2-5A ligands - A new concept for the treatment of prostate cancer

Article abstract of DOI:10.1080/15257770701542652

Several potent prostate specific membrane antigen (PSMA) inhibitors have been described recently. We generated a PSMA-specific 2-5A ligand called RBI 1033 by linking 2-5A to the N-acetylaspartylglutamate (NAAG)-based inhibitor ZJ-24. We measured the inhibitory activity of RBI 1033 to the folate hydrolase activity of PSMA. Amazingly, we found that compared to ZJ-24 (IC50 = 53.9 nM), RBI 1033 was more than 10 times more potent (IC50 = 4.78 nM) as a folate hydrolase inhibitor, while SMCC 2-5A lacking the ZJ-24 part, did not show much activity (IC50 = 1974 nM). Also, RBI 1033's affinity to PSMA was found to be 10 times higher than ZJ-24 itself. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/15257770701542652

This article was downloaded by: [New York University]
On: 18 April 2015, At: 06:02
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic
Acids
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lncn20
2-5A Ligands—A New Concept for the
Treatment of Prostate Cancer
Hagen Cramer a , James R. Okicki a , Taikyun Rho a , Xinning Wang b ,
Robert H. Silverman b & Warren D. W. Heston b
a Ridgeway Biosystems, Inc. , Cleveland, Ohio, USA
b Cleveland Clinic Foundation , Cleveland, Ohio, USA
Published online: 05 Dec 2007.
To cite this article: Hagen Cramer , James R. Okicki , Taikyun Rho , Xinning Wang , Robert
H. Silverman & Warren D. W. Heston (2007) 2-5A Ligands—A New Concept for the Treatment
of Prostate Cancer, Nucleosides, Nucleotides and Nucleic Acids, 26:10-12, 1471-1477, DOI:
10.1080/15257770701542652
To link to this article: http://dx.doi.org/10.1080/15257770701542652
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,
proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Nucleosides, Nucleotides, and Nucleic Acids, 26:1471–1477, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701542652
2-5A LIGANDS—A NEW CONCEPT FOR THE TREATMENT
OF PROSTATE CANCER
Hagen Cramer, James R. Okicki, and Taikyun Rho
Ridgeway Biosystems, Inc.,
2
Cleveland, Ohio, USA
Xinning Wang, Robert H. Silverman, and Warren D. W. Heston
Cleveland
2
Clinic Foundation, Cleveland, Ohio, USA
Several potent prostate specific membrane antigen (PSMA) inhibitors have been described re-
2
cently. We generated a PSMA-specific 2-5A ligand called RBI 1033 by linking 2-5A to the N-
acetylaspartylglutamate (NAAG)-based inhibitor ZJ-24. We measured the inhibitory activity of RBI
1033 to the folate hydrolase activity of PSMA. Amazingly, we found that compared to ZJ-24 (IC50
= 53.9 nM), RBI 1033 was more than 10 times more potent (IC50 = 4.78 nM) as a folate hy-
drolase inhibitor, while SMCC 2-5A lacking the ZJ-24 part, did not show much activity (IC50 =
1974 nM). Also, RBI 1033’s affinity to PSMA was found to be 10 times higher than ZJ-24 itself.
Keywords PSMA; RBI 1033; inhibitory activitty
INTRODUCTION
Ridgeway Biosystems, Inc. is developing novel drugs for targeting cancer
and infectious diseases by harnessing the powerful endoribonuclease RNase
L. RNase L is activated by 2ꢁ,5ꢁ-linked oligoadenylates collectively referred
to as 2-5A. To date the only well-established biochemical function of 2-5A is
activation of RNase L. It was found that 2-5A activation of RNase L leads to
RNA damage-mediated apoptosis in metastatic prostate tumor cell lines.[1]
In this investigation we linked a stabilized 2-5A analog to a ligand, which
specifically recognizes prostate specific membrane antigen (PSMA), thereby
directing 2-5A directly into prostate cancer cells.
PSMA is a unique membrane bound glycoprotein, which is highly
prostate specific and greatly overexpressed on prostate cancer cells and on
tumor vascular endothelium of virtually all solid carcinomas and sarcomas,
H.C. is presently at Girindus America, Inc., 8560 Reading Rd., Cincinnati, OH 45215, USA.
This work was supported in part by NIH grant CA 101069-02.
Address correspondence to Hagen Cramer, Girindus America, Inc., 8560 Reading Rd.,Cincinnati,
OH 45215. E-mail: hcramer@girindus.com
1471

1472
H. Cramer et al.
but not in the vasculature of normal tissues.[2−10] PSMA is a type II integral
cell-surface membrane protein that is not secreted.[11] PSMA, like other cell
surface receptors, undergoes internalization, thereby mediating the inter-
nalization of a putative ligand. This makes PSMA an ideal target for the
treatment of cancer in particular prostate cancer.
RESULTS AND DISCUSSION
We previously have shown that RNase L activation by 2-5A leads to
apoptosis and therefore to the killing of prostate tumor cells.[1] However,
so far delivery to the target organ has not been addressed. It was shown
that small ligands based on the structure of NAAG show very high binding
affinity to the NAALADase domain of prostate specific membrane antigen
(PSMA).[12−15] Structure activity relationship (SAR) studies revealed that a
urea containing at least one glutamate residue plus a second residue bear-
ing a carboxyl group in addition to another group (SR orCO2H) represents
the minimum requirement to achieve effective NAALADase inhibition.[16]
Based on these findings several simple di-peptide ligands were identified
with activities against NAALADase in the low nanomolar range.[12] Such lig-
ands formed the basis of the novel 2-5A ligand presented here.
Synthesis of the 2-5A Ligand RBI1033
RBI 1033 was prepared by linking an amino modified 2-5A moiety to the
thiol group of the dipeptidyl ligand Cys-C(O)-Glu using the bifunctional
linker SMCC (Pierce, WI, USA). The urea-based ligand Cys-C(O)-Glu was
prepared following published procedures.[12]
The amino-functionalized 2-5A moiety (RBI 1024) was synthesized by
means of solid-phase synthesis using a Polygen 10 DNA synthesizer. Thereby
a 5 umol slider was filled with 3ꢁ-Phtalimidyl-Amino Modifier C3 CPG (Glen
Research, VA, USA). The first three couplings were performed using 5ꢁ-O-
DMT-3ꢁ-O-TBDMS-N-Bz-adenosince 2ꢁ-CED-phosphoramidte (ChemGenes,
MA, USA) and the last coupling using a TMT-on phosphorylation reagent,
which was synthesized using published procedures (Guzaev and Manoha-
ran, 2001). Beaucage Reagent was used as sulfurization reagent. The
free amino-functionalized 2-5A moiety was isolated by (1) detritylation on
the column: (2) cleavage from the support and base deprotection using
NH4OH/EtOH 3:1 and; (3) desilylation using a cocktail made up of N-
methyl-pyrrolidinone, triethylamine, TEA x 3HF (1.5/0.75/1.0 v/v/v).
The amino-functionalized 2-5A moiety (RBI 1024) was conjugated to
the ligand Cys-C(O)-Glu using the bifunctional linker ulfosuccinimidyl-
4-(N-maleimidomethyl)-1-carboxylate (SMCC, Pierce, WI, USA) (see
Figure 1). After RP-HPLC purification RBI 1032 was coupled to the ligand
Cys-C(O)-Glu. The final product was purified by HPLC and its mass con-
firmed by mass spectrometry using MALDI.

2-5A Ligands
1473
FIGURE 1 Synthesis of 2-5A ligand RBI 1033. The amino-modified 2-5A moiety RBI 1024 was linked to
the urea-based dipeptidyl peptide Cys-C(O)-Glu using the bifunctional linker SMCC.
RNase L Activation, PSMA Binding, and PSMA Activity Assays
The 2-5A ligand was tested on its ability to activate RNase L in a pre-
viously developed fluorescence resonance energy transfer (FRET) assay,
which is now being used routinely in our lab for screening novel 2-5A
analogs on activity.[17] Thereby, the 2-5A analog was added to a cell-free
system containing purified RNase L and a cleavable 36-nucleotide RNA sub-
strate labeled with fluorescein at the 5ꢁ- and with black hole quencher 1
at the 3ꢁ-end. EC50 (concentration of activator to give 50% maximum acti-
vation) was determined for the 2-5A ligand and found to be about 10 nM
(see Figure 2). Therefore, the activity is somewhat reduced in comparison to
natural 2-5A. However, the activity is similar to other 2’-tailed 2-5A analogs
such as 2-5A antisense. Despite this decreased activity in vitro, such tailed 2-
5A analogs show enhanced activity in vivo due to their increased resistance
towards 2’,5’ ribonucleases.[18,19]
Binding of the novel 2-5A ligand RBI 1033 was compared to the methyl
thioether of the parent ligand, MeS-Cys-C(O)-Glu (ZJ-24) and RBI 1032 (2-
5A devote of the ligand moiety, see Figure 1) toward the active dimeric
form of soluble recombinant hPSMA (see Figure 3). The binding assay

1474
H. Cramer et al.
FIGURE 2 RNase L activation assay. RBI 1033’s ability to activate RNase L in the presence of a fluores-
cence resonance energy transfer (FRET) labeled RNA substrate. 2-5A ligand activates RNase L with an
EC50 of 10 nM.
FIGURE 3 PSMA binding assay. Ability of a novel compound to compete for binding with radiolabeled
ligand. 2-5A ligand binds to PSMA at lower concentrations (EC50 = 1.5 nM) than parent ligand ZJ-24
(EC50 = 15.3 nM).

2-5A Ligands
1475
FIGURE 4 PSMA activity assay: Ability of hPSMA to cleave/hydrolyze a polyglutamte substrate under
presence of a ligand at different concentrations. 2-5A ligand inhibits PSMA at lower concentrations
(EC50 = 0.62 nM) than parent ligand ZJ-24 (EC50 = 56.7 nM).
examines the ability of a novel compound to compete for binding with
radio-labeled ligand ZJ-24.[20] Thereby, different final concentrations of the
inhibitor are incubated in the presence of ZJ-24∗ and the concentration
determined, which is required to inhibit 50% of binding. The ligand con-
centration reducing binding by 50% is considered the EC50 of the ligand
analog.
We determined the ability of hPSMA to cleave/hydrolyze a polyglutamte
substrate under the presence of a ligand at different concentrations.[21,22]
(see Figure 4). Briefly, recombinant PSMA was incubated with ligand ZJ-24,
2-5A ligand RBI 1033, or RBI 1032. The polyglutamate substrate MTXGlu2
(5 nMol) was added and the amount of MTX formed was determined
by HPLC.
From the data presented here, we can conclude that the 2-5A ligand
RBI 1033 is superior in binding and inhibiting PSMA than the parent lig-
and itself. The EC50 for binding was found to be 1.5 nM for RBI 1033
versus 15.3 nM for the parent ligand 6, while the inhibitory activity of
RBI 1033 was 0.62 nM in comparison to 56.7 nM for the parent ligand.
To verify that this increased activity is not due to non-specific binding of
the 2-5A or SMCC part of the molecule, we determined the binding and
inhibitory activity of RBI 1032 to PSMA as well. However, it was found
that the SMCC 2-5A alone bound only weakly to PSMA with an EC50 of
3.5 mM (Figure 3) and the inhibitory activity was only in the millimolar
range.

1476
CONCLUSION
H. Cramer et al.
By chemically linking a 2-5A moiety to a ligand moiety we were able
to create a 2-5A ligand with molecular weights below 1500 Dalton, which
binds to PSMA with extremely high affinities and it is believed to become
internalized into the cancer cell together with the protein. In comparison
to mAb strategies currently in clinical trials for imaging and therapy[23−25]
these molecules are very small and much easier and cheaper to make.
REFERENCES
1. Xiang, Y.; Wang, Z.; Murakami, J.; Plummer, S.; Klein, E.A.; Carpten, J.D.; Trent, J.M.; Isaacs, W.B.;
Casey, G.; Silverman, R.H. Effects of RNase L mutations associated with prostate cancer on apoptosis
induced by 2ꢁ,5ꢁ-oligoadenylates. Cancer Res. 2003, 63 (20), 6795–6801.
2. Murphy, G.P.; Elgamal, A.A.; Su, S.L.; Bostwick, D.G.; Holmes, E.H. Current evaluation of the tis-
sue localization and diagnostic utility of prostate specific membrane antigen. Cancer 1998, 83 (11),
2259–2269.
3. Israeli, R.S.; Powell, C.T.; Corr, J.G.; Fair, W.R.; Heston, W.D. Expression of the prostate-specific
membrane antigen. Cancer Res. 1994, 54 (7), 1807–1811.
4. Wright, G.L., Jr.; Grob, B.M.; Haley, C.; Grossman, K.; Newhall, K.; Petrylak, D.; Troyer, J.; Konchuba,
A.; Schellhammer, P.F.; Moriarty, R. Upregulation of prostate-specific membrane antigen after
androgen-deprivation therapy. Urology 1996, 48 (2), 326–334.
5. Liu, H.; Moy, P.; Kim, S.; Xia, Y.; Rajasekaran, A.; Navarro, V.; Knudsen, B.; Bander, N.H. Monoclonal
antibodies to the extracellular domain of prostate-specific membrane antigen also react with tumor
vascular endothelium. Cancer Res. 1997, 57 (17), 3629–3634.
6. Chang, S.S.; Reuter, V.E.; Heston, W.D.; Bander, N.H.; Grauer, L.S.; Gaudin, P.B. Five different
anti-prostate-specific membrane antigen (PSMA) antibodies confirm PSMA expression in tumor-
associated neovasculature. Cancer Res. 1999, 59 (13), 3192–3198.
7. Chang, S.S.; O’Keefe, D.S.; Bacich, D.J.; Reuter, V.E.; Heston, W.D.; Gaudin, P.B. Prostate-specific
membrane antigen is produced in tumor-associated neovasculature. Clin. Cancer Res. 1999, 5 (10),
2674–2681.
8. Dumas, F.; Gala, J.L.; Berteau, P.; Brasseur, F.; Eschwege, P.; Paradis, V.; Lacour, B.; Philippe, M.;
Loric, S. Molecular expression of PSMA mRNA and protein in primary renal tumors. Int. J. Cancer
1999, 80 (6), 799–803.
9. Chang, S.S.; Reuter, V.E.; Heston, W.D.; Gaudin, P.B. Metastatic renal cell carcinoma neovasculature
expresses prostate-specific membrane antigen. Urology 2001, 57 (4), 801–805.
10. Silver, D.A.; Pellicer, I.; Fair, W.R.; Heston, W.D.; Cordon-Cardo, C. Prostate-specific membrane
antigen expression in normal and malignant human tissues. Clin. Cancer Res. 1997, 3 (1),
81–85.
11. Troyer, J.K.; Beckett, M.L.; Wright, G.L., Jr. Detection and characterization of the prostate-
specific membrane antigen (PSMA) in tissue extracts and body fluids. Int. J. Cancer 1995, 62 (5),
552–558.
12. Kozikowski, A.P.; Nan, F.; Conti, P.; Zhang, J.; Ramadan, E.; Bzdega, T.; Wroblewska, B.; Neale, J.H.;
Pshenichkin, S.; Wroblewski, J.T. Design of remarkably simple, yet potent urea-based inhibitors of
glutamate carboxypeptidase II (NAALADase). J. Med. Chem. 2001, 44 (3), 298–301.
13. Jackson, P.F.; Tays, K.L.; Maclin, K.M.; Ko, Y.S.; Li, W.; Vitharana, D.; Tsukamoto, T.; Stoermer, D.;
Lu, X.C.; Wozniak, K.; Slusher, B.S. Design and pharmacological activity of phosphinic acid based
NAALADase inhibitors. J. Med. Chem. 2001, 44 (24), 4170–4175.
14. Tang, H.; Brown, M.; Ye, Y.; Huang, G.; Zhang, Y.; Wang, Y.; Zhai, H.; Chen, X.; Shen, T.Y.; Tennis-
wood, M. Prostate targeting ligands based on N-acetylated alpha-linked acidic dipeptidase. Biochem.
Biophys. Res. Commun. 2003, 307 (1), 8–14.
15. Majer, P.; Jackson, P.F.; Delahanty, G.; Grella, B.S.; Ko, Y.S.; Li, W.; Liu, Q.; Maclin, K.M.; Po-
lakova, J.; Shaffer, K.A.; Stoermer, D.; Vitharana, D.; Wang, E.Y.; Zakrzewski, A.; Rojas, C.; Slusher,
B.S.; Wozniak, K.M.; Burak, E.; Limsakun, T.; Tsukamoto, T. Synthesis and biological evaluation of

2-5A Ligands
1477
thiol-based inhibitors of glutamate carboxypeptidase II: discovery of an orally active GCP II in-
hibitor. J. Med. Chem. 2003, 46 (10), 1989–1996.
16. Rong, S.B.; Zhang, J.; Neale, J.H.; Wroblewski, J.T.; Wang, S.; Kozikowski, A.P. Molecular modeling
of the interactions of glutamate carboxypeptidase II with its potent NAAG-based inhibitors. J. Med.
Chem. 2002, 45 (19), 4140–4152.
17. Geselowitz, D.A.; Cramer, H.; Wondrak, E.M.; Player, M.R.; Torrence, P.F. Fluorescence resonance
energy transfer analysis of RNase L-catalyzed oligonucleotide cleavage. Antisense Nucleic Acid Drug
Dev. 2000, 10 (1), 45–51.
18. Imai, J.; Johnston, M.I.; Torrence, P.F. Chemical modification potentiates the biological activities of
2-5A and its congeners. J. Biol. Chem. 1982, 257 (21), 12739–12745.
19. Defilippi, P.; Huez, G.; Verhaegen-Lawalle, M.; De Clercq, E.; Imai, J.; Torrence, P.; Content, J. An-
tiviral activity of a chemically stabilized 2-5A analog upon microinjection into HeLa cells. FEBS Lett.
1986, 198 (2), 326–332.
20. Pomper, M.G.; Musachio, J.L.; Zhang, J.; Scheffel, U.; Zhou, Y.; Hilton, J.; Maini, A.; Dannals, R.F.;
Wong, D.F.; Kozikowski, A.P. 11C-MCG: synthesis, uptake selectivity, and primate PET of a probe for
glutamate carboxypeptidase II (NAALADase). Mol. Imaging, 2002, 1 (2), 96–101.
21. Heston, W.D. Characterization and glutamyl preferring carboxypeptidase function of prostate spe-
cific membrane antigen: a novel folate hydrolase. Urology 1997, 49 (3A Suppl), 104–112.
22. Pinto, J.T.; Suffoletto, B.P.; Berzin, T.M.; Qiao, C.H.; Lin, S.; Tong, W.P.; May, F.; Mukherjee, B.;
Heston, W.D. Prostate-specific membrane antigen: a novel folate hydrolase in human prostatic car-
cinoma cells. Clin. Cancer Res. 1996, 2 (9), 1445–1451.
23. Bander, N.H.; Nanus, D.M.; Milowsky, M.I.; Kostakoglu, L.; Vallabahajosula, S.; Goldsmith, S.J. Tar-
geted systemic therapy of prostate cancer with a monoclonal antibody to prostate-specific mem-
brane antigen. Semin. Oncol. 2003, 30 (5), 667–676.
24. Nanus, D.M.; Milowsky, M.I.; Kostakoglu, L.; Smith-Jones, P.M.; Vallabahajosula, S.; Goldsmith, S.J.;
Bander, N.H. Clinical use of monoclonal antibody HuJ591 therapy: targeting prostate specific mem-
brane antigen. J. Urol. 2003, 170 (6 Pt 2), S84–S88; discussion S88–S89.
25. Bander, N.H.; Trabulsi, E.J.; Kostakoglu, L.; Yao, D.; Vallabhajosula, S.; Smith-Jones, P.; Joyce, M.A.;
Milowsky, M.; Nanus, D.M.; Goldsmith, S.J. Targeting metastatic prostate cancer with radiolabeled
monoclonal antibody J591 to the extracellular domain of prostate specific membrane antigen.
J. Urol. 2003, 170 (5), 1717–1721.