Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (RTK) commonly targeted for inhibition by anticancer therapeutics. Current therapeutics target EGFR's kinase domain or extracellular region. However, these types of inhibitors are not specific for tumors over healthy tissue and therefore cause undesirable side effects. Our lab has recently developed a new strategy to regulate RTK activity by designing a peptide that specifically binds to the transmembrane (TM) region of the RTK to allosterically modify kinase activity. These peptides are acidity-responsive, allowing them to preferentially target acidic environments like tumors. We have applied this strategy to EGFR and created the PET1 peptide. We observed that PET1 behaves as a pH-responsive peptide that modulates the configuration of the EGFR TM through a direct interaction. Our data indicated that PET1 inhibits EGFR-mediated cell migration. Finally, we investigated the mechanism of inhibition through molecular dynamics simulations, which showed that PET1 sits between the two EGFR TM helices; this molecular mechanism was additionally supported by AlphaFold-Multimer predictions. We propose that the PET1-induced disruption of native TM interactions disturbs the conformation of the kinase domain in such a way that it inhibits EGFR's ability to send migratory cell signals. This study is a proof-of-concept that acidity-responsive membrane peptide ligands can be generally applied to RTKs. In addition, PET1 constitutes a viable approach to therapeutically target the TM of EGFR.

18FDG-PET predicts pharmacodynamic response to OSI-906, a dual IGF-1R/IR inhibitor, in preclinical mouse models of lung cancer.

PURPOSE: To evaluate 2-deoxy-2-[(18)F]fluoro-d-glucose positron emission tomography imaging ((18)FDG-PET) as a predictive, noninvasive, pharmacodynamic (PD) biomarker of response following administration of a small-molecule insulin-like growth factor-1 receptor and insulin receptor (IGF-1R/IR) inhibitor, OSI-906. EXPERIMENTAL DESIGN: In vitro uptake studies of (3)H-2-deoxy glucose following OSI-906 exposure were conducted evaluating correlation of dose with inhibition of IGF-1R/IR as well as markers of downstream pathways and glucose metabolism. Similarly, in vivo PD effects were evaluated in human tumor cell line xenografts propagated in athymic nude mice by (18)FDG-PET at 2, 4, and 24 hours following a single treatment of OSI-906 for the correlation of inhibition of receptor targets and downstream markers. RESULTS: Uptake of (3)H-2-deoxy glucose and (18)FDG was significantly diminished following OSI-906 exposure in sensitive tumor cells and subcutaneous xenografts (NCI-H292) but not in an insensitive model lacking IGF-1R expression (NCI-H441). Diminished PD (18)FDG-PET, collected immediately following the initial treatment agreed with inhibition of pIGF-1R/pIR, reduced PI3K (phosphoinositide 3-kinase) and MAPK (mitogen activated protein kinase) pathway activity, and predicted tumor growth arrest as measured by high-resolution ultrasound imaging. CONCLUSION: (18)FDG-PET seems to serve as a rapid, noninvasive PD marker of IGF-1R/IR inhibition following a single dose of OSI-906 and should be explored clinically as a predictive clinical biomarker in patients undergoing IGF-1R/IR-directed cancer therapy.

Molecular imaging metrics to evaluate response to preclinical therapeutic regimens.

Molecular imaging comprises a range of techniques, spanning not only several imaging modalities but also many disease states and organ sites. While advances in new technology platforms have enabled a deeper understanding of the cellular and molecular basis of malignancy, reliable non-invasive imaging metrics remain an important tool for both diagnostics and patient management. Furthermore, the non- invasive nature of molecular imaging can overcome shortcomings associated with traditional biological approaches and provide valuable information relevant to patient care. Integration of information from multiple imaging techniques has the potential to provide a more comprehensive understanding of specific tumor characteristics, tumor status, and treatment response.

Quantitative, preclinical PET of translocator protein expression in glioma using 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline.

UNLABELLED: Translocator protein (TSPO), also referred to as peripheral benzodiazepine receptor (PBR), is a crucial 18-kDa outer mitochondrial membrane protein involved in numerous cellular functions, including the regulation of cholesterol metabolism, steroidogenesis, and apoptosis. Elevated expression of TSPO in oncology correlates with disease progression and poor survival, suggesting that molecular probes capable of assaying TSPO levels may have potential as cancer imaging biomarkers. In preclinical PET studies, we characterized a high-affinity aryloxyanilide-based TSPO imaging ligand, 18F-N-fluoroacetyl-N-(2,5-dimethoxybenzyl)-2-phenoxyaniline (18F-PBR06), as a candidate probe for the quantitative assessment of TSPO expression in glioma. METHODS: Glioma-bearing rats were imaged with 18F-PBR06 in a small-animal PET system. Dynamic images were acquired simultaneously on injection of 18F-PBR06 (70-100 MBq/0.2 mL). Over the course of scanning, arterial blood was collected to derive the input function, with high-performance liquid chromatography radiometabolite analysis performed on selected samples for arterial input function correction. Compartmental modeling of the PET data was performed using the corrected arterial input function. Specific tumor cell binding of PBR06 was evaluated by radioligand displacement of 3H-PK 11195 with PBR06 in vitro and by displacement of 18F-PBR06 with excess PBR06 in vivo. Immediately after imaging, tumor tissue and adjacent healthy brain were harvested for assay of TSPO protein levels by Western blotting and immunohistochemistry. RESULTS: 18F-PBR06 was found to preferentially accumulate in tumors, with modest uptake in the contralateral brain, facilitating excellent contrast between tumor and adjacent tissue. Infusion with PBR06 (10 mg/kg) displaced 18F-PBR06 binding by approximately 75%. The accumulation of 18F-PBR06 in tumor tissues and adjacent brain agreed with the ex vivo assay of TSPO protein levels by Western blotting and quantitative immunohistochemistry. CONCLUSION: These preclinical studies illustrate that 18F-PBR06 is a promising tracer for visualization of TSPO-expressing tumors. Importantly, the close correlation between 18F-PBR06 uptake and TSPO expression in tumors and normal tissues, coupled with the high degree of displaceable binding from both tumors and the normal brain, represents a significant improvement over other TSPO imaging ligands previously evaluated in glioma. These data suggest the potential of 18F-PBR06 to elucidate the role of TSPO in oncology, as well as its potential development as a cancer imaging biomarker.