Supplementary Methods from In vivo optical imaging of human lymphoma xenograft using a library-derived peptidomimetic against α4β1 integrin

Supplementary Figures 1-4, Methods from Halogenated Benzimidazole Carboxamides Target Integrin α₄β₁ on T-Cell and B-Cell Lymphomas

Abstract Integrin α4β1 is an attractive but poorly understood target for selective diagnosis and treatment of T-cell and B-cell lymphomas. This report focuses on the rapid microwave preparation, structure-activity relationships, and biological evaluation of medicinally pertinent benzimidazole heterocycles as integrin α4β1 antagonists. We documented tumor uptake of derivatives labeled with 125I in xenograft murine models of B-cell lymphoma. Molecular homology models of integrin α4β1 predicted that docked halobenzimidazole carboxamides have the halogen atom in a suitable orientation for halogen-hydrogen bonding. The high-affinity halogenated ligands identified offer attractive tools for medicinal and biological use, including fluoro and iodo derivatives with potential radiodiagnostic (18F) or radiotherapeutic (131I) applications, or chloro and bromo analogues that could provide structural insights into integrin-ligand interactions through photoaffinity, cross-linking/mass spectroscopy, and X-ray crystallographic studies. Cancer Res; 70(13); 5448–56. ©2010 AACR.

Current cancer chemotherapeutic agents clinically deployed today are designed to be indiscriminately cytotoxic, however, achieving selective targeting of cancer malignancies would allow for improved diagnostic and chemotherapeutic tools. Integrin alpha(4)beta(1), a heterodimeric cell surface receptor, is believed to have a low-affinity conformation in resting normal lymphocytes and an activated high-affinity conformation in cancerous cells, specifically T- and B-cell lymphomas. This highly attractive yet poorly understood receptor has been selectively targeted with the bisaryl urea peptidomimetic antagonist 1. However, concerns regarding its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzothiazole moiety, resulting in an analogue with improved physicochemical properties, solubility, and kidney:tumor ratio while maintaining potency (6; IC(50) = 53 pM). The results presented herein utilized heterocyclic and solid-phase chemistry, cell adhesion assay, and in vivo optical imaging using the cyanine dye Cy5.5 conjugate.

AbstractIncreasing literature suggests that cell adhesion molecule α4β1 integrin plays a pivotal role in autoimmune diseases and cancer development. Noninvasive visualization of α4β1 integrin in vivo will facilitate the understanding of its involvement in disease progression and development of targeted therapies. Due to the lack of high-affinity targeting ligands, molecular imaging of α4β1 integrin is much less explored than that of αvβ3 and αvβ5 integrins. We have recently reported using the one bead–one compound combinatorial library method to identify a peptidomimetic, LLP2A, that preferentially binds to activated α4β1 integrin. Here, we described the use of LLP2A-Cy5.5 conjugate as an in vivo optical imaging probe in a human lymphoma xenograft model. This univalent LLP2A-Cy5.5 conjugate retained the binding activity and specificity to α4β1 integrin as shown by cell binding assays using α4β1-positive Molt-4 T-leukemia cells. The subcutaneous Molt-4 tumor was clearly visualized from 1 to 24 h after tail vein injection of the conjugate. Direct imaging and confocal microscopic examination of excised tumors and organs confirmed the accumulation of LLP2A in tumors and revealed very little or no uptake in normal organs except for lymph nodes. Kidney uptake was high when the whole organ was scanned but it was negative when examined microscopically, suggesting that LLP2A bound to the renal tubules loosely. Tumor uptake of LLP2A-Cy5.5 conjugate was blocked by excess unlabeled LLP2A. This study showed that the combinatorial chemical library-derived peptidomimetic LLP2A can be easily developed into an optical imaging probe for noninvasively monitoring of activated α4β1 integrin in vivo. [Mol Cancer Ther 2008;7(2):432–7]

C121 There is a need to develop cell surface targeting ligands for glioblastoma. The one-bead one-compound (OBOC) combinatorial library method is an enabling technology that allows one to probe tens of thousands to millions of different synthetic compounds against various molecular targets, which include soluble proteins or intact cells. The OBOC chemical library consists of large number of 90 micron polystyrene beads, each displaying a unique peptide, peptidomimetic or small molecule. When the compound-bead library is mixed with intact cancer cells, beads that coated with cancer cells can be physically isolated and chemically decoded (1). Using these methods, we have identified high-affinity peptidic and peptidomimetic ligands against lymphoid cancer (2) and ovarian cancer (3). We have further demonstrated their excellent in vivo targeting potential in xenograft model and canine model. Recently, we applied the same approach to screen OBOC combinatorial cyclic peptide libraries, and through several cycles of discovery and optimization, we succeeded in identifying cyclic peptides (LXY1 and LXY3) that target U-87 human glioblastoma cell line with high affinity and specificity. In vivo optical imaging studies demonstrated that these ligands were able to target both orthotopic and subcutaneous U-87 implant with high specificity. Work is currently underway in our laboratory to develop LXY1 and LXY3 into therapeutic targeting agents, using these cyclic peptides as vehicles to deliver radionuclides and cytotoxic agents to orthotopic and subcutaneous glioblastoma implants. In conclusion, OBOC combinatorial chemistry is a powerful and proven method for the development of cancer targeting agents. References 1. Aina OH, Liu R, Sutcliffe-Goulden JL, Marik J, Pan C, and Lam KS. From Combinatorial Chemistry to Cancer Targeting Peptides. Molecular Pharmaceutics, in press, 2007. 2. Peng L, Liu R, Marik J, Wang X, Takada Y, Lam KS. Combinatorial Chemistry Identifies High-Affinity Peptidomimetics against α4β1 Integrin. Nature Chemical Biology, 2(7). 381-389, 2006. 3. Aina OH, Marik J, Liu R, Lau DH, and Lam KS: Identification of novel targeting peptides for human ovarian cancer cells using OBOC combinatorial libraries. Molecular Cancer Therapeutics, 4:806-813, 2005.

The cell surface receptor alpha 4 beta 1 integrin, activated constitutively in lymphoma, can be targeted with the bisaryl urea peptidomimetic antagonist 1 (LLP2A). However, concerns on its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzimidazole moiety, resulting in improved solubility while maintaining picomolar potency [5 (KLCA4); IC50 = 305 pM]. With exceptional solubility, this finding has the potential for improving PK to help diagnose and treat lymphomas.