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Structural characterization and in vivo evaluation of ?-Hairpin peptidomimetics as specific CXCR4 imaging agents.

ABSTRACT: The CXCR4 chemokine receptor is integral to several biological functions and plays a pivotal role in the pathophysiology of many diseases. As such, CXCR4 is an enticing target for the development of imaging and therapeutic agents. Here we report the evaluation of the POL3026 peptidomimetic template for the development of imaging agents that target CXCR4. Structural and conformational analyses of POL3026 and two of its conjugates, DOTA (POL-D) and PEG12-DOTA (POL-PD), by circular dichroism, two-dimensional NMR spectroscopy and molecular dynamics calculations are reported. In silico observations were experimentally verified with in vitro affinity assays and rationalized using crystal structure-based molecular modeling studies. [(111)In]-labeled DOTA conjugates were assessed in vivo for target specificity in CXCR4 expressing subcutaneous U87 tumors (U87-stb-CXCR4) through single photon emission computed tomography (SPECT/CT) imaging and biodistribution studies. In silico and in vitro studies show that POL3026 and its conjugates demonstrate similar interactions with different micelles that mimic cellular membrane and that the ?-NH2 of lysine(7) is critical to maintain high affinity to CXCR4. Modification of this group with DOTA or PEG12-DOTA led to the decrease of IC50 value from 0.087 nM for POL3026 to 0.47 nM and 1.42 nM for POL-D and POL-PD, respectively. In spite of the decreased affinity toward CXCR4, [(111)In]POL-D and [(111)In]POL-PD demonstrated high and significant uptake in U87-stb-CXCR4 tumors compared to the control U87 tumors at 90 min and 24 h post injection. Uptake in U87-stb-CXCR4 tumors could be blocked by unlabeled POL3026, indicating specificity of the agents in vivo. These results suggest POL3026 as a promising template to develop new imaging agents that target CXCR4.

SUBMITTER: Lesniak WG

PROVIDER: S-EPMC4612581 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Structural characterization and in vivo evaluation of β-Hairpin peptidomimetics as specific CXCR4 imaging agents.

Lesniak Wojciech G WG Sikorska Emilia E Shallal Hassan H Behnam Azad Babak B Lisok Ala A Pullambhatla Mrudula M Pomper Martin G MG Nimmagadda Sridhar S

Molecular pharmaceutics 20150203 3

The CXCR4 chemokine receptor is integral to several biological functions and plays a pivotal role in the pathophysiology of many diseases. As such, CXCR4 is an enticing target for the development of imaging and therapeutic agents. Here we report the evaluation of the POL3026 peptidomimetic template for the development of imaging agents that target CXCR4. Structural and conformational analyses of POL3026 and two of its conjugates, DOTA (POL-D) and PEG12-DOTA (POL-PD), by circular dichroism, two-d ...[more]

PMID: 25590535

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Project description:UNLABELLED:Very-late-antigen-4 (VLA-4, ?4?1 integrin, CD49d/CD29) is a transmembrane adhesion receptor that plays an important role in cancer and immune responses. Enhanced VLA-4 expression has been observed in multiple myeloma (MM) cells and surrounding stroma. VLA-4 conformational activation has been associated with MM pathogenesis. VLA-4 is a promising MM imaging and therapeutic biomarker. METHODS:Specificity of (64)Cu-LLP2A ((64)Cu-CB-TE1A1P-PEG4-LLP2A), a high-affinity VLA-4 peptidomimetic-based radiopharmaceutical, was evaluated in ?4 knock-out mice and by competitive blocking in wild-type tumor-bearing mice. (64)Cu-LLP2A PET/CT (static and dynamic) imaging was conducted in C57BL6/KaLwRij mice bearing murine 5TGM1-GFP syngeneic tumors generated after intravenous injection via the tail. Blood samples were collected for serum protein electrophoresis. Bone marrow and splenic cells extracted from tumor-bearing and control mice (n= 3/group) were coincubated with the optical analog LLP2A-Cy5 and mouse B220, CD4, Gr1, and Mac1 antibodies and analyzed by fluorescence-activated cell sorting. Human radiation dose estimates for (64)Cu-LLP2A were extrapolated from mouse biodistribution data (6 time points, 0.78 MBq/animal, n= 4/group). Ten formalin-fixed paraffin-embedded bone marrow samples from deceased MM patients were stained with LLP2A-Cy5. RESULTS:(64)Cu-LLP2A and LLP2A-Cy5 demonstrated high specificity for VLA-4-positive mouse 5TGM1-GFP myeloma and nonmalignant inflammatory host cells such as T cells and myeloid/monocytic cells. Ex vivo flow cytometric analysis supported a direct effect of myeloma on increased VLA-4 expression in host hematopoietic microenvironmental elements. SUVs and the number of medullar lesions detected by (64)Cu-LLP2A PET corresponded with increased monoclonal (M) protein (g/dL) in tumor-bearing mice over time (3.29 ± 0.58 at week 0 and 9.97 ± 1.52 at week 3). Dynamic PET with (64)Cu-LLP2A and (18)F-FDG demonstrated comparable SUV in the prominent lesions in the femur. Human radiation dose estimates indicated urinary bladder wall as the dose-limiting organ (0.200 mGy/MBq), whereas the dose to the red marrow was 0.006 mGy/MBq. The effective dose was estimated to be 0.017 mSv/MBq. Seven of the ten human samples displayed a high proportion of cells intensely labeled with LLP2A-Cy5 probe. CONCLUSION:(64)Cu-LLP2A and LLP2A-Cy5 demonstrated binding specificity for VLA-4 in an immune-competent murine MM model. (64)Cu-LLP2A displayed favorable dosimetry for human studies and is a potential imaging candidate for overexpressed VLA-4.

Dataset Information

Structural characterization and in vivo evaluation of ?-Hairpin peptidomimetics as specific CXCR4 imaging agents.

Publications

Structural characterization and in vivo evaluation of β-Hairpin peptidomimetics as specific CXCR4 imaging agents.

Similar Datasets

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