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

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

Generally used chelators

CHEMOKINE RECEPTORS

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

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

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

8 Blocking receptor functions Cell Drug CXCL12/HIV

Over expression of CXCR4 receptors Normal cell Cancer cell

CXCR4 antagonists Peptide based Side chains protonated at physiological pH

11 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.

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

Restrict to one configuration Only cis V

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

Copper(II) coordination

MODELING THE INTERACTION

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.

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

MACROCYCLE SYNTHESIS

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

Bisaminal precursors

COMPLEX CHARACTERIZATION

Cu-O12.28(1) Å Cu-O22.90(1) Å Side bridged (SB) Cross bridged (CB) Cu-O11.95(1) Å Cu-O22.66(1) Å

BINDING TO THE PROTEIN

SB EQUATORIAL CB AXIAL

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 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

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

Competitive Binding Studies 35 IC 50 and EC 50 concentrations for CXCR4 antagonists in competition with mAb in Jurkat cells.

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.

BLOCKING SIGNALING PROCESSES

Ca 2+ signaling Signal transduction by chemokine receptors leads to elevation of cytosolic free calcium. Signaling induced by CXCL12 was monitored in CXCR4 transfected U87 cells. IC 50 values were in the range of ng/ml with no signal blocking observed for other chemokine receptors (CCR5)

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.

CALCIUM SIGNALING RESULTS CompoundCalcium signaling Av IC50(nM) Cu 2 (ClO 4 ) 4 (SB) Cu 2 Cl 2 (PF 6 ) 4 (CB) 4.64 AMD Cu 2 AMD

ANTI-CANCER ACTIVITY

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

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

Invasive CXCR4 mutants

Control Drug/ no CXCL12 CXCL12 Drug + CXCL12

Cancer Cell Invasion Assay 47 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.

CONCLUSIONS

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.

Analogues Prepared and (Tested) 50 Metal Complexes: Zn 2+,Cu 2+,Ni 2+,Co 2+ Current Leads: Cu(Ligand 1) Zn(Ligand 7)

Recent Developments Synthesis of Unsubstituted Cross-Bridged Linked Ligands All current cross-bridged analogues have four tertiary nitrogens, due to synthetic method. The presence of a secondary nitrogen may enhance CXCR4 binding through H-bonding.

52 2. If two is good, three is better? C 3 Symmetric Compounds All current linked analogues two macrocycles. The presence of a third macrocycle may enhance CXCR4 binding through another AA.

53 3. Targeting CXCR4 is good, is targeting CXCR4/CCR5 better? None of our current compounds works on CCR5. A Dual CXCR4/CCR5 Antagonist incorporates macrocycles and a 2,6-dichloropyridine pharmacophore.

54 Acknowledgements Funding – OK-INBRE (NIH) – Research Corporation – SWOSU COLLABORTORS Dr. Steve Archibald (Hull) Abid Khan Prof. Erik De Clercq (Leuven) Dr. Christophe Pannecouque(Leuven) Dr. Dominique Schols (Leuven) Prof Tony Ng (KCL) Dr. Gilbert Fruhwirth (KCL) Dr. Jana Barlic (OMRF) Current research group: Courtney Garcia (Pre-Med) Desiray Cannon (Chemistry) Kevin Wilson (Chemistry) Past members: Robert Ullom—University of Kansas (Medicine) TauLyn Snell—Wichita State University (PA) 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) Neil Funwie—OU (Petroleum Engineering) Orry Birdsong—UT Galveston (Medicine) Kimberly Roewe—OSU (Chemistry) Kiet Ngyuen—SWOSU (Pharmacy) Katherine Coats (Chemistry) Josh Priddle—OSU (Medicine)