1. 1 Development and Screening of Transition Metal Complexes as CXCR4 Antagonists Dr. Tim Hubin Department of Chemistry and Physics March 17, 2012 ACS Pentasectional Meeting

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

5. 5 Gerlach et al., 2001

6. 6 Blocking receptor functions Cell Drug CXCL12/HIV

7. 7 Molecular shape Bosnich, B.; Poon, C. K.; Tobe, M. L. Inorg. Chem.,1965, 4,1102

8. 8 Restrict to one configuration Only cis V Only trans-II

9. 9 AMD3100 Lewis, E. A.; Hubin, T. J.; Archibald, S. J. European Patent 1765826A2.

10. 10 Binding to CXCR4 by flow cytometry CXCR4 Drug molecule Receptor specific antibody Fluorescent antibody KeyNameParameter - control.001FL1-H + Control 717.019FL1-H L2 717.010FL1-H L1 717.009FL1-H

11. 11 Summary of mAb 12G5 binding to CXCR4 in the presence of bound antagonists. Inhibition (of CXCL12) Assays

12. 12 Competitive Binding Studies IC 50 concentrations for CXCR4 antagonists in competition with mAb 44717 in Jurkat cells. AntagonistIC 50 (μM) AMD3100 0.360 ± 0.030 Zn-AMD3100 0.278 ± 0.033 Cu-AMD3100 0.439 ± 0.021 Zn-Side Bridged 0.922 ± 0.161 Cu-Side Bridged 0.251 ± 0.074 Zn-Cross Bridged 0.230 ± 0.004 Cu-Cross Bridged0.160 ± 0.004

13. 13 Residence time, Cu-Cross Bridged 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.

14. Antiviral Activity in MT-4 Cells Compound HIV strain Av EC50(μM) a SD b Av CC50(μM) c SI d L1L1 HIV-1 (III B )0.011>225>20,455 [Zn 2 L 1 (OAc) 2 ](OAc) 2 HIV-1 (III B )0.008>225>28,125 L2L2 HIV-1 (III B )6.981.84>225>32 HIV-2 (ROD)23.26.40>225>10 [Zn 2 L 2 (OAc) 2 ](OAc) 2 HIV-1 (III B )0.00250.001060.5624,225 HIV-2 (ROD)0.00400.001360.5615,140 L3L3 HIV-1 (III B )11.300.42>125> 11 HIV-2 (ROD)>113>125>1 [Cu 2 L 3 (OAc) 2 ](OAc) 2 HIV-1 (III B )0.004590.0017>125>27,233 HIV-2 (ROD)0.01710.0011>125>7,331 HIV-1 (III B ) g 0.01170.0023>125>10,730 L1L1 L2L2 L3L3

15. 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 metastasis Normal cellCancer cell

16. 16 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

17. 17 Control Drug/ no CXCL12 CXCL12 Drug + CXCL12

18. 18 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.

19. 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. 19 Stem Cell Mobilization—OMRF (Barlic)

20. Time (h): - 2 4 8 Total leukocyte count (x10 3 /  l) Total neutrophil count (x10 3 /  l) Total monocyte count (x10 3 /  l) saline AMD3100SAJ5 baseline

21. 21 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)

22. 22 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)