

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

13. Restrict to one configuration Only cis V

14. AMD3100 Lewis, E. A.; Hubin, T. J.; Archibald, S. J. Patent WO2005121109, 2005.

15. Copper(II) coordination

17. MODELING THE INTERACTION

18. Molecular modeling studies • Docking studies? • No known X-ray structure of CXCR4 • Develop a homology model to allow evaluation of the full set of interactions in the binding pocket • DFT calculations • Mono-macrocycle compounds at BP86/ TZP level • Bis-macrocycle compounds with mixed treatment at QM/ MM level. G. McRobbie, G. C. Valks, C. J. Empson, A. Khan, J. D. Silversides, C. Pannecouque, E. De Clercq, S. G. Fiddy, A. J. Bridgeman, N. A. Young and S. J. Archibald, Dalton Trans., 2007, 5008.

19. Homology modeling • Predict the structure using X-ray data from a related protein • Align the sequences using conserved regions • Five CXCR4 sequences were used • Disulfide bridges of key importance

21. MACROCYCLE SYNTHESIS

22. Reagents: (a) acetonitrile, RT, 24 h (89%); (b) NaBH4, EtOH reflux, 1 h (65%).

23. Bisaminal precursors

25. COMPLEX CHARACTERIZATION

28. Side bridged (SB) Cross bridged (CB) Cu-O1 1.95(1) Å Cu-O2 2.66(1) Å Cu-O1 2.28(1) Å Cu-O2 2.90(1) Å

30. BINDING TO THE PROTEIN

31. EQUATORIAL AXIAL CB SB

32. Selecting the cell line • Use anti-CXCR4 antibodies to screen cell lines • Two identified Jurkat and Molt-4 (T-cell leukemia) • Four anti-CXCR4 antibodies used (variation in binding epitopes)

33. 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

34. Summary of mAb 12G5 binding to CXCR4 in the presence of bound antagonists.

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

36. 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.

37. BLOCKING SIGNALING PROCESSES

38. Ca2+ signaling • Signal transduction by chemokine receptors leads to elevation of cytosolic free calcium. • Signaling induced by CXCL12 was monitored in CXCR4 transfected U87 cells. • IC50 values were in the range of ng/ml with no signal blocking observed for other chemokine receptors (CCR5)

40. 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.

41. CALCIUM SIGNALING RESULTS

42. ANTI-CANCER ACTIVITY

43. 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 metastasis Normal cell Cancer cell

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

45. Invasive CXCR4 mutants

46. CXCL12 Control Drug/ no CXCL12 Drug + CXCL12

47. Cancer Cell Invasion Assay Invasion of SJSA cells in matrigel with CXCL12 (12.5 nM) and CXCR4 antagonists (20-200 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.

48. CONCLUSIONS

49. Strong and specific CXCR4 antagonism from a cross-bridged bicylam analogue • Axial vs. equatorial coordination makes all the difference in copper(II) containing protein binding drugs. • Promising early anti-metastatic properties in vitro. In vivo testing to follow.

50. Analogues Prepared and (Tested) Current Leads: Cu(Ligand 1) Zn(Ligand 7) Metal Complexes: Zn2+,Cu2+,Ni2+,Co2+