1

Development and Screening of Transition Metal Complexes as CXCR4 Antagonists

Dr. Tim Hubin

Department of Chemistry and Physics

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

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

A

B C

D E

4

Disease states

• Role in disease

– Human Immunodeficiency Virus (along with CCR5)– Tumour growth and metastasis– Stem cell mobilization– Autoimmune disorders (rheumatoid arthritis)

5

• Inhibitors against 5 steps of HIV replication cycle

• Still need for new targets

• Prototype bicyclamsAMD3100

CXCR4-antagonists and HIV

6

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N N

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NH

HN NH

HN

HN

Gerlach et al., 2001

7

Blocking receptor functions

Cell

DrugCXCL12/HIV

8

Plerixafor/ AMD3100

N N

N N

NN

NN

H

H H

H

HH

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.

9

Molecular shape

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

N N

N N

H

H

H

H

trans-I trans-II trans-III trans-IV trans-Vcis-V

10

Restrict to one configuration

N N

N N

N N

N N

H

H

H

H

Only cis V

trans-II

NN X

N

N

R

R

X

Only trans-II

11

Side-Bridged Synthesis

N N

NN

N N

NN

Br- N N

NNBr-

Br

Br

N N

HNN

NH N

NN

NaBH4

H

H

H

H(a)

(b)

H

H

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

12

N N

NN

Br- N N

NNBr-

N N

NN

N N

NN

NaBH4

H

H

H

H

N N

NN

Br- N N

NNBr-

H

H

H

H

MeI

Cross-Bridged Synthesis

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

(a)

(b)

13

N N

N N

NN

NN

H

H H

H

HH

AMD3100

N N

N N

NN

NNH H

N

NN

NN

NN

N

Me Me

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

14

Cu-O1 2.28(1) Å

Side bridged (SB) Cross bridged (CB)

Cu-O1 1.95(1) Å

N N

HNN

NH N

NN

N N

NN

N N

NN

Axial Equatorial

15

Cu

N

N N

Cl

N

Cu

N

N N

N

Cl

Cu

N

N N

Asp

N

Cu

N

N N

N

Asp

shorter /stronger

longer /weaker

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

16

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) A B C

18

Binding by flow cytometry

CXCR4

Drug molecule

Receptor specific antibody

Fluorescent antibody

Key Name Parameter- control.001 FL1-H

+ Control 717.019 FL1-H

L2 717.010 FL1-H

L1 717.009 FL1-H

mAB Inhibition

19

0 20 40 60 80 100 120

CB

SB

AMD3100

[Cu2SB]4+

[Cu2AMD3100]4+

[Cu2CB]4+

[Zn2CB]4+

[Zn2AMD3100]4+

[Zn2SB]4+

mAb Inhibition / %

Anta

goni

st

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

Competitive Binding Studies

20

Antagonist Concentration (μM)

IC50 EC50 CI (95 %) AMD3100 0.360 ± 0.030 0.134 0.067 - 0.267

Zn-AMD3100 0.278 ± 0.033 0.164 0.127 - 0.211

Cu-AMD3100 0.439 ± 0.021 0.303 0.240 - 0.382

Zn-Side Bridged 0.922 ± 0.161 0.740 0.457 - 1.200

Cu-Side Bridged 0.251 ± 0.074 0.251 0.191 - 0.330

Zn-Cross Bridged 0.230 ± 0.004 0.109 0.046 - 0.257

Cu-Cross Bridged 0.160 ± 0.004 0.100 0.086 - 0.116

IC50 and EC50 concentrations for CXCR4 antagonists in competition with mAb 44717 in Jurkat cells.

21

Residence time

1 24 48 72 960

10

20

30

40

50

60

70

80

90

100

Copper 3

AMD3100

Copper AMD3100

hours

% in

hibi

tion

of a

ntibo

dy b

indi

ng

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

Compound Concentration (16 nM)

(τ) (hr) t1/2 diss (hr)

AMD3100 9.7 6.7

Cu2-AMD3100 23.5 16.3

Cu2-Cross Bridged

49.0 33.9

Relative (τ) and t1/2 dissociation constants for compounds binding to CXCR4 receptor in Jurkat cells.

HIV Infection Assays

23

Table 1. Anti-HIV activities, cytotoxicity and selectivity index in MT-4 cells.

aaverage effective concentration to reduce the HIV-induced cytopathic effect by 50% in MT-4 cells. b standard deviation over three assays. c Concentration required to have a cytotoxic effect reducing MT-4 cell viability by 50%. d Selectivity index. AMD3100 data from Este, et al Mol. Pharmocol. 1999, 55(1), 67-73.

Compound HIV strain Av EC50(μM)a SDb Av CC50(μM)c SId Zn2Side-Bridged HIV-1 (IIIB) 0.0025 0.0010 60.56 29,215

HIV-2 (ROD) 0.0040 0.0013 60.56 16,077

Cu2Cross-Bridged AMD3100 Zn2-AMD3100

HIV-1 (IIIB) 0.00459 0.0017 >125 >27,233 HIV-2 (ROD) HIV-1 (IIIB) HIV-1 (IIIB)

0.0171 0.011 0.008

0.0011

>125 >225 >225

7,331 >20,455 >28,125

Ca2+ Ion Signaling Assays

24

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

0 50 100 150

Control1000 ng/ml200 ng/ml40 ng/ml8 ng/ml

Time (sec)

Fluo

resc

ence

Cha

nge

(cou

nts)

Ca-signaling data for AMD3100 CXCR4 experiment by collaborator Schols.

25

Table 2. IC50 (ng/ml) values calculated from Ca-signaling experiments U87.CD4.CXCR4 U87.CD4.CCR5 Cu2-Cross Bridged 4.33 >1000 Zn2-Cross Bridged 5.42 >1000 Cu2-Side Bridged 43.97 >1000 Zn2-Side Bridged <1.6 >1000 AMD3100 16.7 >1000 Cu2-AMD3100 72.67 >1000 Zn2-AMD3100 <1.6 >1000 maraviroc >1000 2.09

AMD3451 Micromolar Micromolar

CXCR4 and Cancer Cell Metastasiso CXCL12 is normally responsible for trafficking of lymphocytes

o CXCL12 is secreted by stromal, lung and liver cells, and lymph nodes

o The interaction at the cell membrane is through CXCR4, which is over-expressed in some cancers

o Potential mechanism of metathesis

Normal cell Cancer cell

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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

Control

Drug/ no CXCL12

CXCL12

Drug + CXCL12

Cancer Cell Invasion Assay

29

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.

• 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)

31

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02468

1012141618

0

1

2

3

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7

0

0.1

0.2

0.3

0.4

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0.7

Time (h): - 2 4 8 Time (h): - 2 4 8

Time (h): - 2 4 8

Tota

l leu

kocy

te c

ount

(x10

3 /ml)

Tota

l neu

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3 /ml)

Tota

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x103 /m

l)

saline AMD3100 SAJ5baseline

34

AcknowledgementsFunding– 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

NN

N N

N N

NNH3C CH3

United StatesWeatherford—synthesis/characterization (Hubin)Oklahoma City—stem cell mobilization, atheroregression, obesity (Barlic)

United KingdomHull—synthesis/characterization, CXCR4 binding, imaging, cancer metastases (Archibald)London—cancer cell imaging (Ng), PET Imaging, pharmacology (Blower)

BelgiumLeuven—anti-HIV properties (DeClercq, Pannecouque), Ca-Signaling (Schols)

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

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

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

38

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